Disposable bioprocess system supporting biological activity

Abstract

To provide a disposable bioprocess system supporting biological activity.SOLUTION: A disposable bioprocess system has a liquid loop to be selectively validated, the liquid loop returning a culture liquid to a culture liquid reservoir 12d through a filter device 16 to the culture liquid reservoir under action of a pumping device 15, wherein: the filter device has a liquid-moving device for making a selected component from a liquid volume through a membrane to another liquid volume; the liquid-moving device has a membrane-cleaning mode for cleaning the membrane at a speed of the culture liquid, which is faster than the speed of a culture liquid for obtaining a product; a flowing direction of the culture liquid in the membrane-cleaning mode is identical to a flowing direction of the culture liquid for obtaining the product; and in the membrane-cleaning mode, the liquid loop is validated to return the liquid loop having passed through the membrane to the culture liquid reservoir.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to the production of microorganisms suspended in a liquid medium at high microbial concentrations for the expression of biological substances. A single-use bioreactor, a single-use pumping device, and a and a single-use microbial-retaining filter, The present invention relates to a method for operating a single-use bioprocess in a continuous process. [Background technology]

[0002] In pharmaceutical bioreactors and fermenters, the goal is to develop new bioreagents for regenerative medicine and therapeutic applications or or the production of biological materials or biomass for medical uses such as medicines, or the production of biological cells or for more typical expression of molecules from microorganisms for therapeutic purposes or as drugs. The first step is to cultivate or ferment microorganisms.

[0003] Reusable, non-disposable stirred tank reactors (STRs) are used in bioreactors or Fermenters, which range in size from less than 1 liter to more than 200 liters, are stainless steel and made of high-quality glass. Typical volumes for laboratory-scale STR versions are: Available in 1 to 20 liters, with a glass housing under a removable stainless steel head plate The entire STR is suspended on a tripod metal structure, with the head plate attached to the External controls to ensure sensor, sampling, gas exchange, medium exchange, and stirring of the medium inside the STR. Multiple internal ports for centrally oriented shafts for connection of externally mounted servo motors Temperature is controlled by the STR's electric heating blanket or water jacket. do.

[0004] Disposable stirred tank reactors are new as recently as the mid-90s. Perhaps even out of fashion. Single-use bioreactors (SUBs) have matured. As a result, the demand for single-use equipment has grown rapidly in the market. The transition has, in many instances, addressed cross-contamination issues, cost savings on steam sterilization and cleaning requirements, and plastics. The motivation is to improve the flexibility of the client, reduce the cost of the final product and shorten the time to market. do.

[0005] Conventional technology Patent documents 1 and 2, both from Millipore Corp., describe rigid plastics. A rigid plastic housing body with a top cover and one or more fluid ports describes a pre-sterilized disposable bioreactor consisting of two parts: a main body; The top cover has an integrated sensor port and all ports have caps.

[0006] Patent document 3 by Corning Inc. - Impeller on a flexible axle and a turbulent flow generating device The container has a baffle with three integrated container walls and a cover without sensor ports. Disposable, symmetrical cylindrical plastic spinner flask container.

[0007] New Brunswick Scientific Fermenter Housing Body Model B The ioFlo310 consists of a metal jacketed bottom assembly, a glass cylinder, and a metal head. All ports except for the two water heating / cooling ports are located on the head plate. Further, a drive motor is disposed on the head plate.

[0008] Patent document 4 by Refined Technology Co. is a liquid filtration system The present invention discloses a cell retention system for high cell density culture. a culture storage container, a hollow fiber filter module, and a septum pump; In this system, the pumping device alternately operates to recirculate the medium without using a valve. do.

[0009] Patent document 5 by Artelis SA describes a disposable culture housing body. The cell culture medium is equipped with at least one outer wall with an integrated sensor, a high cell density cell reservoir, and The culture and transfer zones function as a maintenance system, and a vortex for medium recirculation. Equipped with a pump.

[0010] Patent document 6 by Sartorius Stedim Biotech GmbH One or more aeration tubes with syringes are provided next to the culture container. This document describes a bioreactor that can

[0011] ·DASGIP Information and Process Technol Patent document 7 by ogy GmbH is designed to fit parallel blocks (see Figure 5). (Reference) describes a small disposable bioreactor (STR). This product controls the temperature and also drives the stirring device. Designed to operate four STRs or SUBs in parallel, housed in the bottom of the STR container. will be done.

[0012] Operating a stirred tank reactor in semi-continuous process mode / perfusion mode at high cell densities Additional techniques and equipment, such as cell retention systems, are required for the stirred tank reactor. An externally mounted autonomous alternating flow oriented pump and a cross-flow filter When combined with the s-Flow-Filter device, it can be used for medium to high cell densities and It has been shown to provide excellent results in perfusion mode operation for several weeks. Refined Technology, a well-known and dominant technology company in New Jersey, USA. Alternating Tangential Flow (Alternating-Tangential Flow) The l-Flow concept allows for high cell numbers and the harvesting of a cell-free product. All ATFs offered are reusable stainless steel and glass designs, single-use It is not available as a complete setup for use.

[0013] Despite increasing demand from industry for serial processing and rapid multiplex testing, A stirring tank with an integrated pump and cell retention system in a pre-sterilized, single-use package. The development of reactors has surprisingly not been pushed forward. Replaces traditional glass and steel-based ATFs that require expensive and time-consuming steam sterilization. Reuse of any STR or ATF parts that must be sterile is prohibited. This involves the subject of contamination and costly culture loss. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] U.S. Patent No. 2009 / 0311776 [Patent Document 2] European Patent No. 2141224 [Patent Document 3] U.S. Patent No. 2008 / 0131957 [Patent Document 4] U.S. Patent No. 6,544,424 [Patent Document 5] International Publication No. 2010055143 [Patent Document 6] U.S. Patent No. 7,425,441 [Patent Document 7] European Patent No. 2674480

[0015] Definitions relevant to the present invention The terms "a" or "an" as used herein mean one or more, Or at least one of them.

[0016] The term "alternating" as used herein refers to a bidirectional liquid flow. A specific identical volume moves forward and backward, and the flow direction Valves for controlling the

[0017] The term "ATF" as used in the industry and herein means Alternating Tangential Force This means Alternating Tangential Flow (ALF), , the rated volume of the pump is constant, and this volume alternates, i.e., forward and backward (forward and backward). The concept is that the membrane can only move between two extreme positions, creating a new No fresh fluid or culture medium is added directly to the pump and no valves are included.

[0018] The term "backflow" is used to refer to the return flow of a small amount of material. This increases TMP, or Trans Membrane Pressure. This helps to some extent with the high velocity flow along the membrane to remove the deposits. Although widely used in the membrane industry, "hollow fiber modules" The term "backflush" is not appropriate for elastic membranes such as a "bre module." This should not be confused with "kflush".

[0019] The term "batch operation" is used herein This refers to a method of operation with constant WV, where fresh medium is not added, but used medium and / or Or the liquid is not removed, typically for less than a week when culturing microorganisms such as CHO cells. It takes.

[0020] The term "biomaterial" as used herein means any biological material. Organic compounds, tissues, cellular components, biocompatible fluids, biomass, biocomposites, biocompatible materials This refers to general antibodies, DNA, RNA, proteins, molecules for therapeutic purposes, etc.

[0021] The term "bioreactor" as used herein means The biologically active environment is created by the microorganisms suspended in a liquid medium agitated by an impeller. It refers to a physical device suitable for carrying out the desired process for cultivation.

[0022] The term "culture medium (broth)" as used herein refers to the unfiltered liquid content, The culture broth in the fermenter or bioreactor contains cells, debris, microorganisms, nutrients, The culture medium and raw materials enter the CFF and are then cross-flowed. - Passes through the filter membrane, which results in the culture medium being slightly concentrated at the outlet of the CFF. This becomes a retentate and is returned to the bioreactor, where it is It is remixed with the culture medium.

[0023] The term "CHO" refers to Chinese Hamster Ovary. Ovary cells, which are used as a protein expression platform in the pharmaceutical industry. The most common types of cells are "microbial" and mammalian cell lines.

[0024] The term "container" as used herein means a hollow Housing means a body with an internal reservoir, which may be open or closed, e.g. For example, but not limited to, beakers, flasks, bottles, tubes, vessels A polymeric film bag having walls that form a tank or reservoir. For example, when operating as a stirred tank reactor, the reactor typically has vertical walls and a downward facing and a horizontal bottom wall, so that the liquid or fluid in the container is During operation, the container is primarily maintained inside a cylindrical or conical design. non-cylindrical designs such as cubic designs, flexible film bags, or combinations thereof The container may be of any design, including a SUB, SUP, or CFF. That's fine.

[0025] The term "Container Volume (CV)" is used herein to mean When used in a bioreactor or fermenter or as a mixer, the housing This refers to the total volume of the container itself.

[0026] The term "Cross-Flow-Filter (CFF)" As used herein, a filter device is a separation device that separates a liquid and a selectable component suspended in a liquid, into the separation device having a liquid volume. The desired suspended components can be passed through the opposite end of the device, which allows the desired suspended components to pass through the The device has specific specifications that allow it to be removed without passing through the separation device. The device has a first inlet for the culture medium and a second inlet for the concentrate. Between the first culture medium inlet and the second concentrate outlet, there is a product, a filtrate, There is a third permeate outlet for harvest that has passed through the device.

[0027] The term "cultivation" or "culturing" means Microorganisms, such as mammalian cells, are cultured in a manner that allows expression of a product by the microorganism or growth of the microorganism. This refers to the process of keeping the cells in a bioreactor for the purpose of survival.

[0028] The terms "deposit" or "membrane coating" The terms "filtering" or "filter cake" are used herein. When a filter is used, solutes, particles, and microorganisms are deposited on or further onto a membrane surface, such as a cross-flow filter membrane surface. In the membrane pores, the membrane performance is degraded and the transmembrane pressure is increased to an undesirable level. This refers to the process of depositing materials in such a way that they are easily absorbed by the recirculating medium for cleaning purposes. It can be removed by increased shear forces, such as high velocities of the nutrient solution.

[0029] The term "diaphragm" as used herein means a circular or A sheet of rubber material that is roughly dome-shaped and preferably has a sealing structure on the periphery. The membrane operates within the housing, separating the side wetted by the culture fluid from the SUB, and the side wetted by the driving fluid. Separate from.

[0030] The term "disposable" refers to a product that is preferably low cost and can be broken down after use. Disposable refers to products that are often made of synthetic materials. The bioreactor system is then packaged and pre-sterilized in preparation for use.

[0031] The term "exterior facility (area)" means a laboratory, production facility, or testing facility where a single-use bioprocessing system is used. Downstream processing may also be carried out at external facilities.

[0032] The term "fed-batch" operation, as used herein, refers to Start the process with a small medium volume and add fresh medium to complete the process. This refers to a bioreactor or fermenter in which the liquid is not removed until the minimum culture volume within the housing body is reached. The soil volume is inoculated with microorganisms and passed through the seed column ( Typical operation times are 2-3 times longer than batch operations.

[0033] The term "fermenter" as used herein refers to a vessel in which a liquid medium is cultured. The physics favor the fermentation of microorganisms that undergo fermentation when suspended in a medium and agitated by a turbine. This refers to a device or container.

[0034] The term "fermentation" as used herein refers to the industrial It refers to the maintenance of target living microorganisms such as unicellular organisms, prokaryotes, and bacteria in a fermenter. The product is expressed.

[0035] The term "filter device" is used herein In this case, it refers to a cross-flow filter, also known as a CFF.

[0036] The term "fluid" refers to a gas or liquid, i.e., air or nitrogen with a variable volume. a gas such as water or a liquid such as water and / or oil at a constant volume, or a mixture of gas and liquid Point.

[0037] The term "glass" as used herein refers to a transparent silica-based Refers to amorphous, brittle, solid materials that often have excellent corrosion resistance.

[0038] The term "harvest" as used herein refers to the bioreactor. The predicted product produced by a microorganism cultured in a tank or fermented in a fermentor. The product (e.g., protein) of the culture medium is the product of the membrane. and / or CFF filtration. The permeate is removed from the CFF. , the culture medium is concentrated.

[0039] The term "hollow fiber module" is used in As used herein, porous elastic material such as polyethersulfone or other polymers A bundle of thin-walled tubes made from a material having an outer rigid-wall tube with an inner end cover. The term refers to a device made from a bundle of tubes that are sealed and molded into end covers. This separates the inner tube from the outer tube, thus forming a cross-flow filter. It is attractive as.

[0040] The term "impeller" refers to a wheel used for stirring, mixing, pumping, and circulating liquids. , a low-speed fluid agitator with multiple blades or vanes that rotates in a liquid-filled container Refers to the device.

[0041] The term "liquid suction tube" is used in this specification. When used in this document, it refers to a liquid inlet, pipe or refers to the tubing. The liquid suction tube is used to draw liquid through the inlet valve to the pumping device. Transport to the inlet.

[0042] The terms "media", "growth media", "nutrient "nutrient" as used herein are used interchangeably. Most of the ingredients are water, carbon sources, various gases such as oxygen, as well as vitamins, hormones, and nutrients. Growth factors, animal serum, antibiotics, antioxidants, antifoaming agents, cell stabilizers and for culturing "microorganisms" Refers to a sterile, complex mixture containing additives such as serum and other ingredients for the treatment of ulcerative colitis. Based on several different ingredients, some of which are serum-free, animal-free, protein-free, and protein-free. During cultivation, the medium and the microorganisms and various fats The combination of these is called a "broth."

[0043] The term "membrane" refers to the boundary layer, which acts as a selective barrier. and when acted upon by a driving force (such as that provided by a pump), The porous membrane may be made of a polymer, ceramic, or other material that remains impermeable to the desired particles, molecules, or substances. They are made from a variety of flexible and rigid materials, including plastic and metal. Cross-Flow-Filter (CFF) Device The membrane allows a portion of the culture medium to clear, known as the harvest.

[0044] The term "membrane fouling" is used herein In this case, solids, cells, cell parts, cell membranes, aggregates, etc., form layers, biofilms, and debris cakes. This refers to the effect of deposits on the porous membrane inlet surface. This effect is caused by a combination of the membrane and the deposits. Higher TM to overcome increased resistance to mating and maintain constant flow when needed Membrane deposits can occur due to high shear forces, high broth velocities, and / or backflushing. can be removed by

[0045] The term "microcarrier" refers to attachment-dependent microorganisms. It refers to a microbial support device or growth medium that allows the cultivation of microorganisms, which may be made of gelatin, collagen, or other materials. They are made of polyethylene, cellulose or polymeric materials or glass, and typically range in size from 1 00-1000 μm and can be further functionalized with one or more coatings.

[0046] The terms "microorganism" or "cell" or "biological "Biological cell," as used herein, refers to a cell that is capable of interacting with other cells. They are used interchangeably and are typically divided into: 1. Fungi, algae, and mosses , plankton, yeast, protists, eukaryotes, archaea, microfauna, extremobacteria and plant cells 1. Living unicellular organisms, bacteria, etc.; 2. Animal cells, insect cells, mammalian cells 2. Adherent, semi-adherent or suspended biological cells such as human cells, stem cells; 3. E. coli and various bacteria. Most of these serve specific purposes and product needs. They are genetically modified to do so.

[0047] ·The term “perfusion” or “cell / microbe retention perfusion” cro-organism retention perfusion) mode operation As used herein, the medium is replaced sequentially throughout the culture period to provide fresh nutrients. Adding ingredients sequentially and removing used medium / harvest, operating method for STR, SUB refers to the principle that microorganisms retained in STR and SUB can survive for 4 to 8 times longer than in the batch. The cells survive 2 to 4 times longer than fed-batch cultures.

[0048] The term "permeate" as used herein refers to a mixture, culture medium, or The specific portion of a nutrient solution or feedstock that can pass through a membrane. Also known as filtrate.

[0049] The term "permeable membrane" is used herein. When used in a membrane, it refers to a porous wall, and a "membrane" refers to a membrane that allows the passage of liquid and selected particles ( size separation) through the membrane and specific particles are separated by porous membranes, barriers, Avoid passing through separation walls.

[0050] The term "port," as used herein, refers to a suitable attachment or connection. Holes anywhere in the wall to allow for the installation of associated sensors or general connections Refers to a port selected from the following group: PG13,5 thread port, locking port, pressure Inlet ports or luer lock fittings, connecting fittings, sterilization fittings, hoses, tubing, Ports related to hose barbs, etc.

[0051] The term "PG13,5" refers to conventional sensors with threaded mechanical connectors (RUS ) or port. PG stands for Stahl‐Panzer‐Rohr‐Gewinde It is a well-known technical standard term.

[0052] The term "PCS" or "Process Control System" "Programmable logic control system" as used herein means a programmable logic control system. A central processing unit (CPU) with computing power, Central Processing Unit (CPU) is an electronic device that Embedded personal computers (PCs) and PLC refers to an embedded computer (EC). , for inputs from various sensors and various actuators for process control purposes. The PCS has various inputs and outputs (I / O), with software, algorithms, and The system integrates the process recipe in the built-in memory, which For the control and analysis of culture or fermentation processes, for the alignment of ongoing processes according to The PCS uses a variety of sensors and actuators to vary the process parameters. It corresponds to the computer.

[0053] The term "PTF" as used herein is new industry terminology; The invention presented herein, Pulsating Tangential Flow The PFT process goes through the CFF while the A port having a first inlet valve and a second outlet valve to ensure unidirectional liquid flow. Related to pumps.

[0054] The term "retentate" as used herein refers to a membrane. and do not pass through the membrane due to their size, shape or charge. Refers to parts, microcarriers, particles, "microorganisms," and debris of a mixture.

[0055] The term "scalable" as used herein means a CV and WV refers to the characteristics when it is not limited to fixed glass container dimensions. The ratio between the diameter and height of the rotor can be changed to suit the requirements of the end user. This refers to the following.

[0056] The term "sensor" as used herein refers to a sensor that measures the activity of a given process. Relevant operational quality process variables, e.g., pH level, dissolved oxygen ed oxygen (DO), biomass / cell density, capacitance, conductivity, lysis Carbon dioxide, lactate, glucose, glutamine, glutamic acid, ammonia, pressure, liquid level, flow This refers to a device that can measure mass flow, velocity, temperature, viscosity, etc. The sensor for this purpose may be a proximity sensor, and the distance may be mechanical, optical, or electrical ( For example, a triangular laser sensor, a pressure sensitive level sensor, or a capacitance or ultrasonic Sensors are generally available as reusable or single-use sensors. Available.

[0057] The term "single-use" as used herein means one time use. Products designed for use only and to be discarded after use, typically referred to as "sterile" are delivered "sterilized" and ready for use, e.g., "single-use" Single-Use Bioreactor (SUB) and Single-Use Bioreactor (SUB) The sensor is a single-use sensor (SUS).

[0058] The term "single-use sensor (SUS)" as used herein refers to a sensor that contains an analyte, a protease, or a The process conditions and fluid concentrations can be measured during operation and a signal such as an electrical signal related to the measured concentration can be generated. It refers to a disposable device that can transmit. Inexpensive SUS bodies are mainly double or triple film Pre-installed in SUB or SUF for simultaneous sterilization while all items are contained within the bag It is designed from polymer materials so that it can be easily attached to the body, which is convenient for the end user and This allows end users to skip sterilization before use.

[0059] The term "single-use bioreactor (SUB)" as used herein refers to a ST R or bioreactor or single-use fermentor, and preferably includes a pumping device and one or one or more film bags pre-installed with multiple "single-use sensors" All of these are made from disposable materials and are sterilized and ready to use. This eliminates the traditional home heat sterilization process.

[0060] The term "Single-Use-Pump (SUP)" is used herein to mean a pump. Fluid transfer devices fabricated at least in part from disposable materials when used in For example, peristaltic pump, centrifugal pump, tube pump, diaphragm pump, or piston pump. pump or direct gas-liquid surface-driven air column pump, etc., with disposable wetted parts and It has a reusable non-wetted portion and a valve for controlling the flow direction.

[0061] The term "stainless steel" is used herein When used, it is based mainly on nickel, chromium, vanadium, carbon and iron, and contains at least Refers to an alloy metal characterized by excellent corrosion resistance.

[0062] The term "sterilization" as used herein means Eliminate (remove) or kill (inactivate) any form of life or other biological agent, Sterilization can be achieved by one or more of the following: heat, chemical Materials, irradiation, high pressure and filtration.

[0063] The term "sterilized" as used herein refers to a plastic enclosed in a plastic film bag with a sterilizing agent to ensure the contents of said bag are sterile. This refers to the product that has been subjected to the sterilization method. This product is placed in one or more of the above film bags. and supplied to the end user ready to open and use. The laser does not require the same tedious heat sterilization process as conventional reusable equipment that uses an autoclave. It can be avoided.

[0064] The term "Stirred Tank Reactor (STR)" " is a widely used expression and as used herein refers to the nutrients and An aeration device for forced exchange of gas with "microorganisms" and one or more "Container Volume" incorporating a stirring or mixing device STR refers to a bioreactor or fermenter equipped with a stainless steel and It is made from glass.

[0065] The term "suspension" or "suspended" " as used herein refers to the liquid (culture medium) in a STR or SUB or "container." particles, artificial particles, microcarriers, preferably uniformly suspended or concentrated in a nutrient solution; Refers to microorganisms.

[0066] The term "Tangential Flow Filter" "Teflon (TFF)" as used herein refers to the process of subjecting selected components to a Transfer from one liquid volume to another across a "membrane" (crossflow filter) Some membranes are used in devices that remove some components as they pass through the membrane. Alternatively, the filter device may be incorporated into a cassette or may be a flat or rounded plate. formed in a cartridge as plates, tubes, corrugated tubes or laminated hollow fibers It may also be based on a screen or a sheet of porous material.

[0067] The term "top cover" as used herein means the upper This typically refers to the head plate, e.g., a flat metal disk for a conventional STR. The top cover has been manufactured by manufacturers including, but not limited to, Satorius, Ap Sourced from plikon, Finesse, Broadley-James and other sources Available in PG13, PG14, PG15, PG16, PG17, PG18, PG19, PG20, PG21, PG22, PG23, PG24, PG25, PG26, PG27, PG28, PG29, PG30, PG31, PG32, PG33, PG40, PG41, PG42, PG43, ,5The number of ports is affected by the diameter of the upper cover and is generally 3 to 4 ports. The top cover may take a shape other than a flat disk and may include one or more ports. That's fine.

[0068] The term "TMP" or "Trans-Membrane-Pressure" " as used herein refers to membrane fouling, the accumulation of cells, debris, particles on the membrane surface. It is a good indicator of membrane fouling at a given flow rate as TMP increases. Compensate for:

[0069] The term "Working Volume (WV)" is used herein. If so, the medium volume, process flow rate, and It also refers to the head space volume. " + usable volume = container volume (CV). [Brief explanation of the drawings]

[0070] [Figure 1] Figure 1 shows the complete PTF operating the SUB incorporating the SUP and CFF. [Figure 2] FIG. 2 shows a tube passing through the vertical side wall of a container. [Figure 3] FIG. 3 shows a tube passing through the vertical side wall of a container. [Figure 4] FIG. 4 shows the tubing connected to the one-way valve and pump. [Figure 5] FIG. 5 shows the present invention installed in a parallel operating Bioblock. [Figure 6] FIG. 6 shows a PTF setup with a diaphragm SUP between the SUB and the CFF. [Figure 7]FIG. 7 shows a PTF pump with valves and a crossflow filter installed. [Figure 8] FIG. 8 is a possible flow and process diagram. Summary of the Invention [Means for solving the problem]

[0071] The present invention relates to a disposable bioprocessing system for supporting biological activity, which comprises: A process liquid culture enclosed by a wall that separates the internal process liquid volume from the external facility area a liquid-tight container having a process liquid volume, the process liquid volume being connected to a first liquid valve and a process a conveying liquid pumping device for pumping a process liquid in one direction away from an internal process liquid volume; a second liquid valve and a filter device separating the internal process liquid volume from the external facility area. The system also provides a means for extracting a liquid from the internal process liquid volume of the liquid-tight container. A liquid communication means to the liquid pumping device, and a liquid communication means from the pumping device to the filter device. a further unfiltered liquid communication means extending to the first unfiltered liquid communication port of the filter; into the internal process liquid volume of the container via a second liquid communication port of the filter device. and further means for recirculating the unfiltered concentrate liquid. The process fluid is then transported to the external facility area as a collected product. wherein said first liquid valve is , controlling communication between the internal process liquid volume and the pumping device, and the second liquid valve and a filter device outlet port. The port provides communication between the filter device and an external facility area. DETAILED DESCRIPTION OF THE INVENTION

[0072] An object of the present invention is to provide a disposable bioprocessing system that supports biological activity. This is surrounded by a wall that separates the internal process liquid volume from the external facility area. a liquid-tight container having a process liquid culture volume containing a first a liquid valve for pumping the process liquid in one direction away from the internal process liquid volume; , a first liquid valve and a conveying liquid pumping device, a second liquid valve, and an internal process liquid The body volume is in communication with a second liquid valve and filter device that separates the body volume from the external facility area. The system also includes a process liquid volume (12d) of the liquid-tight container (12a) for discharging the liquid from the process liquid volume (12d). a liquid communication means from the pressure-feeding device (15) to the liquid pressure-feeding device (15), a further unfiltered liquid communication port extending to the first unfiltered liquid communication port of the filter device (16); and a second liquid communication port (16a) of the filter device. a further means for recirculating unfiltered concentrate liquid into the internal process liquid volume of said container; The filter device comprises means for separating the filtered process liquid from the collected product. and at least one third permeate outlet port for transport to said external facility area. wherein the first liquid valve is connected to an internal process liquid volume and pressure the second liquid valve controls communication between the pumping device and the filter device; The filter device outlet port controls communication between the filter device and the It provides communication with external facility areas.

[0073] The present invention relates to a disposable bioprocessing system for the continuous processing of microorganisms in the pharmaceutical industry. This system includes a single-use bioreactor (SUB), a single-use pump (SUP), and and a single-use CFF-based microbial retention device. When operated according to the "Sensitive Tangential Flow" The PTF provides end users with the following benefits, freedoms and features: To reduce costs and increase laboratory throughput, the PTF of the present invention Pre-assembled, pre-sterilized and ready to use right out of the box This eliminates the need for traditional, time-consuming steam sterilization; Pre-assembled and pre-sterilized components useful for both research and development and full-scale production The concept eliminates cross-contamination; PTF cell retention system is used for SUP and CF inside the SUB or outside the SUB container. Integrate F and thereby parallel block or robot design for desired perfusion mode process. Installation in a vise is also possible, minimizing installation time; The working fluid in the SUP (which may be separated by a diaphragm or piston) CFF culture medium for different harvest and wash cycles with sensors measuring the temperature Non-parallel control of velocity and volume; Disposable bioprocessing systems allow for multiple microorganisms to be processed with significant savings in materials and labor. This allows for rapid evaluation of biological cultures and improved productivity of cell line expression.

[0074] The pharmaceutical industry could potentially use DASGIP parallel-operated bioblocks or fully automated TAP balances. Io Systems (currently supplied by Sartorius Stedim Biotech) In parallel processing using a fusion reactor, or in separate implementations of serial processing, improved volume efficiency, and on-the-job analyst measurements, all aimed at microbial retention in perfusion mode operation. They want a SUB platform that supports single-use designs with the capabilities The disposable bioprocessing system product as presented herein is currently So for some reason it doesn't exist.

[0075] The present invention provides a SUB for the cultivation of biomass and selected components or a SU for fermentation. Regarding the single-use bioprocess with F, this has the following advantages (briefly described below): (to be) equipped with: A hollow plastic container with an internal reservoir, optionally with one or more sidewall ports. It has various ports that are mechanically connected to or are an integral part of the antenna. This container can be of any diameter or cross section (e.g. rounded design, cylindrical a cylindrical design (or a non-cylindrical design such as a cube) and height to accommodate the desired internal reservoir and volume. Provide the product; An optional draft tube stator disposed vertically within the container. This is the outer space of the draft tube stator. The fan has no baffles or one or more baffles arranged vertically and radially, and The raft tube stator has a fluid motion stirring means housed in the internal space. or a plurality of internal reservoirs, a fluid agitation device for generating a radial vortex of the medium, and The stator converts this into axial fluid motion and axial vortex for more efficient mixing; A SUB is disposed vertically within the container and extends inward from the side wall of the container. Works with any baffle; Container bottom for magnetic force transmission through the bottom wall driven by external magnetic means a rotating shaft attached to a component having a magnet and a bearing in contact with the rotating shaft; Fluid motion agitation device; A rotating shaft that passes through the top of the container via a bearing / seal arrangement for power input. a fluid motion agitation device disposed on the - Aims to provide an additional gas mixture to ensure safe aeration of the cells a tube disposed vertically within said container; - Fluid communication between the culture medium in the container and the single-use pump device. is a liquid conveying and / or liquid direction control device for facilitating pumping operations. The SUP device may be located within the container or external to the container; Containers, single-use clotting agents, and clotting agents to promote favorable microbial retention under perfusion mode operation. and one or more flow filter devices, i.e., the CFF devices. Constitutes a liquid conveying and / or liquid pumping device and a liquid direction control device. The plurality of CFF devices may be disposed within the container or external to the container; Considered disposable and sterile for cost-effective single-use applications The assembly is fabricated from materials that are supplied to the end user in a sealed state.

[0076] The dimensions of the SUB container depend on the preferred size of the BioBlock or robot assembly in which the STR can be placed. The preferred dimensions may correspond to, but are not limited to, these.

[0077] The container consists of an outer wall and two essentially flat end covers, a top cover and a bottom cover. The container and housing assembly have dimensions of 10 to 15 mm in diameter. 500mm, for example, 20-200mm, and height 10-1000mm, for example, 50-50 0 mm. Alternatively, if you measure the cross section of the container, it will be 1 to 2000 cm2. Alternatively, CV can be measured from a few milliliters up to 2000 liters.

[0078] The disposable bioprocessing system of the present invention further comprises a relay for adjusting the process means. Hollow bodies that act as reservoirs, cross-flow filter devices, aeration devices devices, instruments, sensors, liquid transfer devices, valves, actuators, fluid accumulators, etc. Extending simple batch operation, such as one or more of the above, to more advantageous perfusion mode operation. It also includes a variety of additional devices and features for:

[0079] Furthermore, the disposable bioprocessing system of the present invention is , single-use bioreactors, liquid transfer or pumping devices, and cross-flow filter devices Such CFFs are rigid structures that incorporate thin microporous membranes into honeycomb modules. They may be fabricated from a flexible, semi-permeable, flat or porous ceramic support. C may be made from rounded membranes or hollow fibers bundled in a cartridge. The FF may be placed inside the SUB in liquid contact with the process liquid medium or outside the SUB. The pump is located adjacent to the SUB and is discharged from the SUB above or below the level of the container culture medium. The CFF is placed in liquid contact with the process liquid medium. The liquid supply is delivered by a liquid transfer device from a SUB process liquid reservoir to a valve means. through the CFF concentrate channel and into the concentrate compartment, which is entirely internal. The liquid delivery inlet and CFF outlet are connected to the concentrate compartment. After partially or fully closing both valves, the liquid transport occurs across the CFF membrane to generate TMP. This allows for the isolation of at least a portion of the predicted desired protein product from the process. It is suspended in a medium and passed through a membrane, converting it into a permeate and harvest. The remaining microorganism-rich liquid and suspended debris in the tank are simultaneously converted into a concentrated liquid. The water is then flushed back through the CFF outlet to the reservoir at a rate suitable for liquid transport operation. do.

[0080] The permeate side of the CFF is exposed to a pressure preferably less than the liquid pressure of the concentrate, thereby In fact, the force on the permeate side reduces the flow of liquid through the membrane. It is desirable to help overcome the TMP of semipermeable membranes due to limitations, which are primarily due to typical Specifically, this is caused by membrane fouling and deposition from the culture medium on the concentrate side of the CFF. can be.

[0081] The disposable bioprocessing system of the present invention supports many requirements and is a basic component This facilitates the use of a variety of rigid, semi-rigid or flexible products in the fabrication of the device. Materials such as polymers for forming the components include, but are not limited to, polycarbonate. Polyester, nylon, polyamide, PTFE resin and other fluoropolymers, Acrylic and methacrylic resins and copolymers, polysulfone, polyethersulfone, poly Aryl sulfone, polystyrene, polyetherimide, polyethylene terephthalate , polyvinyl chloride, chlorinated polyvinyl chloride, ABS and its alloys and mixtures, polyolefin Fins, preferably polyethylene (e.g., linear low-density polyethylene, low-density polyethylene , high density polyethylene, ultra-high molecular weight polyethylene and their copolymers), polypropylene and copolymers thereof, and metallocene-produced polyolefins, or and semi-rigid materials such as silicone, rubber and other elastic materials.

[0082] The individual basic components of the container are manufactured by or assembled from: Can be: Injection molding, blow molding or vacuum thermoforming of basic parts; · Welding of basic components; · Molding small pieces of basic parts by injection molding process; One or more elastic components such as an O-ring, flat washer, or bulkhead Toma elements (nitrile rubber, silicone rubber, Viton rubber, latex rubber, EPDM or other elastic material) between the assembly surfaces to seal the basic components; - Basic parts between assembly surfaces with adhesives such as UV curable glue or epoxy sealing; or · Combinations of these.

[0083] In a first embodiment, the disposable bioprocessing system of the present invention includes the following in the liquid loop: The present invention provides a method for the preparation of a microorganism-retaining perfusion system using a disposable material. Provides a container that: a sensor for measuring a process parameter, the sensor being arranged on the outer wall of the first container; Various ports established; Additionally, a liquid suction tube for connection to a second container, which acts as a SUP, for example. Various tubes, hoses, and ports pass through the exterior wall, some of which are into and out of the first container interior volume for various fluid communications with external devices. extending from the container interior volume; Further, the first container may include a second tube having a suitable internal volume and acting as a SUP. a second tubular container having a first inlet port and a second an outlet port, and the container is closed with a bottom cover and a top cover; Equipped with a liquid level sensor; Furthermore, the first container and the second container arranged in a direct current state with respect to the liquid, The CFF device includes a housing, a microporous inner separation wall, a first an inlet, a second outlet and a third harvest port; Furthermore, the first container is provided with the first and second containers and the CFF device. a first valve for unidirectionally directing the culture medium from the first container to the second container; A second valve transports the culture medium in one direction to the CFF device, and a second valve transports the culture medium in one direction to the CFF device. It is accompanied by a fluid connection that returns to the container and closes the fluid loop.

[0084] In a second embodiment, the single-use bioprocessing system of the present invention comprises: Provide a first container suitable for bioperfusion mode: Outside the first container for mounting sensors to measure various process parameters Various PG13,5 ports arranged in the wall; Furthermore, various tubes and hoses pass through the outer wall of the first container, some of which Some of the fluid communication with external devices is to the interior volume of the first container. and extending from the interior volume of the first container; A culture medium suction device to transfer the culture medium to a second container that acts as a SUP. A port disposed on the outer wall of the first container for further mounting a pull tube. an inlet of the medium from the first container with a suction tube inside the container; The penetration of the first container into the upper part of the outer wall is independent of the penetration of the first container into the upper part of the outer wall of the first container. The process system is also independent of the physical dimensions of the first container, which is a beneficial feature of the present invention. characteristic; Further, the first container may include a second tube having a suitable internal volume and acting as a SUP. a second tubular container having a first inlet port and a second an outlet port, and the container is closed with a bottom cover and a top cover; Equipped with a liquid level sensor; Furthermore, the first container and the second container arranged in a direct current state with respect to the liquid, The CFF device includes a housing, a microporous inner separation wall, a first an inlet, a second outlet and a third harvest port; The second outlet port of the CFF delivers the treated liquid via a tube to the first container. and complete the PTF concept; · The third outlet of the CFF is considered a harvest port; SUP and CFF containers arranged independently of each other; A liquid-agitating impeller or turbinate disposed on a shaft within the reservoir of the first container. The shaft is driven in rotation by external means.

[0085] A first liquid transfer variant of the disposable bioprocessing system of the present invention is a liquid culture medium transfer system. a second container disposed in series and fluidly connected with the first container, the second container operating as a transfer pump; The second container has an internal space for storing the first liquid culture medium and the second liquid culture medium. The process broth arrives from this container and is then transported to the drive gas compartment. The second container, a SUP, contains: A first inlet for delivering liquid from a first container, a liquid culture medium reservoir. valve; A second outlet port that delivers liquid culture medium to the first inlet port of the CFF device. Rett valve; A series of, but continuous, one-way movements (as opposed to alternating fluid movements, which are bidirectional fluid movements) The level of the culture medium conveyed by the pumping device due to the reciprocating action of the liquid in the direction of the movement of the culture medium. The volume and velocity of the culture medium are continuously adjusted by an external drive gas control means and a corresponding drive gas connection. Continued; Liquid level or volume measurement sensors, e.g., internally or externally arranged single-use A SUP activity sensor, such as a sensor or a reusable sensor.

[0086] The second outlet side of the CFF device corresponds to an optional third valve, and the outlet of the third valve The tray accommodates the first container, the process medium reservoir, and closes the liquid loop. The SUP container, valves and CFF devices are placed outside the SUB container. Two or more SUPs and CFFs may be installed in the same or different areas to promote the process. Any suitable combination and number may be integrated with or within a SUB.

[0087] If desired, separate the drive gas compartment and the liquid culture compartment in the second container. Between the compartments, there is a membrane, a diaphragm, a pith, one side of which is wet and the other side is dry or semi-dry. Separation devices such as centrifuges, discs, etc. may also be present.

[0088] For the start of the culture, add the desired amount of HCl to cover at least the sensors and stirring devices within the SUB. The medium is inoculated according to the desired recipe. The process then transitions from batch mode operation to maintenance perfusion mode operation.

[0089] The perfusion process of the present invention is a combination of several different operating modes and principles: 1. Culture mode (typically continued for several weeks in SUB); 2. Harvesting - Low in CFF 3. "Membrane conveying mode" at high speed; Debris removal - membrane cleaning mode at high speed in CFF mode).

[0090] The different independent operating modes may be switched off from one another at desired intervals as described herein. Followed by: The process liquid in the SUB is passed through a second pump that ensures a one-way fluid path from the SUB to the SUP. Corresponding to valve 1; The first valve corresponds to one associated liquid-transporting SUP; The liquid transport SUP transports the process medium transported from the SUB through the first valve. The volume of nutrient solution is variable and the pressure applied to the process broth is variable; The liquid transfer SUP transfers the process culture medium through a second valve to one or more of the above into the first inlet of the CFF device and further through the CFF inlet channel, transported unidirectionally (without alternating) across the membrane; - Only one or more of the above CFF devices are fed to the concentrate side at a certain volume and rate. and reducing the pressure to flow along the concentrate membrane side of the CFF device to the opposite side of the CFF device. Pass it through to the second outlet / end on the side; The treated fluid is returned to the SUB. In membrane cleaning mode, A quick flush removes membrane fouling and deposits, which are returned to the SUB; An optional third valve is provided after the concentrate / CFF outlet, which is at least partially closed. When the liquid delivery SUP is in the CFF device, the liquid delivery SUP is concentrated by one or more of the membranes of the CFF device. It is possible to apply a relatively high pressure to the liquid side, and the membrane is Separating organisms and other particles to prevent them from passing through the membrane and preventing the liquid from passing through the membrane If the hole is not opened, it is considered to be a harvested or harvested product, and the harvested product is passed through the hole. conveying; The harvested product is separated from the permeate side of the CFF device and collected externally. and forwarded to downstream processing; Extraction of a certain body mass from the culture medium (cell extract) in the scalable single-use bioreactor. By gradually removing the product as waste, rapid growth is reduced and the desired total biomass is obtained. Maintaining the body at the desired level; Add the corresponding volume of fresh medium to the first container, SUB, and replenish accordingly. do; Complete the cycle.

[0091] A short sub-batch culture time (1 week) and a small number of microorganisms can produce a large amount of biomass (5-10%). It is desirable to obtain a cell density of 10 x 10E6 cells / ml. The cell density is 20-100 x 10E6 cells / milliliter, which is preferably This is measured using a biomass sensor. Generally, higher cell densities result in more SUB. Higher productivity means longer incubation times, higher throughput, and more per cycle. This means less time is spent starting the culture. It takes 1 week + 1 week to clean up and restart the process. After 7-10 days of batch culture, the PTF process The 5-6 week continuous processing time allows for a 5-10x cell density increase compared to the batch. With this invention, it only takes one day to start the next process. Compared to the batch for PTF, PTF has a 30-fold increase in cell production and yield.

[0092] The benefits of the second container with an air column-driven liquid transport SUP (Figure 1) are: low shear stress on the cells; microcarriers are referred to herein as microcarriers or cell None of the cells can be handled without passing through the peristaltic pump; the setup is dead-end free. No issues with CFF clock settings as it is not a cross-filter system; absence of fluid flow; simplified operation; good control of fluid velocity; and most importantly and SUP devices can be designed from plastic and used as disposable bioprocessing systems. The simplicity and low cost of the device allow it to be fully integrated and sterilized.

[0093] The general conditions for the first container, SUB, are to maintain a single microorganism in suspension. or agglomerating large numbers of microorganisms into colonies maintained in suspension, or The goal is to attach the microorganisms to microcarriers that are maintained in suspension.

[0094] A third embodiment includes one or more containers, which may be used to store the containers if desired. The first container has a different diameter and height and an internal space volume. and the second container, and in the latter case, the second container. The tena is surrounded by a first container. The interior space of the first container contains microorganisms. The first container contains a process liquid culture medium, and the second container is a SUP that transports the culture medium. The third, or optional fourth, container is a CFF device. The second container contains an air-driven SUP pumping reservoir. a first process medium inlet, and a second process medium outlet. The first process liquid inlet of the SUP is connected to the first SUB container via a first valve. The process liquid flows through a second valve corresponding to the first reservoir. A second process fluid outlet corresponds to the first inlet on the culture fluid side of one CFF device. The CFF device conveys the culture medium to a second outlet for the concentrate, The CF device is used to extract the sample from all sterile setups. To this end, the microorganism-free liquid is discharged from the third outlet of the permeate through an appropriate connection with an external means. and transport it.

[0095] A controlled sterile driving gas volume and pressure is provided to the headspace of the second SUP container. By supplying a pressure regulator, the pressure in the headspace volume above the process liquid culture medium can be controlled. The pressure of the headspace drive gas is controlled to control the level of the process liquid. The level of the process fluid in one container is lower than the level of the process fluid in the second container. must be transported through the first valve into the interior space volume of the second container. So this is all controlled by the gas pressure in the headspace of the second container. Fluctuations in the gas pressure in the head space regulate the liquid level and, in conjunction with an optional third valve, allow for sampling. for removing deposits on the membrane surface on the concentrate side. At its concentrate outlet, the CFF provides a high-speed process fluid supply. a controlled third valve, which is connected to the process liquid reservoir of the first container. When the third valve is open, the high-velocity liquid is supplied to the membrane cleaning valve. The fluid is returned and transported back to the process fluid reservoir of the first container.

[0096] To operate the perfusion process, the first container contains the process liquid and the second container contains the culture liquid. At least one CFF must be maintained as a culture medium containing microorganisms suspended in the medium. By sequentially separating the microorganisms (or microcarriers) from the process liquid, Operates to maintain the material primarily within the interior space of the first container at any one time. One or more SUPs and one or more CFFs may operate in parallel if desired. and may operate to different specifications and with different control parameters.

[0097] End users can now rely on the only commercially available stainless steel housing. In CFF, process time is limited by extensive film deposition issues. If the only CFF device module loses its transport capability, it will be destroyed under sterile conditions. It is not possible to exchange the CFF and reinsert a new one.

[0098] The present invention facilitates pre-installation of two or more CFFs inside and outside the first container. This feature allows the user to easily extend the incubation time. The process operating time of the first container in the grid was calculated using a number of connected and installed CFF modules. It can be extended by

[0099] According to the present invention, a second SUP container for transporting one liquid can be attached to one or more CFF devices. The performance of each CFF can be measured using, for example, pressure drop (TMP) and mass flow sensors. CFFs may be of different sizes and specifications. Once the lifetime of the first CFF, i.e. the upper limit of the associated transmembrane pressure, is reached, the CFF may be replaced by a second CFF. 3 valve, allowing the next new CFF to be inserted without risk of compromising the sterile setup The first and second valves are preferably low cost valves that are incorporated into the present invention. The third, fourth and further valves are pre-assembled in the complete process loop as shown in Figure 8. Allows for stand-up and pre-sterilization to form one fully sterile component, further The sterilization step may be a controlled hose / pinch valve that completely eliminates the need for local sterilization.

[0100] In a fourth preferred embodiment (see FIG. 6), a free-floating, uncontrolled diaphragm is used as the base. A SUP is provided, which transfers the culture fluid from the SUB to the CFF and through the CFF. The SUP has two housing parts, each with its own internal chamber. The valve has a spring and an O-ring sealing groove on the outer periphery, which are assembled by an elastic element. When the elastic element is inserted, the elastic element separates the two parts and seals them. It is a preformed thin flexible sheet integrated with a sealing O-ring on the periphery. Preferably, it is compatible with the requirements of biological processes. The pump has two ports: a first inlet for connection with the SUB via a first one-way valve; It has an inlet port and a second outlet port for connection to a CFF via a one-way valve. The potentially reusable housing parts on the side are used to connect the vacuum and the compressed air in the store. It has a port for connecting a drive gas, such as a controlled drive gas pressure. The pump is fully inflated to each of the two end positions determined by the internal dimensions of the pump housing. It has two positions, one extended and the other extended.

[0101] The SUB is a container with magnetic stirring transport through the bottom plate of the container. A rotating shaft, such as the one shown, is mounted with a wheel and driven by an external rotational force. Various stirring devices may be added to the SUB. The suction tube is used for suction of the culture medium. The suction tube penetrates the wall of the SUB into the internal medium volume of the SUB. through the first one-way inlet valve located on the outside of the SUP dome, and then through the elastic diaphragm and the upper dome housing to the upper internal liquid chamber of the SUP. The elastic diaphragm separates a sterile, moist environment above from a non-moist, non-sterile driving gas below. This SUB suction tube continues into the hose, which separates the body compartment. The flow continues to the barb at the inlet of the first valve. The first valve is connected to the SUB. The second valve allows one-way flow of medium from the SUP pumping chamber to the SUP chamber. The CFF allows for one-way flow of culture medium from the chamber to the CFF inlet port. The nutrient solution is then passed along the channel to the CFF outlet, from which The hose is then guided back to the SUB where the concentrated liquid is mixed with the culture liquid. This ensures that the liquid cycle is completed.

[0102] In yet a fifth preferred embodiment (see FIG. 7), the S The SUP for the combined operation of UB and CFF is a diaphragm pump. Examples of SUP are: The present invention is described in WO 2010 / 069321, in which a free-floating, A moving, position-controlled elastic diaphragm is provided, which allows the culture medium to pass from the SUB to the CF. The SUP has two housings. Each of the elastic elements has an internal chamber and an O-ring sealing groove on its outer periphery. When assembled by the elastic element, the elastic element is separated and sealed by the elastic element. The elastic element is a pre-formed thin flexible separator integrated with a sealing O-ring on the periphery. The material is preferably compatible with the requirements of the biological process. The curved dome part used has two ports: the first for connection to the SUB via a one-way valve; a first inlet port for connection to the CFF via a one-way valve; and a second outlet port for connection to the CFF via a one-way valve. The lower housing part provides a controlled flow between the vacuum and the compressed air in the store. Provides a port for connecting a drive gas, such as a controlled drive gas pressure. The side pump housing measures the position of the elastic diaphragm in real time, sometimes to within 0.1 mm. The PID algorithm uses data from the position sensor to determine the Proportional valve opening for variable drive gas pressure to the non-wetted side of the membrane. SUP speed and power output are calculated using state-of-the-art electronics for real-time adjustments. The diaphragm changes position accordingly, and the selected diaphragm position is maintained. The diaphragm-based pump operates in series with the valve to move the desired portion of the liquid. Disposed between the SUB and the CFF, one first valve is on the inlet side of the SUP, and one A second valve is located on the outlet side of the pump facing the concentrate inlet side of the CFF. Optionally, one third valve is on the outlet side of the CFF for TMP control. The pump cooperates with and operates in series with the first and second valves, thereby The PTF concept of the present invention can control and guarantee the unidirectional flow. P is the volume and velocity of the culture medium passing through the CFF, as well as the volume of the harvest and the concentrate of the CFF. To overcome the increasing TMP in harvesting situations, Pressure is supplied by the SUP. The speed of the culture fluid to wash the collected membrane deposits successively with water is This restores the transmembrane pressure of the CFF to its original stage.

[0103] Overcoming the increased TMP is achieved by filling the SUP with process medium and the CFF with process medium. Filled with medium, the first valve is closed, the second valve is open, and the optional third valve is partially closed. When the pump's controlled diaphragm expands, the system pressure is The force increases to overcome the TMP and the membrane transports the liquid, thereby transferring the concentrate to the desired The permeate is then converted into a type of harvest.

[0104] One practical procedure is to have the SUP diaphragm in a relaxed stage and the pump chamber and CFF is filled with process medium and only the first valve is closed, An optional third valve located on the opposite side of F is now opened, allowing high velocity liquid (1 ~20 m / s) is pumped through the CFF in one or more pulses. returns to the SUB reservoir through the opened third valve, and from the surface of the concentrated liquid membrane of the CFF, The liquid loop is a collection of all these components, dragging sediment along with the collected cellular material. The film deposition is performed in a closed loop. No alternating liquid flow is required for the removal of material. In order to perform sequential cleaning of the PTF setup of the present invention, the liquid direction is in only one direction. This improves the system in general and CFF performance in particular.

[0105] The SUP of the diaphragm pump embodiment exhibits a high saturation when exposed to a driving gas pressure ranging from vacuum to above atmospheric pressure. A flexible and resilient membrane that can expand to a desired shape and / or return to a desired shape. and / or based on elastic elements. On one side there is a driving gas and on the other side there is a process gas. The movement of the flexible and / or elastic element by the liquid may cause the non-sterile, non-wetted side to move from the wetted side to the Furthermore, the septum may take several different shapes and Either the outside and the inside, or part of a rigid container wall, or part of a container wall that is both the outside and the inside The diaphragm may be in the form of a sheet of elastic material, a sphere, a partial sphere, a tube, or the like. It may be tubular, cylindrical, or partially cylindrical with one end closed.

[0106] For the PTF of the present invention, the first and second valves are known in the art as umbrella valves or ball valves. A passive one-way valve, also known as a duckbill valve or pinch valve, is opened by external means such as a hose valve, seat valve, mushroom valve or a combination of these. The valve may be controlled by a valve.

[0107] Anyone with experience working with membrane filtration knows that deposits, debris, and clogging are significant problems. The deposit on the elastic membrane is controlled by the flow of the culture medium during the process. It can be eliminated by rapid transformation. Spectrum Laboratories Inc (California, USA) and Refined Technology (current name are commercially available cells manufactured by Repligen Corp. (Massachusetts, USA). The pumps used in the holding CFF system cannot achieve rapid changes in nutrient flow rate. Spectrum Laboratories uses centrifugal pumps and Ined Technology limits the flexibility of the diaphragm pump movement.

[0108] The present invention allows for a wide and rapid change in the broth velocity (measured in meters per second) across the CFF membrane surface. Regarding the sampling mode, a low speed of 0.01 to 1 m / s is sufficient. For the cleaning cycle, faster speeds of 1-20 m / s or more facilitate the removal of deposits. The SUP of the system is driven from one pumping cycle to the next between individual pumping cycles. The speed can be varied in a controlled manner from minimum to maximum between pumping cycles. This allows the expressions "harvest cycle" and "cleaning cycle" to be used. A "cleaning cycle" is created. Better CFF performance, and longer process times before accumulating CFF deposits limit process performance. The life span is utilized and the culture is terminated.

[0109] The exchange of thermal energy in the medium reservoir, such as heating for cultivation and cooling for fermentation, , performed by wrapping the SUB in a heating blanket or water jacket system You may.

[0110] Heat regulation can also be used to control the total biomass and cell density in the culture. Mammalian cells such as CHO cells exhibit maximum cell growth rates at 37°C. The cells grow exponentially, dividing approximately once every 24 hours, and eventually block SUB. By reducing the temperature by 3 to 10°C, the number of cells in the SUB can be increased to 20 to 100 × 10E6 cells / Stable cell densities and cell numbers in the milliliter range can be achieved over several weeks.

[0111] Further desktop configurations for insertion of the single-use bioprocessing system of the present invention. Parallel boxes, sockets, workstations and robots for easier sampling, It provides the benefit of complete connection of sensors to wiring and associated PCS. The mount may be any shape that facilitates placement of the SUB body. The number of the rows may be 1 to 48 or more in one setup. An assembly is formed that includes the SUB.

[0112] Alternating Tangential Flow Details Market-Dominating Alternating Tangent by Refined Technology The dual flow concept "ATF" is a two-way flow concept. ATF provides reduced fluid exchange within the cross-flow filter device. This is because the entire internal volume of the CFF is not replaced with fresh fluid on each stroke. Uncontrolled dilution occurs within the CFF.

[0113] The ATF diaphragm pump also provides only two positions for the diaphragm. There is no position between them, so it is a metering pump other than the total volume. The ATF diaphragm pump has one special feature, which is the volume of a specific pump size. This fact makes it difficult to pump different CFF sizes and different STRs. A wide range of pump sizes is required. No mention is made of valves or flow or No valves are used for directional control. The ATF concept pump ensures optimal perfusion. Programming of the exact selected pumping volume for performance is not possible and the CFF life It cannot be combined with high speed for controlled deposit removal, which extends the time required for removal.

[0114] The ATF perfusion system with cell-retaining CFF devices was designed with five different pump sizes. Covered, conventional glass with volumes from 3 liter laboratory scale to 500 liters steelmaking scale / Steel STR operates. Five pump sizes called ATF-2, 4, 5, 8, and 10 Overall, our ATF options cover a scalability factor of approximately 1:55. TF pumps can only provide one complete stroke within their pumping capacity, not fractional strokes. Each pump stroke replaces 0.1 liters of ATF-2. ATF-4 replaces 0.4 liters, ATF-6 replaces 1.3 liters, ATF- The 8 replaces 2.5 liters, and the ATF-10 replaces 5.5 liters. Each pump offers individual scalability of approximately 1:5 relative to STR volume Each ATF pump is designed to have a maximum surface area of ​​±10% (approximately 0.1 or 0.75 or 2.5 or 4.2 or 10 m2) of one specific size CF F. ATF system (pump housing, CFF housing, Neither the ATF nor the ATF (connection) are available for pre-assembled and pre-sterilized disposable designs. Relatively complex with manual installation, CFF module insertion, steam sterilization, STR or large steel vessels Only available in stainless steel due to rough connection.

[0115] Pulsed Tangential Flow Details In comparison, the PTF of the present invention allows for one liquid flow direction in a pulse, a unidirectional, single flow. The present invention utilizes the direction of each pulse from the SUP to the entire culture medium volume of the CFF. Fresh culture medium and liquid are constantly added to the valve-controlled SUP. By integrating the sensor, the measurement accuracy of 0.01 to 20 m / s can be achieved by the SUP. Complete control of speed and volume per second is possible.

[0116] The disposable bioprocessing system of the present invention, operating in PTF mode, is Use the principle.

[0117] In the present invention, the second container SUP (Figs. 1-5) is also a metering pump, which is a sensor When integrated with a condenser, it is possible to flow a desired, pre-controlled volume independently of time. The SUP exchanges a volume of culture fluid equal to the internal volume of the CFF in each pulse. The embodiment shown in FIG. 1 has a stroke resolution of 0.1 mm and a maximum stroke of 150 mm. Using the Loak, there is an astonishing 1:1500 volume change with just one SUP size. Furthermore, one pump size provides a surface area of ​​1:50 and a S in the range of 1:50. It can meet the requirements of a very wide range of CFF modules, such as UB, all of which Reduce end-user expenses.

[0118] The perfusion system (PTF) of the present invention allows for a SUB size of 0.25 liters. It can be reduced to room scale VV and surprisingly, using only one SUP size. It can be scaled up to 500 times larger.

[0119] The present invention provides extremely simple and easy use and extremely high throughput at the end user's location. For your convenience, we offer SUBs, all fully pre-assembled, pre-sterilized and fully functional. Disposable, SUP, wide range of SUS, single use CFF, all hoses and all connections Become one with.

[0120] In the present invention, the diaphragm pump (Figs. 6 and 7) can measure the pumping speed and the measurement value when integrated with a sensor. With an incredible range of pumping volumes, from a few ml / hour to several ml / hour per stroke A desired pre-controlled volume in liters / hour can be flowed independently of time 100mm / 0.5 liters, 150mm / 1.7 liters, 220mm / 5.5 liters Each membrane has a diameter / volume of 1:5000, achieving a dynamic range of 1:5000. In principle, to cover the entire range of CFFs with surface areas between 0.1m2 and 10m2, one All you need is a 220mm SUP.

[0121] How it works Generally, it relates to a bioreactor or fermenter containing suspended microorganisms as biomass. The three most commonly used cultivation, fermentation and operation modes are: A constant working volume (WV) and a concentration of typically 5, and in good cases 10 cells / ml The amount of each batch; Start with the minimum WV and gradually increase the WV by adding process medium sequentially or continuously. Fed-batch culture, which rapidly increases the fermentation mass to increase the effective volume and the amount harvested; Extremely high levels of typically suspended biomass and high levels of microbial retention The effective volume is gradually increased by using a CFF device connected to the This is done continuously and sampling continues, perfusion.

[0122] Perfusion culture - Furthermore, experienced users preferably use the single-use bioprocess of the present invention. The system platform is configured to maintain a desired average amount of microbial biomass in culture. The product is used as a SUB operating in PTF perfusion mode. The microorganisms in the SUB grow exponentially. The cells continue to grow and produce the desired product. The culture medium must be removed from the SUB and fresh medium added. Although sequentially performing the parts results in loss of biomass and product, it is an acceptable method. In this regard, the microbial retention perfusion process is not a continuous process carried out under steady-state conditions. The desired product is extracted from the medium WV containing concentrated microorganisms, nutrients, waste products, products, etc. One or more CFF devices are typically used to separate the concentrate. After being held in the FF, it is returned to the SUB. The suspended microorganisms / concentrate are then sequentially exposed to the process fluid, which at this stage converts the suspended microorganisms / concentrate into The collected microorganisms are separated from the harvest / permeate. The above) may preferably be characterized as single use and may be integrated within the SUB. All components are assembled together and sterilized in a double film bag. The microorganism-containing medium is supplied from the SUB WV to one or more CFF devices. Transport through and along the channel is achieved by one or more fluid transport SUP devices. The pressure difference along and across the CFF is The proteins are then transported to the CFF channel for purification and then transported to the inner compartment of the CFF channel. The permeate side of the CFF is at atmospheric pressure within the container. The membrane may be constantly or sequentially exposed to a pressure lower than the pressure at which the membrane is Deposit / filter cake removal is achieved by a rapid and / or pulsed flow of liquid. The flow is then washed away from the membrane and returned to the WV in the SUB container. The cells are then swept along the membrane surface at high speeds. Typical biomass concentrations for mammalian cell cultures are 20-100 x 10E6 cells / ml. It is more than a liter.

[0123] The SUP is also preferably a disposable component, and the speed, volume and pressure controls of said SUP Use a control device or sensor that can provide operational information to the PCS. The sensor is preferably a low-cost stainless steel, which is discarded with the present invention, or a high-precision Reusable sensors. Lengths from 30 to 200 mm: Dynamic liquid level in SUP tubular cylinders with a response time better than 0.1 seconds; -The dynamic diaphragm position of the SUP body can be measured from below the elastic diaphragm with a resolution of 0.1 mm. Good results were obtained using a high-precision triangular laser distance sensor.

[0124] The first embodiment is shown in FIG. 1 and is supplied with a suitable driving pressure of 1 to 8 bar. A SUP of this size can perform 1 stroke / hour to 1 stroke / second, which allows A hollow fiber tube with 12 fiber tubes, each with an inner diameter of 0.5 to 1 mm and a length of 400 mm. In this example of a CFF module, the tube shown in Figure 1 has an inner diameter of 34 mm. For an air column driven SUP such as the setup shown, a velocity of 20 m / s Achieve volumes of several ml / hour to 150 ml / sec per stroke. Hollow fiber tubing The specifications may vary depending on the SUB volume and CFF surface.

[0125] The second embodiment of the SUP, sized as shown in FIGS. 6 and 7, has a programmable speed Equipped with a precision pump capable of delivering programmable volumes at 1-8 bar. Suitable driving pressure When powered, a SUP can perform 1 stroke / hour to 1 stroke / second, which This allows for velocities of 0.01 to 20+ m / s to be achieved in the connected CFF device. The volume varies depending on the diameter and expansion rate of the diaphragm, ranging from 1 ml to several liters per stroke. It becomes a bottle.

[0126] After capturing the real-time positions of the piston surface, liquid surface, and diaphragm surface, the computer Simple calculations performed by the is obtained.

[0127] The movement of the piston surface, liquid surface, and diaphragm surface occurs at pressures equal to or higher than atmospheric pressure. The pressure is controlled by a pressure regulating valve, preferably a proportional valve. The valve is controlled by the computer or PCS.

[0128] The above-described embodiments of the single-use bioprocessing system of the present invention preferably include a PCS. connected to the process variables, which control the process information, such as that described in the process recipe. Integrate information: The PCS is connected to multiple sensors integrated into the SUB, SUP, and CFF. Continuously collecting data on process variables from these sensors; The operating parameters of the single-use bioprocessing system are for the purpose of The PCS communicates with various actuators and devices outside the process system. So it is constantly changing.

[0129] The PCS contains controls for various input and output channels, such as: Various electrical analog and digital devices for measuring and collecting data from various sensors tal input channel; Pneumatic, gas, electrical analog and digital outputs to a variety of actuators.

[0130] DESCRIPTION OF THE DRAWINGS FIG. 1 illustrates a sterile disposable device of the present invention supplied fully assembled to the end user. An x-ray of the bioprocessing system 11 is shown (shown without the film protection bag). The container 12a of the SUB 12 includes sensors 13a, 13b, and Biomass sensor 13c, a pair of impellers 17c and 17d installed on shaft 17a, and The aeration tube 13e is installed through the upper cover 12b. The UB 12 comprises a container 12a having vertical side walls 12c and a reservoir 12b within the container. The optional SUP suction tube 12f has a 90° angle. The joint is connected to the reservoir 12d through a joint, and the joint is disposed in the reservoir 12d and has a port 12g. The first one-way SUP 15 is connected to the inlet valve 14a in front of the SUP 15 via the The SUP 15 penetrates the vertical side wall 12c of the antenna 12a. b, an electronic liquid level / volume sensor 15a and an external, not shown, drive gas control device. and a sterile filter 15c in series. The CFF device 16 is The CFF 16 is disposed in series with and behind the forward outlet valve 14d. The liquid passes through outlet port 16b, through hose 16c, and down the vertical outer sidewall of SUB 12. 12c through inlet 16d and returns to SUB12, The permeate outlets 16h and 16g of the CFF 16 are decelerated in the nutrient solution reservoir 12d. , and conveys the collected product through tubes 16e and 16f. Four externally disposed hoses 18a, 18b, 18c from the top cover 12b of a 18d allows the end user to connect to external pumps and various media containers (not shown). The SUB 12 passes through the upper cover 12b and into the container 12a via the bearing 17e. The shaft 17a extends from the rotor 17c. The shaft 17a has one or more impellers 17c. , 17d, etc., extending radially from the shaft 17a, provide agitation.

[0131] FIG. 2 shows the vertical container wall 22a and the support shafts disposed on the bottom wall 22e of the container 22. The container 22 is shown in cross section at the bottom, and the inside of the support bearing 27b is The rotating shaft 27a is supported by two impellers 27c. , 27d. The tip of the sensor 23b of PG13.5 size is shown. First valve The body 24a is attached to the outside of the vertical wall 22a of the container 22 by a top cover (not shown). The valve body 24a is disposed between the bottom wall 22e and the wall 22a of the container 22. The support leg 24b is disposed between the support leg 24b and a second support leg 24c on the outside of the container wall 22a. The assembly of the inlet leg 24b, the inlet one-way valve 24, and the outlet leg 24c is The liquid medium is transported in one direction from the reservoir 22d to the SUP15. The suction tube 22f is arranged perpendicular to the nozzle and has its top connected to the joint 2 in the reservoir 22d. 2g and on the container inner side wall 22c at a suitable height from the bottom wall 22e. The suction tube 22f is fixed at a suitable distance 2 above the interior side of the container of the bottom wall 22e. At 2h, it extends in a freely hanging state.

[0132] Figure 3 shows the SUB 32 with the cylindrical container 32a in a cross-sectional view from below. The suction tube 32f is disposed at its upper end in an angled position within the container 32a. The suction tube is fixed to the section body 32g and is also fixed onto the inner side of the container wall 32c. The tube 32f is disposed within the support leg 34b through a port 32d in the container wall 32c. The support legs are disposed on the outside of the container wall 32c, corresponding to the first inlet valve 34a. The support leg 34b further supports the SUP vertical cylinder 35 at the upper outer side.

[0133] FIG. 4 illustrates one embodiment of the present invention in a cross-sectional view of a PTF perfusion system, with the container wall 42 a and a bottom wall support bearing 47b, A rotating shaft 47a is supported within 47b. The shaft 47a is connected to two impellers 4 7c, 47d and a cover for facilitating the installation of a servo motor (not shown) on the outer apex. and a head plate drive bearing support 47e mounted on the bar wall 42h. The 5-base sensor 43b and the similarly sized biomass sensor 43c are attached to the top cover wall. 42h into medium reservoir 42b. The main body of the inlet valve 44a is connected to the SUP 45 and SUB 46 on the outside of the outer wall of the container 42a. 41 and drawn from medium reservoir 42b. The vertically disposed suction tube 42f ensures one-way flow of liquid. a and on the side wall of the container 42a. The SUP cylinder 45 is located outside the SUB 42 and behind the first inlet valve 44a. The medium is supplied from the medium reservoir 42b in the SUB 41 through the first inlet valve 44a. The processed culture medium is received.

[0134] FIG. 5 illustrates one or more disposable bioprocessing systems 51, each of which provides thermal control and mechanical support. 1 shows an embodiment of the present invention installed in a block 59a designed for holding The above blocks (59a, 59b, 59c, 59d) may, if desired, One or more disposable bioprocesses 51 operating in parallel are separately stirred by a SUP55 and CFF56 are designed for the purpose of stirring. All other HOs are shown as installed in SUB59a, which contains only one HO. The devices, accessories and sensors are not shown for simplicity.

[0135] Figure 6 shows the embodiment and recirculation liquid flow path in the PTF setup. 2 transports the culture medium between the SUB60 and the CFF64 and returns the liquid to the SUB60. .

[0136] The SUB60 container is mounted on a shaft 61b that is driven by external means (not shown). The liquid culture medium is continuously stirred using an impeller 61a mounted on the shaft, and the shaft is a rotating magnetic The suction tube 62b is attached to the device 61c. The SUP 62 penetrates the cover wall 60a of the SUB 60 into the medium reservoir 60b. The liquid is supplied from the liquid reservoir 60a of the UB 60 through the tube 62d and the first valve 62a. The culture medium is aspirated and distributed onto the dome wall 62d of the SUP 62 via the hose 62c. When the SUP 62 is in the fill mode, the first valve 62a is The one-way valve 62a of the first stage receives the culture medium from the SUB 60 and further transfers this culture medium volume to the SUP When the SUP 62 is in pumping mode, the second The forward valve 62e receives the culture medium from the outlet of the SUB62 and feeds it to the internal pressure channel of the SUP62. The chamber is opened and the culture medium is transferred from the internal pumping chamber of the SUP62. For the purpose of operation, a driving gas is introduced into the SUP60. The CFF 64 then transfers the culture medium to the liquid inlet port 64a. The liquid is conveyed along an inner porous membrane (not shown) in the tubular container 64b, and the liquid is conveyed along an inner porous membrane (not shown) in the tubular container 64b. Within b, CFF64 converts the culture medium into a retentate and a permeate. The concentrate exits port 64c and returns to SUB 60 via hose 64d, where it is passed through the process stream. The CFF64 is used to close the body loop. The CFF porous membrane can be passed through one or both of the feed ports 64e, 64f. can.

[0137] For simplicity, the sensor 60c is shown passing through the cover wall 60a into the container reservoir 60b. Only the sensor in the SUP64 is shown, other necessary hoses and sensors are not shown. is not shown.

[0138] Figure 7 shows SUP72 assembled with connected CFF74 in Figure 7a; In Figure 7b the individual parts are shown before assembly.

[0139] The SUP 72 includes an inflated, curved elastic diaphragm 72a, which is located in the upper housing dome section. The upper housing part 72b is spaced apart from and suspended between the lower housing part 72c.

[0140] Dome 72b is a reusable element that provides balance and support to the vertically disposed CFF 74. The SUP is mechanically secured by a connection (not shown) to the lower housing part 72c of the SUP. The SUP 72 has a port 72 for the supply of drive gas from an external source (not shown). The outer wall of the dome 72b is a housing for the first inlet valve 72e. and a second outlet valve connected directly to the medium inlet port 74a of the CFF 74. The curved single-use dome 72b is located inside a flat outer wall 72g. , is attached to a thin, elastic, curved diaphragm 72a in a liquid-tight manner, and the diaphragm 72a is The inner sterile, moist environment is separated from the outer non-moist, non-sterile drive gas compartment by 72 hours. The suction tube 62b of the SUB 60 continues into the hose 62c. This is connected to the inlet valve 62a of the SUP 62 / 72 via the first inlet port 72l. The first inlet valve 72e allows the SUB60 to flow through the SUP62 / 72. The second outlet valve 72f of the SUP 72 allows for one-way flow of the culture medium to the The culture medium is then pumped from the pumping chamber 72h through the second outlet port 72m to the CFF 74 can be transported in one direction to the first inlet port 74a. The culture medium flows along the porous membrane surface (not shown) to the second outlet port 74b of the CFF 74. From this second outlet port 74b, the hose 64 discharges the concentrate. The collected material is guided and transported back to the SUB60 reservoir, where it is mixed with the culture medium. Ensures that the PTF liquid cycle is complete. Third permeate outlet of CFF74 port 74c, and a fourth permeate outlet port 74d, which deliver the filtered process liquid to As the product is harvested, it is transported to the external facility area.

[0141] The reusable lower housing part 72c of the SUP 72 controls the movement of the SUP 72. The drive gas connection port 72b for the operation and the outer peripheral seal 72k of the diaphragm 72a are connected in an airtight manner. and a suitable housing flange 72j.

[0142] The culture medium entering the pumping chamber 72h of the SUP 72 is pumped through the elastic free-floating diaphragm 72a and the single The reusable rigid dome upper housing part 72d is enclosed and trapped between the upper housing part 72d.

[0143] The block diagram of FIG. 8 shows a first container SUB80, a second container SUP for transporting liquids, 82, a third container CFF 84 in the closed process liquid loop, A suitable and practical flow diagram for the cultivation and harvesting process is illustrated and explained.

[0144] The first container 80 contains the bioactive process and the culture medium is placed in the second container SUP82. The SUP transports the harvest and the process liquid back to the SUB80 in a return loop. For separation, the culture medium is pumped into the third container CFF 84. B80 is a medium outlet port 80a, a fresh medium pump 80b and an inlet port 80c, a stirring device 80d driven by a servo motor 80e, a monosaccharide sensor 86a, lactate sensor 86b, dual biomass sensor 86c, pH sensor 86d, dissolved acid The temperature of the SUB is measured by a heating element 86g. Fresh medium is added by pump 80b and used (containing cells). The medium and waste are removed via pump 80f. Aeration gas is supplied via control valve 80. g) into the SUB and pour into the liquid volume of the process culture medium 80 h. The exhaust is routed through the Ruta 80i.

[0145] The second container SUP 82 contains the pumping process and contains the culture medium volume 82a and the drive gas. a volume 82b, a drive gas inlet 82c, a second inlet valve 82e associated with the first inlet valve; a first inlet port 82d, a second outlet valve 82g associated with the first inlet port 82d; Toretto port 82f, liquid sensor 82h, pressure sensor 82i, externally installed dual ratio Example valves 82k and 82l (one valve for vacuum and the other for pressure-driven gas) The liquid sensor 82h includes a drive gas port 82j connected to the liquid level. This information is used by the PCS (not shown) to determine the dual valve configuration 82k , 821, the driving gas pressure of SUP82 (pressure equal to atmospheric pressure to pressure higher than atmospheric pressure) Adjust the pressure.

[0146] The third container CFF 84 contains the separation process and has a broth side 84c and a permeate side 84d. 4d, and a separating porous membrane 84a in a CFF housing 84b. The tena 84 has a medium inlet port 84e, dual permeate outlet ports 84f, and 4g, and one concentrate outlet port 84h. Permeate outlet port 84g and A TMP sensor 84i is installed between the CFF 84 and the concentrate outlet port 84h. The outlet port 84h for the concentrated liquid guides the concentrated liquid in the process liquid loop to SUB80. Optionally, a control valve 84j may be provided in the concentrate return line 84k for various process adjustments. The CFF device 84 is connected to the permeate side 84d of the device via an optional controlled pump ( or optional valves) 84n, 84o for product removal via one or more outlets. Equipped with 84f and 84g.

[0147] In general, the mechanical design is not limited and embodiments of the single-use bioprocessing system Does not have any PCS or control package or external fluid reservoir associated with it The state is illustrated.

[0148] While the present invention has been described with respect to specific embodiments thereof, it is to be understood that the invention is not limited to the specific embodiments defined by the appended claims. Numerous changes and modifications can be made without departing from the scope of the invention as defined herein. will be understood by those skilled in the art.

Claims

1. 1. A disposable bioprocessing system, comprising: an electrically controlled pumping device (15) for the delivery liquid; a first electrically controlled liquid valve arranged downstream from said electrically controlled delivery liquid pumping device; a first cross-flow filter (CFF) device located downstream from said first electrically controlled liquid valve; a second electrically controlled liquid valve located downstream from said first cross-flow filter (CFF) device or devices; Equipped with The CFF is a first inlet port; a first outlet port (16a); a second outlet port (16g, 16f); Equipped with the electrically controlled transfer liquid pumping device is configured to transfer liquid from an external facility area to the electrically controlled first liquid valve; the first liquid valve controls communication of the liquid between the pumping device and the first cross-flow filter (CFF) device; The CFF is configured to receive and treat a liquid to concentrate and permeate; an unfiltered liquid communication means extends from the pumping device (15) through the first liquid valve to the first inlet port; the first outlet port (16a) conveys unfiltered concentrate through the second liquid valve, and the unfiltered concentrate is returned to the external facility area; The second outlet port (16g, 16f) provides a filtered permeate that is passed through a membrane in the CFF, the CFF transferring selected components from the liquid to the permeate through the membrane.

2. 10. The single-use bioprocessing system of claim 1, wherein the performance of the first cross-flow filter (CFF) device is monitored by a Trans Membrane-Pressure sensor and / or a mass flow sensor.

3. 3. The disposable bioprocessing system of claim 1, further comprising one or more second cross-flow filter (CFF) devices, wherein the second cross-flow filter (CFF) devices are blocked by a third liquid valve when the first cross-flow filter (CFF) device reaches an upper transmembrane pressure limit that indicates degradation of the cross-flow filter membrane's performance, allowing the second cross-flow filter (CFF) devices to be used without risk of compromising sterility.

4. 10. A method of operating a single-use bioprocessing system according to claim 1, wherein after a perfusion mode controlled by a process control system, a process variable indicative of a quality of the process measured by the sensor is continuously controlled and adjusted, integrating software, algorithms and recipes of the process, comprising: the process control system connects to the sensors integrated into the single-use bioprocessing system to continuously collect data on process variables; The operating parameters of the single-use bioprocessing system are continuously varied by the process control system in communication with actuators and / or pumps for process parameter control; For the initiation of cultivation, a desired amount of medium covering at least the sensor and the stirring device is inoculated with microorganisms according to the desired recipe.

5. 5. The method of claim 4, wherein the process transitions from batch mode operation to perfusion mode operation when a desired biomass and working volume reach a maximum relative to the biomass and working volume at the start of the culture.

6. 5. The method of claim 4, wherein when the desired biomass and effective volume reach a maximum amount relative to the biomass and effective volume at the start of the cultivation, the process transitions to a perfusion mode operation, and wherein the perfusion mode operation performs a membrane cleaning mode operation to remove membrane fouling and deposits from the culture medium in the cross-flow filter (CFF) device, and a membrane transport mode operation to transport a harvested liquid product in the cross-flow filter (CFF) device, the membrane cleaning mode operation and the membrane transport mode operation follow each other at a desired interval.

7. 1. A method for producing a biological material by culturing or fermenting a microorganism in a biological process, comprising: operating the single-use bioprocessing system of claim 1 in a continuous processing mode and / or a perfusion mode; Flowing the culture medium through the transfer liquid pumping device (15) and through the cross-flow filter (CFF) device; Equipped with The flow of the culture medium is unidirectional; The method wherein the single-use bioprocessing system simultaneously produces a microorganism-free biological product as a harvested liquid product.

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