Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/02—Devices for withdrawing samples
  • G01N1/10—Devices for withdrawing samples in the liquid or fluent state
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
  • C12M33/04—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by injection or suction, e.g. using pipettes, syringes, needles
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M37/00—Means for sterilizing, maintaining sterile conditions or avoiding chemical or biological contamination
  • C12M37/02—Filters
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/02—Devices for withdrawing samples
  • G01N1/10—Devices for withdrawing samples in the liquid or fluent state
  • G01N1/20—Devices for withdrawing samples in the liquid or fluent state for flowing or falling materials
  • G01N1/2035—Devices for withdrawing samples in the liquid or fluent state for flowing or falling materials by deviating part of a fluid stream, e.g. by drawing-off or tapping
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T436/00—Chemistry: analytical and immunological testing
  • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
  • Y10T436/2575—Volumetric liquid transfer

Definitions

• the invention relates to a sampling valve for taking samples containing mechanically sensitive material from a reactor and a process analysis system with analysis stations, in particular chromatography systems, biosensors, cell determination devices, the automated, ste ⁇ le removal of a sample from a reactor and gentle transport of the sample containing mechanically sensitive material, in particular cells to the analysis station allows.
• Sterile sampling is a standard procedure in fermentation processes. It is the first step on the way to sample analysis to determine the condition and quality of a bioprocess and especially the resulting products. For this purpose, it has been necessary in many cases for a laboratory technician to manually take a sample. After sample delivery to a central analysis station, sample preparation, i. the biomass separation and aliquoting, and finally the analysis on several different analyzers. The analysis results are printed out to document product quality and manually entered into databases and recorded. In addition, appropriately marked restoring patterns are stored for later detection procedures at low temperatures. The analysis results are checked in quality assurance in order to release the product obtained in a bioprocess or to discard it due to quality deficiencies. All of these steps are very resource-intensive and therefore entail high costs. The control and regulation of the process in the reactor usually takes place after manually entering the analysis results obtained. A complete automation of the process control and regulation is therefore not possible.
• a process chromatography for Bioapphkationen is described in US 2004259241 Al (Groton Biosystems).
• the described apparatus for sampling is limited to bioreactors on a laboratory scale, as not ste ⁇ hsierbar with steam, which is the usual Ste ⁇ hsationsmethode in production.
• Dionex Corporation also offers the process chromatograph DX-800 (product brochure "DX-800 Process Analyzer, Process Analytical Liquid Chromatography”), which can be used for process control of bio-aphcations, providing automated chromatography but no sampling.
• this system is limited to the analysis of cell-free media.
• the two systems are designed to determine several parameters, but they do not provide an integrated sampling solution, especially shear-sensitive material such as cells and materials also no control and regulation of a bioprocess over the obtained data, since important automation units are missing for the connection to a process control system.
• Sampling devices and in particular sampling valves for bioprocesses for the removal of biological material and in particular cells are known in the art and some even commercially available.
• WO 1990012972 A1 by the company Keof ⁇ tt a / s describes a sampling valve, which consists of two parts - a valve body, and a valve head. Within the valve body, two ports are connected by an annular channel around a rubber diaphragm. This design allows the valve to be sterilized before and after use.
• WO 2004044119 A1 of the Sarto ⁇ us BBI Systems GmbH describes a coolable sampling valve with a cylindrical flow channel and a plunger to block the flow channel, wherein the walls of the sampling valve from a Mate ⁇ al germangerer strength usually made of metal, in particular stainless steel, to rapid cooling of the valve, for example to allow for Dampfste ⁇ lisation.
• WO 1990012972 A1 and WO 2004044119 A1 do not describe the transport of the sample taken.
• the disadvantage of the two abovementioned sampling valves from WO 1990012972 A1 and WO 2004044119 A1 is that both metal-metal contact interfaces are located between the head of the sampling valve and the bioreactor.
• the sampling valve which causes a local heating of the medium in the bioreactor in a Dampfste ⁇ lisation the sampling valve, so that at the place of sampling increased cell aggregates may occur (so-called Biofouhng).
• Such aggregates which are also produced in the normal operation of a bioreactor, can be swirled up when opening the sample-free sampling valves and enter the valve. To avoid blockages, transport lines with relatively large diameters are therefore necessary for the transport of the removed cell suspension.
• the volume of the sample is determined solely by adjusting the opening time of the sampling valve. The solution does not allow the precise acquisition of a predefined volume and in particular a small volume. Moreover, so far no sample transport of a small cell-containing volume over a longer distance is known, without that this sample is further falsified during transport, for example by sedimentation during transport or destruction of cells by shearing.
• a first subject of the present invention is a sampling valve which can remove a sample of a defined volume, in particular of biological material, and in particular living cell material under reduced mechanical stress, in particular by shearing forces.
• a particular embodiment of the sampling valve has a preferably cylindrical sample chamber defined volume limited by a front and a rear sealing element.
• the front sealing element is actuated by a connecting shaft.
• the front sealing element m is opened in the direction of the interior of the bioreactor and the rear sealing element is closed against the sample chamber. In this open valve state, the air bubble trapped in the sample chamber escapes into the reactor, with a sample of defined volume flowing from the bioreactor into the sample chamber.
• the rear sealing element limits the volume of the sampling and allows the removal of a defined volume.
• the closing force can be achieved by biasing a spring, preferably a spiral spring, from a pressure plate via a connecting rod to the rear one Sealing element are transmitted, which seals via surface pressure by means of a sealing device, preferably an O-ring, against a valve stem.
• the bias voltage is usually set to a differential pressure between the sample chamber and the reactor of at least + 1.5 bar. Underpressure or vacuum in the bioreactor leads to the said spring preload so little to an unintentional valve opening as compared to the external pressure increased internal pressure in the reactor (eg autoclaving), which can additionally strengthen the closing force in the same direction of force.
• the sampling valve is preferably opened by actuation of a lifting cylinder.
• a control which can be pneumatically (control via compressed air) or electrically (via a pulse) preferably pneumatically, closes the valve in fractions of a second after the issuing of the closing command, this small delay secures the exact volume of the sample.
• a control which can be pneumatically (control via compressed air) or electrically (via a pulse) preferably pneumatically, closes the valve in fractions of a second after the issuing of the closing command, this small delay secures the exact volume of the sample.
• For secure positioning of the sealing surfaces can serve a mounted on the connecting shaft guide rod.
• a membrane On this guide rod usually a membrane is attached, which can be held squeezed between two holding plates.
• the pressure force on the guide rod leads to the deflection of the membrane, which hermetically seals the sample chamber and a rear valve interior against the environment.
• the rear valve interior to reduce the membrane load has a relation to the sample chamber enlarged diameter.
• the sampling valve on the probe head (the opening to the reactor) is protected by a self-cleaning filter, so that no larger aggregates can get into the sample chamber and into the transport line.
• the width of the pores of this filter is usually 0.02 ⁇ m to 2 mm, but preferably 0.45 ⁇ m to 1 mm.
• the filter has a cavity which is filled from inside by a cap around the front sealing element, in the closed state, i. The pores are closed by the cap from the inside. The cap is moved out in the open state in the direction of the reactor, so that forms an open area.
• the membrane preferably consists of a material which is resistant to water vapor, preferably EPDM, silicones, HNBR or PFR plastics.
• the probe head preferably consists of a plastic approved for pharmaceutical applications, preferably of PVDF, PEEK or POM, which has a lower thermal conductivity than stainless steel and adhere to the cells particularly bad. So there is no metal-metal interface with the connected bioreactor, so that a local heating of the bioreactor and Fouling layers on the sealing device, which is preferably an O-ring can be avoided during the cleaning process, for example in a steam sterilization.
• the sampling valve according to the invention is usually analogous to standard probes e.g. installed by means of a screw in standardized fermenter neck, preferably with the diameter of DN25, usually with the aid of a sealing device, preferably with a so-called O-ring.
• a downwardly inclined installation of the sampling valve to the bioreactor wall is advantageous.
• Favorable installation angles are between 0 ° and 90 ° to the horizontal, preferably between 1 ° and 15 °.
• the cylindrical sample chamber can be mounted inclined even against the nozzle axis, preferably between 1 ° and 15 ° to the horizontal.
• the sampling valve according to the invention can be tempered if necessary, preferably for this purpose, the sampling valve is sheathed and tempered with a Peltier element.
• the path to a connected transport line is released.
• the sample is then assayed as a substantially contiguous plug to the target site, e.g. a sample preparation and / or analysis station transported.
• gas or liquid are usually introduced via an addition opening and a channel and slowly displace the sample from the sample chamber into a rear valve interior and to a discharge connection.
• the addition opening can be protected by a valve, preferably a check valve.
• the sampling valve is usually coupled to transport and supply lines, in particular cleaning lines.
• the coupling is preferably carried out by autoclaving and steam sterilizable quick-release couplings, which have a locking mechanism in the decoupled state, which protects the sterile inner surfaces of the two coupling pieces from contamination.
• a cock is integrated between the sampling valve and the transport line, preferably a three-way cock, with which a sample can be manually can be removed, which preferably has a control unit and can be controlled decentrally.
• a further subject matter of the present invention is a process analysis system which has at least one apparatus for taking a sample from a reactor, a sample transport device and at least one sample analysis station, which allows a volume of sample material to be removed from the reactor and transported to the analysis station, wherein the sample material is a suspension of mechanically sensitive, in particular shear-sensitive material, which is subjected to reduced mechanical stress, in particular reduced shear forces.
• the volume is precisely defined and / or free of aggregates.
• Mechanically sensitive, in particular shear-sensitive material in the sense of the present invention is, in particular, biological material, such as e.g. Cells, Bakte ⁇ en, unicellular fungi such as yeasts, viruses, agglomerates of protein precipitates, Protemk ⁇ stalle, native proteins, antibodies, liposomes and especially living tie ⁇ sche and / or plant cells.
• biological material such as e.g. Cells, Bakte ⁇ en, unicellular fungi such as yeasts, viruses, agglomerates of protein precipitates, Protemk ⁇ stalle, native proteins, antibodies, liposomes and especially living tie ⁇ sche and / or plant cells.
• the process analysis system usually has at least one device for sampling and sample transport connected to at least one sample preparation and / or sample analysis station.
• the device for automatic sampling is a sampling valve according to the invention.
• the process analysis system usually controls at least one sample analysis station and has a connection to an automation system, preferably a process control system or programmable logic controllers for guiding, controlling and / or regulating the process in a reactor and in particular a bioreactor.
• an automation system preferably a process control system or programmable logic controllers for guiding, controlling and / or regulating the process in a reactor and in particular a bioreactor.
• the sampling and Probenentransportvor ⁇ chtoder, the sample preparation and the process analysis station are modular.
• the sample preparation station typically includes sample valves, reservoirs, burettes, valves, valves, metering valves, and the like, which are interconnected by transport lines and allow treatment of the sample in one or more ports.
• the process analysis system preferably has at least one sample preparation station.
• the Sch ⁇ tte required for the use of the sample for the analysis are carried out, such as dilution, addition of internal standard, addition of stabilizers (eg Glycerm), marker or detergent, tempering before- Cooling to 4 to 37 0 C, pH adjustment, stripping, Umpuff für für, filtration or De ⁇ va- üstechnik.
• sensors such as pH electrode, conductivity probe, sensors for measuring the optical density, turbidity, pressure, temperature, flow are called.
• the process analysis system includes a sensor-controlled test of the sample which examines whether the properties of the sample (e.g., cell density) are compatible with the given workflow and, in particular, the usage instructions of a required analyzer. Is e.g. If the cell density is too high, an analyzer can become clogged. In addition, the cell density may be outside the range of the analyzer. In this case, the sample is diluted by means of a sensor control until a reliable quantification of the sample is possible. If, in the opposite case, the cell density is too low for a quantification, a sample concentration program is started under sensor control.
• the properties of the sample e.g., cell density
• the sample preparation station preferably has a central collecting vessel in which a sensor for monitoring the sample is integrated in order to regulate and / or control the work-up of the sample in the sample preparation station. If the sample is very diluted, a concentration program is started; if the concentration is too high, a dilution step is initiated. This will prevent measurements from being made on samples or aliquots that are outside the measurement range, specifications, and / or validated area of the analysis station.
• this type of sample preparation can be used to prepare the sample so that an exact analysis in an analyzer in the most sensitive range can be performed. This allows a more sensitive determination of parameters over a larger concentration span of the sample and thus an improved control of bioprocesses.
• the sampling device and the sample analysis stations are connected via transport lines to the sample preparation stations.
• This modular structure has the particular advantage that the automatic sample preparation for different analyzers can be configured without much effort.
• the sample is divided into several aliquots in an aliquoting station. These aliquots either pass through one sample preparation station in succession, alternatively they are transported in parallel to several sample preparation stations and are processed there differently.
• the aliquoting is carried out after sample preparation such as filtration, decantation, concentration or dilution.
• the process analysis system has a central sample preparation station that passes through the aliquots one at a time.
• sample preparation After sample preparation, the sample or aliquot is transported to the various sample analysis stations through the transport lines.
• the process analysis system preferably has self-monitoring, which records and monitors the properties of the sample, preferably temperature, pressure, pH, flow, optical density, conductivity, turbidity, in at least one of the various modules.
• the transport of the sample and in particular flow, optical density or turbidity in the transport lines is monitored and controlled in order to prevent clogging of the lines.
• the process analysis system is designed to be in a safe condition in the event of a power outage or malfunction that prevents the contents of connected bioreactors from being contaminated.
• the sample analysis station may include a variety of sample analyzers (analyzers), e.g. Cell counter, biosensors, spectroscopy systems, chromatography systems such as HPLC, ion, affinity and / or gel permeation chromatography systems, which serve to examine the sample or aliquots.
• sample analyzers e.g. Cell counter
• biosensors e.g., biosensors
• spectroscopy systems e.g. Cell counter
• chromatography systems such as HPLC, ion, affinity and / or gel permeation chromatography systems
• HPLC ion, affinity and / or gel permeation chromatography systems
• the sample analyzer performs biochemical analysis of reaction products or minor components.
• Reaction products are usually the proteins to be prepared, while examples of secondary components are cell state parameters such as mentimine, lactate dehydrogenase or DNA.
• biosensors are incorporated for the control of nutrients and metabolic products such as glucose and lactate.
• biofunctional surfaces such as microtiter plate, glass, biosensor, bead or magnetic bead surfaces are preferably biologically, chemically or biochemical recognition elements such as DNA, RNA, aptamers, receptors to which an analyte specifically binds upon detection by a recognition reaction.
• biochemical analysis using biochemical recognition elements can also be carried out in homogeneous formats in solution, for example in the context of homogeneous time-resolved fluorescence (HTRF).
• the biochemical recognition elements are coupled with signal-generating molecules such as fluorescent dyes or nanoparticles.
• recognition reactions are the binding of ligands to complexes, the complexation of ions, the binding of ligands to (biological) receptors, membrane receptors or ion channels, of antigens or haptens to antibodies (immunoassays), of substrates to enzymes, of DNA or RNA to specific proteins, aptamers or aptmer to their targets, the hybridization of DNA / RNA / PNA or other nucleic acid analogues (DNA assays) or the processing of substrates by enzymes.
• the method of the polymerase chain reaction (PCR) particularly preferably the method of kinetic PCR, can be used with advantage.
• signal amplification for immunoassays can advantageously be used the method of immuno-PCR.
• analytes to be detected are DNA, RNA, PNA, nucleic acid analogs, enzyme substrates, peptides, proteins, potential drugs, drugs, cells, viruses.
• recognition elements to which the analytes to be detected bind are DNA, RNA, PNA, nucleic acid analogs, aptamers, spiegelmers, peptides, proteins, complexing agents for metals / metalhones, cyclodextrins, crown ethers, antibodies or their fragments, anticalme, enzymes , Receptors, Membrane Receptors, Ion Channels, Cell Adhesion Proteins, Ganghosides, Mono- or Oligosaccharides.
• the detection of the detection reaction of the biochemical detection system can be carried out by using optical, electrical, mechanical or magnetic signal conversion methods. Particular preference is given to optical methods such as chemo-cumimensis, electrochemiluminescence, absorption detection of an enzymatically induced color change, fluorescence detection of a enzymatically induced turnover of a fluorogenic substrate, alpha screen or homogeneous time-resolved fluorescence.
• Alpha Screen stands for a homogeneous detection method in which light-induced smglet oxygen is produced on a first bead, which after diffusion to a second bead, which is coupled to the first bead via a biochemical binding reaction, stimulates the chemiluminescence.
• an autosampler which collects samples and cools.
• this modular structure is also reflected in the control program of the process analysis system.
• a driver software with local operation is preferably stored in a decentralized control and supply unit for each module.
• the control program of the automation unit accesses this driver software in order to carry out the steps of sterile sampling, automatic sample preparation, analysis and cleaning according to a user-defined workflow.
• the control and supply unit ensures and regulates the supply of compressed air, steam, cleaning fluids and transport fluids to the sampling valve through the supply and transport lines.
• a plurality of reactors in particular bioreactors, are operated, which operate independently of one another.
• Each sampling device can be controlled remotely via its own control unit and thus independently.
• the process analysis system has a decentralized automation unit with on-site operation on each sampling device, which are coupled to the central PLC via all standard bus systems. This makes it easy to switch off and on individual units.
• the sequence of the control program is defined by user-definable parameters.
• the user can select modules available via a graphical user interface of a standard personal computer and select actions to be performed by them.
• sequence sequences for sampling, sample preparation and sample analysis can be defined in tabular form with the aid of the modules.
• the parameters describing this procedure are then exported from a PC and transmitted to the control unit of the control system. There, these parameters determine the program sequence of the control program. The parameters thus determine the sequence in which the control program calls individual driver programs as well as the control parameters which the control program sends to the driver software in order to cause a specific module to perform a specific action.
• an automation component such as a Simatic S7 from Siemens AG.
• Such an automation component is designed for trouble-free continuous use in an industrial environment and therefore can not "crash” like a conventional PC, in which case the PC, with the aid of which the user enters the sequence, and the control unit during operation of the PC That is, after the parameters defining the program flow have been transferred from the PC to the control unit, the PC can be disconnected from the control unit, thereby enabling operation of the control unit independently of the PC.
• a further element of the process analysis system according to the invention is the transport device whose task is to forward the sensitive sample of a defined volume from the sampling device to the preparation or analysis station smoothly, without clogging and lossless.
• the transport device usually has transport lines, and at least one system for smooth acceleration of the sample or aliquots through the transport lines.
• the subject of the present invention is therefore also a transport device for transporting suspensions containing mechanically sensitive material and in particular ivy cells, comprising transport lines and at least one system for accelerating the sample or aliquots through the transport line from at least two burettes, wherein the burets following steps are operated ' a) a first burette is raised,
• the burets are connected to at least one valve terminal to the transport lines, the burettes and valve terminal are controlled by the automation system, so that an accurate adjustment of the transport speed is guaranteed.
• suspensions of mechanically sensitive material and in particular of living cells adherence to a defined transport speed is preferred because the material at too high transport speed, for. due to shearing forces and could sediment at too low transport speed in the horizontal laid pipe.
• the transport is preferably carried out pneumatically or with liquids.
• the burettes also ensure the aliquoting of the sample.
• cell separation can be accomplished via filtration by adjusting the pore size of the filter directly on the probe head of the sampling valve.
• the sample may be filtered in the preparation station or collected in a vessel to sediment there. After sedimentation can from the supernatant a cell-poor Sampled, filtered and fed to an analyzer.
• a filterless and therefore low-maintenance alternative is the sample transport at a speed of ⁇ 1 m / mm. In the case of lines longer than 5 m, all cells in the pipeline sediment during transport, so that a cell-free sample can be collected in a sample vessel, which does not have to be further filtered for further processing.
• the transport lines can be tempered, preferably with a Peltier element, to a temperature between 0 0 C and 100 0 C, preferably between 4 ° C and 37 ° C.
• a temperature control over a heated double sheathed cable is realized.
• the sample transport device has two burettes, which are each connected by a valve island to the transport lines, which in turn connected to one or more sampling valve, the preparation or analysis stations and sources for air or transport and / or cleaning fluids are.
• the transport lines preferably have at least one control unit, which in particular monitors and controls flow, pressure, optical density or turbidity in the transport lines in order to prevent clogging of the lines.
• the sampling valve can be autoclaved together with the reactor, in particular bioreactor, and its supply lines are closed by means of silicon compounds.
• the device for sampling and sample transport is preferably cleaned with steam, sterilized water or sterilizing solutions.
• the sampling valve and the transport line are preferably rinsed with steam and heated to temperatures usually between 100 to 135 ° C to remove any cell debris and sterilize the system and clean.
• the cleaning can also be done with sterile water or sterilizing rinse solution.
• sterile, dry air is typically delivered through the sampling valve and conduit to cool and dry the sample transport system.
• the sampling valve at the rear valve interior additionally has an attached auxiliary connection, which serves for better in-situ cleanability of the sampling process.
• the auxiliary connection can be used to ensure that no air bubble that is unfavorable for cleaning and thus a dead space forms in the upper part of the rear valve interior.
• the device for sampling and sample transport and especially the sampling valve and transport line are thoroughly cleaned with Clean-m-place media and dried with ste ⁇ ler air. It is important to rinse thoroughly with demineralized water at the last clean-in-place or sterilization-in-place cleaning cycle to prevent the formation of deposits on the areas in contact with the product during the subsequent drying process.
• the present invention enables a fully automated sample analysis including sterile sampling, transport, preparation and analysis under sterile conditions with integration of the obtained analysis values by direct connection to a process control system and / or a PLC for controlling and controlling the process.
• a process control system and / or a PLC for controlling and controlling the process.
• the removal of small, defined sample volumes and gentle transport, in particular cell-containing samples, possibly cell separation and liquid sample preparation and analysis are guaranteed.
• the present invention allows for flexible workflow design and automatic adjustment of sample preparation or aliquoting to the requirements of both the workflows and the analyzers available in the system.
• bioprocess process analysis system Another major advantage of the bioprocess process analysis system described here is the ability to control multiple bioreactors that operate independently of each other. Each valve can be controlled decentrally and thus independently. Furthermore, it is conceivable to operate several reactors, which work with different cell lines and produce different products, with a single process analysis system and thus particularly cost-effectively, since a sophisticated cleaning management prevents the mutual contamination of the individual bioreactors.
• Fig. Ia Sampling valve in the open state on a bioreactor
• Fig. Ib Sampling valve in the closed state on a bioreactor
• Fig. 2 Side view of the sampling valve in the open state
• Fig. 3a Downwardly inclined installation of the sampling valve
• Fig. 3b horizontal installation of the sampling valve with inclined to the nozzle axis
• Fig. 3c 90 ° downwardly inclined installation of the sampling valve
• Fig. 4 Connection of several bioreactors to a central analysis station
• Fig. 5 sample transport, preparation and analysis and control and regulation of a
• Fig.la shows the in a bioreactor (1) built open sampling valve (2) with a sample chamber (3) defined volume into which the sample initially flows when valve opening.
• the rear sealing element (17) limits the volume of the sampling and allows the removal of a small, defined volume.
• the front sealing element (16) prevents further liquid from flowing out of the bioreactor into the sampling valve.
• Fig. Ib After closing the valve (Fig. Ib), the path to a connected transport line (4) is released.
• the sample is then transported as a substantially continuous plug (6) to the target site (7), a central sample preparation and / or analysis station.
• the preferred use of two burettes (8) allows, by suitable interaction, a continuous transport speed and thus particularly gentle and loss-free transport through the line.
• the cell suspension is collected in a sample vessel.
• the sampling valve (2) is installed analogously to standard probes by means of a screw connection (27) into standardized fermenter stubs (14) with the diameter of DN25.
• the sampling valve (2) is closed in the energy-free state to the reactor chamber with a sealing, rear sealing element (16).
• the closure force is transmitted by biasing the spring (15), preferably a coil spring, from the pressure plate (21) via the Verbmdungsstange (18) on the rear sealing element (17).
• the rear sealing element (17) seals via surface pressure by means of a sealing device (30), preferably an O-arm, against the valve stem (42).
• the air bubble enclosed in the sample chamber (3) escapes into the bioreactor (1), with a sample of defined volume flowing from the bioreactor into the sample chamber (3).
• the sampling valve is replaced by a self-cleaning filter (39) protected, so that no larger aggregates can get into the sample chamber (3) and in the transport line (4).
• This filter (39) surrounds part of the probe head and prevents aggregates from entering the valve.
• the width of the pores (43) of the filter is 0.5 mm to 2 mm, preferably 1 mm.
• a filter surface with a pore width of 0.02-2 ⁇ m, preferably 0.45 ⁇ m, can be used.
• a cap (41) has moved out in the open state in the direction of the fermenter, so forms an open area. Some pores (43) are shown as white boxes, through which a sample can enter the valve from all directions. After relieving the Hubzylmders (22) via a control (33), which is pneumatic, the valve closes. The cap (41) is moved in the direction of the valve and fills the cavity of the filter (39) from the inside, ie the pores (43) are closed by the cap (41) from the inside.
• the guide (34) mounted on the connecting shaft serves to secure the sealing surfaces. For sample transport, gas or liquid is introduced via the feed opening (24) and an inlet channel (31) and slowly displaces the sample from the sample chamber (3) into the rear valve chamber (36) and to the outlet pipe (25).
• the addition opening is protected by a valve (35), preferably a non-return valve.
• the additional valve crevices (36) additionally attached auxiliary cusps (26) serves for better m-situ cleanability.
• the rear Ventilmnenraum (36) has a relation to the sample chamber (3) has an enlarged diameter to reduce the membrane load.
• the auxiliary connection (26) ensures that no unfavorable for the cleaning air bubble and thus a dead space in the upper part of the rear Ventilmnenraums (36) form.
• the sample is fed under gas pressure or with a liquid via a drainage spigot (25) into the transport line (4).
• Fig. 3a shows a preferred embodiment with downwardly inclined installation of the sampling valve (2) to the bioreactor wall.
• Fig. 3b shows a further preferred embodiment of the valve, wherein the cylindrical sample chamber (3) is mounted inclined even against the nozzle axis.
• Fig. 3c shows a further preferred embodiment of the valve with a 90 ° downwardly inclined installation of Probe Spotifyventiis (2) on the bioreactor wall.
• sampling valve 4 shows a possibility of coupling the sampling valve to the line system, consisting of supply, remaning and transport line by autoclaving and steam-adjustable quick-release couplings (37), which have a closing mechanism in the decoupled state, the sterile inner surfaces the two coupling pieces (38) protects against contamination.
• the sample is transported to a central sample preparation and / or sample analysis station (7).
• bioreactors can be controlled, which operate on each other depending on each other.
• Each valve can be controlled decentrally and thus independently.
• the process analysis system has a decentralized control and supply unit (46) with decentralized local operation (47) on each sampling system. This communicates via a central automation unit (48).
• the decentralized control and supply unit (46) regulates the supply of compressed air, steam, cleaning fluids and transport fluids.
• a control unit (49) is integrated in the transport line between the sampling system (2) and a three-way cock (44).
• the three-way cock (44) allows manual sampling (50).
• Two burettes (51, 52) are controlled by the automation system to ensure the transport and aliquoting (70) of the sample.
• the sample is transported to a central collection vessel (53).
• a probe (54) characterizes the sample
• a stirrer (55) is additionally integrated.
• a signal is sent to the automation system to decide whether the sample can be further diluted, concentrated or untreated for further processing.
• the sample is distributed to different analyzers: an unfiltered sample is analyzed in a cell number determiner (56) or in analyzer 1 (57). Another portion of the sample passes through a filter (62) and is distributed to analyzer 2 (58), chromatography (59) and / or biosensor (60).
• Fig. 6 shows a flowchart. After sampling from the bioreactor, the entire sample is gently transported to a central collection vessel. In a preliminary examination, a sensor characterizes the sample. If the concentration is too high, a sensor-controlled sample dilution program will be started. This is repeated until the sample is in the desired concentration range. Thereafter, the sample preparation program is started and the sample is measured in the various analyzers. The analysis results are transferred to a process control system. After completion of all measurements, the steam sterilization and the cleaning of the valve and the lines started. The process control system uses the analysis results to adjust the process.
• a 12 ml sample of SF9 insect cells was removed from a bioreactor using the sample valve according to the invention and transported through a tube (1.5 mm inner diameter, 10 m length) at a speed of 3 m / min in the sample analysis system according to FIG.
• a sample was taken by hand from the bioreactor and measured, a sample was taken with the sampling valve, transported by the sample transport device in and measured.
• the CEDEX analyzer Innovatis company
• the cell determination station 56
• the aid of the analyzer CEDEX it was found that the reuse rate of the cells was> 90%. This proved the applicability of the sample analysis system according to the invention for the removal and transport of living cells.
• a 10 ml sample of hybridoma cells was produced by means of the inventive sampling valve, which produce antibodies for the control of tumors, and through a tube (1.5 mm inner diameter, 5 m length) at a speed of 3 m / min in the Pro analysis system according to Fig. 5 transported.
• One sample was removed by hand from the bioreactor and measured, another sample was taken with the sampling valve, transported by the sample transport device in the process analysis system and measured.
• the analyzer CEDEX Innovatis AG
• the cell determination station 56) with the aid of the analyzer CEDEX, it was found that the recovery rate of the cells was> 95%. This proved the applicability of the sample analysis system according to the invention for the removal and transport of living cells.

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