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
  • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
  • G01N1/40—Concentrating samples
  • G01N1/4077—Concentrating samples by other techniques involving separation of suspended solids
• • 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
  • C12M33/14—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus with filters, sieves or membranes
• • 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
  • G01N1/14—Suction devices, e.g. pumps; Ejector devices
• • 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
  • G01N2001/1062—Sampling under constant temperature, pressure, or the like
  • G01N2001/1081—Storing samples under refrigeration

Definitions

• the present invention relates to an automatic sampling system for a biological fluid with sterile sampling.
• the system described in the above-mentioned article also implements a recirculation loop and steam sterilization of the sampling circuits up to the filtration module must be provided.
• the various systems previously described are, most of the time, usable under limited working conditions, fermentation of yeasts or large living cells.
• the most frequently encountered problems are linked to the more or less rapid polarization and clogging depending on the type of filtration membrane used, especially in the case of frontal filtration, to the lag time to obtain a representative sample in the case of filtration.
• tangential to the size of the pores which, too large, does not allow working with bacteria, to the complexity of the systems which include either a large number of pumps, or of complex steam sterilization systems, to the too large volume of the recycling loop in relation to the volume of a small laboratory reactor or fermenter, of the order of two liters, as well as to the reliability of the sterile sample taking and therefore to respect for sterility.
• the purpose of the automatic sampler system for a biological liquid with sterile sampling which is the subject of the invention, is to remedy all of the aforementioned drawbacks.
• Another object of the present invention is also the implementation of a system making it possible to ensure the taking of samples under conditions of satisfactory reliability and sterility in the absence of effective sterilization of the corresponding circuits during successive direct debits.
• the system for automatically sampling a biological liquid with sterile sampling which is the subject of the invention, comprises a module for sampling from a reactor for biological fluid, a filtration module connected to a sampling pot, a distribution circuit for the or samples to analyzer means and control and calculation means interconnected respectively to the sampling module, to the filtration module, to the sampling pot and to the distribution circuit of the sample (s) to ensure a sequential control of the taking of 'samples.
• the filtration module is placed at the end of the sampling module in the reactor, the sampling pot being directly connected at the outlet of the sampling module and, on the other hand, what it includes a circuit for injecting control fluids of the sampling circuits constituted by the sampling module, the sampling pot, the circuit for injecting control fluids being controlled sequentially by the control and calculation.
• FIG. 1 represents a general view of the automatic sampler system for a biological fluid with sterile sample • object of the invention
• . 2 shows a sectional view along a longitudinal plane of symmetry of a sampling pot for the realization of the system according to the invention, as shown in Figure 1,
• FIGS. 3 and ⁇ represent in section along a plane of longitudinal symmetry respectively of a filtration module and of a filter cartridge or sleeve, in accordance with the object of the present invention
• FIG. 5 represents a simplified flow diagram making it possible to carry out one or more sequential sampling, in accordance with the system according to the invention.
• microorganisms bacteria, yeasts, fungi, plant and animal cells
• the complex metabolic reactions involved in microorganisms (bacteria, yeasts, fungi, plant and animal cells) during the "fermentation" processes are sensitive to many external parameters, such as pH, temperature, pressure, and so on!> than the age or physiological state of the cells.
• the sampler system object of the invention, further comprises control and calculation means 5 interconnected respectively to the sampling module 2 and to the filtration module 1 to the sampling pot 3 and to the distribution circuit CD of the sample or samples to ensure sequential control of the aforementioned sample taking.
• the filtration module 1 is placed at the end of the sampling module 2 in the reactor R or fermenter.
• the sampling jar in accordance with another particularly advantageous aspect of the system which is the subject of the invention, is then directly connected to the output of the sampling module 2, in the absence of a loop for recycling the biological fluid sampled.
• the system the sampler also includes a circuit 40, 41, 42 for injecting fluids for controlling the sampling circuits constituted by the filtration module 1, the sampling module 2, the sampling pot 3.
• the circuit for injecting control fluids is controlled sequentially by the control means and calculation 5.
• the circuit for injecting fluids for controlling the sampling circuits advantageously comprises, as well as represented in FIG. 1, a first circuit 40 for injecting an inert gas with the biological liquid to be sampled.
• This circuit advantageously allows pre-cleaning and / or unclogging of the filtration module l by injection of the inert gas towards the reactor or fermenter R.
• the inert gas with respect to the biological liquid to perform the pre-cleaning or unclogging functions can be constituted, depending on the nature of the biological liquid considered to be sampled, with air, nitrogen , carbon dioxide for example.
• the circuit for injecting fluids for controlling the sampling circuits comprises a second circuit 41 for injecting rinsing water from the circuits ensuring the connection between the reactor or fermenter R and the sampling pot 3.
• a third circuit 42 for injecting dry air is shown in the circuits ensuring the connection between the reactor or fermenter R and the pot sampling 3.
• the circuit 42 previously mentioned is therefore also one of the constituent elements of the circuit for injecting control fluids, in accordance with the object of the present invention, this circuit 42 making it possible to dry the circuits ensuring the connection between reactor R and the sampling pot 3.
• the first 40, second 41 and third 42 injection circuits can advantageously be constituted by a first three-way solenoid valve 421 interconnected in connection between the end of the module of sampling connected to the filtration module 1 and the sampling pot 3.
• This first three-way solenoid valve 421 receives on an auxiliary input the rinsing water or the drying air, as will be described below .
• the second injection circuit 41 can, as shown in FIG. 1, advantageously be constituted by a second three-way solenoid valve 422 connected in connection between the rinsing water supply circuit and the auxiliary input of the first solenoid valve 421 previously mentioned.
• the second three-way solenoid valve 422 receives on an auxiliary input the drying air intended to ensure the above-mentioned drying.
• a third three-way solenoid valve 423 is provided, this third solenoid valve being connected in connection between the first solenoid valve 421, at the output thereof, and the sampling pot 3.
• An auxiliary input of the third solenoid valve 423 receives the inert gas with the biological liquid to perform the pre-cleaning or unclogging function mentioned above.
• the third solenoid valve 423 thus constitutes the inlet to the first injection circuit 40 mentioned above.
• the supply of inert gas with respect to the biological liquid can be carried out from a reservoir of inert gas under pressure.
• this previously mentioned solenoid valve 422 is supplied from a distilled water tank, which distributes the previously mentioned rinsing water.
• the sampling means 2 also comprise a pump of the peristaltic pump type 20 ensuring the interconnection between the third solenoid valve 423 and the sampling pot 3.
• the peristaltic pump 20 is associated with a valve or solenoid valve 22 mounted by bypass on the aforementioned pump.
• the abrupt opening of the solenoid valve 22 makes it possible to unclog the filtration module 1 during sampling. Indeed, this opening causes the suction stop upstream of the pump 20, and therefore a takeoff of the particles which would have agglomerated on the outer layer or membrane 113 of the filtration module.
• junction between the sampling element 2 and the filtration module 1 can advantageously be carried out by means of a manual valve 23.
• This manual valve makes it possible to provide a safety function in order to totally isolate, if necessary, the reactor R, and the biological medium contained in the latter, from the entire installation.
• the sampling pot 3 is a refrigerated pot.
• the sampling pot 3 has a double wall, in which a stream of refrigerant liquid is formed, making it possible to maintain the sample of biological fluid taken at a predetermined temperature depending on the nature of the biological fluid considered.
• the refrigeration system of the sampling pot 3 will not be described because it can be produced by any means usually used in the laboratory allowing refrigeration conditions compatible with obtaining and maintaining the sterility of the sample or samples taken. A more detailed description of an advantageous embodiment of a sampling pot 3 will be given in connection with FIG. 2.
• FIG. 2 above represents a sectional view along a longitudinal plane of symmetry of a sampling pot 3 according to an advantageous embodiment of the system which is the subject of the invention.
• the sampling pot comprises an orifice 30 for entering the sample, this orifice 30 being directly connected at the outlet of the peristaltic pump 20.
• the orifice for entering the sample 30 is itself connected to a sample inlet chamber 31, this inlet chamber being provided with a distributor 310.
• a vent 31 1 is provided, this vent being in communication with the aforementioned inlet chamber 31.
• the distributor 310 can advantageously consist of a conical element of revolution provided with through holes parallel to the longitudinal axis of the sampling pot 3.
• the aforementioned through holes are in communication with a sample receiving chamber 32.
• the sample reception 32 is itself in communication with a sample outlet orifice 33, which is itself connected to the distribution circuit CD, as will be described later in the description.
• the sample reception chamber 32 is provided with a plurality of electrodes 320, 321, 322.
• the above-mentioned electrodes constitute probes of the level of sampled biological liquid.
• the electrode 320 can correspond to a minimum volume of samples taken
• the electrode 321 can correspond to an intermediate volume of samples
• the electrode 322 can correspond to a maximum volume of samples .
• the body of the sampling pot can then be made of a material inert to biological fluid, such as for example food plastics or the like.
• the outlet orifice 35 of the sampling pot is connected to the distribution circuit CD by means of a two-way solenoid valve 35.
• This same two-way solenoid valve tracks 35 is itself interconnected to a three-way solenoid valve 36, one input of which is connected to the two-way valve 35, a first direct outlet is connected to a drain or sewer pipe and a second controlled outlet is connected to a distribution tube or to a sample fraction collector.
• the vent 31 1 in Figure 2 is itself connected via a three-way solenoid valve 37, on the one hand, to a pipe discharge or sewer in direct connection and, on the other hand, in controlled connection to a pressure source of an inert gas or biological liquid sampled.
• the connection between the first three-way solenoid valve 421 and the end of the sampling module connected to the filtration module 1 is carried out by the 'Intermediate of a multi-path selection valve 21.
• the multiple inputs of the selection valve 21 can be connected sequentially to a different determined reactor, each reactor may contain different biological solutions or different batches of the same biological solution.
• the output of the multi-way selection valve 21 is directly connected to the first three-way solenoid valve 421.
• the two-way solenoid valves and the three-way solenoid valves have been represented symbolically, the two-way solenoid valves comprising an inlet inlet A and a discharge outlet r, the energization of the solenoid valve causing admission by the inlet ⁇ and the outlet by the outlet r of the fluid in question.
• the three-way solenoid valves have been represented as comprising an inlet inlet A, a discharge outlet or inlet r and an auxiliary inlet P, the absence of voltage control of the three-way solenoid valve considered allowing the transmission of a fluid between the intake inlet A and the discharge outlet r or vice versa, while the voltage control of the three-way solenoid valve considered has the effect of transmitting of a different fluid between the auxiliary inlet P and the discharge outlet r.
• the filtration module 1 advantageously comprises a central filter body 10 forming a mounting axis for a filtration element and a removable filter element 1 1 formed by a sleeve made of ceramic material.
• the removable filter element II is engaged on the mounting pin 10.
• the connection between the cross-section edges of the filter element 11 and the bearing surfaces of the mounting pin 10 is carried out by means of seals denoted 100, 110 in FIG. 3.
• the seals can advantageously consist of silicone seals.
• the filtration element 11 is constituted by a cylindrical sleeve 112, as shown in FIG. 4, in section along a longitudinal plane of symmetry of the sleeve.
• This cylindrical sleeve 1 12 can advantageously be made of ceramic with an average pore diameter of the order of 10 ⁇ m.
• an inner layer 1 14 and an outer layer 113 of grafted ceramic are provided on the inner and outer side surfaces of the aforementioned cylindrical sleeve.
• the internal 114 and external 113 grafted layers have an average pore diameter less than or equal to 0.2 ⁇ m and act as a filter membrane.
• an enameling or deposition of a layer of epoxy resin 1 15, 1 16 is provided at the two ends of the filter element 11.
• the enameling or the aforementioned deposit can cover the aforementioned ends over a length of about 1 cm.
• the outer 113 and inner 114 layers are made of alumina with an average pore diameter of between 0.1 and 0.4 ⁇ m, preferably 0.2 ⁇ m, and the cylindrical sleeve 112 of ⁇ alumina with a pore diameter of 10 ⁇ m to approximately 15 ⁇ m.
• the cylindrical sleeve 112 and the internal layers 1 14 and external 113 are bonded monolithically by sintering.
• the mounting axis 10 advantageously comprises a central internal pipe 105 leading to the opposite part of the distal part of the above-mentioned mounting axis 10.
• the central internal pipe 105 thus opens up inside the filtration element 11 or the aforementioned cylindrical sleeve 112 and constitutes the end of the sampling means 2.
• the orifices through which the central internal channel 105 opens up in the sleeve 112 are noted 1050 and 1051.
• control and calculation means 5 will be given in conjunction with FIGS. 1 and 5.
• control and calculation means 5 can advantageously be constituted by a microprocessor CPU interconnected to interface circuits 51 and 52 and further comprising a program memory 50.
• the aforementioned interface circuits advantageously comprise a first series 51 of sampling control interface circuits.
• These interface circuits of the first series 51 may each consist of a digital analog conversion circuit delivering to each of the three-way solenoid valves previously described and to the peristaltic pump 20 a voltage for controlling on or off for example.
• the second series 52 of sample level detection interface circuits may consist, for each of the interfaces, in an analog-to-digital conversion circuit delivering to the microprocessor CPU corresponding messages of sample level in the pot d '' sampling 3.
• the program memory 50 can advantageously be constituted by a ROM read-in memory in which a program for sequentially conducting the sampling is installed.
• the program for sequential sampling of the sample can advantageously include a first sub-program 1000 making it possible to ensure a pre-cleaning phase of the filter element 1 by admitting a inert gas to reactor or fermenter R.
• the third three-way solenoid valve is voltage-controlled, solenoid valve 423, and the first and second three-way solenoid valves are at rest, solenoid valves 421, 422.
• the first subroutine 1000 is then followed by a second subroutine 1001 allowing the system, object of the invention, to be placed in a waiting phase for a duration of a few seconds for example. In this case, none of the sampling elements is excited.
• the second subroutine 1000 previously mentioned is then followed by a third subroutine 1002 making it possible to ensure a sampling phase proper.
• the peristaltic pump 20 is then periodically controlled in voltage and the stopping of the sampling phase is carried out upon detection by the microprocessor CPU of the corresponding messages of sample level in the sampling pot 3.
• the third sub-program 1002 is then followed by a fourth sub-program 1003 making it possible to ensure a phase of transfer of the sample contained in the sampling pot 2 to the analyzer circuits or, at least, to the distribution circuits CD via the solenoid valves 35 and 36.
• the fourth sub-program 1003 allows for example, following the opening of the solenoid valves 35 and 36, to control the voltage of the solenoid valve 37 so as to send to the sampling pot 3 at the upper part thereof relative to the sample contained in the aforementioned sampling pot an overpressure of inert gas with the biological liquid constituting the sample Ion. This overpressure ensures transfer of the entire sample to the fractionation collector according to a predetermined sequence.
• the fourth subroutine 1003 previously described is then followed by a fifth subroutine 1004 making it possible to ensure rinsing with water by voltage control of the first solenoid valve 421, the second solenoid valve 422 in the non-excited state delivering the rinsing water to the sampling pot 3 via the peristal ⁇ tick pump 20 which is itself tensioned. Rinsing, of course, can be carried out continuously for a fixed period.
• a seventh sub-program 1006 for drying the sampling pot is provided in order to ensure the drying of the aforementioned sample pot and the pipes for a few minutes by means of the voltage control of the first 421 and second three-way solenoid valve 422 and of the peristaltic pump 20.
• a subroutine can be called arbitrarily so as to constitute an appropriate sequence according to the application considered.
• the analyzer circuits 1003 it is possible between the sampling phase sub-program 1002 and the sample transfer phase sub-program to the analyzer circuits 1003 to insert a call from the unclogging phase sub-program 1000 to constitute a new unclogging of the element. filtration 1.
• a summary table of a preferred automatic sampling cycle is given below in the description, this table indicating by way of nonlimiting example, on the one hand, the sub-programs called, the numbers of the corresponding sequences, the operations units corresponding to each sequence, the duration of these operations and the solenoid valves and system elements shown in Figure 1 controlled in voltage.
• the aforementioned durations are given as an indication and are liable to be modified for adaptation to the different types of culture or biological media.
• the duration of the sequence 5 of unclogging can be increased up to 3 to 4 seconds if necessary and the sequence 6 of transfer can be of the order of 10 seconds. That of sequence 9 is very fast and that of sequence 10 is arbitrary.
• the microprocessor CPU can advantageously be constituted by an 8 bit microprocessor normally available on the market.
• the microprocessor CPU can be interconnected by a bus type link to a microcomputer, which makes it possible to ensure interactive operation of the system object of the invention with a user, the system, object of the aforementioned invention, being able to then be programmed.
• each bit of the 8 bit word processed by the microprocessor considered is assigned to one of the elements such as two-way or three-way solenoid valve or peristaltic pump of the device represented in FIG. 1 via the corresponding control interface 51.
• each sequence simply corresponds to an 8-bit word constituting the sub-program 1000 to 1006 represented in FIG. 5.
• Each bit of value 0 or 1 can then make it possible, via the aforementioned control interfaces 51, to control voltage or, on the contrary, to remove this voltage control for the solenoid valves or the peristaltic pump considered.
• the microprocessor CPU and the interface circuits are replaced by a programmable controller.
• the programmable controller used was a L ⁇ CKNER-MOELLER brand programmable controller type PS3-DC.
• Such an embodiment allows a more flexible use of the system.
• the system which is the subject of the invention allows installation "in situ" in the absence of a recycling loop. Then the system, object of the invention, ensures a sample collection in the absence of constraint on animal cells, plants or microorganisms in the biological environment in which the sample is to be taken, the system according to the invention , being non-destructive in this.
• the system according to the invention makes it possible to obtain sterile behavior in the absence of the need for steam sterilization in accordance with the devices of the prior art.
• a filter element with a porosity of 0.2 ⁇ m or below in an external layer, single or double, makes it possible to sample a biological medium comprising bacteria.
• Another advantage of the system, object of the invention is the permanent control of the clogging of the filtration element by a first pre-cleaning sequence then a unclogging sequence, following the sampling sequence.
• the system which is the subject of the invention allows the choice of a constant sampling volume whatever the quantity of dissolved and desorbing gas, the concentration of cells of the biological medium considered, the size of these cells or else the internal pressure in reactor R.
• the cartridges of the removable filter element 1 are easily reusable and interchangeable.
• the system, object of the invention also allows modular use from the size of small reactors or fermenters of laboratory capacity of two liters up to industrial size.
• the use of the system, as represented in FIG. 1, is facilitated by the overpressure of the reactor or fermenter R, which makes it possible to envisage a use with relatively viscous biological media or liquids.
• a precise analysis of volatile products can be carried out by refrigerating the sampling pot or molecules with little or no volatility.
• Saccharomyces cerevisiae bacteria like Escherichia coli and

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• 1989
  • 1989-05-22 FR FR8906647A patent/FR2647213B1/fr not_active Expired - Lifetime
• 1990
  • 1990-05-21 JP JP2508392A patent/JPH04507456A/ja active Pending
  • 1990-05-21 WO PCT/FR1990/000356 patent/WO1990014586A1/fr not_active Ceased
  • 1990-05-21 EP EP19900908539 patent/EP0473676A1/de not_active Withdrawn

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