INST FOR POLIOMYELITIS, CHUMAKOV FED SCI CTR R&D IMMUNE&BIOLOGICAL PRODS, RUSSIAN ACAD OF SCIENCES

Abstract

The sampler (1) for connection to a bioreactor (2) filled with a cell culture aqueous solution includes a conduit (1) having a first end (101) for connection to a port (21) of the bioreactor (2), said port being intended for collecting samples of the cell culture aqueous solution, and a second free end (102), a hydrophobic air filter (11) for a free egress of air from the conduit (10) to an exterior through the second free end (102), when the conduit (10) is filled with the cell culture aqueous solution, the hydrophobic air filter (11) being impermeable to the cell culture aqueous solution, and a transparent solid container (12) incorporated into the conduit (10) between the first end (101) and the hydrophobic air filter (11). The transparent solid container (12) is adapted for filling with the cell culture aqueous solution and for a contactless analysis of the solution.

Claims

1. A sampler idi adapted in an operational state for connection to a bioreactor filled with a cell culture aqueous solution, the sampler comprising: a conduit having a first end adapted for connection to a port of the bioreactor, the port being intended for collecting samples of the cell culture aqueous solution, and a second free end opposite the first end, a hydrophobic air filter disposed within the conduit between the first end and the second free end, the hydrophobic air filter being adapted for a free egress of air from the conduit to an exterior through the second free end under an effect of a pressure differential between the first end and the second free end, when the conduit of the sampler is filled, in the operational state, with the cell culture aqueous solution from the bioreactor, the hydrophobic air filter being impermeable to the cell culture aqueous solution, and a transparent solid container incorporated into the conduit between the first end and the hydrophobic air filter, the transparent solid container being adapted for filling with the cell culture aqueous solution and for a contactless analysis of the cell culture aqueous solution with the help of an analyzer.

2. The sampler according to claim 1, wherein the transparent solid container has a shape of a quartz cuvette, the quartz cuvette being adapted to be placed in a spectrophotometer.

3. The sampler according to claim 1, wherein the transparent solid container is adapted to be placed on a slide of an optical microscope.

4. The sampler according to claim 1, wherein the transparent solid container comprises an analysis zone having a shape of a flat slit extending along an axis of the conduit, an inner thickness of the flat slit being comprised in a first interval from 1 mm to 4 mm.

5. The sampler according to claim 1, wherein the sampler is adapted for disposable use.

6. The sampler according to claim 1, wherein the sampler is adapted for sterilizing in an autoclave at a temperature of at least 120 C. during at least 1 hour.

7. The sampler according to claim 1, wherein the sampler is adapted for steam sterilizing at a temperature comprised in a second interval from 120 C. to 160 C. during at least 1 hour.

8. The sampler according to claim 1, wherein the sampler is adapted for sterilizing with ionizing gamma radiation.

9. The sampler according to claim 1, wherein the second free end is adapted for connection to an air pump, the sampler in the operational state being adapted for operating under a pressure in the conduit comprised in a third interval from 0.1 bar to 2.0 bar.

10. The sampler according to claim 1, wherein the sampler comprises a sterilizing air filter arranged inside the conduit between the hydrophobic air filter and the second free end of the conduit, the sterilizing air filter being adapted for sterilizing an external air forced inside the conduit through the second free end by the air pump when the sampler is emptied back into the bioreactor after the contactless analysis of the cell culture aqueous solution.

11. The sampler according to claim 10, wherein the sterilizing air filter has a porosity comprised in a fourth interval from 0.1 micrometer to 0.2 micrometer.

12. The sampler according to claim 1, wherein the sampler comprises a first locking element arranged between the transparent solid container and the first end of the conduit and adapted for locking the conduit from a side of the first end of the conduit.

13. The sampler according to claim 1, wherein the sampler comprises a second locking element arranged between the hydrophobic air filter and the second free end of the conduit and adapted for locking the conduit from a side of the second free end of the conduit.

14. The sampler according to claim 1, wherein the sampler comprises a retainer associated with the transparent solid container and adapted to secure the transparent solid container to the analyzer of the cell culture aqueous solution from the bioreactor.

15. The sampler according to claim 1, wherein the first end of the conduit is adapted for connection by welding to the port of the bioreactor, the port being intended for collecting samples of the cell culture aqueous solution.

16. The sampler according to claim 2, wherein the transparent solid container is adapted to be placed on a slide of an optical microscope.

17. The sampler according to claim 16, wherein the transparent solid container comprises an analysis zone having a shape of a flat slit extending along an axis of the conduit, an inner thickness of the flat slit being comprised in a first interval from 1 mm to 4 mm.

18. The sampler according to claim 2, wherein the transparent solid container comprises an analysis zone having a shape of a flat slit extending along an axis of the conduit, an inner thickness of the flat slit being comprised in a first interval from 1 mm to 4 mm.

19. The sampler according to claim 3, wherein the transparent solid container comprises an analysis zone having a shape of a flat slit extending along an axis of the conduit, an inner thickness of the flat slit being comprised in a first interval from 1 mm to 4 mm.

20. The sampler according to claim 2, wherein the sampler is adapted for disposable use.

Description

[0065] Other distinctive features and advantages of the invention are clearly derived from the description set forth below for illustration and without being restrictive, with reference to the accompanying figures, in which:

[0066] .sub.1FIG. 1 schematically shows in a simplified top view (in a plane perpendicular to gravity G) the sampler according to the invention in its operating state E, i.e. connected to the bioreactor;

[0067] FIG. 2 schematically depicts in a simplified top view (in a plane perpendicular to gravity G and coinciding with the plane of the slide of an optical microscope used for visual analysis of samples) a transparent solid container of the sampler, according to a first embodiment thereof;

[0068] FIG. 3 schematically depicts in a simplified side view (in a plane coincident with the force of gravity G and perpendicular to the plane of the slide of the optical microscope used for visual analysis of samples) a transparent solid container of the sampler, according to a first embodiment thereof;

[0069] FIG. 4 schematically depicts in a simplified top view (in a plane perpendicular to the force of gravity G and coinciding with the plane of the slide of an optical microscope used for visual analysis of samples) a transparent solid container of the sampler, according to a second embodiment thereof;

[0070] FIG. 5 schematically depicts in a simplified side view (in a plane coinciding with the force of gravity G and perpendicular to the plane of the slide of the optical microscope used for visual analysis of samples) a transparent solid container of the sampler, according to the second embodiment, wherein the analysis zone has the form of a flat slit extended along the axis of the pipeline.

[0071] .sub.1As mentioned previously and illustrated in FIGS. 1 to 5, the invention relates to a sampler 1 adapted in operating condition E for connection to a bioreactor 2. The latter enables the multiplication of pathogenic microorganisms, such as viruses, on a culture of cells contained within the bioreactor 2 in a nutrient medium (aqueous solution) under controlled conditions in terms of sterility, in terms of stirring intensity, in terms of continuous purging with sterile air, and in terms of constant temperature. The bioreactors 2 may be in various designs (disposable, e.g., in the form of a plastic bag, or reusable, e.g., in the form of a glass tank or flask). Bioreactors 2 can be designed in various volumes, ranging from a few hundred milliliters to thousands of liters. Cells can be cultured on suspension cells or on microcarriers, for example in the form of polymer beads.

[0072] In a non-operational state (not shown), the sampler 1 is disconnected from the bioreactor 2. It may be in this inoperative state as a separate accessory until it is connected to bioreactor 2 (i.e., until a sample is taken). Alternatively, the sampler 1 may remain in a non-operational state after disconnection from the bioreactor 2 (i.e., after sample collection) pending further sterilization and/or or disposal.

[0073] As shown in FIG. 1, the sampler 1 contains: [0074] conduit 10, being: [0075] A first end 101 adapted to connect to a port 21 of the bioreactor 2 for sampling an aqueous solution with cell culture, and [0076] A second free end 102 opposite the first end 101, [0077] A hydrophobic air filter 11 disposed within the conduit 10 between its first end 101 and its second free end 102.
.sub.1In this case, the hydrophobic air filter 11 is adapted to allow air to freely escape from the pipeline 10 to the outside through the second free end 102, under the effect of a pressure differential between the first end 101 and the second free end 102, when the pipeline 10 of the sampling device 1 is filled (e.g. by gravity G) in the working condition E with an aqueous solution with cell culture from the bioreactor 2. Under these conditions, the hydrophobic air filter 11 is impermeable to the aqueous solution with cell culture: this prevents the sample from spilling out of the second free end to the outside of the working space (not shown).

[0078] According to the invention (FIG. 1), the sampling device 1 comprises a transparent solid container 12 cut into the pipeline 10 between the first end 101 and the hydrophobic air filter 11. In these circumstances, the transparent solid container 12 is adapted to be filled with this aqueous solution with cell culture and to analyze this aqueous solution with cell culture in a non-contact manner. This analysis is performed by an operator using an analyzer 40 (FIG. 1), that is, using one or more instruments that are adapted to measure the properties, composition, structure of the sample. Such analyzer 40 may be, for example, a spectrophotometer. In this case, the transparent solid container 12 may be in the form of a quartz cuvette (not shown). This quartz cuvette is adapted to be placed in the cuvette holder of a spectrophotometer (not shown).

[0079] Also, the analyzer 40 may be, for example, an optical microscope (not shown). In this case, the transparent solid container 12 may be made of glass or plastic (FIGS. 2-5). This transparent solid container 12 may be adapted to be placed on a slide (not shown) of the optical microscope.

[0080] A first embodiment of the transparent solid container 12 is shown in FIGS. 2-3. In this first embodiment, the transparent solid container 12 has the shape of a cylinder (FIG. 3) extending along the axis AB of the pipeline 10, which in the analysis zone contains a flat (in the plane perpendicular to the gravity force G in FIG. 2) elliptically shaped extension. In this case, the inner diameter of this cylinder (measured, as shown in FIG. 3, along the force of gravity G) is contained within the range of 1 mm to 6 mm: 1 mm 6 mm, and the flat elliptical extension has a characteristic maximum dimension (measured, as shown in FIG. 2, in a plane perpendicular to the force of gravity G) exceeding the inner diameter approximately (in order of magnitude) four times: 4. The outer diameter of this cylinder (measured, as shown in FIG. 3, along the force of gravity G) is contained within the range of 3 mm to 8 mm: 3 mm 8 mm, and the thickness of the walls of the transparent solid container 12 is determined by the formula=()/2. In practice, this thickness of the walls is less than or equal to 1 millimeter: 1 mm.

[0081] An advantage of the first embodiment of the transparent solid container 12 is the simplicity and rapidity of its manufacture in mass production: such a shape is easily obtained by glass blowing or thermal molding of corresponding transparent thermoplastic plastics.

[0082] As shown in FIGS. 2-3, opposing ends of the cylinder may include thickenings 120 (e.g., in the form of a ring molded onto the outer surface of the transparent solid container 12) designed to securely fasten the transparent solid container 12 to the conduit 10. In this case, the outer diameter of the thickening 120 (measured, as shown in FIG. 2, perpendicular to the force of gravity G) corresponds approximately (in order of magnitude) to two inner diameters of the cylinder: 2. This increases operator safety when handling the sampler 1.

[0083] A second embodiment of the transparent solid container 12 is shown in FIGS. 4-5. Analogous to the first embodiment described above, in this second embodiment, the transparent solid container 12 has the shape of a cylinder (FIG. 5) extending along the axis AB of the pipeline 10, which in the analysis zone contains a flat (in the plane perpendicular to the force of gravity G in FIG. 4) elliptically shaped extension. In this case, the inner diameter of this cylinder (measured as shown perpendicular to gravity G in FIG. 4) is contained within the range of 1 mm to 6 mm: 1 mm 6 mm, and the elliptically shaped flat extension has a characteristic maximum dimension (measured as shown in FIG. 4, in the plane perpendicular to gravity G) exceeding the inner diameter by a factor of about (in order of magnitude) four: 4. The outer diameter of this cylinder (measured, as shown in FIG. 3, along the force of gravity G) is contained within the range of 3 mm to 8 mm:3 mm 8 mm, and the thickness of the walls of the transparent solid container 12 is determined by the formula=()/2. In practice, this wall thickness is less than or equal to 1 millimeter: 1 mm. As shown in FIG. 5, in this second embodiment, the analysis zone which contains, as indicated above, a flat (in a plane perpendicular to gravity G in FIG. 4) elliptically shaped extension (FIG. 4), is additionally a flat slot extending along the axis AB of the pipeline 10. In this case, the inner thickness of this flat slot is smaller than the inner diameter of the cylinder (i.e. <) and is predominantly in the first range of one to four millimeters: 1 mm 4 mm.

[0084] Advantageously, the sampler 1 is adapted for disposable use.

[0085] The conduit 10 may be made of a flexible material.

[0086] The sampling device 1 can be adapted: [0087] for sterilization in an autoclave at a temperature T of at least 120 C. for at least 1 hour, and/or. [0088] for steam sterilization at a temperature T contained in the second range of 120 C. to 160 C. (120 C. T 160 C.) for at least 1 hour, and/or [0089] for sterilization by ionizing gamma radiation.

[0090] As shown in FIG. 1, the second free end 102 may be adapted to be connected to an air pump 3 (e.g. in the form of a medical syringe of appropriate volume). .sub.1In this case, the sampler 1 in the operating state E is adapted to operate at a pressure P in the pipeline 10 contained in the third interval between 0.1 bar and 2.0 bar: 0.1 bar P 2.0 bar.

[0091] As shown in FIG. 1, the sampler 1 may comprise a sterilizing air filter 13 positioned within the pipeline 10 between the hydrophobic air filter 11 and the second free end 102 of the pipeline 10. This sterilizing air filter 13 is adapted to sterilize external air forced inside the conduit 10 through the second free end 102 by the air pump 3 when the sampler 1 is emptied back into the bioreactor 2 after non-contact analysis of the aqueous solution containing the cell culture.

[0092] Advantageously, the sterilizing air filter 13 has a porosity, contained in the fourth interval from 0.1 micrometer to 0.2 micrometer: 0.1 micrometer 0.2 micrometer.

[0093] As shown in FIG. 1, the sampling device 1 may comprise a first locking element 14 disposed between the transparent solid container 12 and the first end 101 of the conduit 10 and adapted to lock the conduit 10 at a side of the first end 101 of the conduit 10.

[0094] As shown in FIG. 1, the sampling device 1 may include a second locking element 15 disposed between the hydrophobic air filter 11 and the second end of the conduit 102 and adapted to lock the conduit 10 to the side of the second end 102 of the conduit 10.

[0095] Advantageously, the second locking element 15 is arranged on the side of the second free end 102 of the conduit 10 between the hydrophobic air filter 11 and the sterilizing air filter 13.

[0096] As shown in FIG. 1, the sampler 1 may include a retainer 16 associated with the transparent solid container 12 and adapted to secure the transparent solid container 12 to the analyzer 40 of an aqueous solution containing cell culture from the bioreactor 2.

[0097] As shown in FIG. 1, the sampling device 1 may include a first connector 17 disposed at a first end 101 of the conduit 10 and adapted for first quick-release connection of the conduit 10 to a port 21 of the bioreactor 2 for sampling an aqueous solution containing a cell culture.

[0098] As shown in FIG. 1, the sampling device 1 may include a second connector 18 disposed at the second end 102 of the conduit 10 and adapted for a second quick-release connection of the conduit 10 to the air pump 3.

[0099] The first end 101 of the conduit 10 may be adapted to be connected by heat welding (not shown) to a port 21 of the bioreactor 2 for sampling an aqueous solution with cell culture.

[0100] Advantageously, the length L of the pipeline 10 between its first end 101 and the transparent solid container 12 does not exceed 10 meters: L 10 m.

[0101] Advantageously, the length L of the conduit 10 between its first end 101 and the transparent solid container 12 is contained in a spot range from 0.5 meters to 1.5 meters: 0.5 m L 1.5 m.

[0102] Advantageously, the conduit 10 is a polished and chemically neutral inner surface.

[0103] Two examples of the operation of the sampler 1 according to the invention are given below.

Example 1

[0104] The operator takes the sampler 1 in a disposable and inoperable state from the accessory box for the bioreactor 2. This sampler 1 contains a conduit 10 (in the form of a flexible hose with a polished and chemically neutral inner surface), a transparent solid container 12, a hydrophobic air filter 11, and a sterilizing air filter 13. The operator locks the conduit 10 with a first locking element 14 on the side of the first end 101, and with a second locking element 15 on the side of the second free end 102. .sub.1Next, the operator brings the sampler 1 into the operating state E, shown in FIG. 1, by connecting the first end 101 of the conduit 10 to the bioreactor 2 using the first connector 17 and the second end 102 of the conduit 10 to the air pump 3 (in the form of an empty sterile syringe of 50 mL to 100 mL volume) using the second connector 18. Then, the operator creates, by activating the air pump 3 (i.e., by applying hand pressure to the syringe piston), a predetermined vacuum in the conduit 10. After that, the operator unclamps the first locking element 14 and the second locking element 15: under the action of the predetermined vacuum, the aqueous solution with the cell culture rushes from the bioreactor 2 into the pipeline 10 and further into a transparent solid container 12 in the form of a quartz cuvette (not shown). After filling this cuvette with sample, the operator places the cuvette in the cuvette holder (otherwise known as the nest) of the spectrophotometer. Next, the operator performs a spectrophotometric analysis of the contents of the bioreactor 2. After the end of the analysis by activating the air pump 3 (i.e. by reverse stroke of the syringe piston) the cell suspension with pathogen is brought from the sampler 1 back into the bioreactor 2. Next, the operator locks the conduit 10 with the first locking element 14 at the side of the first end 101. Thereafter, the operator places the sampler 1 in an inoperable state by disconnecting the first end 101 of the pipeline 10 from the bioreactor 2 using the first connector 17. Finally, the disposable sampler 1 together with the air pump 3 connected thereto is disposed.

Example 2

[0105] The operator takes a reusable and inoperable sampler 1 from the bioreactor accessory box 2. This sampler 1 contains a pipeline 10 (in the form of a flexible hose with a polished and chemically neutral inner surface), a transparent solid container 12, a hydrophobic air filter 11, and a sterilizing air filter 13. The operator locks the conduit 10 with a first locking element 14 on the side of the first end 101, and with a second locking element 15 on the side of the second free end 102. .sub.1Next, the operator brings the sampler 1 into the operating state E, shown in FIG. 1, by connecting the first end 101 of the conduit 10 to the bioreactor 2 using the first connector 17 and the second end 102 of the conduit 10 to the air pump 3 (in the form of an empty sterile syringe of 50 mL to 100 mL volume) using the second connector 18. Then, the operator creates, by activating the air pump 3 (i.e., by applying hand pressure to the syringe piston), a predetermined vacuum in the conduit 10. After that, the operator unclamps the first locking element 14 and the second locking element 15: under the action of the predetermined discharge of aqueous solution with cell culture rushes from the bioreactor 2 into the pipeline 10 and then into a transparent solid container 12 in the form of a cylinder (FIG. 5), extended along the axis AB of the pipeline 10, which in the analysis zone contains a flat (in the plane perpendicular to the force of gravity G in FIG. 4) elliptical expansion (FIG. 4). This elliptically shaped extension holds a narrow flat slot extending along the axis AB of the pipeline 10, as shown in FIG. 5.

[0106] The operator fills of the entire volume of the transparent solid container 12 with the sample. Next, the operator re-locks the conduit 10 with a first locking element 14 on the side of the first end 101, and with a second locking element 15 on the side of the second free end 102. Then, the operator secures the one-third filled transparent solid container 12 to the optical microscope slide by means of a retainer 16 (in this example, by means of two rubber bands located on either side of the transparent solid container 12 along the axis AB of the conduit 10). The operator then visually analyzes the state of the cell culture within the transparent solid container 12 using the optical microscope.

[0107] After the analysis is completed, the operator unclamps the first locking element 14 and the second locking element 15, and then by activating the air pump 3 (i.e. by reversing the stroke of the syringe piston) the cell suspension with the pathogen is introduced from the sampler 1 back into the bioreactor 2. Next, the operator re-locks the conduit 10 with a first locking element 14 on the side of the first end 101, and with a second locking element 15 on the side of the second free end 102. Thereafter, the operator places the sampler 1 in an inoperable state by disconnecting the first end 101 of the pipeline 10 from the bioreactor 2 using the first connector 17 and the second end 102 of the pipeline 10 from the air pump 3 using the second connector 18. Finally, the reusable sampler 1 is sterilized by ionizing gamma radiation for further use.

[0108] The main advantages of the sampler 1 according to the invention are summarized below: [0109] minimizes the risk of release of infectious agent into the environment and, in particular, on the operator, on measuring instruments, in the working room, because during any manipulations with the sample (taking from the bioreactor 2/analysis of the sample/return of the sample to the bioreactor 2) the cell culture studied by the operator does not come into contact with the external environment. Thus, the risk of contamination is reduced and, consequently, the working conditions and safety of all working personnel operating the bioreactor 2 and, in particular, the operator who performs routine operations of sample collection, analysis and subsequent return to the bioreactor 2 are improved; [0110] the risk of contamination of the bioreactor 2 itself is minimized when the sample is returned to the bioreactor 2 after it has been analyzed by an operator using an analyzer (e.g., an optical microscope with a magnification of 5, 10, 20, and/or a spectrophotometer), because even during the analysis of the sample there is no contact of the contaminated cell culture (i.e., infectious agent) with the environment; [0111] minimizes the consumption of supplies for daily monitoring of infected cells, reduces waste in routine work to monitor the multiplication of pathogens, such as viruses, on cell culture media (aqueous solution) under controlled conditions in bioreactor 2.