One-way separator for retaining and recirculating cells

Abstract

The invention relates to a separator for retaining and recirculating cells in a continuous-flow or batch-flow type plastic bag or bottle, which can preferably be operated outside of a bioreactor. Additionally, the invention relates to a method for retaining and recirculating cells within or outside a bioreactor. The invention further relates to a method for producing the separator according to the invention.

Claims

1. A solids separator for retaining and recirculating solids from a reactor mixture, comprising a flow-bearing sterilizable plastic bag or plastic bottle and, within the plastic bag or plastic bottle: in the upper region, one or more passages/internals for withdrawing from a harvest stream collection region a harvest stream that is separated from the solids, in the upper segment of a central region, a separation region having a separation area, which, during operation, is inclined at an angle of 0° to 80° to the horizontal, in the lower segment of the central region, one or more passages or internals for a uniform horizontal flow distribution of the reactor mixture over an introduction surface, wherein the separation area is situated above the introduction surface, in the lower region a solids collection region that is tapered at the bottom for collecting the solids using gravity.

2. The solids separator of claim 1, wherein solids collection region has one or more passage or internals for withdrawing the solids.

3. The solids separator of claim 2, wherein the plastic bag or plastic bottle has a rectangular cross section, wherein the downwardly tapered solids collection region ends in a neck which is closed with a cover or stopper, wherein the cover or stopper has all passages.

4. The solids separator of claim 1, wherein the solids collection region is tapered downwards conically or pyramidally.

5. The solids separator of claim 1, which comprises at least one single-use sensor in the interior.

6. The solids separator of claim 1, wherein the separation region consists of a multiplicity of adjacently arranged channels in a lamellae pack and the separation area during operation is inclined at an angle to the horizontal of 30° to 80°.

7. The solids separator of claim 6, wherein the lamellae pack consists of a plurality of ridgeplates stacked one above the other which form the channels of the lamellae pack.

8. The solids separator of claim 7, wherein the ratio of ridge height to channel width hs/d is 0.01≦hs/d ≦5 with the restriction.

9. The solids separator of claim 6, wherein the channels have a channel length L of 30% to 95% of a length LK of the plastic bag or plastic bottle.

10. The solids separator of claim 1, wherein the plastic bag is polyhedral or conical and wherein the plastic bag, during operation, is placed such that the solids collection region which is tapered at the bottom is formed by the walls of the plastic bag and the apex or corner of the polyhedron or of the cone.

11. The solids separator of claim 10, wherein the plastic bag is a disphenoid, a regular pyramid, an octahedron or a cube.

12. The solids separator of claim 1, comprising a container for receiving the plastic bag, wherein the container comprises at least: (a) an interior for receiving the plastic bag, wherein the interior is adapted to the shape of the plastic bag by means of walls which are adapted to the shape of the plastic bag and enclose the interior and delimit it from the exterior, and (b) an opening for introducing the plastic bag from the top into the container.

13. The solids separator of claim 1, wherein the separation area does not comprise plates.

14. A bioreactor system comprising a bioreactor connected to a solids separator as claimed in claim 1.

15. A method for retaining and recirculating solids in a flow-bearing vessel of the solids separator of claim 1, comprising supplying solids-containing medium continuously or batchwise to the vessel, and removing solids-free medium from the vessel, wherein the vessel is the flow-bearing sterilizable plastic bag or plastic bottle which, in the lower region, comprises faces set at an incline, favorably the solids collection region, which is tapered conically at the bottom for collecting the solids with the aid of gravity, wherein the solids comprise cells.

Description

(1) Hereinafter, exemplary embodiments of the invention are described in more detail with reference to drawings without restricting the invention thereto.

(2) FIG. 1 Schematic depiction of the single-use solids separators according to the invention with lamellae pack.

(3) FIG. 2 Schematic depiction of a lamellae pack 1 (longitudinal section)

(4) FIG. 3 Schematic depiction of a lamellae pack 1 (longitudinal section)

(5) FIG. 4 Diagram of the structure of various lamellae packs (cross section AA′ of FIG. 3)

(6) FIG. 5 Diagram of the application of the plastic bag 50 on a lamellae pack 1 (cross section AA′ of FIG. 3)

(7) FIG. 6 and FIG. 7 Stretching and fastening of the plastic bag 50 on a lamellae pack 1 (cross section)

(8) FIG. 8 and FIG. 9 Alternative stretching and fastening of the plastic bag 50 on a lamellae pack 1 with the aid of frame 130 and cover 110 (cross section)

(9) FIG. 10 Side views of the solids separators according to the invention with lamellae pack 1 on stand 140.

(10) FIG. 11 Front views of the solids separators according to the invention with lamellae pack 1 on stand 140.

(11) FIG. 12 Longitudinal sections of the solids separators according to the invention with lamellae pack 1 on stand 140 with frame 130 and cover 110.

(12) FIG. 13 Front views of the solids separators according to the invention with lamellae pack 1 on stand 140 thereof with frame 130 and cover 110.

(13) FIG. 14 Schematic three-dimensional depiction of the solids separators according to the invention in the disphenoidal embodiment

(14) FIG. 15 Schematic longitudinal section of the solids separators according to the invention in the disphenoidal embodiment with flow inverter 81

(15) FIGS. 16 and 17 Schematic depiction of the solids separators according to the invention in the cubic embodiment.

(16) FIG. 18 Process diagram of a perfusion reactor. In order to reduce the respiratory activity of the cells in the bioreactor sequence, the temperature thereof is decreased to a lower level in a cooling device as soon as possible after the takeoff. In this manner, the cells in the cell separator are prevented from having too long a residence in an oxygen-limited state, which could damage the cells physiologically. In the example shown, the separator 640 consists of a separation bag 620 and an integrated cooling device 600. The liquid streams between bioreactor 610 and separator 640 are established via the low-shear pumps 630 and 631. Other circuitry, e.g. the positioning of one of the two pumps 630 and 631 in the bioreactor sequence, are likewise conceivable.

(17) FIG. 19 Comparison of the separation systems

(18) FIG. 20 Comparison of the separator volumes per unit separation area.

(19) FIG. 21 Longitudinal sections of the bottle separator with trays as separation area 1, horizontal flow distributor 85 and stopper 220.

(20) FIG. 22 Lateral section of the bottle separator according to FIG. 21 on its stand.

(21) FIG. 23 Details of a stopper 220 with collar 230 and flow distributor 85 with downwardly directed inlet flows

(22) FIG. 24 Front views of a suspended plastic bag (=BagSettler) as solids separator and lateral section on stand.

(23) FIG. 25 shows the effect of various flow distributors 85 on the retention performance R under a varying effective ascension velocity v=q/Aeff.

REFERENCE SIGNS

(24) 1 Lamellae pack/separator area 5 Ridge width 8 Plate separation 10 Angle 13 Length 15 Width 18 Height 30 Support plate 50 Plastic bag or plastic bottle 51 Neck 52 Excess/fold 55 Weld seam 56 Harvest stream collection region 57 Solids collection region 58 Angle 59 Angle 60 Fastening strip 70 Harvest stream (harvest) 74 Bioreactor mixture/feed 79 Recirculation 80 Passage 81 Flow inverter 84 Passage 85 Flow distributor in particular horizontal distributor or flow distributor having downwardly directed inlet flows 86 Inlet flow 88 Central removal by suction 89 Passage 90 Connection plate 100 Housing 110 Cover 112 Elongation 115 Fastening element 130 Frame 140 Stand 142 Projection 145 Stand foot 148 Support 200 Vibrator 210 Assembly plate 220 Cover or stopper 230 Collar nut 240 O ring
Profiles of a Lamellae Pack 311 Lamellae pack 320 Rectangular profile 321 Lamellae pack 330 Round profile 331 Lamellae pack 340 Round profile 341 Lamellae pack 350 Hexagonal profile 351 Lamellae pack 500 Theoretical maximum separator area 501 Separator region 510 Inlet surface 600 Cooling device 610 Bioreactor 620 Separation device 630, 631 Pumps 640 Separator=Separation bag+cooling device optionally integrated in the stand or container 650 Culture medium

(25) Hereinafter, studies are described on the applicability of the devices according to the invention without restricting it thereto.

(26) Particle System

(27) For the simulation of cells, the particle system polyacrylonitrile X polymer “PAN-X” is used. The water-insoluble polymer is principally used in the clothing industry for producing fibers. Hereinafter is an extract from the product data sheet of the manufacturer Dralon GmbH, Dormagen.

(28) TABLE-US-00001 Name PAN-X Chemical formula [C.sub.3H.sub.3N].sub.n Administration form Powder Particle form spherical Color white Density 1.18 g/m.sup.3 Particle size distribution 97 vol % ≦ 50 μm Solubility in water insoluble Ignition temperature 485° C.

(29) The particle size distribution shows the most frequent particle diameter between 15 and 30 μm measured using the Mastersizer laser diffraction measuring instrument from Malvern, which corresponds roughly to eukaryotic cells (CHO, BHK).

(30) Separation Systems

(31) Inclined Channel Separator

(32) For purposes of comparison, a study was made of a large and a small plate separator made of stainless steel according to WO03/020919 having theoretical separation areas of A.sub.th=1.42 m.sup.2 and A.sub.th=0.027 m.sup.2 as a model of the separator according to the invention according to FIG. 2. The large separator has 20 plates which are accommodated in a separator volume of 17.4 l. The small lamellae separator consists of 4 plates in a separator volume of 0.3 l.

(33) Cube Separator

(34) Two cubes were produced as a hydrodynamic model having the edge lengths D=200 mm and D=400 mm from Plexiglass plates. The upper corner of the cube had an opening for the passage of flexible tubes. One internal for flow distribution (also termed (flow) distributor 85) of the particle suspension in the form of a T-piece or Y-piece (in each case two inlets) fastened to a flexible tube was inserted up to the center (h=50% HK) of the plastic model. The width of the distributors c was varied. Via a further flexible tube extending to the lower cone apex (solids collection region 57 also collector), the sediment was taken off vertically upwards in a flow-inverting manner. Via a further passage, a plastic tube was fastened for collecting the clear phase=passage 80, in such a manner that the clear phase (=harvest stream 70) was taken off from the surface directed downwards (U-tube) in a flow-inverting manner. The gradual cross section expansions towards the lower and upper separator apex permit a good flow harmonization and already function thereby as flow collectors (solids collection region 57 at the bottom, harvest stream collection region 56 at the top), which should also function sufficiently well without flow-inverting internals.

(35) Tetrahedral Separators

(36) The tetrahedron was produced as a Plexiglass model from equilateral triangles having an edge length D=400 mm Opposite the corner, which was selected as conical solids collection region 57, the solids separator was open at the top. Via the openings, various passages for introduction and takeoff of suspension (=feed 74), sediment and clear run (=harvest stream 70) could be installed. At the passages, generally flexible tubes, distributor and plastic tube for collecting the clear phase (=passage 80) were constructed and positioned in an analogous manner to the cube.

(37) The tetrahedron differs in its flow characteristics compared with the cube primarily in that a flow-favoring tapering is only present downwards to the return collector and the clear phase is collected at the point of the largest surface area. For uniform takeoff, in the tetrahedron, therefore, a flow distributor 85 placed in this maximum separation area is used.

(38) The separation area A.sub.th in the unit studied was 0.069 m.sup.2 at a volume of 7.6 1.

(39) Vertical Separator with Conical Feed—Dortmund Tank Type

(40) This vertical separator is frequently used in the wastewater industry. The separator studied consisted of a cylindrical shell having a cross section of 145 mm which forms a collection funnel in the lower region and, in the upper region, has a centrally placed conical feed having a cross section of 51 mm. Both elements were fabricated from glass. The non-clarified liquid was introduced via the conical feed from the top into the cylindrical region (vertical separation region) and ascended into the separation region, whereas the suspended matter sedimented in the collection funnel. At the top end of the separation region, the clear phase was collected at four points. The vertical separator studied had a volume of 1.7 l of a separator area A.sub.th of 0.014 m.sup.2.

(41) Methods

(42) Analytical Method

(43) The sample is filtered off using a Buchner funnel (pore size<2 μm), the filter paper is dried at 140° C. and weighed. A drying balance (Sartorius MA45) was used therefor.

(44) Experimental Procedure

(45) PAN-X 3 g/l was provided in the reservoir tank and introduced into the respective separator by means of peristaltic pumps (Watson-Marlow Du323). The desired ascension velocity v (v=q/Ath, wherein q is the harvest stream with which the separator is loaded for a given perfusion rate and bioreactor volume V) is set via the pump rate of the peristaltic pumps q.

(46) The particle suspension is first pumped in circulation. After a waiting time of two hydrodynamic residence times, for establishing stationary conditions, the sampling from the harvest stream was started.

(47) The sample volume is oriented according to the particle mass on the filter. This should be approximately 100 mg±25 mg within the limits of measurement accuracy. Sample volumes result therefrom of 40 to 800 ml for determining the particle concentration, which were measured in triplicate.

(48) Results

(49) TABLE-US-00002 TABLE 1 R = w = w_eff = 1 − c.sub.H/c q/Ath w/ηDo Separator Geometry 1 m/h m/h Tetrahedron A m.sup.2 0.0692 0.91 0.03 0.0294 d mm 400 0.82 0.05 0.0588 VS L 6.42 0.74 0.10 0.1176 ηDo — 0.8 0.51 0.20 0.2353 Cube 1 A m.sup.2 0.052 0.96 0.03 0.0208 d mm 200 0.92 0.05 0.0417 VS L 8.00 0.83 0.10 0.0833 ηDo — 1.2 0.63 0.20 0.1667 Cube 2 A m.sup.2 0.208 0.95 0.03 0.0208 d mm 400 0.91 0.05 0.0417 VS L 64 0.81 0.10 0.0833 ηDo — 1.2 0.61 0.20 0.1667 PLA 1 A m.sup.2 0.0274 0.95 0.03 0.0227 Z 1 4 0.90 0.05 0.0455 VS L 0.375 0.76 0.10 0.0909 ηDo — 1.1 0.61 0.20 0.1818 PLA 2 A m.sup.2 1.42 0.96 0.03 0.0227 Z — 21 0.92 0.05 0.0455 VS L 18 0.79 0.10 0.0909 ηDo — 1.1 0.59 0.20 0.1818 Dortmund A m.sup.2 0.0142 0.93 0.03 0.9286 tank d mm 145 0.87 0.05 0.8704 VS L 1.7 0.75 0.10 0.7506 ηDo — 1 0.56 0.20 0.5617

(50) Comparison of the separation systems shows the expected fall in the degree of retention R with increasing media stream or harvest stream q or the effective ascension velocity v=q/A.sub.eff (FIG. 19). The effective ascension velocity results by introducing the efficiency coefficient η1, which identifies the differing retention performance of the maximum area used of the separators compared with the Dortmund tank. FIG. 19 indicates that the separators, after addition of this efficiency coefficient, can be described by a joint line of best fit.

(51) The performance of the separators is compared in FIG. 20. This presentation shows how many separator volumes are necessary in order to accommodate the effective separation area Small separator volumes are desirable in cell cultures in order to minimize residence times outside the fermentation space supplied. In this comparison, the inclined channel separators come out favorably, which can be operated, independently of the scale, with very high separation areas per unit separator volume greater than 50 l/m. This example makes clear the outstanding scalability of these separator systems. In contrast thereto, in the case of the vertical separators, considerably greater volume is required in order to develop therein the horizontal separation area. In addition, the efficiency of accommodation in the scale enlargement falls with V=A.sup.3/2. Surprisingly, the efficiency of the single-use models, the cubes and the tetrahedron is considerably superior to the standard system of the Dortmund tank, and so these very simple and inexpensive systems can be used in considerably larger bioreactors (up to approximately 6-times) than the Dortmund tank. A further adaptation (geometry and/or position) of the feed distributors and harvest stream collectors could lead to an optimization of the degree of retention R.

(52) The work which led to this application was funded in accordance with the financial aid agreement “Bio.NRW: MoBiDik—Modular bioproduction—disposable and continuous” (funding code w1004ht022a) under the European regional development fund (ERDF).