BIOREACTOR WITH FILTER UNIT AND METHOD FOR TREATING A CELL BROTH

Abstract

A bioreactor having a filter unit and a method for treating a cell broth. The filter unit has a supply channel (2), a first filter medium (4), a retentate channel (1), a second filter medium (5) and a permeate channel (3), arranged so that the first filter medium delimits the supply channel and the retentate channel from one another; and the second filter medium delimits the retentate channel and the permeate channel from one another. The supply channel is connected to an inlet for a supply medium; the retentate channel is connected to an inlet for a cell broth and to an outlet for the cell broth; the permeate channel is connected to an outlet for a permeate; and the interior of the bioreactor is connected to the inlet for the cell broth and to the outlet for the cell broth.

Claims

1. A method for treating a cell broth comprising: (A) providing a filter unit, wherein the filter unit comprises at least one supply channel, at least one first filter medium, at least one retentate channel, at least one second filter medium and at least one permeate channel, arranged such that the first filter medium delimits the supply channel and the retentate channel from one another, and the second filter medium delimits the retentate channel and the permeate channel from one another, wherein the supply channel is connected in a fluid conducting manner to at least one inlet for a supply medium and is connected via the first filter medium to the retentate channel with a first pressure gradient, the retentate channel is connected in a fluid conducting manner to at least one inlet for the cell broth and to at least one outlet for the cell broth and is connected via the second filter medium to the permeate channel with a second pressure gradient, the permeate channel is connected in a fluid conducting manner to at least one outlet for a permeate; (B) feeding the supply medium into the inlet for the supply medium; (C) feeding the cell broth into the inlet for the cell broth; (D) discharging the cell broth from the outlet for the cell broth; and (E) discharging the permeate from the outlet for the permeate, and wherein the first filter medium introduces the supply medium into the retentate channel while the second filter medium filters the cell broth between the retentate channel and the permeate channel.

2. The method as claimed in claim 1, wherein the treating of the cell broth comprises washing cells in the cell broth, adding or exchanging a culture medium, changing a concentration of the cells in the cell broth, fractionating the cells and/or gassing the cells.

3. The method as claimed in claim 1, wherein in said step (A), a bioreactor is provided with the filter unit; in said step (C), the cell broth is fed from an interior of the bioreactor into the inlet for the cell broth; and in said step (D), the cell broth is discharged from the outlet for the cell broth into the interior of the bioreactor.

4. The method as claimed in claim 3, wherein the filter unit is arranged inside the interior of the bioreactor.

5. The method as claimed in claim 3, wherein the filter unit is arranged outside the interior of the bioreactor.

6. The method as claimed in claim 1, wherein a ratio of a volume flow of the supply medium, fed in said step (B), to a volume flow of the cell broth, fed in said step (C), is in a range of 1:10 to 10:1.

7. The method as claimed in claim 1, wherein a ratio of a volume flow of the cell broth, fed in said step (C), to a volume flow of the cell broth, discharged in said step (D), is in a range of 1:10 to 10:1.

8. The method as claimed in claim 1, wherein the method further comprises (C) feeding the cell broth into the outlet for the cell broth and (D) discharging the cell broth from the inlet for the cell broth.

9. The method as claimed in claim 1, wherein the supply medium is fed at a pressure of 0.1 to 4 bar.

10. The method as claimed in claim 1, wherein the supply medium is fed at a pressure that is greater than an outlet pressure of the retentate.

11. The method as claimed in claim 1, further comprising: providing further, mutually independent filter units to thereby provide a plurality of the filter units, connecting the plurality of the filter units in series such that the outlet for the cell broth of a respective upstream filter unit is connected in a fluid conducting manner to the inlet for the cell broth of a downstream filter unit, and feeding the cell broth in accordance with said step (C) into the inlet for the cell broth of a first one of the filter units, upstream of which no other filter unit is connected, and discharging the cell broth in accordance with said step (D) from the outlet for the cell broth of a final one of the filter units, downstream of which no other filter unit is connected.

12. The method as claimed in claim 1, further comprising: providing further, mutually independent filter units to thereby provide a plurality of the filter units, and connecting the plurality of the filter units in parallel.

13. The method as claimed in claim 1, wherein the cell broth discharged in said step (D) is at least partially fed back directly into the inlet for the cell broth.

14. The method as claimed in claim 1, operated under the following conditions: $\mathrm{PEVK}=\mathrm{PER};$ $x=\mathrm{VEVK}/\mathrm{VER};\mathrm{and}$ $k=\mathrm{VER}/\mathrm{VAR};$ where PEVK is the pressure at which the supply medium is added, PER is a retentate inlet pressure at which the cell broth is added to the filter unit, VAR is a volume flow of the discharged cell broth, VEVK is a volume flow of the supply medium, VER is a volume flow of the supplied cell broth, x is an exchange ratio, and k is a concentration factor.

15. The method as claimed in claim 14, wherein x=1 and k=1.

16. The method as claimed in claim 14, wherein 3x10.

17. The method as claimed in claim 1, wherein at least one further filter unit is connected in a fluid conducting manner to the filter unit by way of the permeate outlet such that the permeate, which is discharged in said step (E), is provided to the at least one further filter unit.

18. The method as claimed in claim 1, wherein the filter unit is a flat filter module, spiral wound fiber module or hollow fiber module.

19. The method as claimed in claim 1, wherein the inlet for the supply medium is connected in a fluid conducting manner to a pump for the supply medium.

20. The method as claimed in claim 1, wherein the inlet for the cell broth is connected in a fluid conducting manner to a pump for the cell broth.

21. The method as claimed in claim 1, wherein the outlet for the cell broth comprises a valve.

22. The method as claimed in claim 1, wherein the outlet for the cell broth comprises a pump.

23. The method as claimed in claim 1, wherein the outlet for the permeate comprises a valve.

24. The method as claimed in claim 1, wherein the outlet for the permeate comprises a bidirectional pump.

25. The method as claimed in claim 1, wherein the introduction of the supply medium is performed simultaneously with the filtration of the cell broth.

26. The method as claimed in claim 1, wherein the filter unit further comprises: a further second filter medium, a further retentate channel, and a further first filter medium, arranged such that the further second filter medium delimits the permeate channel and the further retentate channel from one another, and the further first filter medium delimits the further retentate channel, wherein the supply channel, the one first filter medium, the one retentate channel, the one second filter medium, the further second filter medium, the further retentate channel, and the further first filter medium form a stacked arrangement forming a filter cartridge.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0095] FIGS. 1 and 2 show exemplary embodiments of the present invention, wherein FIG. 1 shows a filter unit for treating a cell broth, and FIG. 2 shows a filter unit that permits some cell broth impurities to pass.

[0096] FIGS. 3 and 4 show exemplary embodiments of the present invention, wherein FIG. 3 shows a filter unit located inside a bioreactor, and FIG. 4 shows a filter unit located outside a bioreactor.

[0097] FIG. 5 shows an exploded view of one possible design of a filter unit.

[0098] FIG. 6 shows an exemplary embodiment of the present invention, in which a further filter unit is connected in a fluid conducting manner to a first filter unit by way of a permeate outlet.

DETAILED DESCRIPTION

[0099] FIGS. 1 to 4 show in schematic form exemplary embodiments of a method according to the present invention for treating a cell broth as well as a (filter unit of an) exemplary embodiment of the bioreactor according to the present invention, wherein the flow patterns of materials and the flow directions are indicated by arrows.

[0100] FIGS. 1 and 2 show filter units as well as methods for the treatment of a cell broth in exemplary experimental setups. The second filter medium (5) in FIG. 2 retains the cells (10) and allows impurities (11) to pass through, so that the cell broth leaves the retentate channel (1) in a cleaned state. The cell broth is fed into the filter unit by way of the inlet (7) to the retentate channel (1). A fluid (liquid/gas) is pumped into the filter unit by way of the inlet (6) to the supply channel (2). The fluid (supply medium) is introduced into the retentate channel (1) by way of a pressure gradient; and at the same time a filtration takes place in the permeate channel (3) by way of the pressure gradient between the retentate channel (1) and the permeate channel (3). The pressure gradient can be controlled, for example, with a valve and/or a pump at the outlet (8) of the retentate channel. The cells (10) are retained by selecting a suitable separation limit of the second filter medium and do not pass through the second filter medium. Smaller molecules, such as proteins, on the other hand, pass through the filter medium. The same also applies to the culture medium (supply medium). Smaller components (constituents of the cell broth and the supply medium that can pass through the second filter material) are discharged through the permeate outlet (9). As a result, the concentration of smaller components in the cell broth/in the retentate decreases accordingly in the direction of the retentate outlet (8).

[0101] Further exemplary embodiments of the present invention are shown in FIGS. 3 and 4 and will be explained in the following. In the embodiment of FIG. 3, the filter unit (13) is located inside the interior of the bioreactor (12). In the case of an arrangement of the filter unit in the interior of the bioreactor, the bioreactor has preferably an outer connection (16) for the supply medium. Supply medium can be supplied to the supply channel from the outside by way of the connection (16). In the embodiment of FIG. 4, the filter unit (20) is located outside the interior of a tank (19).

Filter Unit, integrated in a Bioreactor for obtaining Monoclonal Antibodies and Culture Medium Exchange

[0102] This embodiment is illustrated in FIG. 3.

[0103] The filter unit (13) is integrated in a bioreactor (12) (for example, in a perfusion bioreactor for producing a monoclonal antibody (=product) using ovarian cells of the Chinese dwarf hamster). The cell broth (34, 40) in the bioreactor (12) is fed by a pump (14) into the retentate channel (1) of the filter unit (13) and leaves the retentate channel again directly into the perfusion bioreactor (12) by way of the retentate outlet (15). In so doing, the cells (10) pass only through the retentate channel (1) without passing into the supply (2) or permeate channel (3). The product and the contaminants pass through the filter medium (4) into the permeate channel (3).

[0104] Culture medium and/or washing buffer is/are optionally fed through a connection (16) to the perfusion bioreactor. The procedure also makes it possible to change the supplied media in different phases by way of the connection (16). Optionally the permeate outlet (18) can be closed, for example, in the phase of supplying fresh culture medium. In an additional phase the permeate outlet (18) is opened again; and washing buffer is supplied.

[0105] The filtration pressure is adjusted by using a valve (17) or a pump (not shown in FIG. 3) at the outlet (15) of the retentate channel. Product and contaminants (by-products, cell constituents, etc.) are discharged through the permeate outlet (18) for further work-up.

Method for Concentrating and for Washing a Cell Broth

[0106] This embodiment is shown in FIG. 4.

[0107] The retentate channel (1) of the filter unit (20) is connected to a tank (19), which contains a cell broth, through a pump (21). The cell broth from the tank (19) is fed to the retentate channel (1) of the filter unit (20). The filtration pressure is adjusted by through a valve (22) or a pump (not shown in FIG. 4) at the outlet of the retentate channel (23), and the volume flow at the outlet (23) is regulated in relation to the inlet volume flow. A solution is fed as a supply medium (38) through the connection (24) to the supply channel (2). Contaminants as well as the major portion of the solution are discharged through the permeate outlet (25). Cells (10) in the retentate can be fed through the retentate outlet (23) and then, as the case may be, to the next step or to a further tank or back to the original tank (19). The cell broth is concentrated by a factor of preferably 2 to 10.

[0108] The present invention is explained through the following example, but is not limited thereto.

EXAMPLE

Cell Washing of a Yeast Cell Suspension with Bovine Serum Albumin as the Product

[0109] The underlying test setup is shown in FIGS. 1 and 2. A membrane, made of PES and having a pore size of 0.1 m, was used as the first (4) and second (5) filter medium. The membrane area was 0.216 m.sup.2 in each case. A cell broth (30 g/l of baker's yeast, 10.6 g/l of bovine serum albumin (protein) and 0.9% NaCl solution in water) was introduced into the inlet (7). Tap water was fed as a supply medium into the supply channel (2). The protein concentration and mass of the respective total volume of the cell broth before and after the washing as well as the protein concentration and mass of the permeate were determined. The results are shown in Tables 1 and 2.

TABLE-US-00001 TABLE 1 t P.sub.ER V.sub.AR V.sub.AP V.sub.EVK V.sub.ER V.sub.ER:V.sub.AR V.sub.EVK:V.sub.ER [min] [bar] [ml/min] [ml/min] [ml/min] [ml/min] [] [] 5 0.12 64 64 61 64 1.0 1.0 12 0.13 66 66 62 65 1.0 1.0 20 0.17 66 66 71 62 0.9 0.9 30 0.24 66 66 61 69 1.0 1.1 50 0.37 66 64 61 68 1.0 1.1 t time of measurement P.sub.ER pressure, at which the cell broth was added V.sub.AR volume flow at the outlet of the retentate channel V.sub.AP volume flow at the outlet of the permeate channel V.sub.EVK volume flow at the inlet of the supply channel V.sub.ER volume flow at the inlet of the retentate channel

TABLE-US-00002 TABLE 2 Mass Mass Conductivity Volume Concentration (Protein) [mS/cm] [1] (Protein) [g/l] [g] Cell broth 14.55 5 10.6 53.0 before washing Permeate 10.52 5.1 6.72 34.3 Cell broth 3.93 4.95 3.1 15.3 after washing

[0110] The result shows a decrease in the NaCl and protein concentration in the cell suspension (retentate).

[0111] FIG. 5 shows an exploded view of one possible design of a filter unit, which is suitable for the bioreactor (12) of the present invention and for the method of the present invention, with a flat first filter medium (4) and a flat second filter medium (5), where in this case the flow patterns of the supply medium (38), the cell broth (34, 40) and the permeate (36) are illustrated by arrows. The supply channel (2), the retentate channel (1) and the permeate channel (3) are kept open by spacers (32) for the supply medium (38), the cell broth (34, 40) or, as the case may be, the permeate (36). In the exploded drawing of FIG. 5, the components of the filter unit are offset from one another, in order to provide greater clarity. In reality, the components/layers of this embodiment are arranged one above the other such that the holes (passage openings) in the edge regions form channels.

[0112] FIG. 6 shows a further exemplary embodiment of the present invention, wherein at least one further filter unit (20) is connected in a fluid conducting manner to the filter unit (20) by way of the permeate outlet (25).

LIST OF REFERENCE NUMERALS

[0113] 1 retentate channel [0114] 2 supply channel [0115] 3 permeate channel [0116] 4 first filter medium [0117] 5 second filter medium [0118] 6 inlet for supply medium [0119] 7 inlet for cell broth [0120] 8, 15, 23, (23) outlet for cell broth (or outlet for retentate) [0121] 9, 18, 25, 25 outlet for permeate [0122] 10 cells [0123] 11 impurities [0124] 12 (perfusion) bioreactor [0125] 13, 20, 20 filter unit [0126] 14, 21, 21 pump [0127] 16 connection to inlet for supply medium [0128] 17, 22, 22 valve [0129] 19 tank [0130] 24, 24 connection for supply medium [0131] 32 spacer [0132] 34, 40 cell broth [0133] 36 permeate [0134] 38 supply medium