CELL CULTURES COMPRISING POLY(OXAZOLINE) STABILIZERS AND USE OF POLY(OXAZOLINES) FOR STABILIZING CELL CULTURES

Abstract

Cell cultures comprising poly(oxazoline) stabilizers and use of poly-(oxazolines for stabilizing cell cultures

Described are cell cultures containing in a cell culture medium one or more water-soluble poly(oxazolines).

The water-soluble poly(oxazoline) acts as a stabilizer for the cells and reduces the mechanical stress exerted by agitating the cell culture medium on the cells. This leads to an improved survival rate of the cells compared to the unstabilized state.

Claims

1. Cell cultures comprising one or more water-soluble poly(oxazolines) in a cell culture medium, provided that the cell culture medium contains essentially no serum.

2. Cell cultures according to claim 1, characterized in that the cell culture medium is essentially free of animal components.

3. Cell cultures according to claim 1, characterized in that the used cell culture medium is serum-free and/or protein-free.

4. Cell cultures according to claim 1, characterized in that the cell culture medium is a chemically defined medium.

5. Cell cultures according to claim 1, characterized in that these contain cells, which are suspended in the cell culture medium.

6. Cell cultures according to claim 1, characterized in that the cells are suspensions-adapted cells.

7. Cell cultures according to claim 1, characterized in that these are used for protein production, virus and/or virus particle production, investigation of metabolism, division and/or other cellular processes.

8. Cell cultures according to claim 1, characterized in that these contain cell lines selected from at least one of the following groups: 293-T, A431, A549, BCP1, bEnd.3, BHK-21, BxPC-3, BY-2, CHO, CMT, COS-1, COS-7, CV-1, EPC, HDMEC-T, HEK, HeLa, HepG2, HL-60, HMEC-1, HUVEC-T, HT-1080, Jurkat, K562, LNCaP, MCF-7, MCF-10A, MDCK II, MDT-1A, MyEnd, Neuro-2A, NIH-3T3-T, NTERA-2 cl.D1, P19, PANC-1, Peer, RTL-W1-T, Sf-9, Saos-2, T2, T84 or U-937.

9. Cell cultures according to claim 8, characterized in that the cell lines are selected from the group consisting of CHO or HEK, in particular HEK 293.

10. Cell cultures according to claim 1, characterized in that these contain hybridoma cells.

11. Cell cultures according to claim 1, characterized in that these contain stem cells.

12. Cell cultures according to claim 1, characterized in that these contain 0.01 to 15 wt. %, preferably 0.075 to 15 wt. %, more preferred 0.05 to 10 wt. %, highly preferred 0.1 to 10 wt. % of water-soluble poly(oxazolines) in relation to the total amount of cell culture.

13. Cell cultures according to claim 1, characterized in that the water-soluble poly(oxazoline) contains at least 80 wt. %, based on its total mass, of repeat structural units of formula I and/or formula II
(I), NR.sup.1CH.sub.2CH.sub.2CH.sub.2(II), in which R.sup.1 means a residue of formula COR.sup.2, R.sup.2 is selected from the group consisting of hydrogen, methyl, ethyl, C.sub.mH.sub.2mX or (C.sub.nH.sub.2nO).sub.o-(C.sub.pH.sub.2pO).sub.qR.sup.3, R.sup.3 is hydrogen or C.sub.1C.sub.6-alkyl, m is an integer from 1 to 6, X is selected from the group consisting of hydroxyl, alkoxy, amino, N-alkylamino, N,N-dialkylamino, carboxyl, carboxylic ester, sulfonyl, sulfonic acid ester or carbamate, n and p independently of one another are integers from 2 to 4, wherein n is unlike p, and o and q independently of one another are integers from 0 to 60, wherein at least one of o or q is unlike 0.

14. Cell cultures according to claim 13, characterized in that the water-soluble poly(oxazoline) has at least 90 wt.-%, based on its total mass, of repeat structural units of formula I, wherein R.sup.2 means methyl or ethyl.

15. Cell cultures according claim 1, characterized in that these contain one or more active ingredients, in particular pharmaceutically active ingredients and/or vaccines.

16. A method for protein production, virus and/or viral particle production, investigation of metabolism, division and/or other cellular processes comprising the cultivation of cell cultures comprising one or more water-soluble poly(oxazolines) in a cell culture medium, provided that the cell culture medium contains essentially no serum.

17. The method according to claim 16, characterised in that it also contains the following measures: (i) filling a bioreactor with one of the cell cultures, and (ii) agitating the contents of the bioreactor.

18. The method according to claim 16, characterized in that the duration of the cultivation of the cells in the case of a batchwise method is 5 hours to 30 days, preferably 5 hours to 21 days and in particular 10 hours to 15 days and in the case of a continuous process is 10 days to 180 days, preferably 10 days to 60 days and in particular 15 days to 35 days.

19. The method according to claim 17, characterized in that the content of the bioreactor is stirred.

20. The method according to claim 17, characterized in that the bioreactor contains baffles for the generation of turbulence.

21. (canceled)

22. (canceled)

23. (canceled)

24. (canceled)

25. (canceled)

Description

Example 1A

Manufacture of poly(2-oxazolines) (PO.SUB.x.) in the Microwave

[0126] The synthesis of poly(2-oxazolines) has already been described in the literature (see e.g. F. Wiesbrock et al. Macromolecular Rapid Communications 2004, 25, 1895-1899). The procedure is therefore described as an example for poly (2-ethyl-2-oxazoline) with a degree of polymerization (DP) of 61 (P(EtO.sub.x).sub.61).

[0127] In a microwave reaction vessel, 2-ethyl-2-oxazoline (6.06 mL, 60.0 mmol), methyltosylate (0.15 mL, 0.1 mmol) and acetonitrile (8.79 mL) were mixed under inert conditions. The reaction vessel was then heated in a synthesis microwave for 14 min to 140 C. Subsequently, the reaction was terminated by the addition of 0.5 mL of deionized water and stirred overnight at room temperature. The resulting solution was purified by diluting with dichloromethane and then by precipitation in an excess of ice-cold diethyl ether. The precipitated polymer was then filtered and dissolved in dichloromethane. The solvent was then removed in a rotary evaporator and the polymer was dried in the high vacuum until completely solvent-free. The final product was available as a crystalline white solid.

[0128] .sup.1H-NMR (CDCl.sub.3, 300 MHz): =4.34 (0.1H, s, backbone-OH), 3.44 (4.0H, s, backbone), 3.02 (0.3H, s, CH.sub.3-backbone), 2.4 (1.7H, m, CH.sub.2 (EtO.sub.x)), 1.11 (2.5H, s, CH.sub.3 (EtO.sub.x)) ppm.

[0129] SEC (eluent: DMAc.sup.1), 0.21% LiCl, PS.sup.2)-Standard): M.sub.n=11.200 g mol.sup.1, M.sub.w=12.200 g mol.sup.1, D=1.09.

[0130] .sup.1) DMA.sub.c=dimethyl acetamide

[0131] .sup.2) PS=polystyrene

[0132] FIG. 1 shows a .sup.1H-NMR (300 MHz, CDCl.sub.3) of the purified P(EtO.sub.x).sub.61.

[0133] FIG. 2 shows an SEC elugramme (DMA.sub.c, 0.21% LiCl, PS calibration) of the purified P(EtO.sub.x).sub.61.

EXAMPLE 1B

Manufacture of PO.SUB.x .in the Reactor

[0134] In the reactor, 2-ethyl-2-oxazoline (4.04 L, 40.0 mol), methyltosylate (100 mL, 0.67 mol) and acetonitrile (5.86 L) were mixed under inert conditions. The reactor was then heated under reflux for 6 hrs. Reaction progress was monitored by taking samples at regular intervals. Subsequently, the reaction was terminated by the addition of 270 mL of deionized water and stirred overnight at room temperature. The resulting solution was cleaned in five portions. For this purpose, the solvent was removed in the rotary evaporator. Subsequently, the polymer was dissolved in 4 L dichloromethane. Then the organic phase was washed with 2 L of a saturated sodium bicarbonate solution and then twice with 2 L of a saturated sodium chloride solution. The organic phase was dried over sodium sulfate and the solvent was evaporated in the rotary evaporator. Then the polymer was dried in the high vacuum until completely solvent-free. The final product was available as a crystalline white solid.

EXAMPLE 2

Cell Growth in a Baffled Flask

[0135] Suspension-adapted HEK-F cells were cultured by means of dynamic cell culture in serum free medium (deficiency variant of the HEK TF medium (serum and protein free, without Pluronic F68) (order no. 861 (Xell AG, Bielefeld)) for at least 5 days without the addition of new medium. In a baffled flask (250 mL), the cells with a cell density of 0.310.sup.6 cells per mL were applied in 30-40 mL minimal medium with correspondingly added surfactant (750 mg L.sup.1). Cultivation took place in the incubator under shaking at 128 rpm, 37 C. and 5% CO.sub.2. Every day, cell density and cell viability were determined by counting using trypan blue. A cell viability below 60% is considered as an abort criterion.

TABLE-US-00001 TABLE 1 Cell concentrations of living cells expressed in cells per millilitre day Pluronic F68 P(EtOx).sub.61 P(MeOx).sub.57 0 300000 300000 300000 1 980000 720000 1400000 2 1644000 1816000 2012000 3 3045000 3308000 3044000 4 3728000 4304000 4904000 5 5296000 6152000 6040000 6 6030000 6960000 6488000 7 5670000 7288000 6752000 8 8020000 7640000 7820000

TABLE-US-00002 TABLE 2 Cell survival rate, expressed in % day Pluronic F68 P(EtOx).sub.61.sup.a) P(MeOx).sub.57.sup.b) 0 100 100 100 1 96.1 97.3 93.3 2 97.2 96.8 97.7 3 95.4 98.2 98.0 4 96.5 98.3 95.8 5 96.2 95.1 94.0 6 93.6 95.1 97.7 7 89.4 95.6 96.8 8 92.7 90.4 93.3 .sup.a)poly(2-ethyl-2-oxazoline) with a polymerization degree (DP) of 61 .sup.b)poly(2-methyl-2-oxazoline) with a polymerisation degree (DP) of 57

[0136] FIG. 3 shows the cell concentrations and viability of HEK F cells when cultivated with different surfactants.

EXAMPLE 3

Transfection of Suspension Cells

[0137] HEK-F cells were pre-cultivated in the corresponding minimal medium deficiency variant of the HEK TF medium (serum and protein free, without Pluronic F68) (order no. 861 (Xell AG, Bielefeld)) with surfactant (750 mg L.sup.1). On the day of transfection, the cells were centrifuged and the cell density was adjusted with fresh medium to 310.sup.6 cells mL.sup.1. Transfection at N/P 20 (amine to phosphate ratio) was carried out in a 2 mL preparation as follows: First addition of 15 g mL.sup.1 pDNA (EGFP reporter gene) to the cell suspension followed by pivoting this, then adding polyethyleneimine (PEI, 1 mg mL.sup.1) and repeated pivoting of the culture. The preparation was incubated for 4 h under shaking in the incubator at 37 C., 5% CO.sub.2. After this incubation period, the cells were transferred to 6-well plates and diluted with the same volume of fresh medium and incubated for a further 48 h. Transfection efficiency was determined by flow cytometry.

[0138] Table 3 shows the results of transfection experiments of HEK-F cells in minimal medium

TABLE-US-00003 incubation cell transfected average time viability cells intensity of [hours] [%] [%] fluorescence negative control 48 98.4 0.7 0.775 minimal medium 48 95.7 11.7 37.700 Pluoronic F68 48 96.6 49.0 267.000 P(EtO.sub.x).sub.61 48 96.3 60.3 344.000 P(MeO.sub.x).sub.57 48 97.7 74.8 482.000

[0139] FIG. 4 shows the average fluorescence intensity of the transfected HEK F cells.

[0140] FIG. 5 shows an overview of the number of transfected cells, expressed in %, when using different culture media.

EXAMPLE 4

Variation of the Concentration Range of PO.SUB.x

[0141] In order to be able to estimate the concentration range in which PO.sub.x can be used as a surfactant in cell culture processes, additional cultivations were carried out. HEK F cells were cultivated for this purpose in a deficiency variant of the HEK TF medium (serum and protein free, without Pluronic F68) (order no. 861 (Xell AG, Bielefeld)) with the addition of the current standard surfactant Pluronic F68 or P(EtO.sub.x). For Pluronic F68 concentrations of 0.75 g/L and 1 g/L (most commonly used concentrations) were used and P(EtO.sub.x) was tested in a larger concentration range from 0.75 g/L to 15 g/L. In order to investigate also the protective property against increased shear forces, this test was carried out both in shaking flasks without baffle and with baffle. The results are shown in the lower graphs in FIGS. 6 to 8.

[0142] FIGS. 6 to 13 refer to P(EtO.sub.x) as A60.

[0143] FIG. 6 provides an overview of the growth curves and the viability course (dashed lines) of the HEK F cells in HEK TF medium. Different concentrations Pluronic F68 (0.75 g/L and 1 g/L) and P(EtO.sub.x) (0.75-15 g/L) were used as surfactants. A shaking flask with baffle (baffled, dotted lines) and a shaking flask without baffle (plain, solid lines) were used to expose the cells to different shear forces.

[0144] FIG. 6 shows an overall overview of the concentration testing of Pluronic F68 against P(EtO.sub.x). In general, the maximum cell densities in the shaking flasks without baffles were significantly higher than those in shake flasks with baffles (9.6-12.9-10.sup.6 cells/mL compared to 0.2-5.610.sup.6 cells/mL). It is noticeable here that the cells in cultivation with P(EtO.sub.x) consistently achieved higher cell densities than in cultivations with Pluronic F68. Especially under high shear loads, the cultures with the surfactant PO.sub.x showed significantly increased maximum cell densities, increased viability and a longer culture duration compared to the so far used Pluronic F68. The viability of the cultures with Pluronic F68 decreased to less than 60% within 3 and 4 days, respectively.

[0145] With regard to the concentration range in which P(EtO.sub.x) was tested here, there were hardly any differences in the maximum live cell density (10.8-12.6-10.sup.6 cells/mL) in the shaking flasks without baffle. The cultures with increased shear stress showed maximum live cell densities of 3.5-5.610.sup.6 cells/mL, with no direct relation to the concentration used.

[0146] For a better insight, the cultures in FIGS. 7 and 8 are again listed separately by shear stress.

[0147] FIG. 7 shows growth curves (solid lines) and development of viability (dashed lines) of the HEK F cells in HEK TF medium in shaking flasks without baffles. Different concentrations Pluronic F68 (0.75 g/L and 1 g/L) and P(EtO.sub.x) (0.75-15 g/L) were used as surfactants.

[0148] FIG. 8 shows growth curves (solid lines) and development of viability (dashed lines) of the HEK F cells in HEK TF medium in shake flasks with baffles (baffled). Different concentrations Pluronic F68 (0.75 g/L and 1 g/L) and P(EtO.sub.x) (0.75-15 g/L) were used as surfactants.

EXAMPLE 5

PO.SUB.x .Lot Testing

[0149] In another preparation, HEK F cells were cultivated in HEK TF medium (serum and protein free, without Pluronic F68) (order no. 861 (Xell AG, Bielefeld)) with the addition of the various lots of P(EtO.sub.x) compared to two lots of Pluronic F68 to identify possible effects of the upscaling of the production process. In order to investigate again the protective property against increased shear forces, this test was carried out both in shaking flasks without baffle and with baffle. The results of this test are shown in FIGS. 9 and 10.

[0150] FIG. 9 shows the growth curve and the development of viability of the HEK F cells in HEK TF, adding various lots of P(EtO.sub.x) from the upscaling of the production process in shake flasks without baffle.

[0151] FIG. 9 shows the results in shaking flasks without baffles. With the exception of the Pluronic Lot 1 culture (7.810.sup.6 cells/mL), all cultures showed comparable maximum live cell densities of 9-10, 110.sup.6 cells/mL and no significant differences between the different P(EtO.sub.x) lots could be detected.

[0152] FIG. 10 shows the growth curve and the development of viability of the HEK F cells in HEK TF, adding various lots to P(EtO.sub.x) from the upscaling of the production process in shake flasks with baffles.

[0153] Cultivations in shake flasks with baffles again showed that cells with the addition of P(EtO.sub.x) have a higher shear tolerance than those with Pluronic F68 as surfactant; the cultures with Pluronic F68 showed a decline of viability by 20% (lot 1) and 10% (lot 2) after 24 hours. The cultures with different P(EtO.sub.x) lots reached maximum live cell densities of 6.4-910.sup.6 cells/mL, with a lot from the 0.5 L production scale showing a higher growth compared to the other lots. Under increased shear stress, only slight differences of the different P(EtO.sub.x) lots from the upscaling of the production process arose, which were not recognizable in this test under conditions without increased shear stress.

EXAMPLE 6

Transfection Efficiency

[0154] In order to check the transfection efficiency, two shaking tubes of the cultures from the shaking flasks without baffles were applied and the HEK F cells were transfected with a CFP plasmid by means of polyethyleneimine (PEI). Forty-eight hours after transfection, cell densities, viabilities and the transfection efficiency of these cultures were recorded. The results of these measurements are shown in FIGS. 11 to 13.

[0155] FIG. 11 shows mean values of the transfection efficiencies of HEK F cells in HEK TF medium. Two Pluronic F68 lots and the various lots of the scaling up of P(EtO.sub.x) production were used as surfactants for cultivation in the shaking tube followed by transfection of a CFP plasmid by means of PEI.

[0156] FIG. 11 shows that the transfection efficiencies of the cultures with the different P(EtO.sub.x) lots were all over 90% (93.5%-96.7%). No significant differences in transfection efficiencies were found between the individual scaling steps. The cultures of the two Pluronic F68 lots could not be successfully transfected in this experiment, the efficiencies were less than 1%.

[0157] FIG. 12 shows mean values of live cell densities 48 h after transfection of HEK F cells in HEK TF medium. Two Pluronic F68 lots and the various lots of the scaling up of P(EtO.sub.x) production were used as surfactants for cultivation in the shaking tube followed by transfection of a CFP plasmid by means of PEI.

[0158] FIG. 12 shows the live cell densities 48 h after transfection. Here it can be seen that the mean values of the live cell densities of the cultures with the different P(EtO.sub.x) lots did not differ significantly. The values varied between 4.410.sup.6 cells/mL and 5.110.sup.6 cells/mL. The two Pluronic F68 lots had live cell densities of 1.810.sup.6 cells/mL (lot 1) and 3.510.sup.6 cells/mL (lot 2).

[0159] FIG. 13 shows mean values of the viabilities 48 h after transfection of the HEK F cells in HEK TF medium. Two Pluronic F68 lots as well as the various lots of the scaling up of the P(EtO.sub.x) production were used as surfactants for the cultivation in the shaking tube with subsequent transfection of a CFP plasmid by means of PEI.

[0160] The mean values of the viabilities of the transfected cultures 48 h after transfection can be found in FIG. 13. It can be seen that the PD.sub.X cultures showed a high viability between 97.5% and 98.2% and that there was no significant difference between the P(EtO.sub.x) lots. The viability of the Pluronic F68 cultures was 82.4% (lot 1) and 93.6% (lot 2).