A METHOD FOR STABILIZING CELL CULTURE SYSTEMS USING AN AMPHIPHILIC GRAFT COPOLYMER AS CELL CULTURE REAGENT

Abstract

A method for stabilizing cells in cell culture production, the method comprising culturing a cell line capable of expressing proteins in cell culture media, and supplementing said cell culture media with a graft polymer in which N-vinyl caprolactam and vinyl acetate moieties are grafted on a polyethylene glycol backbone.

Claims

1. A method for stabilizing cells in cell culture production comprising culturing a cell line capable of expressing proteins in cell culture media, and supplementing said cell culture media with a graft polymer in which N-vinyl caprolactam (VCap) and vinyl acetate (VAc) moieties are grafted on a polyethylene glycol (PEG) backbone.

2. The method according to claim 1, wherein the amount of grafted N-vinyl caprolactam and vinyl acetate moieties in the graft polymer ranges from 10 to 90% b.w. of the total weight.

3. The method according to claim 1, wherein the graft polymer is a product of i) 20 to 80 wt.-% N-vinyl caprolactam moieties, ii) 10 to 50 wt.-% vinyl acetate moieties, and iii) 10 to 50 wt.-% of a polyethylene glycol, with the proviso, that the total i), ii), and iii) equals 100% b.w.

4. The method according to claim 1, wherein the graft polymer is a product of i) 30 to 70 wt.-% N-vinyl caprolactam moieties, ii) 15 to 35 wt.-% vinyl acetate moieties, and iii) 10 to 30 wt.-% of a polyethylene glycol.

5. The method according to claim 1, wherein the graft polymer is a product of i) 40 to 60 wt.-% N-vinyl caprolactam moieties, ii) 15 to 35 wt.-% vinyl acetate moieties, and iii) 10 to 30 wt.-% of a polyethylene glycol.

6. The method according to claim 1, wherein the PEG-VCap-VAc graft polymer is a product of 57% by weight N-vinylcaprolactam, 30% by weight vinyl acetate and 13% by weight polyethylene glycol PEG 6000.

7. The method according to claim 1, wherein the PEG-VCap-VAc graft polymer is used in an amount of from 0.01 to 10 wt.-% based on the volume of the supplemented culture media.

8. (canceled)

9. (canceled)

10. The method according to claim 7, wherein the graft polymer is used in an amount of 0.05 to 5% by weight.

11. The method according to claim 7, wherein the graft polymer is used in an amount of 0.5 to 3% by weight.

12. The method according to claim 1, wherein the graft polymer is used in combination with a second stabilizer.

13. The use method according to claim 12, wherein the second stabilizer is a shear protectant.

Description

EXAMPLES

[0066] The small-scale cell culture experiment was designed to evaluate the performance of Soluplus and mixtures of Soluplus with poloxamer P188 as shear protectant in suspension culture of Chinese hamster ovary (CHO) cell lines capable of producing monoclonal antibodies. The test below was carried out to identify statistically significant performance differences at a confidence levels of >95%.

[0067] Pluronic® F68, a commercially available poloxamer P188, was used as a reference standard.

[0068] The PEG-VCap-VAc graft polymer was a commercially available product obtained from 13% by weight polyethylene glycol PEG 6000, 57% by weight N-vinylcaprolactam and 30% by weight vinyl acetate (Soluplus®, BASF SE).

[0069] Materials & Methods

[0070] Cell Line:

[0071] CHO-S-IgG1

[0072] Seeding Density:

[0073] 3×10.sup.5 cells/mL; Cells were passaged without poloxamer 188 in the media for at least 5 passages prior to setting up the experiment.

[0074] Volume after Inoculation: 50 mL

[0075] Shake flasks: 500 mL, baffled, Nalgene sterile vented shake flasks (Fisher Cat #10-531)

[0076] Agitation: 225 rpm

[0077] Cell culture Medium: HyClone™ HyCell™ CHO Medium (General Electric Health Care, USA, Cat #SH30933.01)—supplied without shear protectant

[0078] Run Duration: 4 days

[0079] Incubator: Infors HT Multitron

[0080] Shear protectant:

[0081] Pluronic® F68 (reference standard)

[0082] Commercially available PEG-VCap-VAc graft copolymer: 57% by weight N-vinylcaprolactam, 30% by weight vinyl acetate and 13% by weight polyethylene glycol PEG 6000, MW in the range of 90.000 to 140.000 g/mole; Soluplus®, Fa. BASF SE Concentration of shear protectant: 0.25 g/L

[0083] Analysis

[0084] Viable Cell Counts, Total Cell Counts and % Viability: 300 μL sample were run on a Roche

[0085] CEDEX automated cell counter using Trypan Blue exclusion.

[0086] Cell viability and growth rate were calculated as follows:

[00001]$\mathrm{Total}.Math..Math.\mathrm{Cell}.Math..Math.\mathrm{Density}.Math..Math.\left(\mathrm{TDC}\right)\left[1.Math.\text{/}.Math.\mathrm{mL}\right]=\frac{\mathrm{Number}.Math..Math.\mathrm{of}.Math..Math.\mathrm{total}.Math..Math.\mathrm{cells}.Math.[-]}{\mathrm{Sample}.Math..Math.\mathrm{volume}.Math.\left[\mathrm{mL}\right]}$$\mathrm{Viable}.Math..Math.\mathrm{Cell}.Math..Math.\mathrm{Density}.Math..Math.\left(\mathrm{VDC}\right)\left[1.Math.\text{/}.Math.\mathrm{mL}\right]=\frac{\mathrm{Number}.Math..Math.\mathrm{of}.Math..Math.\mathrm{viable}.Math..Math.\mathrm{cells}.Math.[-]}{\mathrm{Sample}.Math..Math.\mathrm{volume}.Math.\left[\mathrm{mL}\right]}$$\mathrm{Viability}.Math.\left[\%\right]=1.00-\left(\frac{\mathrm{Number}.Math..Math.\mathrm{of}.Math..Math.\mathrm{dead}.Math..Math.\mathrm{cells}}{\mathrm{Total}.Math..Math.\mathrm{cell}.Math..Math.\mathrm{count}}\right).Math.100$$\mathrm{Specific}.Math..Math.\mathrm{Growth}.Math..Math.\mathrm{rate}.Math..Math.\left({\mathrm{day}}^{-1}\right)=\left(\ln .Math.\frac{\mathrm{Viable}.Math..Math.\mathrm{cell}.Math..Math.\mathrm{density}.Math..Math.\left(t\right)}{\mathrm{Viable}.Math..Math.\mathrm{cell}.Math..Math.\mathrm{density}.Math..Math.\left(t.Math..Math.0\right)}\right)$

[0087] Cumulative growth rate: calculated for 3 and 4 days.

[0088] Ln(Viable Cell Density Day 3,4/Viable Cell Density Day 0)/3

[0089] Statistical analysis: Unpaired Student t-test to determine if differences are significant (>95% confidence)

[0090] Results

TABLE-US-00001 TABLE 1 AVERAGE: Cumulative Via- STANDARD DEVIATION: Cu- ble Cell Growth Rate (day-1) mulative Viable Cell Growth from Day 0 to Day X Rate(day-1) X = D3 X = D4 X = D3 X = D4 F68 0.80 1.03 0.01 0.02 F68/PEG-VCap- 0.74 0.98 0.01 0.02 VAc 3:1 F68/PEG-VCap- 0.82 1.04 0.05 0.05 VAc 1:3 PEG-VCap-VAc 0.78 1.01 0.03 0.03 Without shear −0.27 −0.55 0.3 0.5 protectant

[0091] The results show that Soluplus has a performance as shear protectant comparable to the reference standard.

[0092] Aldehyde Content

[0093] The aldehyde contents of the different samples were tested according to the following method:

[0094] The aldehydes were determined by reversed phase HPLC after reaction of the sample with 2,4-dinitrophenylhydrazine as the respective dinitrophenyl hydrazones. For quantification, an external standard was applied using UV detection at 370 nm.

[0095] Sample derivatization: 60 mg of polymer (poloxamer 188 or PEG-PVCap-PVAc graft copolymer were weighed (accurate to 0.01 mg) into a 10 mL volumetric flask, dissolved in 1 mL acetonitrile, and derivatized by addition of 1-2 mL reagent solution followed by heating to 60° C. for 5 min. After cooling down to ambient temperature, the flask is filled up to the mark with acetonitrile/water (1:1)

[0096] Reagent solution: Approx. 4 g with 2,4-dinitrophenylhydrazine (stabilized with 50% water) re weighed into a 1 L Erlenmeyer flask. 800 mL water and 200 mL concentrated hydrochloric acid were added. The mixture is stirred until it is clear.

[0097] Stationary phase: Symmetry Shield RP 18-5 μm, Waters (2.1 mm diameter, stainless steel)

[0098] Calibration solutions: 20 mg of aldehyde dinitrophenylhydrazones were weighed, accurate to 0.01 mg, and dissolved in acetonitrile. Dilutions were adjusted in such a way that the concentration of hydrazine is within the following ranges:

[0099] Formaldehyde derivative 0.0021-0.43 mg/10 mL injection solution

[0100] Acetaldehyde derivative 0.0024-0.47 mg/10 mL injection solution

[0101] Propionic aldehyde derivative Approx. 0.0024-0.47 mg/10 mL injection solution

[0102] Mobile phase: water (A)/acetonitrile (B) gradient:

TABLE-US-00002 t/min A [%] B [%] 0 60 40 25 30 70 35 30 70 36 60 40 45 60 40

[0103] Flow: 0.4 mL/min

[0104] Injection volume: 5 μL

[0105] Temperature: 45° C.

[0106] Detection: UV/VIS, lambda=370 nm

[0107] The total aldehyde contents listed in the tables for the starting materials were calculated based on the hydrazone derivatives content.

[0108] Results:

TABLE-US-00003 TABLE 2 Formaldehyde Acetaldehyde Propionic aldehyde Sample [ppm] [ppm] [ppm] Poloxamer 188 <20 163 257 PEG-VCap-VAc <20 <5 <5