WO1995012664A1 - Adaption of mammalian cell lines to high cell densities - Google Patents
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- WO1995012664A1 WO1995012664A1 PCT/US1994/011535 US9411535W WO9512664A1 WO 1995012664 A1 WO1995012664 A1 WO 1995012664A1 US 9411535 W US9411535 W US 9411535W WO 9512664 A1 WO9512664 A1 WO 9512664A1
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0018—Culture media for cell or tissue culture
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- C12N2500/00—Specific components of cell culture medium
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- C12N2500/32—Amino acids
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- C12N2510/00—Genetically modified cells
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Definitions
- the present invention relates to improved methods of expressing proteins through culture of mammalian cell lines.
- the present invention relates to methods of improving the productivity of mammalian cell lines through adaption to otherwise growth-limiting conditions.
- the present invention provides methods by which the growth-limiting factors present for a particular cell line can be overcome without first conducting time-consuming testing to identify the specific growth-limiting inhibitors.
- the above objects are largely achieved by providing methods for adapting mammalian cell lines to culture at increased cell densities.
- the methods of the present invention comprise adapting mammalian cell lines to grow at increased cell densities, by (a) initiating a passage by diluting a culture containing mammalian cells with a suitable growth medium by a dilution factor suitable for the passage duration; (b) maintaining pH, dissolved oxygen, and nutrients at non-limiting levels during the passage; and repeating steps (a) and (b) at least about 5 times. In a preferred embodiment of the invention, the steps are repeated about 5 to about 20 times.
- the present invention further comprises methods for adapting CHO cell lines to grow to increased cell densities, comprising: • a) initiating a passage of duration aproximately 1 to 5 days by diluting a culture containing CHO cells at a density of at least approximately 1 x 10 6 cells/ml with a suitable growth medium, the dilution factor being suitable to the passage duration; (b) maintaining pH, dissolved oxygen, and nutrients in non-limiting levels during the passage; and c) repeating steps (a) and (b) at least about 5 times.
- the present invention further comprises methods for adapting CHO cell lines to grow to increased cell densities comprising: (a) initiating a passage of duration approximately 3 to 4 days by diluting a culture containing CHO cells at a density of at least approximately 1 x 10° cells/ml with a suitable growth medium, the dilution factor being suitable to the passage duration; (b) maintaining pH, dissolved oxygen, and nutrients at non-limiting levels during the passage; and (c) repeating steps (a) and (b) at least about 5 times.
- the present invention comprises a method for adapting mammalian cell lines to culture at increased cell densities, said method comprising continuously or periodically diluting a cell culture, containing mammalian cells, with a suitable growth medium, for between approximately 10 and 60 days, while maintaining pH, dissolved oxygen and nutrients at non-limiting levels.
- kits for the present invention comprise adapting CHO cell lines to culture at increased cell densities, said method comprising continuously or periodically diluting a culture containing CHO cells, at a density of at least approximately 1 x 10 6 cells/ml with a suitable growth medium, the dilution rate being less than approximately 0.029 hr 1 , while maintaining pH, dissolved oxygen and nutrients at non-limiting levels.
- Preferred dilution rates are between approximately O.Ol ⁇ hr 1 and 0.026hr 1 .
- Mammalian cell lines are used for the production of commercially useful proteins. Some mammalian cell lines which are commonly used include Chinese hamster ovary (CHO) cell lines, hybridomas, monkey COS-1 cells, HeLa cells, melanoma cell lines such as the Bowes cell line, hybridoma cell lines, mouse L cells, mouse fibroblasts, mouse NIH 3T3 cells and the CV-1 cell line. In the present invention, these and other mammalian cell lines may be adapted for culture at high cell densities.
- Suitable growth media for the present invention include any medium which provides nutrients at non-limiting levels. Nutrients will generally be at non-limiting levels if raising concentrations of all nutrients results in no increase in growth rate. Nutrient concentrations may be maintained at non-limiting levels by either providing excess amounts of all nutrients in the fresh medium or by adding nutrients to the culture as they are taken up by the cells or degraded.
- a suitable growth medium for mammalian cell lines is disclosed in Ling et al., Experimental Cell Research: 52:469-489 (1968). Accordingly, one preferred growth medium contains the amino acid nutrients in the concentrations disclosed in Table 1. TABLE 1
- nutrients which may be addded to the medium include inorganic salts, such as chlorides, phosphates, sulfates and nitrates, sugars, vitamins, and additives such as glutamine, pyruvate, linoleic, thioctic, selenite, hydrocortisone, insulin.
- inorganic salts such as chlorides, phosphates, sulfates and nitrates
- sugars such as glutamine, pyruvate, linoleic, thioctic, selenite, hydrocortisone, insulin.
- growth media suitable for mammalian cell lines include a medium containing the components described in Table 2 below.
- the magnitude of ⁇ , ⁇ may be estimated without precise measurement in a variety of ways.
- an estimate of ⁇ ⁇ a may be generated as follows. First the maximum cell density attainable in a spinner flask using a common medium (such as a 1:1 mixture of DME and F12) is determined by suspending growth phase cells in this medium in the spinner flask and measuring the cell density on each subsequent day until cell density no longer rises. Next, growth phase cells are suspended in fresh medium in another spinner flask at a starting density approximately 10-fold below the maximum attainable density and cultured for approximately 2 days. This culture is diluted with fresh medium to the same starting cell density every two days for several passages. The estimate of ⁇ . ⁇ is the growth rate observed during these passages, calculated using the following formula:
- a suitable dilution factor for a given duration of passage may be as follows: If the passage is approximately 1 day, a suitable dilution factor is less than about 2, preferably from about 1.5 to about 2. If the passage duration is approximately 2 days, a suitable dilution factor is less than about 4, preferably from about 2 to about 4. If the passage duration is approximately 3 days, a suitable dilution factor is less than about 8, preferably from about 3 to about 7. If the passage duration is approximately 4 days, suitable dilution factors are less than about 16, preferably from about 5 to about 13. If the passage duration is approximately 5 days, a suitable dilution factor is less than about 32, preferably from about 9 to about 23. For other mammalian cell lines, suitable dilution factors may be calculated on the basis of the maximum growth rate of the cell line. The maximum growth rate for a cell line may be determined as described above.
- relatively constant levels of pH, dissolved oxygen, and nutrients are maintained at non-limiting levels during the passage. This may preferably be accomplished by performing the adaption process in a bioreactor. pH may be maintained at the proper pH by addition of a suitable alkaline or acidic additive or buffer, for example sodium carbonate and sodium bicarbonate. Dissolved oxygen may be maintained by introduction of oxygen or air bubbles. If necessary, nutrient levels may be maintained by the addition of those nutrients which are depleted, or by addition of fresh growth medium.
- mammalian cell lines such as CHO cell lines
- a suitable cell density which may be approximately 1 x 10 6 cells/ml, in a suitable growth medium, and may be diluted in accordance with the above description.
- EXAMPLES The recombinant Chinese hamster ovary cell (CHO) line E5F3G expresses recombinant human M-CSF, as described in Clark et al., United States Patents 4,868,119 and 4,879,227. As described below, the E5F3G cell line was adapted to grow to increased cell densities, and thereby generate higher concentrations of rhM-CSF. E5F3G cells from a spinner flask were grown to a density of 1.24 x 10 6 cells/ml in approximately 1000 ml of a nutrient-rich medium (Table 2) in a 2-L bioreactor (passage 1 in Table 3).
- passage 12 For example, in passage 12, which was started at a density of 0.50 x 10° cells/ml, cell density reached 4.90 x 10 6 cells/ml, and rhM-CSF titer reached 32.6 ug/ml. In contrast, in passage 4, which had been started at a higher cell density (0.59 x 10 6 cells/ml), cell density had reached only 2.44 x 10 6 cells/ml and rhM-CSF titer had reached only 14.9 ug/ml.
Abstract
Methods and nutrient media are disclosed useful for adapting mammalian cell lines to culture at increased cell densities.
Description
ADAPTION OF MAMMALIAN CELL LINES TO HIGH CELL DENSITIES
FIELD OF THE INVENTION
The present invention relates to improved methods of expressing proteins through culture of mammalian cell lines. In particular, the present invention relates to methods of improving the productivity of mammalian cell lines through adaption to otherwise growth-limiting conditions.
BACKGROUND OF THE INVENTION It is known that various factors may be responsible for limiting the growth of cells at high cell densities. These factors include absence of sufficient amounts of nutrients needed by the cells for sustained growth, as well as the presence of growth-limiting concentrations of inhibitors that may be secreted by the cells in culture. One inhibitor that is secreted by mammalian cells is ammonia. See Miller et al., Bioprocess Engineering. 3:113-122 (1988); Inlόw et al., United States Patent 5,156,964 describes a method for generating tolerance to ammonia that involves culturing cells in a medium to which ammonia has been added. Similarly, Schumpp et al. , Cytotechnology. 8:39-44 (1992) describe a method for generating cell lines tolerant of both ammonia and lactic acid by culturing cells in a medium to which both ammonia and lactic acid had been added.
The previous methods have several drawbacks. First, in order to generate tolerance to an inhibitor according to the above methods, it is first necessary to determine that a particular inhibitor is a growth-limiting factor for cells and then to develop a protocol for generating tolerance to that inhibitor. Second, the growth of cell lines which are generated with tolerance to a particular inhibitor according to the above methods may then be limited by a second, different inhibitor. Repeated experiments may be necessary to generate tolerance to multiple growth-limiting inhibitors in order to achieve significant increases in cell densities.
SUMMARY OF THE INVENTION
According to the present invention, many of the drawbacks of the above prior art are overcome. The present invention provides methods by which the growth-limiting factors present for a particular cell line can be overcome without first conducting time-consuming testing to identify the specific growth-limiting inhibitors.
It is one object of the present invention to provide methods of improving the productivity of mammalian cell lines.
It is another object of the present invention to provide methods for adapting cell lines to high cell densities. It is yet another object of the present invention to provide nutrient-rich growth media in which nutrients are present in sufficient quantity so that they are not expected to limit cell growth.
According to the present invention, the above objects are largely achieved by providing methods for adapting mammalian cell lines to culture at increased cell densities. The methods of the present invention comprise adapting mammalian cell lines to grow at increased cell densities, by (a) initiating a passage by diluting a culture containing mammalian cells with a suitable growth medium by a dilution factor suitable for the passage duration; (b) maintaining pH, dissolved oxygen, and nutrients at non-limiting levels during the passage; and repeating steps (a) and (b) at least about 5 times. In a preferred embodiment of the invention, the steps are repeated about 5 to about 20 times.
The present invention further comprises methods for adapting CHO cell lines to grow to increased cell densities, comprising: • a) initiating a passage of duration aproximately 1 to 5 days by diluting a culture containing CHO cells at a density of at least approximately 1 x 106 cells/ml with a suitable growth medium, the dilution factor being suitable to the passage duration; (b) maintaining pH, dissolved oxygen, and nutrients in non-limiting levels during the passage; and c) repeating steps (a) and (b) at least about 5 times.
The present invention further comprises methods for adapting CHO cell lines to grow to increased cell densities comprising: (a) initiating a passage of duration approximately 3 to 4 days by diluting a culture containing CHO cells at a density of at least approximately 1 x 10° cells/ml with a suitable growth medium, the dilution factor being suitable to the passage duration; (b)
maintaining pH, dissolved oxygen, and nutrients at non-limiting levels during the passage; and (c) repeating steps (a) and (b) at least about 5 times.
In a preferred embodiment, the present invention comprises a method for adapting mammalian cell lines to culture at increased cell densities, said method comprising continuously or periodically diluting a cell culture, containing mammalian cells, with a suitable growth medium, for between approximately 10 and 60 days, while maintaining pH, dissolved oxygen and nutrients at non-limiting levels.
Other preferred methods of the present invention comprise adapting CHO cell lines to culture at increased cell densities, said method comprising continuously or periodically diluting a culture containing CHO cells, at a density of at least approximately 1 x 106 cells/ml with a suitable growth medium, the dilution rate being less than approximately 0.029 hr1, while maintaining pH, dissolved oxygen and nutrients at non-limiting levels. Preferred dilution rates are between approximately O.Olδhr1 and 0.026hr1.
DETAILED DESCRIPTION OF THE INVENTION Mammalian cell lines are used for the production of commercially useful proteins. Some mammalian cell lines which are commonly used include Chinese hamster ovary (CHO) cell lines, hybridomas, monkey COS-1 cells, HeLa cells, melanoma cell lines such as the Bowes cell line, hybridoma cell lines, mouse L cells, mouse fibroblasts, mouse NIH 3T3 cells and the CV-1 cell line. In the present invention, these and other mammalian cell lines may be adapted for culture at high cell densities.
Suitable growth media for the present invention include any medium which provides nutrients at non-limiting levels. Nutrients will generally be at non-limiting levels if raising concentrations of all nutrients results in no increase in growth rate. Nutrient concentrations may be maintained at non-limiting levels by either providing excess amounts of all nutrients in the fresh medium or by adding nutrients to the culture as they are taken up by the cells or degraded. A suitable growth medium for mammalian cell lines is disclosed in Ling et al., Experimental Cell Research: 52:469-489 (1968). Accordingly, one preferred growth medium contains the amino acid nutrients in the concentrations disclosed in Table 1.
TABLE 1
Column I Column II Column in
NUTRIENT CONCENTRATION OPTIMAL RANGE CONCENTRATION (MG/L) (MG/L)
L-asparagine H20 30-360 540
L-aspartic acid 69-798 266
Glycine 30-450 60
L-isoleucine 79-948 472
L-leucine 158-1890 681
L-lysine HC1 229-2742 728
L-methionine 75-894 238
L-serine 79-948 630
L-threonine 90-1074 381
L-tryptophan 31-366 131
L-tyrosine 2Na 2H2O 65-783 418
L-valine 141-1686 374
Other nutrients which may be addded to the medium include inorganic salts, such as chlorides, phosphates, sulfates and nitrates, sugars, vitamins, and additives such as glutamine, pyruvate, linoleic, thioctic, selenite, hydrocortisone, insulin.
Other preferred growth media suitable for mammalian cell lines include a medium containing the components described in Table 2 below.
TABLE 2 NUTIOENT COMPOSITION OF MEDIUM
Column I Column π Column HI Column IV
Components Medium Medium used Preferred proposed by for adaptation non-limiting
Ling et al. in Example medium
(mg/L) (mg/L) (mg/L)
(1968) sodium chloride 7000 4600 4400 potassium chloride 375 624 310 calcium chloride, anhydrous 156 232 58 sodium phosphate, dibasic, anhydrous 142 sodium phosphate, monobasic, hydrate 125 130 magnesium chloride, anhydrous 57 magnesium sulfate, anhydrous 120 98 84 cupric sulfate, anhydrous 185 0.0016 0.0018 ferrous sulfate, anhydrous 0.68 0.91 ferric nitrate, nonahydrate 1.2 0.10 zinc sulfate, septahydrate 0.86 0.86 0.92 sodium selenite 0.010 0.010 sodium bicarbonate 2440 2400
L-alanine 45-534 36 71
L-arginine 218-2616 600 760
L-asparagine hydrate 30-360 180 540
L-aspartic acid 67-798 133 270
L-cysteine hydrochloride hydrate 282 700
L-cystine dihydrochloride 23-281 125
L-glutamic acid 103-1236 59 120
L-glutamine 212-2544 1168 1200
Components Medium Medium used Preferred proposed by for adaptation non-limiting
Ling et al. in Example medium
(mg/L) (mg/L) (mg/L)
(1968) glycine 38-450 60 60
L-histidine hydrochloride hydrate 105-1260 126 290
L-isoleucine 79-948 210 470
L-leucine 158-1890 260 680
L-lysine hydrochloride 229-2742 291 730
L-methionine 75-894 104 240
L-phenylalanine 99-1188 165 330
L-proline 86-1032 138 280
L-serine 79-948 315 630
L-threonine 90-1074 190 380
L-tryptophan 31-366 33 130
L-tryosine disodium dihydrate 57-678 262 420
L-valine 141-1686 187 370 biotin 0.03 0.41 1.6
D-calcium pantothenate 5.0 4.5 18 choline chloride 350 18 72 folic acid 0.10 5.3 21 i-inositol 35 25 100 nicotinamide 20 4.0 16 pyridoxine hydrochloride 0.062 16 pyridoxal hydrochloride 2.5 4.0 riboflavin 1.5 0.44 1.8 thiamine hydrochloride 1.0 4.3 18 vitamin B12 0.003 1.6 5.6
Components Medium Medium used Preferred proposed by for adaptation non-limiting
Ling et al. in Example medium
(mg/L) (mg/L) (mg/L)
(1968)
D-glucose 2000 6000 6200 sodium pyruvate 110 linoleic acid 0.21 0.084 0.17 thioctic acid 0.70 0.21 0.42 putrescine dihydrochloride 2.2 2.0 polyvinyl alcohol 2400 2400 insulin or Nucellin 1.0 10 10 hydrocortisone 0.072 0.072 methotrexate 1.3 soybean phospholipid 10 fetal bovine serum 5000
B-glycerophosphate, disodium 1000
D-sorbitol 100 oxalacetic acid 65 thymidine 10 deoxycytidine 11 homocysteine thiolactate 8-90 glutathione, reduced 31-372 sodium molybdate, dihydrate 0.015 vitamin A acetate 1.0 vitamin D3 0.005 a-tocopherol 7.0 oleic acid 0.2 arachidonate, methyl 0.02
Components Medium Medium used Preferred proposed by for adaptation non-limiting
Ling et al. in Example medium
(mg/L) (mg/L) (mg/L)
(1968) cholesterol 5 ovo-lecithin 25 ethanol 2000
Suitable dilution factors (for passaging) and suitable dilution rates (for continuous culture) appropriate for adapting a particular mammalian cell line to grow to increased cell densities may be calculated using the formulas: dilution factor = w dilution rate = μ where t is the duration in hours of the upcoming passage and μ is any quantity less than μ→^, preferably a quantity between approximately (0.6 x μ^ and approximately (0.9 x /*„,«). « in hour1, is the specific growth rate of the cell line when none of the following extracellular factors limits growth: pH, dissolved oxygen, nutrient depletion and cell-generated inhibitors.
The magnitude of μ→,^ may be estimated without precise measurement in a variety of ways. For example, an estimate of μ^a may be generated as follows. First the maximum cell density attainable in a spinner flask using a common medium (such as a 1:1 mixture of DME and F12) is determined by suspending growth phase cells in this medium in the spinner flask and measuring the cell density on each subsequent day until cell density no longer rises. Next, growth phase cells are suspended in fresh medium in another spinner flask at a starting density approximately 10-fold below the maximum attainable density and cultured for approximately 2 days. This culture is diluted with fresh medium to the same starting cell density every two days for several passages. The estimate of μ.^ is the growth rate observed during these passages, calculated using the following formula:
/ = (In Xf - In X;)/t where Xf is the cell density at the end of a typical passage, X; is the cell density at the beginning
of the same passage, and t is the duration of the passage in hours.
For CHO cell lines, a suitable dilution factor for a given duration of passage may be as follows: If the passage is approximately 1 day, a suitable dilution factor is less than about 2, preferably from about 1.5 to about 2. If the passage duration is approximately 2 days, a suitable dilution factor is less than about 4, preferably from about 2 to about 4. If the passage duration is approximately 3 days, a suitable dilution factor is less than about 8, preferably from about 3 to about 7. If the passage duration is approximately 4 days, suitable dilution factors are less than about 16, preferably from about 5 to about 13. If the passage duration is approximately 5 days, a suitable dilution factor is less than about 32, preferably from about 9 to about 23. For other mammalian cell lines, suitable dilution factors may be calculated on the basis of the maximum growth rate of the cell line. The maximum growth rate for a cell line may be determined as described above.
In the method of the present invention, relatively constant levels of pH, dissolved oxygen, and nutrients are maintained at non-limiting levels during the passage. This may preferably be accomplished by performing the adaption process in a bioreactor. pH may be maintained at the proper pH by addition of a suitable alkaline or acidic additive or buffer, for example sodium carbonate and sodium bicarbonate. Dissolved oxygen may be maintained by introduction of oxygen or air bubbles. If necessary, nutrient levels may be maintained by the addition of those nutrients which are depleted, or by addition of fresh growth medium. In the present invention, mammalian cell lines, such as CHO cell lines, may be cultured at a suitable cell density, which may be approximately 1 x 106 cells/ml, in a suitable growth medium, and may be diluted in accordance with the above description.
The present invention is illustrated by the following examples. These examples do not limit the invention in any manner. It is contemplated that minor improvements and variations may be made which are part of the present invention.
EXAMPLES The recombinant Chinese hamster ovary cell (CHO) line E5F3G expresses recombinant human M-CSF, as described in Clark et al., United States Patents 4,868,119 and 4,879,227. As described below, the E5F3G cell line was adapted to grow to increased cell densities, and thereby generate higher concentrations of rhM-CSF.
E5F3G cells from a spinner flask were grown to a density of 1.24 x 106 cells/ml in approximately 1000 ml of a nutrient-rich medium (Table 2) in a 2-L bioreactor (passage 1 in Table 3).
These cells were then cultured for an additional ten 3-day or 4-day passages in the 2-L bioreactor (passages 2 through 11) in the nutrient-rich medium. During each passage, pH was maintained at between 7.0 and 7.2 by addition of sodium carbonate and sodium bicarbonate and dissolved oxygen was maintained at between 20% and 60% of air saturation by introduction of oxygen bubbles. Each 3-day passage was started by diluting the culture from the preceding passage by a factor between 5.1 and 6.3, while each 4-day passage was started by diluting the culture from the preceding passage by a factor between 6.0 and 14.3.
The beneficial effect on the cell line was evident during two subsequent passages
(passages 12 and 13). For example, in passage 12, which was started at a density of 0.50 x 10° cells/ml, cell density reached 4.90 x 106 cells/ml, and rhM-CSF titer reached 32.6 ug/ml. In contrast, in passage 4, which had been started at a higher cell density (0.59 x 106 cells/ml), cell density had reached only 2.44 x 106 cells/ml and rhM-CSF titer had reached only 14.9 ug/ml.
Table 3: Adaptation of E5F3G cell line to increased cell densities
Passage Passage Dilution Initial Final density Final titer number length (days) ratio density (lOVml) (ug/ml)
(lOVml)
1 4 - 0.12 1.24 11.6
2 3 5.4 0.23 1.96 14.3
3 3 6.3 0.31 3.00 16.5
4 3 5.1 0.59 2.44 14.9
5 4 12.2 0.20 1.79 -
6 4 6.0 0.30 3.50 -
7 3 5.0 0.70 2.25 12.2
8 3 5.2 0.43 2.70 15.6
9 4 12.3 0.22 4.30 20.2
10 4 14.3 0.30 5.90 29.2
11 3 5.9 1.00 5.70 33.5
12 3 11.4 0.50 4.90 32.6
13 4 16.3 0.30 5.30 34.2
Claims
1. A method for adapting mammalian cell lines to grow at increased cell densities, said method comprising: a) initiating a passage by diluting a culture containing mammalian cells with a suitable growth medium, the dilution factor being suitable for the passage duration; b) maintaining pH, dissolved oxygen, and nutrients at non-limiting levels during the passage; and c) repeating steps (a) and (b) at least about 5 times.
2. The method of claim 1, wherein steps (a) and (b) are repeated about 5 to about 20 times.
3. A method for adapting CHO cell lines to grow to increased cell densities, said method comprising: a) initiating a passage of duration aproximately 1 to 5 days by diluting a culture containing CHO cells at a density of at least approximately 1 x 106 cells/ml with a suitable growth medium, the dilution factor being suitable for the passage duration; b) maintaining pH, dissolved oxygen, and nutrients in non-limiting levels during the passage; and c) repeating steps (a) and (b) at least about 5 times.
4. The method of claim 3, wherein steps (a) and (b) aye repeated about 5 to about 20 times.
5. A method for adapting CHO cell lines to grow to increased cell densities, said method comprising: a) initiating a passage of duration approximately 3 to 4 days by diluting a culture containing CHO cells at a density of at least approximately 1 x 106 cells/ml with a suitable growth medium, the dilution factor being suitable to the passage duration; b) maintaining pH, dissolved oxygen, and nutrients at non-limiting levels during the passage; and c) repeating steps (a) and (b) at least about 5 times.
6. The method of claim 5, wherein steps (a) and (b) are repeated about 5 to about 20 times.
7. A method for adapting mammalian cell lines to culture at increased cell densities, said method comprising continuously or periodically diluting a cell culture, containing mammalian cells, with a suitable growth medium, for between approximately 10 and 60 days, while maintaining pH, dissolved oxygen and nutrients at non-limiting levels.
8. A method for adapting CHO cell lines to culture at increased cell densities,m said method comprising continuously or periodically diluting a culture containing CHO cells, at a density of at least approximately 1 x 106 cells/ml with a suitable growth medium, the dilution rate being less than approximately 0.029 hr1, while maintaining pH, dissolved oxygen and nutrients at non-limiting levels.
9. The method of claim 8, wherein the dilution rate is between approximately O.Olδhr1 and 0,026hr1.
10. A medium according to claim 10, wherein the nutrients are present in the following concentrations: L-asparagine H2O, about 540 mg/1; L-aspartic acid, about 266 mg/1; glycine, about 60 mg/1; L-isoleucine, about 472 mg/1; L-leucine, about 681 mg/1; L-lysine HCl, abou 728 mg/1; L-methionine, about 238 mg/1; L-serine, about 630 mg/1; L-threonine, about 381 mg/1; L-tryptophan, about 131 mg/1; L-tyrosine 2Na2H2O, about 418 mg/1; L-valine, about 374 mg/1.
11. A medium suitable for the culture of mammalian cells at high cell densities, comprising components in the concentrations disclosed at columns HI or IV of Table 2.
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| Application Number | Priority Date | Filing Date | Title |
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| AU80154/94A AU8015494A (en) | 1993-11-03 | 1994-10-12 | Adaption of mammalian cell lines to high cell densities |
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| US08/146,860 USH1532H (en) | 1993-11-03 | 1993-11-03 | Adaption of mammalian cell lines to high cell densities |
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| CA2099876C (en) * | 1991-01-21 | 2002-03-26 | Michael L. Hayes | Production of enzymatically active glucocerebrosidase from recombinant cells |
| GB9118664D0 (en) * | 1991-08-30 | 1991-10-16 | Celltech Ltd | Cell culture |
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| EP1824964A2 (en) * | 2004-11-19 | 2007-08-29 | Biogen Idec MA, Inc. | Methods for producing mammalian cells |
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| EA012840B1 (en) * | 2005-07-05 | 2009-12-30 | Арес Трейдинг С.А. | Use of serum-free culture medium comprising an antioxidant for reducing levels of oxidized forms of recombinant dimeric gonadotropin |
| AU2006264917B2 (en) * | 2005-07-05 | 2011-04-28 | Ares Trading S.A. | Serum-free culture medium for the production of recombinant gonadotropins |
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Also Published As
| Publication number | Publication date |
|---|---|
| USH1532H (en) | 1996-05-07 |
| AU8015494A (en) | 1995-05-23 |
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