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WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




PCT 

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification 6 ; 
C12N 5/00 



Al 



(11) International Publication Number: 
(43) International Publication Date: 



WO 95/12664 

11 May 1995 (11.05.95) 



(21) International Application Number: PCT/US94/ 11535 

(22) International Filing Date: 12 October 1994 (12.10.94) 



(30) Priority Data: 

08/146,860 



3 November 1993 (03.11.93) US 



(71) Applicant: GENETICS INSTITUTE, INC. [US/US]; 87 Cam- 

bridgePark Drive, Cambridge, MA 02140 (US). 

(72) Inventors: ADAMSON, S., Robert; 15 Cathy Road, Chelms- 

ford, MA 01824 (US). DRAPEAU, Denis; 15 Norwood 
Road, Salem, NH 03079 (US). LUAN, Yen-Tung; 3 Ar- 
mand Drive, Chelmsford, MA 01824 (US). MILLER, Dou- 
glas, Alan; 6 Alexander Avenue, Salem, NH 03079 (US). 

(74) Agent: LAZAR, Steven, R.; Genetics Institute, Inc., 87 
CambridgePark Drive, Cambridge, MA 02140 (US). 



(81) Designated States: AU, CA, FI, JP, KP, KR, NO, European 
patent (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, 
MC, NL, PT, SE), OAPI patent (BF, BJ, CF, CG, CI, CM, 
GA, GN, ML, MR, NE, SN, TD, TG). 



Published 

With international search report. 



(54) Title: ADAPTION OF MAMMALIAN CELL LINES TO HIGH CELL DENSITIES 
(57) Abstract 

Methods and nutrient media are disclosed useful for adapting mammalian cell lines to culture at increased cell densities. 



FOR THE PURPOSES OF INFORMATION ONLY 



Codes used to identify States party to the PCT on the front pages of pamphlets publishing international 
applications under the PCT. 



AT 


Austria 


GB 


United Kingdom 


MR 


Mtnifitunra 


AU 


Australia 


GE 


Georgia 


MW 


Malawi 


BB 


Barbados 


GN 


Oninfa 


NE 


Niger 


BE 


Belgium 


GR 


Greece 


NL 


Netherlands 


BF 


Burkina Peso 


HU 


Hungary 


NO 


Norway 


BG 


Bulgaria 


IE 


Ireland 


NZ 


New Zealand 


BJ 


Benin 


IT 


Italy 


PL 


Poland 


BR 


Brazil 


JP 


Japan 


FT 


Portugal 


BY 


Belarus 


KE 


Kenya 


RO 


Romania 


CA 


Canada 


KG 


Kyrgystan 


RU 


Russian Federation 


CF 


Central African Republic 


KP 


Democratic People's Republic 


SD 


Sudan 


CG 


Congo 




of Korea 


SE 


Sweden 


CH 


Switzerland 


KR 


Republic of Korea 


SI 


Slovenia 


CI 


Cote d*lvoire 


KZ 


Kazakhstan 


SK 


Slovakia 


CM 


Cameroon 


LI 


Liechtenstein 


SN 


Senegal 


CN 


China 


LK 


Sri Lanka 


TD 


Chad 


CS 


Czechoslovakia 


LU 


Luxembourg 


TG 


Togo 


cz 


Czech Republic 


LV 


Latvia 


TJ 


Tajikistan 


DE 


Germany 


MC 


Monaco 


TT 


Trinidad and Tobago 


DK 


Denmark 


MD 


Republic of Moldova 


UA 


Ukraine 


ES 


Spain 


MG 


Madagascar 


US 


United States of America 


FI 


Finland 


ML 


Mali 


uz 


Uzbekistan 


FR 


France 


MN 


Mongolia 


VN 


Viet Nam 


GA 


Gabon 











WO 95/12664 



PCT/US94/11535 



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-Umiting 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 Engineerin g, 3:113-122 (1988); 
Inlbw 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., Cytotechnplpgy, 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. 



WO 95/12664 



PCT/US94/11535 



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 
5 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. 

10 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 
15 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 
20 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 
25 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 S 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 
30 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) 



WO 95/12664 



PCTA3S94/11S35 



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 
5 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 

10 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\ while 
maintaining pH, dissolved oxygen and nutrients at non-limiting levels. Preferred dilution rates 
are between approximately 0.01 8hr* and 0.026hr ! . 

DETAILED DESCRIPTION OF THE INVENTION 

15 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 

20 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 

25 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. 



3 



WO 95/12664 


TABLE 1 


PCTAJS94/11S35 


Column I 


Column II 


Column m 


NT7TRTFNT 


CONCENTRATION 
RANGE 
(MG/L) 


OPTIMAL 
CONCENTRATION 
(MG/L) 


L-asparagine H 2 0 


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 HQ 


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 2H 2 0 


£LG TOO 


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 
20 containing the components described in Table 2 below. 



4 



WO 95/12664 



PCT/US94/11535 



TABLE 2 

NUTRIENT COMPOSITION OF MEDIUM 





f^nlumti T 


Column II 


Column in 


Column IV 




components 


Medium 
proposed by 
Ling et al. 

(mg/L) 

(1968) 


1 Medium used 
1 for adaptation 
in Example 
(mg/L) 


1 

Preferred 

non-limitino 

*L\J1 1 UUUUUL 

medium 
(mg/L) 


5 


sodium chloride 


7000 


4600 


44UU 




potassium chloride 


375 




J1U 




calcium chloride, anhydrous 


156 




JO 




sodium phosphate, dibasic, anhydrous 




142 






sodium phosphate, monobasic, hydrate 






1 OA 

130 


10 


magnesium chloride, anhydrous 




57 

j / 






magnesium sulfate, anhydrous 


120 




84 




cupric sulfate, anhydrous 


185 


u.uuio 


r\ g\r\ « a 

0.0018 




ferrous sulfate, anhydrous 




n ac 


0.91 




ferric nitrate, nonahydrate 


1 1 


a in 




15 


zinc sulfate, septahydrate 


u.OD 


A Oil 

U.oo 


0.92 




sodium selenite 




U.U1U 


0.010 




sodium bicarbonate 






2400 




L-alanine 


45-534 


^A 


71 




L-arginine 


218-2616 


OVA/ 


760 


20 


L-asparagine hydrate 


30-360 


180 






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 


25 


L-glutamine 


212-2544 


1168 


1200 



5 



WO 95/12664 



PCT/US94/11535 



Components 


Medium 
proposed by 
Ling et al. 
(mg/L) 
(1968) 


Medium used 
for adaptation 
in Example 
(mg/L) 


Preferred 
non-limiting 
medium 
(mg/L) 


glycine 




OU 


OU 


jL-msnaine nyarociuonae nyaraic 




12o 


ion 
290 


L-isoleucine 


/y-y*fo 


210 


470 


L-leucine 




2o0 


680 


L-lysine hydrochloride 


ion 

229-2742 


291 


730 


L-methionine 


75-894 


104 


240 


L-phenylalanine 


99-1188 


165 


330 


L-proline 


©0-1U32 


138 


Ann 

280 


L-serine 


79-948 


3 15 


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 




1 o 

18 


72 


folic acid 


n in 


5.3 


21 


i-inositol 


35 


25 


100 


nicotinamide 


20 


4.0 


16 


pyridoxine hydrochloride 




0.062 


16 


pynuuxdi iiyuruciiiuiiuc 








riboflavin 


1.5 


0.44 


1.8 


thiamine hydrochloride 


1.0 


4.3 


18 


vitamin B12 


0.003 


1.6 


5.6 



6 



WO 95/12664 PCT/US94/1 1535 



Components 


Medium 
proposed by 
Ling et al. 
(mg/L) 
] (1968) 


Medium used 
for adaptation 
in Example 
(mg/L) 


Preferred 
non-limiting 
medium 
(mg/L) 


D-plucose 




oUOO 


6200 


sodium nvruvflte 




1 1 A 

110 




linoleie acid 


U.Zl 


A AO A 

0.084 


0.17 


thioetic ariH 


U. /U 


0.21 


0.42 


puucocmc uuiyurocmoriuc 




2.2 


2.0 


polyvinyl aiconoi 




2400 


2400 


insulin or iNUCciiin 


1.0 


10 


10 


11 Vn TYV^OTtl Crtfl O 

Jiyui UMIllC 




0.072 


0.072 


JliwUIUU CAdlC 




1.3 




auyucdJi pnospnoupiu 




10 




fetal bovine serum 




5000 




B-glycerophosphate, disodium 


1000 






l>soroitol 


100 






oxaiaceuc acid 


65 






inymiuine 


10 






ucuAycyuQinc 


11 






iiuiiiucy 2> icinc uiioiaciaie 


o aa 

8-90 






giuiatnione, reouceo 


31-372 






dooium uioiyooaic, Ginyorate 


n Air 

0.015 






viuirnin a acetate 


1.0 






vitamin D3 


0.005 






a-tocopherol 


7.0 






oleic acid 


0.2 






arachidonate, methyl 


0.02 







7 



WO 95/12664 



PCT/US94/11535 



Components 


Medium 
proposed by 
Ling et al. 
(mg/L) 
(1968) 


Medium used 
for adantation 
in Example 
(mg/L) 


Preferred 

nnn-limitinp 

medium 
(mg/L) 


cholesterol 


5 






ovo-lecithin 


25 






ethanol 


2000 







5 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 = e 0 * 0 

10 dilution rate = \i 

where t is the duration in hours of the upcoming passage and ji is any quantity less than /w> 
preferably a quantity between approximately (0.6 x /x^J and approximately (0.9 x aw). ^ 
in hour 1 , is the specific growth rate of the cell line when none of the following extracellular 

15 factors limits growth: pH, dissolved oxygen, nutrient depletion and cell-generated inhibitors. 

The magnitude of aw may be estimated without precise measurement in a variety of 
ways. For example, an estimate of aw 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 

20 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 jw is the growth rate observed during these passages, 

25 calculated using the following formula: 

AW = (lnX f -lnXi)/t 

where X f is the cell density at the end of a typical passage, Xj is the cell density at the beginning 



WO 95/12664 



PCT/US94/11535 



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 10 6 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. 



WO 95/12664 



PCT/US94/11535 



E5F3G cells from a spinner flask were grown to a density of 1.24 x 10^ 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 s 
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. 



10 



WO 95/12664 



PCT/US94/11S35 



Table 3: Adaptation of E5F36 cell line to increased cell densities 





Passage 


Passage 

icugui yjBysj 


Dilution 
ratio 


Initial 

density 

(107ml) 


Final density 
(107ml) 


Final titer 
(ug/ml) 




i 


4 


- 


0.12 


1.24 


11.6 


5 


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 




10 


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 


15 


12 


3 


11.4 


0.50 


4.90 


32.6 




13 


4 


16.3 


0.30 


5.30 


34.2 



11 



WO 95/12664 



PCTAJS94/11535 



CLAIMS 

We claim: 

1. A method for adapting mammalian cell lines to grow at increased cell densities, said 
method comprising: 

5 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. 

10 

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: 

15 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 for the passage duration; 

b) maintaining pH, dissolved oxygen, and nutrients in non-limiting levels during the 
passage; and 

20 c) repeating steps (a) and <b) at least about 5 times. 

4. The method of claim 3, wherein steps (a) and (b) aie repeated about 5 to about 20 

times. 

25 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 10 6 cells/ml with a suitable 
growth medium, the dilution factor being suitable to the passage duration; 
30 b) maintaining pH, dissolved oxygen, and nutrients at non-limiting levels during the 

12 



WO 95/12664 



PCT/US94/11535 



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 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. 

9. The method of claim 8, wherein the dilution rate is between approximately 0.01 8hr* 
andO.CEehr 1 . 

10. A medium according to claim 10, wherein the nutrients are present in the following 
concentrations: L-asparagine H 2 0, 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 HC1, about 
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 2Na2H 2 0, 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 m or IV of Table 2. 



13 



INTERNATIONAL SEARCH REPORT 



later. aa) Application No 

PCT/US 94/11535 



A. CLASSIFICATION OF SUBJECT MATTER 

IPC 6 C12N5/00 






According to International Patent Classification (IPC) or to both national classification and IPC 




B. FIELDS SEARCHED 


Minimum documentation searched (classification system followed by classification symbols) 

IPC 6 C12N 


Documentation searched other than minimum documentation to the extent that each documents an included in the fields searched 


Electronic d 


ata base consulted during the international search (name of data base and, where practical, search terms used) 




C. DOCUMENTS CONSIDERED TO BE RELEVANT 


Category* 


Citation of document, with indication, where appropriate, of the relevant passages 


Relevant to claim No. 


X 


US, A, 3 850 748 (COOK, R.A.) 1974 
see the whole document 




l 


X 


GB,A,2 251 249 (MOGAM BIOTECHNOLOGY 
RESEARCH INSTITUTE) 1992 
see page 8 


10 


A 


US, A, 5 122 469 (MATHER, J. P.) 1992 
see the whole document 


l-n 


A 


US,A,5 156 964 (INLOW, D:) 1992 
cited in the application 
see the whole document 


■/" 


l-n 


j Further documents arc listed in the continuation of box C. 


[y j Patent family members are listed in annex. 


* Special categories of cited documents : 

'A* document defining the general state of the art which is not 
considered to be of particular relevance 

earlier document but published on or after the international 
filing date 

"1/ document which may throw doubts on priority daim(s) or 
which is cited to establish the publication date of another 
citation or other special reason (as specified) 

*0" document referring to an oral disclosure, use, exhibition or 
other means 

"P* document published prior to the international filing date but 
later than the priority date claimed 


T later document published after the international filing date 
or priority date and not in conflict with the application but 
cited to understand the principle or theory underlying the 
invention 

"X" document of particular relevance; the dairoed invention 
cannot be considered novel or cannot be considered to 
involve an inventive step when the document is taken alone 

*Y" document of particular relevance; the claimed invention 
cannot be considered to involve an inventive step when the 
document is combined with one or more other such docu- 
ments, such combination being obvious to a person skilled 
in the art. 

document member of the same patent farniiy 


Date of the actual completion of the international search 

6 February 1995 


Date of mailing of the international search report 

2 2 -t)2- 1995 


Name and i 


nailing address of the ISA 

European Patent Office, P.B. 581 8 Patcntlaan 2 

NL\2280HVRijswijk 

Tel. (■+ 31-70) 340-2040, Tx. 31 631 epo nl, 

Fax: ( + 31-70) 340-3016 


Authorized officer 

Fernandez y Branas,F 



Form PCT/I&AO10 (second theel) (July 1993) 



page 



1 of 2 



INTERNATIONAL SEARCH REPORT 



Inter. .ial Application No 

PCT/US 94/11535 



(^Continuation) DOCUMENTS CONSIDERED TO BE RELEVANT 



Category " Citation of document, with indication* where appropriate, of the relevant passages 



Relevant to daim No. 



EXPERIMENTAL CELL RESEARCH, 
vol .52, 1968, NEW YORK 
pages 469 - 489 

LING C.T. ET AL 'Chemically characterised 
concentrated corodies for comtinuous cell 
culture (the 7C'S culture media). 1 
cited in the application 
see the whole document 

R. IAN FRESHNEY 'Culture of Animal cells' 
1987 , ALAN R. LISS, INC. , NEW YORK 
see page 127 - page 136 



1-11 



1-11 



Form PCT/1SA/310 (continuation of second inert) (July 1992) 



page 2 of 2 



INTERNATIONAL SEARCH REPORT 

information on patent family member* 



Inten lal Application No 

PCT/US 94/11535 



Patent document 
cited in search report 


1 Publication 
1 date 


Patent family 
member(s) 


1 Publication 
1 date 


US-A-3850748 


26-11-74 


NONE 




GB-A-2251249 


01-07-92 


FR-A- 2671098 


03-07-92 


US-A-5122469 


16-06-92 


NONE 




US-A-5156964 


20-10-92 


NONE 





Form PCT/ISA/210 (patent family annex) (July 1993) 



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