HIGH TITER ANTIBODY PRODUCTION
The present invention provides, in part, methods for recombinantly expressing proteins at a high level along with cell culture media for doing the same.
1 . A method for producing a protein comprising inoculating an initial mammalian cell growth medium with host cells expressing the protein and adding supplements comprising:
Glucose;
L-glutamine;
Soy hydrolysate or wheat hydrolysate or both;
Adenine sulfate;
Adenosine;
ammonium vanadate;
Biotin;
Choline Chloride;
Cobalt chloride;
Cupric sulfate;
Cytidine;
D-Calcium Pantothenate;
Ethanolamine HCl;
Flavin Adenine Dinucleotide;
Folic Acid;
Glycine;
Guanosine;
Hypoxanthine;
i-Inositol;
L-alanine;
L-arginine;
L-asparagine;
L-aspartic acid;
L-citrulline;
L-cysteine-HCl;
L-cystine;
L-glutamic acid;
L-histidine;
Lipoic Acid;
L-isoleucine;
L-leucine;
L-lysine;
L-methionine;
L-ornithine-HCl;
L-phenylalanine;
L-proline;
L-serine;
L-threonine;
L-tryptophan;
L-tyrosine;
L-valine;
Manganese chloride Tetrahydrate;
Niacin;
Nickel dichloride hexahydrate;
Progesterone;
Putrescine 2HCl;
Pyridoxine HCl;
Riboflavin;
Sodium molybdate dehydrate;
Sodium phosphate monobasic;
Sodium selenite;
Thiamine HCl;
Thymidine;
Tin chloride dehydrate;
Uridine;
Vitamin B12;
Vitamin E; and
Zinc sulfate;
to the medium.
2 . The method of claim 1 wherein the final concentration of the components added to the medium from the supplements are about those set forth below:
Adenine sulfate:
1.632
mg/liter
Adenosine:
17.6
mg/liter
Ammonium vanadate:
0.00078
mg/liter
Biotin:
0.28
mg/liter
Choline Chloride:
50.2
mg/liter
Cobalt chloride:
0.0025
mg/liter
Cupric sulfate:
0.0032
mg/liter
Cytidine:
17.6
mg/liter
D-Calcium Pantothenate:
23.8
mg/liter
Ethanolamine HCl:
4.4
mg/liter
Flavin Adenine Dinucleotide:
0.05
mg/liter
Folic Acid:
4.6
mg/liter
Glycine
72
mg/liter
Guanosine:
17.6
mg/liter
Hypoxanthine:
11.8
mg/liter
i-Inositol:
73.2
mg/liter
L-alanine:
8.9
mg/liter
L-arginine
312.4
mg/liter
L-asparagine:
842
mg/liter
L-aspartic acid
97.6
mg/liter
L-citrulline:
12.6
mg/liter
L-cysteine-HCl
224
mg/liter
L-cystine:
34
mg/liter
L-glutamic acid
155.4
mg/liter
L-histidine
167
mg/liter
Lipoic Acid:
0.52
mg/liter
L-isoleucine
422
mg/liter
L-leucine
384
mg/liter
L-lysine
365
mg/liter
L-methionine
147.2
mg/liter
L-ornithine-HCl:
25.6
mg/liter
L-phenylalanine
207
mg/liter
L-proline
239
mg/liter
L-serine:
281
mg/liter
L-threonine
211.6
mg/liter
L-tryptophan
109.2
mg/liter
L-tyrosine
234
mg/liter
L-valine
308.8
mg/liter
Manganese chloride tetrahydrate:
0.0003
mg/liter
Niacin:
31.4
mg/liter
Nickel dichloride hexahydrate:
0.0004
mg/liter
Progesterone:
0.015
mg/liter
Putrescine 2HCl:
0.4
mg/liter
Pyridoxine HCl:
3
mg/liter
Riboflavin:
1.86
mg/liter
Sodium molybdate dehydrate:
0.00016
mg/liter
Sodium phosphate monobasic:
288.2
mg/liter
Sodium selenite:
0.01426
mg/liter
Thiamine HCl:
16
mg/liter
Thymidine:
7.8
mg/liter
Tin chloride dehydrate:
0.00008
mg/liter
Uridine:
17.6
mg/liter
Vitamin B12:
3.4
mg/liter
Vitamin E:
0.376
mg/liter
Zinc sulfate:
1.08
mg/liter
Glucose
1.5
g/liter
L-glutamine
150
mg/liter
3 . The method of claim 1 wherein the supplements are added from an amino acid feed that comprises amino acids at about the following concentrations:
L-arginine:
6.32
g/liter
L-cystine:
1.7
g/liter
L-histidine:
2.1
g/liter
L-isoleucine:
2.6
g/liter
L-leucine:
2.6
g/liter
L-lysine:
3.6
g/liter
L-Methionine:
0.76
g/liter
L-phenylalanine:
1.65
g/liter
L-threonine:
2.38
g/liter
L-tryptophan:
0.51
g/liter
L-tyrosine:
1.8
g/liter
L-valine:
2.34
g/liter
4 . The method of claim 3 wherein about 20 ml amino acid feed is added per liter of culture medium.
5 . The method of claim 1 wherein the supplements are added from an amino acid feed that comprises amino acids at about the following concentrations:
L-alanine:
0.89
g/liter
L-asparagine:
1.5
g/liter
L-aspartic acid:
1.33
g/liter
L-glutamic acid:
1.47
g/liter
Glycine:
0.75
g/liter
L-proline:
1.15
g/liter
L-serine:
1.05
g/liter
6 . The method of claim 5 wherein about 10 ml amino acid feed is added per liter of culture medium.
7 . The method of claim 1 wherein the supplements are added from a nutrient feed that comprises supplements at about the following concentrations:
L-asparagine:
40.6
g/liter
L-proline
10.81
g/liter
L-isoleucine
18.53
g/liter
L-cysteine-HCl
11.19
g/liter
L-leucine
16.58
g/liter
L-threonine
8.2
g/liter
L-tyrosine
9.9
g/liter
L-arginine
9.29
g/liter
L-aspartic acid
3.56
g/liter
L-glutamic acid
6.28
g/liter
Glycine
2.83
g/liter
L-histidine
6.23
g/liter
L-methionine
6.58
g/liter
L-tryptophan
4.93
g/liter
L-lysine
14.66
g/liter
L-phenylalanine
8.64
g/liter
L-valine
13.08
g/liter
L-serine:
13
g/liter
Sodium phosphate Monobasic:
14.41
g/liter
Zinc sulfate:
0.054
g/liter
Cupric sulfate:
0.00016
g/liter
Ammonium vanadate:
0.000039
g/liter
Cobalt chloride:
0.000125
g/liter
Nickel dichloride Hexahydrate:
0.00002
g/liter
Sodium molybdate dehydrate:
0.000008
g/liter
Tin chloride dehydrate:
0.000004
g/liter
Manganese chloride: tetrahydrate:
0.000015
g/liter.
8 . The method of claim 7 wherein about 20 ml nutrient feed is added per liter of culture medium.
9 . The method of claim 1 wherein the supplements are added from a vitamin/salt feed that comprises supplements at about the following concentrations:
Sodium selenite:
7.13 × 10 −4
g/liter
Adenine sulfate:
0.0816
g/liter
Adenosine:
0.88
g/liter
Cytidine:
0.88
g/liter
Guanosine:
0.88
g/liter
Uridine:
0.88
g/liter
Hypoxanthine:
0.59
g/liter
L-citrulline:
0.63
g/liter
L-ornithine-HCl:
1.28
g/liter
Biotin:
0.014
g/liter
Flavin Adenine Dinucleotide:
0.0025
g/liter
Folic Acid:
0.23
g/liter
Lipoic Acid:
0.026
g/liter
Niacin:
1.57
g/liter
Pyridoxine HCl:
0.15
g/liter
Riboflavin:
0.093
g/liter
Thiamine HCl:
0.8
g/liter
Vitamin E:
0.0188
g/liter
Vitamin B12:
0.17
g/liter
Choline Chloride:
2.51
g/liter
Ethanolamine HCl:
0.22
g/liter
i-Inositol:
3.66
g/liter
Thymidine:
0.39
g/liter
Putrescine 2HCl:
0.02
g/liter
Progesterone:
0.00075
g/liter
D-Calcium Pantothenate:
1.19
g/liter
10 . The method of claim 9 wherein about 20 ml vitamin/salt feed is added per liter of culture medium.
11 . The method of claim 1 further comprising harvesting the medium from the host cells; wherein said protein is secreted from said cells into the medium.
12 . The method of claim 13 wherein the culture medium is harvested from the host cells when viability of the cells is below about 60%.
13 . The method of claim 11 further comprising purifying the culture medium from the cells by centrifuging the medium and/or depth filtering the medium and/or filtering the medium through a 0.2 micron filter.
14 . The method of claim 1 wherein the protein is one or more immunoglobulin chains of an antibody or antigen-binding fragment thereof.
15 . The method of claim 14 wherein the antibody or fragment binds specifically to IGF1R.
16 . The method of claim 15 wherein the antibody or fragment comprises a heavy immunoglobulin chain comprising the amino acid sequence set forth in a member selected from the group consisting of SEQ ID NOs: 1, 3,-6, 13, 21 and 26; or a mature fragment thereof or one or more CDRs therefrom; and/or
a light immunoglobulin chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 7-9, 17 and 25, or a mature fragment thereof or one or more CDRs therefrom.
17 . The method of claim 16 wherein the antibody or fragment secreted from the host cell is an antibody that comprises a light chain immunoglobulin comprising amino acids 20-128 of SEQ ID NO: 3, 4, 5, or 6; and a heavy chain immunoglobulin comprising amino acids 20-137 of SEQ ID NO: 8 or 9.
18 . The method of claim 17 wherein the light chain immunoglobulin is linked to a kappa constant immunoglobulin chain and the heavy chain immunoglobulin is linked to a gamma-1 constant immunoglobulin chain.
19 . The method of claim 1 wherein the initial mammalian cell growth medium to which the supplements are added comprises HEPES, sodium bicarbonate buffers, inorganic salts, non-essential amino acids, recombinant human insulin, trace elements and surfactants; and does not comprise L-glutamine, antibiotics, antimycotics or animal-derived components.
20 . A method for producing an antibody comprising inoculating an initial mammalian cell growth medium, pre-warmed to about 37° C.; which initial medium comprises HEPES, sodium bicarbonate buffers, inorganic salts, non-essential amino acids, recombinant human insulin, trace elements and surfactants; and which does not comprise L-glutamine, antibiotics, antimycotics or animal-derived components; with CHO DXB11 host cells expressing the antibody light chain immunoglobulin and heavy chain immunoglobulin, to a cell density of about 2.5-5×10 5 cells/ml; and, adding the following supplements to the medium before, simultaneously with or immediately after said inoculation: soy hydrolysate to a final concentration of about 10 g/liter; and, optionally, an amino acid feed wherein the concentration of the components added by said amino acid feed are approximately those set forth below:
L-arginine:
126.4
mg/liter
L-cystine:
34
mg/liter
L-histidine:
42
mg/liter
L-isoleucine:
52
mg/liter
L-leucine:
52
mg/liter
L-lysine:
72
mg/liter
L-Methionine:
15.2
mg/liter
L-phenylalanine:
33
mg/liter
L-threonine:
47.6
mg/liter
L-tryptophan:
10.2
mg/liter
L-tyrosine:
36
mg/liter
L-valine:
46.8
mg/liter
L-alanine:
8.9
mg/liter
L-asparagine:
30
mg/liter
L-aspartic acid:
26.6
mg/liter
L-glutamic acid:
29.4
mg/liter
glycine:
15
mg/liter
L-proline:
23
mg/liter
L-serine:
21
mg/liter;
and, when viable cell density reaches over about 1.2×10 6 cells/ml, adding supplement feeds wherein the concentration of the components added by said supplement feeds are approximately those set forth below:
Sodium selenite:
0.01426
mg/liter
Adenine sulfate:
1.632
mg/liter
Adenosine:
17.6
mg/liter
Cytidine:
17.6
mg/liter
Guanosine:
17.6
mg/liter
Uridine:
17.6
mg/liter
Hypoxanthine:
11.8
mg/liter
L-citrulline:
12.6
mg/liter
L-ornithine-HCl:
25.6
mg/liter
Biotin:
0.28
mg/liter
Flavin Adenine Dinucleotide:
0.05
mg/liter
Folic Acid:
4.6
mg/liter
Lipoic Acid:
0.52
mg/liter
Niacin:
31.4
mg/liter
Pyridoxine HCl:
3
mg/liter
Riboflavin:
1.86
mg/liter
Thiamine HCl:
16
mg/liter
Vitamin E:
0.376
mg/liter
Vitamin B12:
3.4
mg/liter
Choline Chloride:
50.2
mg/liter
Ethanolamine HCl:
4.4
mg/liter
i-Inositol:
73.2
mg/liter
Thymidine:
7.8
mg/liter
Putrescine 2HCl:
0.4
mg/liter
Progesterone:
0.015
mg/liter
D-Calcium Pantothenate:
23.8
mg/liter
L-asparagine:
812
mg/liter
L-proline
216
mg/liter
L-isoleucine
370
mg/liter
L-cysteine-HCl
224
mg/liter
L-leucine
332
mg/liter
L-threonine
164
mg/liter
L-tyrosine
198
mg/liter
L-arginine
186
mg/liter
L-aspartic acid
71
mg/liter
L-glutamic acid
126
mg/liter
Glycine
57
mg/liter
L-histidine
125
mg/liter
L-methionine
132
mg/liter
L-tryptophan
99
mg/liter
L-lysine
293
mg/liter
L-phenylalanine
174
mg/liter
L-valine
262
mg/liter
L-serine:
260
mg/liter
Sodium phosphate monobasic:
288.2
mg/liter
Zinc sulfate:
1.08
mg/liter
Cupric sulfate:
0.0032
mg/liter
Ammonium vanadate:
0.00078
mg/liter
Cobalt chloride:
0.0025
mg/liter
Nickel dichloride hexahydrate:
0.0004
mg/liter
Sodium molybdate dehydrate:
0.00016
mg/liter;
and, maintaining glucose concentration in the medium at about 1.5 g/liter and maintaining L-glutamine concentration in the medium at about 150 mg/liter; and during cell growth maintaining O 2 concentration at about 60%; pH at about 6.8±0.02 and temperature at about 36.5° C.±0.5° C.; and, optionally, removing the host cells from the medium when cell viability is below about 60%.
21 . The method of claim 20 wherein the antibody comprises a heavy immunoglobulin chain comprising the amino acid sequence set forth in a member selected from the group consisting of SEQ ID NOs: 1, 3,-6, 13, 21 and 26; or a mature fragment thereof or one or more CDRs therefrom; and/or
a light immunoglobulin chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 7-9, 17 and 25, or a mature fragment thereof or one or more CDRs therefrom.
22 . The method of claim 21 wherein the antibody or fragment comprises an antibody secreted from the host cells that comprises a light chain immunoglobulin comprising amino acids 20-128 of SEQ ID NO: 3, 4, 5, or 6; and a heavy chain immunoglobulin comprising amino acids 20-137 of SEQ ID NO: 8 or 9.
23 . The method of claim 22 wherein the light chain immunoglobulin is linked to a kappa constant immunoglobulin chain and the heavy chain immunoglobulin is linked to a gamma-1 constant immunoglobulin chain.
24 . The method of claim 23 further comprising recovering the culture medium from the cells by disk-stack centrifuging the medium, depth filtering the medium and filtering the medium through a filter with about a 0.2 micron pore size.
25 . The method of claim 24 further comprising purifying the immunoglobulins from the medium by column chromatographic fractionation.
26 . An aqueous liquid cell culture medium comprising:
about 10 g/liter soy hydrolysate,
about 1.5 g/liter glucose,
about 150 mg/liter L-glutamine,
pH of about 6.8±0.02,
HEPES,
Sodium bicarbonate buffers,
Inorganic salts,
Non-essential amino acids,
Recombinant human insulin,
Trace elements,
Surfactants,
an amino acid feed wherein the concentration of the components added by said amino acid feed are approximately those set forth below:
L-arginine:
126.4
mg/liter
L-cystine:
34
mg/liter
L-histidine:
42
mg/liter
L-isoleucine:
52
mg/liter
L-leucine:
52
mg/liter
L-lysine:
72
mg/liter
L-Methionine:
15.2
mg/liter
L-phenylalanine:
33
mg/liter
L-threonine:
47.6
mg/liter
L-tryptophan:
10.2
mg/liter
L-tyrosine:
36
mg/liter
L-valine:
46.8
mg/liter
L-alanine:
8.9
mg/liter
L-asparagine:
30
mg/liter
L-aspartic acid:
26.6
mg/liter
L-glutamic acid:
29.4
mg/liter
Glycine:
15
mg/liter
L-proline:
23
mg/liter
L-serine:
21
mg/liter;
wherein the medium does not comprise antibiotics, antimycotics or animal-derived components.
27 . An aqueous liquid culture medium produced by a process comprising inoculating an initial mammalian cell growth medium, pre-warmed to about 37° C.; which medium comprises HEPES, sodium bicarbonate buffers, inorganic salts, non-essential amino acids, recombinant human insulin, trace elements and surfactants; and which does not comprise L-glutamine, antibiotics, antimycotics or animal-derived components; with CHO DXB11 host cells expressing an antibody light chain immunoglobulin and heavy chain immunoglobulin, to a cell density of about 2.5-5×10 5 cells/ml; and, adding the following supplements to the medium before, simultaneously with or immediately after said inoculation:
soy hydrolysate to a final concentration of about 10 g/liter;
and, optionally, an amino acid feed wherein the concentration of the components added by said amino acid feed are approximately those set forth below:
L-arginine:
126.4
mg/liter
L-cystine:
34
mg/liter
L-histidine:
42
mg/liter
L-isoleucine:
52
mg/liter
L-leucine:
52
mg/liter
L-lysine:
72
mg/liter
L-Methionine:
15.2
mg/liter
L-phenylalanine:
33
mg/liter
L-threonine:
47.6
mg/liter
L-tryptophan:
10.2
mg/liter
L-tyrosine:
36
mg/liter
L-valine:
46.8
mg/liter
L-alanine:
8.9
mg/liter
L-asparagine:
30
mg/liter
L-aspartic acid:
26.6
mg/liter
L-glutamic acid:
29.4
mg/liter
Glycine:
15
mg/liter
L-proline:
23
mg/liter
L-serine:
21
mg/liter;
and, when viable cell density reaches over about 1.2×10 6 cells/ml, adding supplement feeds wherein the concentration of the components added by said supplement feeds are approximately those set forth below:
Sodium selenite:
0.01426
mg/liter
Adenine sulfate:
1.632
mg/liter
Adenosine:
17.6
mg/liter
Cytidine:
17.6
mg/liter
Guanosine:
17.6
mg/liter
Uridine:
17.6
mg/liter
Hypoxanthine:
11.8
mg/liter
L-citrulline:
12.6
mg/liter
L-ornithine-HCl:
25.6
mg/liter
Biotin:
0.28
mg/liter
Flavin Adenine Dinucleotide:
0.05
mg/liter
Folic Acid:
4.6
mg/liter
Lipoic Acid:
0.52
mg/liter
Niacin:
31.4
mg/liter
Pyridoxine HCl:
3
mg/liter
Riboflavin:
1.86
mg/liter
Thiamine HCl:
16
mg/liter
Vitamin E:
0.376
mg/liter
Vitamin B12:
3.4
mg/liter
Choline Chloride:
50.2
mg/liter
Ethanolamine HCl:
4.4
mg/liter
i-Inositol:
73.2
mg/liter
Thymidine:
7.8
mg/liter
Putrescine 2HCl:
0.4
mg/liter
Progesterone:
0.015
mg/liter
D-Calcium Pantothenate:
23.8
mg/liter
L-asparagine:
812
mg/liter
L-proline
216
mg/liter
L-isoleucine
370
mg/liter
L-cysteine-HCl
224
mg/liter
L-leucine
332
mg/liter
L-threonine
164
mg/liter
L-tyrosine
198
mg/liter
L-arginine
186
mg/liter
L-aspartic acid
71
mg/liter
L-glutamic acid
126
mg/liter
Glycine
57
mg/liter
L-histidine
125
mg/liter
L-methionine
132
mg/liter
L-tryptophan
99
mg/liter
L-lysine
293
mg/liter
L-phenylalanine
174
mg/liter
L-valine
262
mg/liter
L-serine:
260
mg/liter
Sodium phosphate monobasic:
288.2
mg/liter
Zinc sulfate:
1.08
mg/liter
Cupric sulfate:
0.0032
mg/liter
Ammonium vanadate:
0.00078
mg/liter
Cobalt chloride:
0.0025
mg/liter
Nickel dichloride hexahydrate:
0.0004
mg/liter
Sodium molybdate dehydrate:
0.00016
mg/liter;
and, maintaining glucose concentration in the medium at about 1.5 g/liter and maintaining L-glutamine concentration in the medium at about 150 mg/liter; and during cell growth maintaining O 2 concentration at about 60%; pH at about 6.8±0.02 and temperature at about 36.5° C.±0.5° C.
28 . The aqueous liquid culture medium of claim 27 wherein the host cells comprise a vector encoding the immunoglobulins of the antibody wherein the antibody is secreted into the medium.
29 . The aqueous liquid culture medium of claim 28 wherein the host cell viability is about 60% or lower and/or wherein host cell growth has proceeded for about 14 to 24 days.
30 . The aqueous liquid culture medium of claim 29 wherein the host cells are removed from the medium.
31 . The aqueous liquid culture medium of claim 30 wherein the host cells are removed from the medium by centrifuging the medium and/or depth filtering the medium and/or filtering the medium through a 0.2 micron filter.
32 . The aqueous liquid culture medium of claim 31 wherein the antibody binds specifically to IGF1R.
33 . The aqueous liquid culture medium of claim 32 wherein the heavy immunoglobulin chain comprises the amino acid sequence set forth in a member selected from the group consisting of SEQ ID NOs: 1, 3,-6, 13, 21 and 26; or a mature fragment thereof or one or more CDRs therefrom;
and a light immunoglobulin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 7-9, 17 and 25, or a mature fragment thereof or one or more CDRs therefrom.
34 . The aqueous liquid culture medium of claim 33 wherein the antibody or fragment is an antibody that is secreted from the host cell that comprises a light chain immunoglobulin comprising amino acids 20-128 of SEQ ID NO: 3, 4, 5, or 6; and a heavy chain immunoglobulin comprising amino acids 20-137 of SEQ ID NO: 8 or 9.
35 . The aqueous liquid culture medium of claim 34 wherein the light chain immunoglobulin is linked to a kappa constant immunoglobulin chain and the heavy chain immunoglobulin is linked to a gamma-1 constant immunoglobulin chain.
36 . A vessel comprising the aqueous liquid culture medium of claim 27 .
37 . The vessel of claim 36 which is a flask, a bioreactor, a tank bioreactor, a bag bioreactor or a disposable bioreactor.
38 . The vessel of claim 37 which bioreactor is a stirred tank bioreactor, a bubble column bioreactor, an air lift bioreactor, a fluidized bed bioreactor or a packed bed bioreactor.