The present invention provides, inter alia, an isolated cell line, 3M as well as methods for making such a cell line and methods of using such a cell line, e.g., to produce a protein such as an immunoglobulin.
1. An isolated Chinese hamster ovary cell deposited at the American Type Culture Collection under deposit number PTA-10481.
2. The cell of claim 1 in an aqueous liquid cell culture medium.
3. The cell of claim 1 in a vessel.
4. The cell of claim 1 which comprises a vector.
5. The cell of claim 4 wherein the vector comprises a polynucleotide encoding one or more proteins.
6. The cell of claim 1 comprising a protein that is an immunoglobulin comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-8, 21, 22, 42, 43, 58-69, 79, 80, 81, 85, 89, 93 and 101 or a mature fragment thereof or an immunoglobulin comprising one or more CDRs from said immunoglobulin; optionally linked to an immunoglobulin constant chain; or one or both chains of etanercept.
7. The cell of claim 1 comprising a protein that is an immunoglobulin chain comprising:
(1)
CDR-L1:
(SEQ ID NO: 9)
KASKKVTIFGSISALH,
CDR-L2:
(SEQ ID NO: 10)
NGAKLES,
and
CDR-L3:
(SEQ ID NO: 11)
LQNKEVPYT;
(2)
CDR-H1:
(SEQ ID NO: 12)
SYGIT,
CDR-H2:
(SEQ ID NO: 13)
ENYPRSGNTYYNEKFKG,
and
CDR-H3:
(SEQ ID NO: 14)
CEFISTVVAPYYYALDY
or
(SEQ ID NO: 15)
SEFISTVVAPYYYALDY
or
(SEQ ID NO: 16)
AEFISTVVAPYYYALDY
or
(SEQ ID NO: 17)
VEFISTVVAPYYYALDY
or
(SEQ ID NO: 18)
SEFISTVMAPYYYALDY
or
(SEQ ID NO: 19)
SEFTSTVVAPYYYALDY;
(3)
CDRH1:
(SEQ ID NO: 23)
Gly Phe Thr Phe Ser Ser Tyr Thr Met Ser,
CDRH2:
(SEQ ID NO: 24)
Thr Ile Ser Ser Gly Gly Thr Tyr Thr Tyr Tyr Pro
Asp Ser Val Lys Gly,
and
CDRH3:
(SEQ ID NO: 25)
Asp Asn His Ala Tyr Asp Arg Gly Pro Phe Phe Asp
Tyr;
(4)
CDRL1:
(SEQ ID NO: 26)
Lys Ser Ser Gln Asn Leu Phe Tyr Arg Ser Asn Gln
Lys Asn His Leu Ala,
CDRL2:
(SEQ ID NO: 27)
Trp Thr Ser Thr Arg Glu Ser,
and
CDRL3:
(SEQ ID NO: 28)
Gln Gln Tyr Tyr Ser Tyr Pro Pro Thr;
(5)
CDRH1:
(SEQ ID NO: 29)
Ala Tyr Gly Met Asp,
CDRH2:
(SEQ ID NO: 30)
Ser Ile Ser Pro Ser Gly Gly Arg Thr Lys Tyr Ala
Asp Ser Val Lys Gly,
and
CDRH3:
(SEQ ID NO: 31)
Asp Leu Gly Gly Gly Tyr Tyr Tyr Tyr Tyr Gly Met
Asp Val;
(6)
CDRL1:
(SEQ ID NO: 32)
Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr
Val Ser,
CDRL2:
(SEQ ID NO: 33)
Glu Val Ser Asn Arg Pro Ser,
and
CDRL3:
(SEQ ID NO: 34)
Ser Ser Tyr Thr Ser Ser Ser Thr Leu Phe Tyr Val;
(7)
CDRH1:
(SEQ ID NO: 35)
GKTFWSWGIN,
CDRH2:
(SEQ ID NO: 36)
YIYIGTGYTEPNPKYKG,
and
CDRH3:
(SEQ ID NO: 37)
IGGYYGNFAD
or
(SEQ ID NO: 38)
IGGYYGNFDQ;
(8)
CDRL1:
(SEQ ID NO: 39)
RSSQSLLISGGNTYLN,
CDRL2:
(SEQ ID NO: 40)
LVSKLDQ,
and
CDRL3:
(SEQ ID NO: 41)
WQGTYFPLT;
(9)
CDRH1:
(SEQ ID NO: 44)
TYWMH
or
(SEQ ID NO: 45)
TYWMH,
CDRH2:
(SEQ ID NO: 46)
EINPTNGHTNYNEKFKS,
(SEQ ID NO: 47)
EINPTNGHTNYNPSFQG,
or
(SEQ ID NO: 48)
EINPTNGHTNYNQKFQG,
and
CDRH3:
(SEQ ID NO: 49)
NYVGSIFDY
or
(SEQ ID NO: 50)
NYVGSIFDY;
(10)
CDRL1:
(SEQ ID NO: 51)
KASENVVSYVS
or
(SEQ ID NO: 52)
KASENVVSYVS,
CDRL2:
(SEQ ID NO: 53)
GASNRNT,
(SEQ ID NO: 54)
GASNRNT
or
(SEQ ID NO: 55)
GASNRES,
and
CDRL3:
(SEQ ID NO: 56)
GQSYNYPYT
or
(SEQ ID NO: 57)
GQSYNYPYT;
(11)
CDRH1:
(SEQ ID NO: 82)
GYTFSSYGMY;
CDRH2:
(SEQ ID NO: 83)
WIDPGSGGTKYNEKFKG;
and
CDRH3:
(SEQ ID NO: 84)
ERYGYYFDY;
(12)
CDRL1:
(SEQ ID NO: 86)
RASQYVGSYLN;
CDRL2:
(SEQ ID NO: 87)
DASNRAT;
and
CDRL3:
(SEQ ID NO: 88)
QVWDSSPPVV;
(13)
CDRL1:
(SEQ ID NO: 90)
RASQDVSRYLT;
CDRL2:
(SEQ ID NO: 91)
AASSLQS;
and
CDRL3:
(SEQ ID NO: 92)
QAYDYSLSGYV;
(14)
CDRH1:
(SEQ ID NO: 94)
GYTFSSYWMH;
CDRH2:
(SEQ ID NO: 95)
RIDPYNGGTKYNEKFKG;
and
CDRH3:
(SEQ ID NO: 96)
YGYYLGSYAMDY;
(15)
CDRH1:
(SEQ ID NO: 98)
GYTFTDYYMN;
CDHR2:
(SEQ ID NO: 99)
DINPNNGGAIYNQKFKG;
and
CDRH3:
(SEQ ID NO: 100)
GIITEIAEDF;
or
(16)
CDRL1:
(SEQ ID NO: 102)
KASQNVGTNVV;
CDRL2:
(SEQ ID NO: 103)
SASYRYS;
and
CDRL3:
(SEQ ID NO: 104)
QQYKTYPYT;
optionally linked to an immunoglobulin constant chain.
8. A method for making one or more polypeptides; or antibodies or antigen-binding fragments thereof that comprise said polypeptides; comprising introducing one or more polynucleotides encoding said polypeptides into the cell of claim 1 and culturing the cell in conditions under which the polypeptides are produced.
9. The method of claim 8 further comprising isolating the polypeptide.
10. The method of claim 8 wherein the polypeptide is an immunoglobulin.
11. The method of claim 10 wherein the immunoglobulin comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-8, 21, 22, 42, 43, 58-69, 79, 80, 81, 85, 89, 93 and 101 or a mature fragment thereof or an immunoglobulin comprising one or more CDRs from said immunoglobulin; optionally linked to an immunoglobulin constant chain.
12. The method of claim 10 wherein the immunoglobulin comprises:
(1)
CDR-L1:
(SEQ ID NO: 9)
KASKKVTIFGSISALH,
CDR-L2:
(SEQ ID NO: 10)
NGAKLES,
and
CDR-L3:
(SEQ ID NO: 11)
LQNKEVPYT;
(2)
CDR-H1:
(SEQ ID NO: 12)
SYGIT,
CDR-H2:
(SEQ ID NO: 13)
ENYPRSGNTYYNEKFKG,
and
CDR-H3:
(SEQ ID NO: 14)
CEFISTVVAPYYYALDY
or
(SEQ ID NO: 15)
SEFISTVVAPYYYALDY
or
(SEQ ID NO: 16)
AEFISTVVAPYYYALDY
or
(SEQ ID NO: 17)
VEFISTVVAPYYYALDY
or
(SEQ ID NO: 18)
SEFISTVMAPYYYALDY
or
(SEQ ID NO: 19)
SEFTSTVVAPYYYALDY;
(3)
CDRH1:
(SEQ ID NO: 23)
Gly Phe Thr Phe Ser Ser Tyr Thr Met Ser,
CDRH2:
(SEQ ID NO: 24)
Thr Ile Ser Ser Gly Gly Thr Tyr Thr Tyr Tyr Pro
Asp Ser Val Lys Gly,
and
CDRH3:
(SEQ ID NO: 25)
Asp Asn His Ala Tyr Asp Arg Gly Pro Phe Phe Asp
Tyr;
(4)
CDRL1:
(SEQ ID NO: 26)
Lys Ser Ser Gln Asn Leu Phe Tyr Arg Ser Asn Gln
Lys Asn His Leu Ala,
CDRL2:
(SEQ ID NO: 27)
Trp Thr Ser Thr Arg Glu Ser,
and
CDRL3:
(SEQ ID NO: 28)
Gln Gln Tyr Tyr Ser Tyr Pro Pro Thr;
(5)
CDRH1:
(SEQ ID NO: 29)
Ala Tyr Gly Met Asp,
CDRH2:
(SEQ ID NO: 30)
Ser Ile Ser Pro Ser Gly Gly Arg Thr Lys Tyr Ala
Asp Ser Val Lys Gly,
and
CDRH3:
(SEQ ID NO: 31)
Asp Leu Gly Gly Gly Tyr Tyr Tyr Tyr Tyr Gly Met
Asp Val;
(6)
CDRL1:
(SEQ ID NO: 32)
Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asn Tyr
Val Ser,
CDRL2:
(SEQ ID NO: 33)
Glu Val Ser Asn Arg Pro Ser,
and
CDRL3:
(SEQ ID NO: 34)
Ser Ser Tyr Thr Ser Ser Ser Thr Leu Phe Tyr Val;
(7)
CDRH1:
(SEQ ID NO: 35)
GKTFWSWGIN,
CDRH2:
(SEQ ID NO: 36)
YIYIGTGYTEPNPKYKG,
and
CDRH3:
(SEQ ID NO: 37)
IGGYYGNFAD
or
(SEQ ID NO: 38)
IGGYYGNFDQ;
(8)
CDRL1:
(SEQ ID NO: 39)
RSSQSLLISGGNTYLN,
CDRL2:
(SEQ ID NO: 40)
LVSKLDQ,
and
CDRL3:
(SEQ ID NO: 41)
WQGTYFPLT;
(9)
CDRH1:
(SEQ ID NO: 44)
TYWMH
or
(SEQ ID NO: 45)
TYWMH,
CDRH2:
(SEQ ID NO: 46)
EINPTNGHTNYNEKFKS,
(SEQ ID NO: 47)
EINPTNGHTNYNPSFQG,
or
(SEQ ID NO: 48)
EINPTNGHTNYNQKFQG,
and
CDRH3:
(SEQ ID NO: 49)
NYVGSIFDY
or
(SEQ ID NO: 50)
NYVGSIFDY;
(10)
CDRL1:
(SEQ ID NO: 51)
KASENVVSYVS
or
(SEQ ID NO: 52)
KASENVVSYVS,
CDRL2:
(SEQ ID NO: 53)
GASNRNT,
(SEQ ID NO: 54)
GASNRNT
or
(SEQ ID NO: 55)
GASNRES,
and
CDRL3:
(SEQ ID NO: 56)
GQSYNYPYT
or
(SEQ ID NO: 57)
GQSYNYPYT;
(11)
CDRH1:
(SEQ ID NO: 82)
GYTFSSYGMY;
CDRH2:
(SEQ ID NO: 83)
WIDPGSGGTKYNEKFKG;
and
CDRH3:
(SEQ ID NO: 84)
ERYGYYFDY;
(12)
CDRL1:
(SEQ ID NO: 86)
RASQYVGSYLN;
CDRL2:
(SEQ ID NO: 87)
DASNRAT;
and
CDRL3:
(SEQ ID NO: 88)
QVWDSSPPVV;
(13)
CDRL1:
(SEQ ID NO: 90)
RASQDVSRYLT;
CDRL2:
(SEQ ID NO: 91)
AASSLQS;
and
CDRL3:
(SEQ ID NO: 92)
QAYDYSLSGYV;
(14)
CDRH1:
(SEQ ID NO: 94)
GYTFSSYWMH;
CDRH2:
(SEQ ID NO: 95)
RIDPYNGGTKYNEKFKG;
and
CDRH3:
(SEQ ID NO: 96)
YGYYLGSYAMDY;
(15)
CDRH1:
(SEQ ID NO: 98)
GYTFTDYYMN;
CDHR2:
(SEQ ID NO: 99)
DINPNNGGAIYNQKFKG;
and
CDRH3:
(SEQ ID NO: 100)
GIITEIAEDF;
or
(16)
CDRL1:
(SEQ ID NO: 102)
KASQNVGTNVV;
CDRL2:
(SEQ ID NO: 103)
SASYRYS;
and
CDRL3:
(SEQ ID NO: 104)
QQYKTYPYT;
optionally linked to an immunoglobulin constant chain.
13. The method of claim 10 wherein the immunoglobulin is a light chain immunoglobulin linked to a kappa or lambda constant immunoglobulin chain and/or wherein the immunoglobulin is a heavy chain immunoglobulin linked to a gamma-1, gamma-2, gamma-3 or gamma-4 constant immunoglobulin chain.
14. 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 one or more cells of claim 1 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%.
15. The method of claim 14 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.
16. The method of claim 14 further comprising purifying the immunoglobulins from the medium by column chromatographic fractionation.
17. A master cell bank or working cell bank comprising the cell of claims 1 .
18. The cell of claim 1 in a cell freezing medium.
19. The cell of claim 18 wherein the cell freezing medium comprises dimethylsulfoxide.
20. A method for making a Chinese hamster ovary cell comprising adapting CHO-DXB11 cells into animal-component free medium in suspension for 83 days; then subcloning the cells into said medium twice.