US20070212346A1 - Highly Concentrated Stabilized Igm Solution - Google Patents
Highly Concentrated Stabilized Igm Solution Download PDFInfo
- Publication number
- US20070212346A1 US20070212346A1 US10/575,192 US57519204A US2007212346A1 US 20070212346 A1 US20070212346 A1 US 20070212346A1 US 57519204 A US57519204 A US 57519204A US 2007212346 A1 US2007212346 A1 US 2007212346A1
- Authority
- US
- United States
- Prior art keywords
- solution
- igm
- nacl
- ion
- polyvalent cationic
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- -1 cationic ion Chemical class 0.000 claims abstract description 44
- 239000000243 solution Substances 0.000 claims description 150
- 238000000034 method Methods 0.000 claims description 71
- 108090000623 proteins and genes Proteins 0.000 claims description 65
- 108060003951 Immunoglobulin Proteins 0.000 claims description 21
- 102000018358 immunoglobulin Human genes 0.000 claims description 21
- 239000008194 pharmaceutical composition Substances 0.000 claims description 21
- 102000004169 proteins and genes Human genes 0.000 claims description 18
- 238000004519 manufacturing process Methods 0.000 claims description 12
- 235000000346 sugar Nutrition 0.000 claims description 9
- 239000004475 Arginine Substances 0.000 claims description 8
- 239000007864 aqueous solution Substances 0.000 claims description 8
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 8
- 150000008163 sugars Chemical class 0.000 claims description 8
- 229910001425 magnesium ion Inorganic materials 0.000 claims description 7
- 230000000087 stabilizing effect Effects 0.000 claims description 7
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 claims description 6
- 230000008014 freezing Effects 0.000 claims description 6
- 238000007710 freezing Methods 0.000 claims description 6
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 abstract description 46
- 229910001629 magnesium chloride Inorganic materials 0.000 abstract description 23
- 239000000654 additive Substances 0.000 abstract description 7
- 238000004220 aggregation Methods 0.000 abstract description 3
- 230000002776 aggregation Effects 0.000 abstract description 3
- 229960003589 arginine hydrochloride Drugs 0.000 abstract description 2
- 150000001875 compounds Chemical class 0.000 abstract description 2
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 abstract 1
- 230000000996 additive effect Effects 0.000 abstract 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 117
- 239000011780 sodium chloride Substances 0.000 description 59
- 239000001509 sodium citrate Substances 0.000 description 36
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 36
- 239000000872 buffer Substances 0.000 description 35
- 239000000203 mixture Substances 0.000 description 28
- 108091008146 restriction endonucleases Proteins 0.000 description 27
- 239000012634 fragment Substances 0.000 description 25
- 210000004027 cell Anatomy 0.000 description 22
- 239000013598 vector Substances 0.000 description 20
- 230000000694 effects Effects 0.000 description 19
- 239000000178 monomer Substances 0.000 description 18
- 230000006641 stabilisation Effects 0.000 description 18
- 238000011105 stabilization Methods 0.000 description 18
- 239000001488 sodium phosphate Substances 0.000 description 17
- 229910000162 sodium phosphate Inorganic materials 0.000 description 17
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 17
- 101000840258 Homo sapiens Immunoglobulin J chain Proteins 0.000 description 16
- 238000009472 formulation Methods 0.000 description 16
- 108091034117 Oligonucleotide Proteins 0.000 description 15
- 238000006243 chemical reaction Methods 0.000 description 15
- 238000001641 gel filtration chromatography Methods 0.000 description 13
- 238000003860 storage Methods 0.000 description 13
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 12
- 229930006000 Sucrose Natural products 0.000 description 12
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 12
- 235000018102 proteins Nutrition 0.000 description 12
- 239000005720 sucrose Substances 0.000 description 12
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 11
- KWTQSFXGGICVPE-UHFFFAOYSA-N 2-amino-5-(diaminomethylideneamino)pentanoic acid;hydron;chloride Chemical compound Cl.OC(=O)C(N)CCCN=C(N)N KWTQSFXGGICVPE-UHFFFAOYSA-N 0.000 description 11
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 11
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 11
- 102100029571 Immunoglobulin J chain Human genes 0.000 description 10
- 238000012181 QIAquick gel extraction kit Methods 0.000 description 10
- 238000002347 injection Methods 0.000 description 10
- 239000007924 injection Substances 0.000 description 10
- 239000002244 precipitate Substances 0.000 description 10
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 9
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 9
- 150000001413 amino acids Chemical class 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 9
- 150000003839 salts Chemical class 0.000 description 9
- 238000000502 dialysis Methods 0.000 description 8
- 230000014509 gene expression Effects 0.000 description 8
- 239000013612 plasmid Substances 0.000 description 8
- 239000000244 polyoxyethylene sorbitan monooleate Substances 0.000 description 8
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 8
- 229920000053 polysorbate 80 Polymers 0.000 description 8
- 229940068968 polysorbate 80 Drugs 0.000 description 8
- 230000001629 suppression Effects 0.000 description 8
- 241001465754 Metazoa Species 0.000 description 7
- 210000003719 b-lymphocyte Anatomy 0.000 description 7
- 230000008901 benefit Effects 0.000 description 7
- 230000001900 immune effect Effects 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 238000001542 size-exclusion chromatography Methods 0.000 description 7
- WVDDGKGOMKODPV-UHFFFAOYSA-N Benzyl alcohol Chemical compound OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 6
- 239000012228 culture supernatant Substances 0.000 description 6
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 6
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 6
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 6
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 6
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 6
- 239000002552 dosage form Substances 0.000 description 6
- 239000013604 expression vector Substances 0.000 description 6
- BRZYSWJRSDMWLG-CAXSIQPQSA-N geneticin Chemical compound O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](C(C)O)O2)N)[C@@H](N)C[C@H]1N BRZYSWJRSDMWLG-CAXSIQPQSA-N 0.000 description 6
- 239000002773 nucleotide Substances 0.000 description 6
- 125000003729 nucleotide group Chemical group 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 238000010839 reverse transcription Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 241000282412 Homo Species 0.000 description 5
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 5
- 239000007832 Na2SO4 Substances 0.000 description 5
- 102000005686 Serum Globulins Human genes 0.000 description 5
- 108010045362 Serum Globulins Proteins 0.000 description 5
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 5
- 229960003121 arginine Drugs 0.000 description 5
- 235000009697 arginine Nutrition 0.000 description 5
- 150000001768 cations Chemical class 0.000 description 5
- 239000003085 diluting agent Substances 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 229910052938 sodium sulfate Inorganic materials 0.000 description 5
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 4
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 4
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 4
- 229940024606 amino acid Drugs 0.000 description 4
- 235000001014 amino acid Nutrition 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 239000000539 dimer Substances 0.000 description 4
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- 229940072221 immunoglobulins Drugs 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 238000013112 stability test Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 3
- 101150074155 DHFR gene Proteins 0.000 description 3
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 229920001213 Polysorbate 20 Polymers 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 3
- 108010084455 Zeocin Proteins 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 239000011543 agarose gel Substances 0.000 description 3
- 238000012197 amplification kit Methods 0.000 description 3
- 210000004102 animal cell Anatomy 0.000 description 3
- 230000000692 anti-sense effect Effects 0.000 description 3
- 239000012512 bulk drug substance Substances 0.000 description 3
- 239000007979 citrate buffer Substances 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 3
- CWCMIVBLVUHDHK-ZSNHEYEWSA-N phleomycin D1 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC[C@@H](N=1)C=1SC=C(N=1)C(=O)NCCCCNC(N)=N)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C CWCMIVBLVUHDHK-ZSNHEYEWSA-N 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000003757 reverse transcription PCR Methods 0.000 description 3
- 230000007017 scission Effects 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 239000001632 sodium acetate Substances 0.000 description 3
- 235000017281 sodium acetate Nutrition 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 239000003381 stabilizer Substances 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 2
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 239000008351 acetate buffer Substances 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 150000001298 alcohols Chemical class 0.000 description 2
- 150000001450 anions Chemical class 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000012752 auxiliary agent Substances 0.000 description 2
- 235000019445 benzyl alcohol Nutrition 0.000 description 2
- SESFRYSPDFLNCH-UHFFFAOYSA-N benzyl benzoate Chemical compound C=1C=CC=CC=1C(=O)OCC1=CC=CC=C1 SESFRYSPDFLNCH-UHFFFAOYSA-N 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 238000002659 cell therapy Methods 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- PFJKOHUKELZMLE-VEUXDRLPSA-N ganglioside GM3 Chemical compound O[C@@H]1[C@@H](O)[C@H](OC[C@@H]([C@H](O)/C=C/CCCCCCCCCCCCC)NC(=O)CCCCCCCCCCCCC\C=C/CCCCCCCC)O[C@H](CO)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@]2(O[C@H]([C@H](NC(C)=O)[C@@H](O)C2)[C@H](O)[C@H](O)CO)C(O)=O)[C@@H](O)[C@@H](CO)O1 PFJKOHUKELZMLE-VEUXDRLPSA-N 0.000 description 2
- 238000005227 gel permeation chromatography Methods 0.000 description 2
- 238000002523 gelfiltration Methods 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 238000000569 multi-angle light scattering Methods 0.000 description 2
- 239000002736 nonionic surfactant Substances 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 229920000136 polysorbate Polymers 0.000 description 2
- 229950008882 polysorbate Drugs 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- UQSHZBSQKMVQBS-YDALLXLXSA-M sodium;(2s)-2-acetamido-3-(1h-indol-3-yl)propanoate Chemical compound [Na+].C1=CC=C2C(C[C@H](NC(=O)C)C([O-])=O)=CNC2=C1 UQSHZBSQKMVQBS-YDALLXLXSA-M 0.000 description 2
- 229960002920 sorbitol Drugs 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000013589 supplement Substances 0.000 description 2
- 239000003981 vehicle Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- HOMROMWVNDUGRI-RVZXSAGBSA-N (2s)-2-aminopentanedioic acid;(2s)-2,6-diaminohexanoic acid Chemical compound NCCCC[C@H](N)C(O)=O.OC(=O)[C@@H](N)CCC(O)=O HOMROMWVNDUGRI-RVZXSAGBSA-N 0.000 description 1
- RVEWUBJVAHOGKA-WOYAITHZSA-N Arginine glutamate Chemical compound OC(=O)[C@@H](N)CCC(O)=O.OC(=O)[C@@H](N)CCCNC(N)=N RVEWUBJVAHOGKA-WOYAITHZSA-N 0.000 description 1
- KWTQSFXGGICVPE-WCCKRBBISA-N Arginine hydrochloride Chemical compound Cl.OC(=O)[C@@H](N)CCCN=C(N)N KWTQSFXGGICVPE-WCCKRBBISA-N 0.000 description 1
- 238000012935 Averaging Methods 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- CTKXFMQHOOWWEB-UHFFFAOYSA-N Ethylene oxide/propylene oxide copolymer Chemical compound CCCOC(C)COCCO CTKXFMQHOOWWEB-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 241000701044 Human gammaherpesvirus 4 Species 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- FXAGBTBXSJBNMD-UHFFFAOYSA-N acetic acid;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound CC(O)=O.OC(=O)CC(O)(C(O)=O)CC(O)=O FXAGBTBXSJBNMD-UHFFFAOYSA-N 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 239000000730 antalgic agent Substances 0.000 description 1
- 210000000628 antibody-producing cell Anatomy 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 229960000686 benzalkonium chloride Drugs 0.000 description 1
- 229960002903 benzyl benzoate Drugs 0.000 description 1
- CADWTSSKOVRVJC-UHFFFAOYSA-N benzyl(dimethyl)azanium;chloride Chemical compound [Cl-].C[NH+](C)CC1=CC=CC=C1 CADWTSSKOVRVJC-UHFFFAOYSA-N 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000003093 cationic surfactant Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000004540 complement-dependent cytotoxicity Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- KAKKHKRHCKCAGH-UHFFFAOYSA-L disodium;(4-nitrophenyl) phosphate;hexahydrate Chemical compound O.O.O.O.O.O.[Na+].[Na+].[O-][N+](=O)C1=CC=C(OP([O-])([O-])=O)C=C1 KAKKHKRHCKCAGH-UHFFFAOYSA-L 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229930195712 glutamate Natural products 0.000 description 1
- 229940022353 herceptin Drugs 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 239000000644 isotonic solution Substances 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 239000012669 liquid formulation Substances 0.000 description 1
- 239000012931 lyophilized formulation Substances 0.000 description 1
- 235000018977 lysine Nutrition 0.000 description 1
- 229960003646 lysine Drugs 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 150000002482 oligosaccharides Polymers 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 229940021222 peritoneal dialysis isotonic solution Drugs 0.000 description 1
- 239000008177 pharmaceutical agent Substances 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229940127557 pharmaceutical product Drugs 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229920001993 poloxamer 188 Polymers 0.000 description 1
- 229940044519 poloxamer 188 Drugs 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229940068977 polysorbate 20 Drugs 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- MFDFERRIHVXMIY-UHFFFAOYSA-N procaine Chemical compound CCN(CC)CCOC(=O)C1=CC=C(N)C=C1 MFDFERRIHVXMIY-UHFFFAOYSA-N 0.000 description 1
- 229960004919 procaine Drugs 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 229940124272 protein stabilizer Drugs 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 229960004641 rituximab Drugs 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000008159 sesame oil Substances 0.000 description 1
- 235000011803 sesame oil Nutrition 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 230000010473 stable expression Effects 0.000 description 1
- 239000008174 sterile solution Substances 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 150000005846 sugar alcohols Polymers 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 239000013638 trimer Substances 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/3076—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties
- C07K16/3084—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells against structure-related tumour-associated moieties against tumour-associated gangliosides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
- A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
- A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7016—Disaccharides, e.g. lactose, lactulose
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
- A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K39/39591—Stabilisation, fragmentation
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/02—Inorganic compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/12—Carboxylic acids; Salts or anhydrides thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
- A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0087—Galenical forms not covered by A61K9/02 - A61K9/7023
- A61K9/0095—Drinks; Beverages; Syrups; Compositions for reconstitution thereof, e.g. powders or tablets to be dispersed in a glass of water; Veterinary drenches
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
Definitions
- the present invention relates to highly concentrated and stabilized IgM solutions, and production thereof.
- IgG immunoglobulins
- IgA immunoglobulin A
- IgM immunoglobulin M
- IgD immunoglobulin D
- IgE immunoglobulin E
- Each immunoglobulin class differs in properties such as size, charge, amino acid composition, and sugar content.
- IgM accounts for approximately 10% of all plasma immunoglobulins.
- IgM is the major component of early antibodies produced against cell-membrane antigens, infectious microorganisms, or soluble antigens, which have a complex antigenicity.
- Human IgMs usually have a pentameric structure. Each of the five subunits constituting this pentameric structure has a four-stranded structure similar to that of IgG.
- the amino acid sequence of the ⁇ chain, which is the heavy chain of IgM, is different from that of the ⁇ chain, which is the heavy chain of IgG. The following differences can also be seen:
- the ⁇ chain has an extra constant domain than the ⁇ chain.
- the ⁇ chain has four more oligosaccharide chains than the ⁇ chain.
- IgM has a polypeptide chain called the J chain, which is not found in IgG.
- the J chain is considered to assist the association of ⁇ chains prior to secretion of IgM from antibody producing cells.
- Chimeric antibodies are antibodies having a structure in which the variable regions have been replaced with variable regions derived from a different species.
- “chimeric antibodies” comprising variable regions of non-human antibodies and the constant regions of human antibodies (Non-Patent Document 1/Proc. Natl. Acad. Sci. U.S.A., (1984) 81:6851) are known.
- humanized antibodies in which the complementarity determining regions (CDR) of other animal species are transferred into human immunoglobulins (Non-Patent Document 2/Nature (1986) 321:521).
- antitumor antibodies are the anti-CD20 human chimeric antibody Rituxan (IDEC), and the anti-HER2/neu humanized antibody Herceptin (Genentech), which have completed clinical trials and have already been approved. These antibodies are now commercially available.
- ADCC antibody-dependent cellular cytotoxicity
- CDC complement-dependent cytotoxicity
- IgM is also very unstable compared to IgG and has a low solubility. Therefore, the production of a highly concentrated and stable IgM solution is difficult.
- Cytotherapy, 2001, 3(3), 233-242 Non-Patent Document 5
- IgM easily aggregates and precipitates during storage. It was especially difficult to ensure an IgM stability sufficient enough to withstand pharmaceutical use only through optimization of pH and buffer type.
- Patent Document 1 discloses formulations for stabilizing highly concentrated antibodies that comprise acidic ingredients. This method uses MgCl 2 and CaCl 2 as additives to stabilize the antibodies, but the stabilization is carried out to prepare IgG formulations, and IgM formulations are not mentioned. As described above, unlike IgG, IgM exists as a multimer, and unlike intrinsically stable IgG, IgM readily aggregates. Therefore, IgM has the distinctive problem of being very difficult to be highly concentrated.
- Non-Patent Document 3 and Journal of Immunological Methods, 111 (1988) 17-23 (Non-Patent Document 5) reported that IgM precipitates at a low salt concentration, and redissolves at a high salt concentration in a phosphate buffer and Tris-hydrochloride buffer that are weakly alkaline.
- Clin. Chem. Lab. Med. 2000; 38(8): 759-764 reports that, near pH 5, IgM readily precipitates and is difficult to handle, suggesting a pessimistic outlook for IgM solutions in weakly acidic buffers.
- Non-Patent Document 5 a buffer comprising 0.1 M Tris-HCl and 1 M NaCl (pH 8) is used to redissolve euglobulin precipitates.
- the resulting recovery rate of IgM varies from 40% to >90% depending on antibodies or batch, indicating a low reproducibility.
- the Methods section describes that 5 to 10 mg/mL of purified antibodies were stored at 4° C. and ⁇ 20° C.
- the Results section only describes that the antibodies could be stored for a few months at ⁇ 20° C. without loss of function, and does not mention anything regarding storage at 4° C. or higher, at which temperature it is usually difficult to ensure stability. Accordingly, this report suggests the difficulty of reproducing precipitate reconstitution and the difficulty of ensuring stability during storage, when trying to provide a highly concentrated solution of IgM as a pharmaceutical product or a bulk drug substance.
- Non-Patent Document 4 BIOTECHNOLOGY 1993, 11, 512-515 (Non-Patent Document 4) and Journal of Immunological Methods, 111 (1988) 17-23 (Non-Patent Document 5) also describe the reconstitution of insoluble aggregates of antibodies as euglobulin precipitates, but the solubility is 10 mg/mL or less, indicating low solubility of IgM. There is no description at all regarding the stabilization of water-soluble aggregates.
- Non-Patent Document 6 discloses stabilization of IgM by PVP addition, but does not disclose the stabilization of highly concentrated antibodies. Journal of Immunological Methods 1995, 181(1), 37-43 (Non-Patent Document 7) discloses lyophilized formulations produced by addition of trehalose, but in this report, the antibody stability is insufficient and there is no description relating to stabilization of highly concentrated antibodies.
- the present invention was achieved in view of the above circumstances.
- An objective of the present invention is to stabilize highly concentrated IgM in solutions. More specifically, the present invention aims to provide methods for stabilizing highly concentrated IgM, solutions in which highly concentrated IgM is stabilized, and methods for preparing the solutions.
- the present invention provides an aqueous solution in which increase of water-soluble aggregates is suppressed.
- Another preferred embodiment provides a highly concentrated IgM formulation that is stable enough to be used as a pharmaceutical.
- the present inventors discovered that, by using compounds comprising polyvalent cationic ions such as magnesium chloride and arginine hydrochloride as additives, aggregation of IgM in solutions may be suppressed and stable highly concentrated IgM solutions may be prepared.
- compounds comprising polyvalent cationic ions such as magnesium chloride and arginine hydrochloride as additives, aggregation of IgM in solutions may be suppressed and stable highly concentrated IgM solutions may be prepared.
- the present invention relates to methods for stabilizing highly concentrated IgM, solutions in which highly concentrated IgM is stabilized, and methods for preparing the solutions. More specifically, the present invention provides the following:
- step (b) freezing or lyophilizing the solution stabilized in step (a);
- step (b) freezing or lyophilizing the solution produced in step (a).
- FIG. 1 shows “ ⁇ aggregate” values obtained by subtracting the initial aggregate content from the aggregate content after one month of storage at 25° C. (25° C.-1 month) and the aggregate content after 2 months of storage at 25° C. (25° C.-2 months) for each sample.
- FIG. 2 shows the aggregate content (%) in each sample in the initial state and at 4° C.-3 months.
- FIG. 3 shows the aggregate content (%) in each sample in the initial state, at 4° C.-4 months, and 25° C.-4 months.
- FIG. 4 shows the aggregate content (%) according to size exclusion chromatography (SEC) in each sample in the initial state, liquid/40° C.-8 days, liquid/40° C.-8 days+4° C.-3 days, lyophilized/40° C.-8 days, lyophilized/40° C.-8 days+4° C.-3 days, and lyophilized/40° C.-8 days+4° C.-3 days+relyophilized/50° C.-8 days.
- SEC size exclusion chromatography
- FIG. 5 shows the aggregate content (%) according to SEC in each sample in the initial state, and lyophilized/40° C.-2 months.
- FIG. 6 shows the chromatogram obtained by gel permeation chromatography (GPC)-MALLS analysis of MABON-01 and the molecular weight determined by calculation.
- the term “IgM” refers to an immunoglobulin that comprises constant regions of the ⁇ chain as the constant regions of the heavy chains, and forms a pentameric or hexameric structure.
- the origin of the variable regions constituting the IgM of the present invention is not limited. Therefore, in addition to a variable region derived from the ⁇ chain, the IgM of the present invention may comprise a variable region derived from IgG, or a partial structure thereof.
- the partial structure of a variable region can comprise the framework and CDR.
- the “IgM” in the present invention refers to expression products of exogenous IgM genes introduced into cells for transformation.
- the constant regions constituting the IgM of the present invention may be derived from any animal species. That is, the IgM of the present invention comprises an IgM constant region derived from any type of animal species carrying an IgM-type immunoglobulin.
- the IgM of the present invention comprises an IgM constant region derived from any type of animal species carrying an IgM-type immunoglobulin.
- at least its constant regions are preferably derived from an animal species same as the species to which the IgM is administered. Therefore, when the IgM is administered to humans, at least its constant regions are preferably derived from humans.
- IgM composed of constant regions derived from humans, and variable regions derived from another animal species or another human, is called a chimeric antibody.
- IgM for administration to humans is an IgM whose variable region framework is derived from humans, in addition to the constant regions.
- Human antibodies which have retained the variable region framework structure, but only the CDR has been replaced with that of an antibody from another animal species are called humanized antibodies.
- the phrase “highly concentrated immunoglobulin (IgM)” means that the IgM content in a solution is higher than 1 mg/mL. Solutions of the present invention preferably have an IgM content of 1 mg/mL to 200 mg/mL. According to the present invention, IgM can be stabilized even at concentrations higher than 10 mg/mL (for example, 20 mg/mL or more, 25 mg/mL or more, 40 mg/mL or more, or 50 mg/mL or more).
- a “polyvalent cationic ion” that may be used in the present invention is a divalent or higher valence cationic ion.
- Mg ++ , Ca ++ , Zn ++ , Fe ++ , or a basic amino acid can be used.
- basic amino acids arginine, lysine, L-lysine L-glutamate, L-arginine L-glutamate, and such may be used.
- the polyvalent cationic ion is Mg ++ or arginine.
- cationic ions that may be used in the present invention are monovalent cationic ions, examples being Na + and K + .
- the concentration of cationic ions or polyvalent cationic ions added to solutions is usually 1 mM to 1,000 mM, preferably 10 mM to 500 mM, and more preferably 50 mM to 200 mM.
- Solutions of the present invention may comprise sugars in addition to cationic ions or polyvalent cationic ions.
- Preferred sugars include trehalose, sucrose, and sorbitol.
- Types of buffers that may be used in the present invention include phosphate buffers, acetate buffers, and citrate buffers.
- stabilization in the present invention refers to suppressing the increase of water-soluble IgM aggregates produced during storage, and/or suppressing the increase of insoluble IgM aggregates (precipitates) produced during storage, and/or maintaining the function of water-soluble IgM.
- stabilization refers to suppressing the increase of water-soluble IgM aggregates produced during storage.
- water-soluble aggregates in the present invention refers to water-soluble multimers such as dimers or trimers of IgM.
- the aggregate increase suppression rate for the solutions of the present invention one month after addition of polyvalent cationic ions to the solution comprising a high concentration of IgM is preferably 10% or more, more preferably 30% or more, even more preferably 50% or more, and yet even more preferably 80% or more.
- Solutions of the present invention are preferably those not comprising human-derived proteins other than IgM. More preferable are solutions not comprising proteins other than IgM in amounts that may make the proteins effective as stabilizers, or in amounts greater than that.
- solutions of the present invention are pharmaceutical formulations, solutions not comprising human-derived proteins other than IgM, in amounts acceptable for a pharmaceutical and/or for a bulk drug substance of a pharmaceutical are preferable, or amounts greater than that.
- the dosage form of pharmaceutical formulations of the present invention is not particularly limited, and any discretionary dosage form is possible.
- Examples of the dosage form include a solution formulation and a lyophilized formulation.
- Examples of the solution formulations include formulations stored in a cold place, formulations stored at room temperature, and frozen formulations.
- the pharmaceutical formulations may thus be administered either orally or parenterally depending on the purpose of use.
- Specific dosage forms for parenteral administration include injections, and dosage forms for nasal administration, pulmonary administration, and transdermal administration.
- Systemic or local injections can be carried out by intravenous injections, intramuscular injections, peritoneal injections, subcutaneous injections, or such.
- IgM stabilized by methods of the present invention can be administered as pharmaceutical agents formulated by well-known pharmaceutical methods.
- the stabilized IgM can be used as sterile solutions prepared with water or other pharmaceutically acceptable liquid, or as injections of suspensions.
- it may be formulated by, for example, appropriately combining with pharmaceutically acceptable carriers or media, such as sterilized water, saline, emulsifiers, suspending agents, surfactants, stabilizers, vehicles, and preservatives, and mixing them at a unit dosage form required for generally accepted pharmaceutical practice.
- pharmaceutically acceptable carriers or media such as sterilized water, saline, emulsifiers, suspending agents, surfactants, stabilizers, vehicles, and preservatives, and mixing them at a unit dosage form required for generally accepted pharmaceutical practice.
- the amount of active ingredient in these formulations can be adjusted so that an appropriate dose within an indicated range can be acquired.
- Sterile compositions for injections can be formulated according to usual pharmaceutical practice using vehicles such as distilled water for injections.
- aqueous solutions used for injections include physiological saline and isotonic solutions comprising glucose and other auxiliary agents.
- the auxiliary agents may be sucrose, D-sorbitol, D-mannose, D-mannitol, sodium chloride, and such.
- Suitable solubilizers may also be added to pharmaceutical compositions.
- alcohols and non-ionic surfactants are preferred solubilizers.
- Specific examples of alcohols comprise ethanol, polyalcohols such as propylene glycol and polyethylene glycol.
- non-ionic surfactants may be Polysorbate80, Polysorbate20, Poloxamer188, HCO-50, and such.
- Cationic surfactants such as benzalkonium chloride may also be used.
- Oily fluids may be, for example, sesame oil and soybean oil, and may be used together with benzyl benzoate or benzyl alcohol as a solubilizer.
- buffers such as phosphate buffer and sodium acetate buffer, analgesic agents such as procain hydrochloride, stabilizers such as benzyl alcohol and phenol, and antioxidants may be combined.
- the prepared injections are usually loaded into suitable vials or ampules.
- the administration dose of the pharmaceutical formulations can be appropriately selected according to the disease to be treated, and age and symptoms of the patient.
- a single dose can be selected within the range of 0.0001 mg to 1,000 mg per 1 kg body weight.
- the dose can be selected within the range of 0.001 to 100,000 mg/body of patient.
- doses of the pharmaceutical formulations of the present invention are not limited to these.
- MABON-01 recombinant anti-ganglioside GM3 human antibody produced in Reference Example 1
- the MABON-01 solution was concentrated to prepare a highly concentrated solution of approximately 9 mg/mL. For buffer replacement, this solution was dialyzed against buffers 1 to 6 as listed below using a dialysis membrane, SLIDE-A-LYZER Dialysis Cassette 10000MWCO (PIERCE).
- the resulting solutions were collected, and the concentration of MABON-01 in each sample was adjusted to 8.4 mg/mL.
- Each sample was placed in a storage container, Multiply-Safecup 0.1 ml Biosph. (SARSTEDT). Stability tests were carried out on these samples at 4° C. The samples were evaluated at the initial state and after storing at 4° C. for 2 months (hereafter referred to as, for example “4° C.-2 months”). The stability of each sample was evaluated based on changes in the residual monomer ratio determined by gel filtration chromatography. G4000SW XL (TOSOH) was used as the column for gel filtration chromatography.
- the MABON-01 solution was concentrated to prepare a highly concentrated solution of approximately 18 mg/mL. This solution was dialyzed using a dialysis membrane, SLIDE-A-LYZER Dialysis Cassette 10000MWCO (PIERCE), to replace the buffer with a solution comprising 20 mM citric acid and 300 mM NaCl (pH 5.5) (the buffer type and pH were optimized under conditions without additives). This highly concentrated MABON-01 solution was dialyzed against the following buffers 1 to 9 using EasySep (TOMY) to replace the buffer.
- TOMY EasySep
- the resulting solutions were collected, and the concentration of MABON-01 in each sample was adjusted to 18.5 mg/mL.
- Each sample was placed in a storage container, Multiply-Safecup 0.1 ml Biosph. (SARSTEDT). Stability tests were carried out on these samples at 25° C. The samples were evaluated at the initial state, at 25° C.-1 month, and at 25° C.-2 months. The stability of each sample was evaluated based on changes (increase) in aggregate content determined by gel filtration chromatography. G4000SW XL (TOSOH) was used as the column for gel filtration chromatography. A solution comprising 50 mM sodium phosphate and 500 mM KCl (pH 7.4) was used as the mobile phase.
- the aggregate content in the samples was calculated from the values of aggregate peak area and monomer peak area obtained from gel filtration chromatography. Values obtained by subtracting the aggregate content in the initial state from the aggregate content at 25° C.-1 month and at 25° C.-2 months for each sample are shown in FIG. 1 as ⁇ aggregate values.
- samples 2 [NaCl (comprising 900 mM NaCl)], 3 [MgCl 2 (comprising 300 mM NaCl+200 mM MgCl 2 )], and 4 [Na 2 SO 4 (comprising 300 mM NaCl+200 mM Na 2 SO 4 )] all have the same ionic strength (each has an ionic strength of 0.9 M), MgCl 2 comprising a divalent cation showed a significant stabilization effect, while sample 4 [Na 2 SO 4 ] comprising a divalent anion showed a stabilization effect similar in magnitude to that in sample 2 [NaCl].
- sample 5 sodium L-glutamate
- sample 6 Li-arginine hydrochloride
- MABON-01 solution was concentrated to prepare a highly concentrated MABON-01 solution of approximately 19 mg/mL.
- the solution was dialyzed against the following buffers 1 to 9 using a dialyzer membrane, EasySep (TOMY), to replace the buffer.
- TOMY EasySep
- the MABON-01 solution was concentrated to prepare a highly concentrated MABON-01 solution of approximately 27 mg/mL.
- the solution was dialyzed against the following buffers 1 to 3 using a dialyzer membrane, EasySep (TOMY), to replace the buffer.
- TOMY EasySep
- the resulting solutions were collected, and the concentration of MABON-01 in each sample was adjusted to 26.8 mg/mL.
- Each sample was placed in a storage container, Multiply-Safecup 0.1 ml Biosph. (SARSTEDT). Stability tests were carried out on these samples. The samples were evaluated at the initial state, at 4° C.-4 months, and at 25° C.-4 months. The stability of each sample was evaluated based on changes (increases) in aggregate content determined by gel filtration chromatography. G4000SW XL (TOSOH) was used as the column for gel filtration chromatography. A solution comprising 50 mM sodium phosphate and 500 mM KCl (pH 7.4) was used as the mobile phase.
- the aggregate content in the samples was calculated from the values of aggregate peak area and monomer peak area obtained from gel filtration chromatography.
- the aggregate content of each sample at the initial state, at 4° C.-4 months, and at 25° C.-4 months are shown in FIG. 3 .
- MgCl 2 was observed to have a stabilization effect.
- trehalose which is known to be a conventional protein stabilizer and which has been found to have a stabilization effect on IgM during lyophilizing as reported in Journal of Immunological Methods 1995, 181(1), 37-43 ( FIG. 3 ).
- a large-scale dialysis of MABON-01 was carried out in “50 mM sodium citrate, 180 mM NaCl, pH 5.5, 5% sucrose” buffer, “50 mM sodium citrate, 180 mM ArgHCl, pH 5.5, 5% sucrose” buffer, or “50 mM sodium citrate, 180 mM MgCl 2 , pH 5.5, 5% sucrose” buffer.
- the solutions were concentrated by filter centrifugation. Centrifugation was carried out using VIVASPIN6 5000MWCO (VIVASCIENCE, VS061) on himac CF8DL (Hitachi, No. SZGEQ054) at 3,000 rpm.
- Stability of the lyophilized material was higher with ArgHCl or MgCl 2 than with NaCl at the same concentration, even during incubation at 50° C.
- a gene encoding the heavy chain of a human antibody that binds to ganglioside GM3 was amplified by RT-PCR using total RNAs extracted from human B cells transformed with Epstein-Barr virus (hereinafter, denoted as anti-ganglioside GM3 human antibody-expressing B cells).
- RNAs were extracted from 1 ⁇ 10 7 anti-ganglioside GM3 human antibody-expressing B cells using RNeasy Plant Mini Kit (QIAGEN).
- Two oligonucleotides (LMH-f3 and LMH-r3) were designed based on the nucleotide sequence of anti-ganglioside GM3 human antibody gene reported by Hoon et al. (Cancer Research 1993; 53: 5244-5250).
- LMH-f3 SEQ ID NO: 7
- LMH-r3 SEQ ID NO: 8
- RNAs Using 1 ⁇ g of total RNAs, gene fragments were amplified separately for the 5′ end and the 3′ end by SMART RACE cDNA Amplification Kit (CLONTECH). Synthetic oligonucleotides LMH-r3 and LMH-f3 were used for amplifying the 5′ and 3′ ends of the gene, respectively. Reverse transcription reaction was carried out at 42° C. for 1.5 hours.
- composition of the PCR reaction solution (50 ⁇ L) is shown below:
- PCR products were purified from agarose gel using QIAquick Gel Extraction Kit (QIAGEN), and then cloned into pGEM-T Easy vector (Promega). After sequencing, an approximately 1.1 kbp fragment was obtained by digesting the vector comprising the 5′ end of the gene using restriction enzymes ApaI (Takara Shuzo) and SacII (Takara Shuzo), while an approximately 1.1 kbp fragment was obtained by digesting the vector comprising the 3′ end of the gene using restriction enzymes ApaI (Takara Shuzo) and NotI (Takara Shuzo). The fragments were then mixed, and cloned into pBluescript KS+ vector (TOYOBO) to obtain a full-length anti-ganglioside GM3 human antibody heavy chain gene.
- LMH-fxho SEQ ID NO: 11
- LMH-rsal SEQ ID NO: 12
- SEQ ID NO: 12 is a reverse primer designed to hybridize to the 3′ end of the anti-ganglioside GM3 human antibody heavy chain gene, and to comprise a SalI restriction enzyme recognition sequence.
- composition of the PCR reaction solution (50 ⁇ L) is shown below:
- the amplified gene fragment was cloned by digesting with the XhoI restriction enzyme (Takara Shuzo) and the SalI restriction enzyme (Takara Shuzo), then purifying using QIAquick PCR Purification Kit (QIAGEN), and linking to the XhoI restriction enzyme site of pUCAG.
- This pUCAG vector is obtained by: linking the 2.6 kbp fragment obtained by digesting pCXN (Niwa et al., Gene 1991; 108: 193-200) using the BamHI restriction enzyme to the BamHI restriction enzyme site of pUC19 vector (TOYOBO).
- the obtained plasmid was named pUCAG/L612H.
- the nucleotide sequence and amino acid sequence of the anti-ganglioside GM3 human antibody heavy chain in this plasmid are shown in SEQ ID NOs: 1 and 2, respectively.
- a gene encoding the light chain of anti-ganglioside GM3 human antibody was amplified by RT-PCR using total RNAs extracted from the anti-ganglioside GM3 human antibody-expressing B cells.
- the total RNAs were extracted from the anti-ganglioside GM3 human antibody-expressing B cells in a manner similar to that mentioned above.
- Two oligonucleotides (LML-f1 and LML-r1) were designed based on the nucleotide sequence of anti-ganglioside GM3 human antibody gene reported by Hoon et al. (Cancer Research 1993; 53: 5244-5250).
- LML-f1 SEQ ID NO: 9
- LML-r1 SEQ ID NO: 10 were synthesized in the sense and antisense directions, respectively.
- RNAs Using 1 ⁇ g of total RNAs, gene fragments were amplified separately for the 5′ end and the 3′ end by the SMART RACE cDNA Amplification Kit (CLONTECH). Synthetic oligonucleotides LML-r1 and LML-f1 were used for amplifying the 5′ and 3′ ends of the gene, respectively. Reverse transcription reaction was carried out at 42° C. for 1.5 hours.
- composition of the PCR reaction solution (50 ⁇ L) is shown below:
- PCR product was purified from the agarose gel using QIAquick Gel Extraction Kit (QIAGEN), and then cloned into pGEM-T Easy vector (Promega). After sequencing, an approximately 0.7 kbp fragment was obtained by digesting the vector comprising the 5′ end of the gene using the EcoRI restriction enzyme (Takara Shuzo), while an approximately 0.9 kbp fragment was obtained by digesting the vector comprising the 3′ end of the gene using the EcoRI restriction enzyme (Takara Shuzo). The two fragments were mixed, and used to amplify the full-length gene fragment using synthetic oligonucleotides LML-feco and LML-rnot.
- LML-feco (SEQ ID NO: 13) is a forward primer, and was designed to hybridize to the 5′ end of the anti-ganglioside GM3 human antibody light chain gene, and to comprise an EcoRI restriction enzyme recognition sequence and a Kozak sequence.
- LML-rnot (SEQ ID NO: 14) is a reverse primer, and was designed to hybridize to the 3′ end of the anti-ganglioside GM3 human antibody light chain gene, and to comprise a NotI restriction enzyme recognition sequence.
- composition of the PCR reaction solution (50 ⁇ L) is shown below:
- the amplified gene fragment was cloned by digesting with the EcoRI restriction enzyme (Takara Shuzo) and the NotI restriction enzyme (Takara Shuzo), then purifying using QIAquick PCR Purification Kit (QIAGEN) and linking to the EcoRI and NotI restriction enzyme cleavage sites of pCXND3.
- the pCXND3 vector was constructed as follows: DHFR- ⁇ E-rvH-PM1-f (see WO 92/19759) was digested at the EcoRI/SmaI restriction enzyme site to separate their antibody heavy chain gene and vector region. Only the vector portion was then collected, into which the EcoRI-NotI-BamHI adaptor (Takara Shuzo) was cloned. This vector was named pCHOI.
- pCXND3 A vector in which the DHFR gene expression site of pCHOI is cloned into the HindIII restriction enzyme site of pCXN (Niwa et al., Gene 1991; 108:193-200) was named pCXND3. Furthermore, the light-chain gene fragment was cloned into pCXND3 and the obtained plasmid was named pCXND3/L612L.
- the nucleotide sequence and amino acid sequence of anti-ganglioside GM3 human antibody light chain in this plasmid are shown in SEQ ID NOs: 3 and 4, respectively.
- pUCAG/L612H was digested with the HindIII restriction enzyme (Takara Shuzo), and the resulting approximately 4.0 kbp fragment was linked to the HindIII restriction enzyme cleavage site of pCXND3/1612L.
- the obtained plasmid was named pCXND3/L612IgM. This plasmid expresses the neomycin-resistance gene, DHFR gene, and anti-ganglioside GM3 human antibody gene in animal cells.
- a gene encoding the J chain of anti-ganglioside GM3 human antibody was amplified by RT-PCR using total RNAs extracted from anti-ganglioside GM3 human antibody-expressing B cells. Total RNAs were extracted from anti-ganglioside GM3 human antibody-expressing B cells in a manner similar to that mentioned above.
- Two oligonucleotides (J-f1 and J-r1) were designed and synthesized based on the nucleotide sequence of the human antibody J chain gene registered in GenBank (GenBank accession number: M12759).
- J-f1 SEQ ID NO: 15
- J-r1 hybridizes to the human antibody J chain gene Exon 4 in the antisense direction.
- RNAs Using 1 ⁇ g of total RNAs, gene fragments were amplified separately for the 5′ end and the 3′ end by the SMART RACE cDNA Amplification Kit (CLONTECH). Synthetic oligonucleotides J-r1 and J-f1 were used for amplifying the 5′ and 3′ ends of the gene, respectively. Reverse transcription reaction was carried out at 42° C. for 1.5 hours.
- composition of the PCR reaction solution (50 ⁇ L) is shown below:
- PCR product was purified from the agarose gel using QIAquick Gel Extraction Kit (QIAGEN), and then cloned into pGEM-T Easy vector (Promega).
- an approximately 0.5 kbp fragment was obtained by digesting the vector comprising the 5′ end of the gene using the EcoRI restriction enzyme (Takara Shuzo), and an approximately 1.0 kbp fragment was obtained by digesting the vector comprising the 3′ end of the gene using the EcoRI restriction enzyme (Takara Shuzo).
- the two fragments were mixed, and used to amplify the full-length gene fragment using synthetic oligonucleotides J-feco and J-rxba.
- J-feco (SEQ ID NO: 17) is a forward primer designed to hybridize to the 5′ end of the anti-ganglioside GM3 human antibody J chain gene, and to comprise an EcoRI restriction enzyme recognition sequence and a Kozak sequence.
- J-rxba (SEQ ID NO: 18) is a reverse primer designed to hybridize to the 3′ end of the anti-ganglioside GM3 human antibody J chain gene, and to comprise an XbaI restriction enzyme recognition sequence.
- composition of the PCR reaction solution (50 ⁇ L) is shown below:
- the amplified gene fragment was cloned by digesting with the EcoRI restriction enzyme (Takara Shuzo) and the XbaI restriction enzyme (Takara Shuzo), then purifying using QIAquick PCR Purification Kit (QIAGEN), and linking to the EcoRI and XbaI restriction enzyme cleavage sites of pCOSII-Zeo.
- This pCOSII-Zeo vector is obtained by removing the DHFR gene expression site of pCHOI, and cloning the Zeocin-resistant gene expression site thereto.
- the obtained plasmid was named pCOSII-Zeo/J chain.
- the nucleotide sequence and amino acid sequence of anti-ganglioside GM3 human antibody J chain in this plasmid are shown in SEQ ID NOs: 5 and 6, respectively.
- Stable expression cell lines derived from CHO cells were produced as described below.
- the electroporated cells were suspended in 40 mL of CHO—S—SFMII medium (Invitrogen) comprising 1 ⁇ HT Supplement (Invitrogen).
- a 50-fold diluted solution was further prepared using the same medium, and then aliquoted at 100 ⁇ L/well into a 96-well culture plate. After incubation for 24 hours in a CO 2 incubator (5% CO 2 ), Geneticin (Invitrogen) was added to the wells at 0.5 mg/mL and cultured for 2 weeks.
- the IgM levels in the culture supernatants of wells in which colonies of Geneticin-resistant transformants were found were measured by the concentration assay described in Reference Example 1.6.
- Cell lines highly expressing the anti-ganglioside GM3 human antibodies were successively expanded to obtain anti-ganglioside GM3 human antibody-expressing stable cell lines CA02, CA15, CA19, CA20, and CA24.
- the electroporated cells were suspended in 40 mL of CHO—S—SFMII medium (Invitrogen) comprising 1 ⁇ HT Supplement (Invitrogen).
- a 50-fold diluted solution was further prepared using the same medium and aliquoted at 100 ⁇ L/well into a 96-well culture plate. After incubation for 24 hours in a CO 2 incubator (5% CO 2 ), 0.5 mg/mL Geneticin (Invitrogen) and 0.6 mg/mL Zeocin (Invitrogen) were added to wells, and cultured for 2 weeks. The IgM levels in the culture supernatants of wells in which colonies of Geneticin- and Zeocin-resistant transformants were found were measured by the concentration assay described in Reference Example 1.6. Cell lines highly expressing the anti-ganglioside GM3 human antibodies were successively expanded to obtain anti-ganglioside GM3 human antibody-expressing stable cell lines (CJ15, CJ25, CJ38, CJ45, and CJ67).
- IgM concentration in the culture supernatants was measured as described below.
- Anti-Human IgM (BIOSOURCE) was diluted using a coating buffer (0.1 M NaHCO 3 and 0.02% NaN 3 ) to prepare a 1 ⁇ g/mL solution.
- the diluted solution was added to a 96-well ELISA plate at 100 ⁇ L/well, and then reacted at 4° C. for 24 hours or longer to coat the plate.
- Rinse Buffer After washing the wells with Rinse Buffer, blocking was carried out by adding 200 ⁇ L/well of Diluent Buffer and reacting at room temperature for 1 hour or longer. Compositions of the Rinse Buffer and Diluent Buffer are shown below.
- culture supernatant suitably diluted with Diluent Buffer was added to the wells at 100 ⁇ L/well, and allowed to react at room temperature for 1 hour.
- alkaline phosphatase-conjugated goat anti-human IgM (BIOSOURCE) diluted 4,000 times with Diluent Buffer was added at 100 ⁇ L/well, and reacted at room temperature for 1 hour.
- wells were washed with Rinse Buffer, and alkaline phosphatase substrate (SIGMA) was added thereto.
- the absorbance was determined at the 405 nm measurement wavelength and 655 nm reference wavelength using Benchmark Plus absorption spectrometer (BioRad).
- the concentration of IgM was calculated by comparing with a purified anti-ganglioside GM3 human antibody (Hoon et al., Cancer Research 1993; 53: 5244-5250).
- Each type of stable cell line expressing anti-ganglioside GM3 human antibodies was cultured in a 75 cm 2 -culture flask at an initial cell density of 2 ⁇ 10 5 cells/mL.
- the IgM concentration in the culture supernatants was measured by the method described above. The results are shown in Table 4.
- the amount of IgM produced was approximately 20 mg/L on the third day and approximately 50 mg/L on the seventh day.
- the productivity indicating the production ability of a single cell was 5 to 19 pg/cell/day. Since IgM is a type of immunoglobulin that forms multimers, expression level of IgM in recombinants is low, and therefore, its large-scale preparation was considered difficult.
- Table 6 shows the result of calculating the aggregate peak area rate and the monomer peak area rate relative to the total peak area value.
- the average molecular weights were obtained by averaging the molecular weights corresponding to 21.5 to 22.0 minutes for peak 2, and 24.5 to 25.0 minutes for peak 1.
- the average molecular weight values for peak 1 and peak 2 are close to the theoretical molecular weight values for the monomer and dimer, respectively.
- the average molecular weight value for peak 2 is approximately two times that of peak 1. Accordingly, peak 1 was found to comprise the MABON-01 monomer, and peak 2 was found to comprise the dimer.
- the present invention enabled stabilization of highly concentrated IgM in solutions. Since the present invention enables stable long-term storage of pharmaceutical formulations comprising IgM as an active ingredient, it can significantly contribute to particularly the preparation of antibody formulations.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Oncology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Inorganic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicinal Preparation (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The present inventors discovered that stable and highly concentrated IgM solutions can be prepared by using, as an additive, a compound comprising a polyvalent cationic ion, such as magnesium chloride or arginine hydrochloride, to suppress IgM aggregation in solutions.
Description
- This application is the National Stage of International Application No. PCT/JP2004/014935, filed on Oct. 8, 2004, which claims the benefit of Japanese Patent Application Serial No. 2003-351388, filed on Oct. 9, 2003. The contents of both of the foregoing applications are hereby incorporated by reference in their entireties.
- The present invention relates to highly concentrated and stabilized IgM solutions, and production thereof.
- Many higher animals have five different classes of immunoglobulins, IgG, IgA, IgM, IgD, and IgE. Each immunoglobulin class differs in properties such as size, charge, amino acid composition, and sugar content. Of these classes, IgM accounts for approximately 10% of all plasma immunoglobulins. IgM is the major component of early antibodies produced against cell-membrane antigens, infectious microorganisms, or soluble antigens, which have a complex antigenicity.
- Human IgMs usually have a pentameric structure. Each of the five subunits constituting this pentameric structure has a four-stranded structure similar to that of IgG. The amino acid sequence of the μ chain, which is the heavy chain of IgM, is different from that of the γ chain, which is the heavy chain of IgG. The following differences can also be seen:
- The μ chain has an extra constant domain than the γ chain.
- The μ chain has four more oligosaccharide chains than the γ chain.
- IgM has a polypeptide chain called the J chain, which is not found in IgG. The J chain is considered to assist the association of μ chains prior to secretion of IgM from antibody producing cells.
- With advances in monoclonal antibody technology and recombinant DNA technology, large-scale production of pure immunoglobulins has become possible in recent years. Furthermore, gene recombination techniques have enabled production of chimeric antibodies and humanized antibodies. Chimeric antibodies are antibodies having a structure in which the variable regions have been replaced with variable regions derived from a different species. For example, “chimeric antibodies” comprising variable regions of non-human antibodies and the constant regions of human antibodies (Non-Patent
Document 1/Proc. Natl. Acad. Sci. U.S.A., (1984) 81:6851) are known. Also known are humanized antibodies in which the complementarity determining regions (CDR) of other animal species are transferred into human immunoglobulins (Non-PatentDocument 2/Nature (1986) 321:521). - Actual examples of antitumor antibodies are the anti-CD20 human chimeric antibody Rituxan (IDEC), and the anti-HER2/neu humanized antibody Herceptin (Genentech), which have completed clinical trials and have already been approved. These antibodies are now commercially available. Antibody-dependent cellular cytotoxicity (hereinafter referred to as ADCC) activity and complement-dependent cytotoxicity (hereinafter referred to as CDC) activity are known as effector functions of IgG and IgM. Since IgM has a higher CDC activity compared to IgG, it has an extremely high chance of becoming an anti-tumor antibody having CDC activity as its main effect. However, as described above, unlike IgG, IgM forms a multimer. Therefore, industrial scale production of recombinant IgM had been considered difficult.
- IgM is also very unstable compared to IgG and has a low solubility. Therefore, the production of a highly concentrated and stable IgM solution is difficult. For example, Cytotherapy, 2001, 3(3), 233-242 (Non-Patent Document 5) reports that, even when IgM had been stored at −20° C., precipitation and decrease of activity occurred upon thawing. Furthermore, according to the report, IgM easily aggregates and precipitates during storage. It was especially difficult to ensure an IgM stability sufficient enough to withstand pharmaceutical use only through optimization of pH and buffer type.
- Therefore, various attempts are being made to stabilize antibodies by methods other than optimization of pH and buffer type. For example, WO 2002/096457 (Patent Document 1) discloses formulations for stabilizing highly concentrated antibodies that comprise acidic ingredients. This method uses MgCl2 and CaCl2 as additives to stabilize the antibodies, but the stabilization is carried out to prepare IgG formulations, and IgM formulations are not mentioned. As described above, unlike IgG, IgM exists as a multimer, and unlike intrinsically stable IgG, IgM readily aggregates. Therefore, IgM has the distinctive problem of being very difficult to be highly concentrated.
- Clin. Chem. Lab. Med. 2000; 38(8): 759-764 (Non-Patent Document 3) and Journal of Immunological Methods, 111 (1988) 17-23 (Non-Patent Document 5) reported that IgM precipitates at a low salt concentration, and redissolves at a high salt concentration in a phosphate buffer and Tris-hydrochloride buffer that are weakly alkaline. Clin. Chem. Lab. Med. 2000; 38(8): 759-764 (Non-Patent Document 3) reports that, near
pH 5, IgM readily precipitates and is difficult to handle, suggesting a pessimistic outlook for IgM solutions in weakly acidic buffers. This report thus gives no indication of the possibility of providing a highly concentrated IgM solution as a pharmaceutical or a bulk drug substance. This document also reports that when human sera comprising a high concentration of IgM are diluted with water, insoluble aggregates are generated as euglobulin precipitates, increasing the turbidity of the solution; but when the salt concentration is then raised by adding NaCl, Arginine, or such, the euglobulin precipitates redissolve. However, this report relates to the reconstitution of euglobulin precipitates, and does not provide any disclosures relating to suppression of increase in water-soluble aggregates of IgM. Furthermore, since patient-derived unpurified sera comprising various serum proteins are used in this document, the resulting insoluble aggregates may comprise proteins other than IgM. The effects on IgM solution in the absence of the other proteins are not described. - In Journal of Immunological Methods, 111 (1988) 17-23 (Non-Patent Document 5), a buffer comprising 0.1 M Tris-HCl and 1 M NaCl (pH 8) is used to redissolve euglobulin precipitates. However, the resulting recovery rate of IgM varies from 40% to >90% depending on antibodies or batch, indicating a low reproducibility. Additionally, although the Methods section describes that 5 to 10 mg/mL of purified antibodies were stored at 4° C. and −20° C., the Results section only describes that the antibodies could be stored for a few months at −20° C. without loss of function, and does not mention anything regarding storage at 4° C. or higher, at which temperature it is usually difficult to ensure stability. Accordingly, this report suggests the difficulty of reproducing precipitate reconstitution and the difficulty of ensuring stability during storage, when trying to provide a highly concentrated solution of IgM as a pharmaceutical product or a bulk drug substance.
- BIOTECHNOLOGY 1993, 11, 512-515 (Non-Patent Document 4) and Journal of Immunological Methods, 111 (1988) 17-23 (Non-Patent Document 5) also describe the reconstitution of insoluble aggregates of antibodies as euglobulin precipitates, but the solubility is 10 mg/mL or less, indicating low solubility of IgM. There is no description at all regarding the stabilization of water-soluble aggregates.
- Pharmaceutical Research 1994, 11(5), 624-632 (Non-Patent Document 6) discloses stabilization of IgM by PVP addition, but does not disclose the stabilization of highly concentrated antibodies. Journal of Immunological Methods 1995, 181(1), 37-43 (Non-Patent Document 7) discloses lyophilized formulations produced by addition of trehalose, but in this report, the antibody stability is insufficient and there is no description relating to stabilization of highly concentrated antibodies.
- Patent Document 1: WO 2002/096457
- Non-Patent Document 1: Proc. Natl. Acad. Sci. U.S.A, (1984) 81:6851
- Non-Patent Document 2: Nature (1986) 321:521
- Non-Patent Document 3: Clin. Chem. Lab. Med. 2000; 38 (8):759-764
- Non-Patent Document 4: BIOTECHNOLOGY 1993, 11, 512-515
- Non-Patent Document 5: Journal of Immunological Methods, 111 (1988), 17-23
- Non-Patent Document 6: Pharmaceutical Research 1994, 11(5), 624-632
- Non-Patent Document 7: Journal of Immunological Methods 1995, 181, 37-43
- Non-Patent Document 8: Cytotherapy, 2001, 3(3), 233-242
- Problems to be Solved by the Invention
- The present invention was achieved in view of the above circumstances. An objective of the present invention is to stabilize highly concentrated IgM in solutions. More specifically, the present invention aims to provide methods for stabilizing highly concentrated IgM, solutions in which highly concentrated IgM is stabilized, and methods for preparing the solutions.
- In a preferred embodiment of solutions in which highly concentrated IgM is stabilized, the present invention provides an aqueous solution in which increase of water-soluble aggregates is suppressed. Another preferred embodiment provides a highly concentrated IgM formulation that is stable enough to be used as a pharmaceutical.
- Means to Solve the Problems
- As a result of dedicated research to solve the above-mentioned problems, the present inventors discovered that, by using compounds comprising polyvalent cationic ions such as magnesium chloride and arginine hydrochloride as additives, aggregation of IgM in solutions may be suppressed and stable highly concentrated IgM solutions may be prepared.
- Specifically, the present invention relates to methods for stabilizing highly concentrated IgM, solutions in which highly concentrated IgM is stabilized, and methods for preparing the solutions. More specifically, the present invention provides the following:
- (1) a solution wherein a high concentration of immunoglobulin is stabilized, and wherein the immunoglobulin is IgM;
- (2) the solution of (1), comprising IgM at a concentration higher than 1 mg/mL;
- (3) the solution of (1), which is an aqueous solution;
- (4) the solution of (1), which is a pharmaceutical formulation;
- (5) the solution of (1), comprising a polyvalent cationic ion;
- (6) the solution of (5), comprising the polyvalent cationic ion at a concentration of 1 mM to 1,000 mM;
- (7) the solution of (5), wherein the polyvalent cationic ion is a Mg ion or an Arg ion;
- (8) the solution of (5), further comprising sugars;
- (9) the solution of (1), which is
pH 5 topH 8; - (10) the solution of (1), wherein the solution does not intrinsically comprise human-derived proteins other than IgM;
- (11) the solution of (1), wherein the solution does not intrinsically comprise proteins other than IgM;
- (12) a pharmaceutical formulation obtained by freezing or lyophilizing the solution of any one of (1) to (11);
- (13) a method for stabilizing a solution comprising a high concentration of immunoglobulin, wherein the immunoglobulin is IgM and wherein the method comprises adding a polyvalent cationic ion to the solution;
- (14) the method of (13), wherein the solution comprises IgM at a concentration higher than 1 mg/mL;
- (15) the method of (13), wherein the solution is an aqueous solution;
- (16) the method of (13), wherein the solution is a pharmaceutical formulation;
- (17) the method of (13), which comprises adding a polyvalent cationic ion to the solution such that the solution comprises the polyvalent cationic ion at a concentration of 1 mM to 1,000 mM;
- (18) the method of (13), wherein the polyvalent cationic ion is a Mg ion or an Arg ion;
- (19) the method of (13), further comprising addition of sugars;
- (20) the method of (13), wherein the pH of the solution is 5 to 8;
- (21) the method of (13), wherein the solution does not intrinsically comprise human-derived proteins other than IgM;
- (22) the method of (13), wherein the solution does not intrinsically comprise proteins other than IgM;
- (23) a method for stabilizing a pharmaceutical formulation, which comprises the steps of:
- (a) performing the method of any one of (13) to (22); and
- (b) freezing or lyophilizing the solution stabilized in step (a);
- (24) a method for producing a solution comprising a high concentration of stabilized immunoglobulin, wherein the immunoglobulin is IgM and wherein the method comprises the step of adding a polyvalent cationic ion to the solution;
- (25) the method of (24), wherein the solution comprises IgM at a concentration higher than 1 mg/mL;
- (26) the method of (24), wherein the solution is an aqueous solution;
- (27) the method of (24), wherein the solution is a pharmaceutical formulation;
- (28) the method of (24), which comprises the step of adding a polyvalent cationic ion to the solution such that the solution comprises the polyvalent cationic ion at a concentration of 1 mM to 1000 mM;
- (29) the method of (24), wherein the polyvalent cationic ion is a Mg ion or an Arg ion;
- (30) the method of (24), which further comprises the step of adding sugars;
- (31) the method of (24), wherein the pH of the solution is 5 to 8;
- (32) the method of (24), wherein the solution essentially does not comprise human-derived proteins other than IgM;
- (33) the method of (24), wherein the solution essentially does not comprise proteins other than IgM;
- (34) a solution which is produced by the method of any one of (24) to (33); and
- (35) a method for producing a pharmaceutical formulation, wherein the method comprises the steps of:
- (a) performing the method of any one of (24) to (33); and
- (b) freezing or lyophilizing the solution produced in step (a).
-
FIG. 1 shows “Δaggregate” values obtained by subtracting the initial aggregate content from the aggregate content after one month of storage at 25° C. (25° C.-1 month) and the aggregate content after 2 months of storage at 25° C. (25° C.-2 months) for each sample. -
FIG. 2 shows the aggregate content (%) in each sample in the initial state and at 4° C.-3 months. -
FIG. 3 shows the aggregate content (%) in each sample in the initial state, at 4° C.-4 months, and 25° C.-4 months. -
FIG. 4 shows the aggregate content (%) according to size exclusion chromatography (SEC) in each sample in the initial state, liquid/40° C.-8 days, liquid/40° C.-8 days+4° C.-3 days, lyophilized/40° C.-8 days, lyophilized/40° C.-8 days+4° C.-3 days, and lyophilized/40° C.-8 days+4° C.-3 days+relyophilized/50° C.-8 days. -
FIG. 5 shows the aggregate content (%) according to SEC in each sample in the initial state, and lyophilized/40° C.-2 months. -
FIG. 6 shows the chromatogram obtained by gel permeation chromatography (GPC)-MALLS analysis of MABON-01 and the molecular weight determined by calculation. - In the present invention, the term “IgM” refers to an immunoglobulin that comprises constant regions of the μ chain as the constant regions of the heavy chains, and forms a pentameric or hexameric structure. The origin of the variable regions constituting the IgM of the present invention is not limited. Therefore, in addition to a variable region derived from the μ chain, the IgM of the present invention may comprise a variable region derived from IgG, or a partial structure thereof. The partial structure of a variable region can comprise the framework and CDR. The “IgM” in the present invention refers to expression products of exogenous IgM genes introduced into cells for transformation.
- Furthermore, the constant regions constituting the IgM of the present invention may be derived from any animal species. That is, the IgM of the present invention comprises an IgM constant region derived from any type of animal species carrying an IgM-type immunoglobulin. When IgM is administered in vivo, at least its constant regions are preferably derived from an animal species same as the species to which the IgM is administered. Therefore, when the IgM is administered to humans, at least its constant regions are preferably derived from humans. IgM composed of constant regions derived from humans, and variable regions derived from another animal species or another human, is called a chimeric antibody. A more preferable IgM for administration to humans is an IgM whose variable region framework is derived from humans, in addition to the constant regions. Human antibodies which have retained the variable region framework structure, but only the CDR has been replaced with that of an antibody from another animal species are called humanized antibodies.
- In the present invention, the phrase “highly concentrated immunoglobulin (IgM)” means that the IgM content in a solution is higher than 1 mg/mL. Solutions of the present invention preferably have an IgM content of 1 mg/mL to 200 mg/mL. According to the present invention, IgM can be stabilized even at concentrations higher than 10 mg/mL (for example, 20 mg/mL or more, 25 mg/mL or more, 40 mg/mL or more, or 50 mg/mL or more).
- In the present invention, when suppressing an increase of water-soluble aggregates, it is preferable to add a polyvalent cationic ion. A “polyvalent cationic ion” that may be used in the present invention is a divalent or higher valence cationic ion. For example, Mg++, Ca++, Zn++, Fe++, or a basic amino acid can be used. As basic amino acids, arginine, lysine, L-lysine L-glutamate, L-arginine L-glutamate, and such may be used. Preferably, the polyvalent cationic ion is Mg++ or arginine. In addition to polyvalent cationic ions, cationic ions that may be used in the present invention are monovalent cationic ions, examples being Na+ and K+.
- The concentration of cationic ions or polyvalent cationic ions added to solutions is usually 1 mM to 1,000 mM, preferably 10 mM to 500 mM, and more preferably 50 mM to 200 mM.
- Solutions of the present invention may comprise sugars in addition to cationic ions or polyvalent cationic ions. Preferred sugars include trehalose, sucrose, and sorbitol.
- Types of buffers that may be used in the present invention include phosphate buffers, acetate buffers, and citrate buffers.
- The term “stabilization” in the present invention refers to suppressing the increase of water-soluble IgM aggregates produced during storage, and/or suppressing the increase of insoluble IgM aggregates (precipitates) produced during storage, and/or maintaining the function of water-soluble IgM. Preferably, “stabilization” refers to suppressing the increase of water-soluble IgM aggregates produced during storage.
- The term “water-soluble aggregates” in the present invention refers to water-soluble multimers such as dimers or trimers of IgM. The water-soluble aggregates can be detected, for example, by gel filtration chromatography. Stabilization of highly concentrated IgM solutions can be measured, for example, from the aggregate increase suppression rate, which can be calculated by using the following formula:
Suppression ratio of aggregate increase=(A−B)/A×100 - A: Percent increase in aggregates in highly concentrated IgM solution without polyvalent cationic ion (control)
- B: Percent increase in aggregates in highly concentrated IgM solution with polyvalent cationic ion (test sample)
- The aggregate increase suppression rate for the solutions of the present invention one month after addition of polyvalent cationic ions to the solution comprising a high concentration of IgM is preferably 10% or more, more preferably 30% or more, even more preferably 50% or more, and yet even more preferably 80% or more.
- Solutions of the present invention are preferably those not comprising human-derived proteins other than IgM. More preferable are solutions not comprising proteins other than IgM in amounts that may make the proteins effective as stabilizers, or in amounts greater than that. When the solutions of the present invention are pharmaceutical formulations, solutions not comprising human-derived proteins other than IgM, in amounts acceptable for a pharmaceutical and/or for a bulk drug substance of a pharmaceutical are preferable, or amounts greater than that.
- The dosage form of pharmaceutical formulations of the present invention is not particularly limited, and any discretionary dosage form is possible. Examples of the dosage form include a solution formulation and a lyophilized formulation. Examples of the solution formulations include formulations stored in a cold place, formulations stored at room temperature, and frozen formulations. There are no particular limitations on the administration route for the pharmaceutical formulations of the present invention; any administration route can be used. The pharmaceutical formulations may thus be administered either orally or parenterally depending on the purpose of use.
- Specific dosage forms for parenteral administration include injections, and dosage forms for nasal administration, pulmonary administration, and transdermal administration. Systemic or local injections can be carried out by intravenous injections, intramuscular injections, peritoneal injections, subcutaneous injections, or such.
- In addition to administering directly to patients as is, IgM stabilized by methods of the present invention can be administered as pharmaceutical agents formulated by well-known pharmaceutical methods. For example, the stabilized IgM can be used as sterile solutions prepared with water or other pharmaceutically acceptable liquid, or as injections of suspensions. Furthermore, it may be formulated by, for example, appropriately combining with pharmaceutically acceptable carriers or media, such as sterilized water, saline, emulsifiers, suspending agents, surfactants, stabilizers, vehicles, and preservatives, and mixing them at a unit dosage form required for generally accepted pharmaceutical practice. The amount of active ingredient in these formulations can be adjusted so that an appropriate dose within an indicated range can be acquired.
- Sterile compositions for injections can be formulated according to usual pharmaceutical practice using vehicles such as distilled water for injections. Examples of aqueous solutions used for injections include physiological saline and isotonic solutions comprising glucose and other auxiliary agents. Specifically, the auxiliary agents may be sucrose, D-sorbitol, D-mannose, D-mannitol, sodium chloride, and such. Suitable solubilizers may also be added to pharmaceutical compositions. For example, alcohols and non-ionic surfactants are preferred solubilizers. Specific examples of alcohols comprise ethanol, polyalcohols such as propylene glycol and polyethylene glycol. Examples of non-ionic surfactants may be Polysorbate80, Polysorbate20, Poloxamer188, HCO-50, and such. Cationic surfactants such as benzalkonium chloride may also be used.
- Oily fluids may be, for example, sesame oil and soybean oil, and may be used together with benzyl benzoate or benzyl alcohol as a solubilizer. Furthermore, buffers such as phosphate buffer and sodium acetate buffer, analgesic agents such as procain hydrochloride, stabilizers such as benzyl alcohol and phenol, and antioxidants may be combined. The prepared injections are usually loaded into suitable vials or ampules.
- When the solutions of the present invention are made into pharmaceutical formulations, their pH is preferably 5 to 8, and particularly preferably 5 to 7.
- The administration dose of the pharmaceutical formulations can be appropriately selected according to the disease to be treated, and age and symptoms of the patient. For example, a single dose can be selected within the range of 0.0001 mg to 1,000 mg per 1 kg body weight. Alternatively, for example, the dose can be selected within the range of 0.001 to 100,000 mg/body of patient. However, doses of the pharmaceutical formulations of the present invention are not limited to these. One can refer to WO 2002/096457 for the preparation of liquid formulations and such of the present invention.
- All prior art literature cited herein are incorporated herein by reference.
- Hereinafter, the present invention is specifically illustrated with reference to Examples, but it is not to be construed as being limited thereto.
- In the following Examples, recombinant anti-ganglioside GM3 human antibody produced in Reference Example 1 (hereinafter, referred to as “MABON-01”) was used as the IgM. The MABON-01 solution was concentrated to prepare a highly concentrated solution of approximately 9 mg/mL. For buffer replacement, this solution was dialyzed against
buffers 1 to 6 as listed below using a dialysis membrane, SLIDE-A-LYZER Dialysis Cassette 10000MWCO (PIERCE). - 1. 20 mM sodium acetate, 300 mM NaCl, pH 5.0/acetate pH 5.0
- 2. 20 mM sodium acetate, 300 mM NaCl, pH 5.5/acetate pH 5.5
- 3. 20 mM sodium acetate, 300 mM NaCl, pH 6.0/acetate pH 6.0
- 4. 20 mM sodium citrate, 300 mM NaCl, pH 5.0/citrate pH 5.0
- 5. 20 mM sodium citrate, 300 mM NaCl, pH 5.5/citrate pH 5.5
- 6. 20 mM sodium citrate, 300 mM NaCl, pH 6.0/citrate pH 6.0
- The resulting solutions were collected, and the concentration of MABON-01 in each sample was adjusted to 8.4 mg/mL. Each sample was placed in a storage container, Multiply-Safecup 0.1 ml Biosph. (SARSTEDT). Stability tests were carried out on these samples at 4° C. The samples were evaluated at the initial state and after storing at 4° C. for 2 months (hereafter referred to as, for example “4° C.-2 months”). The stability of each sample was evaluated based on changes in the residual monomer ratio determined by gel filtration chromatography. G4000SWXL (TOSOH) was used as the column for gel filtration chromatography. A solution comprising 50 mM sodium phosphate and 500 mM KCl (pH 7.4) was used as the mobile phase. The residual monomer ratio in a sample was calculated from the values of aggregate peak area and monomer peak area obtained as a result of gel filtration chromatography. The residual monomer ratio in each sample at 4° C.-2 months when the residual monomer ratio at the initial state was defined as 100% is summarized in Table 1.
TABLE 1 Residual Monomer Ratio (%) pH Acetate Citrate 5.0 99.33 99.16 5.5 99.51 99.3 6.0 98.92 98.9 - Thus stable, highly concentrated MABON-01 solutions could be prepared in citrate or acetate buffers (between pH 5.0 and pH 6.0) comprising 300 mM NaCl.
- The following Examples describe stabilized and highly concentrated MABON-01 solutions, in which increase of water-soluble aggregates is further suppressed by adding polyvalent cations to solutions comprising citrate buffer (pH 5.5).
- The MABON-01 solution was concentrated to prepare a highly concentrated solution of approximately 18 mg/mL. This solution was dialyzed using a dialysis membrane, SLIDE-A-LYZER Dialysis Cassette 10000MWCO (PIERCE), to replace the buffer with a solution comprising 20 mM citric acid and 300 mM NaCl (pH 5.5) (the buffer type and pH were optimized under conditions without additives). This highly concentrated MABON-01 solution was dialyzed against the following
buffers 1 to 9 using EasySep (TOMY) to replace the buffer. - 1. 20 mM sodium citrate, 300 mM NaCl, pH 5.5/no additives
- 2. 20 mM sodium citrate, 900 mM NaCl, pH 5.5/NaCl
- 3. 20 mM sodium citrate, 300 mM NaCl, 200 mM MgCl2, pH 5.5/MgCl2
- 4. 20 mM sodium citrate, 300 mM NaCl, 200 mM Na2SO4, pH 5.5/Na2SO4
- 5. 20 mM sodium citrate, 300 mM NaCl, 100 mM sodium L-glutamate, pH 5.5/sodium L-glutamate
- 6. 20 mM sodium citrate, 300 mM NaCl, 100 mM L-arginine hydrochloride, pH 5.5/L-arginine hydrochloride
- 7. 20 mM sodium citrate, 300 mM NaCl, 100 mM sodium N-acetyltryptophanate, pH 5.5/sodium N-acetyltryptophanate
- 8. 20 mM sodium citrate, 300 mM NaCl, 10 mM urea, pH 5.5/urea
- 9. 20 mM sodium citrate, 300 mM NaCl, 100 mM trehalose, pH 5.5/trehalose
- The resulting solutions were collected, and the concentration of MABON-01 in each sample was adjusted to 18.5 mg/mL. Each sample was placed in a storage container, Multiply-Safecup 0.1 ml Biosph. (SARSTEDT). Stability tests were carried out on these samples at 25° C. The samples were evaluated at the initial state, at 25° C.-1 month, and at 25° C.-2 months. The stability of each sample was evaluated based on changes (increase) in aggregate content determined by gel filtration chromatography. G4000SWXL (TOSOH) was used as the column for gel filtration chromatography. A solution comprising 50 mM sodium phosphate and 500 mM KCl (pH 7.4) was used as the mobile phase. The aggregate content in the samples was calculated from the values of aggregate peak area and monomer peak area obtained from gel filtration chromatography. Values obtained by subtracting the aggregate content in the initial state from the aggregate content at 25° C.-1 month and at 25° C.-2 months for each sample are shown in
FIG. 1 as Δaggregate values. - As a result, increase of aggregates was suppressed in sample 2 [NaCl (comprising 900 mM NaCl)], when compared to sample 1 [no additives (comprising 300 mM NaCl)]. This showed that increase of aggregates can be suppressed by increasing the NaCl concentration.
- Meanwhile, although samples 2 [NaCl (comprising 900 mM NaCl)], 3 [MgCl2 (comprising 300 mM NaCl+200 mM MgCl2)], and 4 [Na2SO4 (comprising 300 mM NaCl+200 mM Na2SO4)] all have the same ionic strength (each has an ionic strength of 0.9 M), MgCl2 comprising a divalent cation showed a significant stabilization effect, while sample 4 [Na2SO4] comprising a divalent anion showed a stabilization effect similar in magnitude to that in sample 2 [NaCl].
- Although sample 5 (sodium L-glutamate) and sample 6 (L-arginine hydrochloride) both comprise ionic amino acids,
sample 5 comprising sodium L-glutamate did not show a stabilization effect, whereassample 6 comprising L-arginine hydrochloride, which comprises a divalent cation, showed a stabilization effect similar to that of sample 3 (MgCl2). - The results revealed that increasing the ionic strength by adding salts such as NaCl leads to aggregate suppression. Moreover, at the same ionic strength, use of divalent cations such as magnesium or arginine leads to stronger aggregate suppression effect. However, a strong aggregate suppression effect cannot be obtained by using divalent anions such as sulfate ion and glutamate. More specifically, interaction of a divalent cation such as magnesium ion or arginine with MABON-0 1 significantly suppressed the aggregation of MABON-01, enabling the preparation of stable and highly concentrated solutions.
- MABON-01 solution was concentrated to prepare a highly concentrated MABON-01 solution of approximately 19 mg/mL. The solution was dialyzed against the following
buffers 1 to 9 using a dialyzer membrane, EasySep (TOMY), to replace the buffer. - 1. 20 mM sodium citrate, 300 mM NaCl, pH 5.5
- 2. 20 mM sodium citrate, 300 mM NaCl, 10 mM MgCl2, pH 5.5
- 3. 20 mM sodium citrate, 300 mM NaCl, 50 mM MgCl2, pH 5.5
- 4. 20 mM sodium citrate, 300 mM NaCl, 200 mM MgCl2, pH 5.5
- 5. 20 mM sodium citrate, 300 mM NaCl, 200 mM MgCl2, 100 mM trehalose, pH 5.5
- 6. 20 mM sodium citrate, 300 mM NaCl, 10 mM L-arginine hydrochloride, pH 5.5
- 7. 20 mM sodium citrate, 300 mM NaCl, 50 mM L-arginine hydrochloride, pH 5.5
- 8. 20 mM sodium citrate, 300 mM NaCl, 100 mM L-arginine hydrochloride, pH 5.5
- 9. 20 mM sodium citrate, 300 mM NaCl, 100 mM L-arginine hydrochloride, 100 mM trehalose, pH 5.5
- The resulting solutions were collected, and the concentration of MABON-01 in each sample was adjusted to 18.9 mg/mL. Each sample was placed in a storage container, Multiply-Safecup 0.1 ml Biosph. (SARSTEDT). Stability tests were carried out on these samples. The samples were evaluated at the initial state and at 4° C.-3 months. The stability of each sample was evaluated based on changes (increases) in aggregate content determined by gel filtration chromatography. G4000SWXL (TOSOH) was used as the column for gel filtration chromatography. A solution comprising 50 mM sodium phosphate and 500 mM KCl (pH 7.4) was used as the mobile phase. The aggregate content in the samples was calculated from the values of the aggregate peak area and the monomer peak area obtained from gel filtration chromatography. The aggregate content of each sample at the initial state and at 4° C.-3 months are shown in
FIG. 2 . - As a result, a MgCl2 and L-arginine hydrochloride concentration-dependent aggregate suppression effect was observed. More specifically, a significant stabilization effect could be obtained by increasing MgCl2 and L-arginine hydrochloride concentrations. Furthermore, while trehalose alone was not effective (Examples 2 and 4), addition of 100 mM trehalose in the presence of 200 mM MgCl2 or 100 mM L-arginine hydrochloride was effective for stabilization.
- The MABON-01 solution was concentrated to prepare a highly concentrated MABON-01 solution of approximately 27 mg/mL. The solution was dialyzed against the following
buffers 1 to 3 using a dialyzer membrane, EasySep (TOMY), to replace the buffer. - 1. 20 mM sodium citrate, 300 mM NaCl, pH 5.5/no additives
- 2. 20 mM sodium citrate, 300 mM NaCl, 200 mM MgCl2, pH 5.5/MgCl2
- 3. 20 mM sodium citrate, 300 mM NaCl, 100 mM trehalose, pH 5.5/trehalose
- The resulting solutions were collected, and the concentration of MABON-01 in each sample was adjusted to 26.8 mg/mL. Each sample was placed in a storage container, Multiply-Safecup 0.1 ml Biosph. (SARSTEDT). Stability tests were carried out on these samples. The samples were evaluated at the initial state, at 4° C.-4 months, and at 25° C.-4 months. The stability of each sample was evaluated based on changes (increases) in aggregate content determined by gel filtration chromatography. G4000SWXL (TOSOH) was used as the column for gel filtration chromatography. A solution comprising 50 mM sodium phosphate and 500 mM KCl (pH 7.4) was used as the mobile phase. The aggregate content in the samples was calculated from the values of aggregate peak area and monomer peak area obtained from gel filtration chromatography. The aggregate content of each sample at the initial state, at 4° C.-4 months, and at 25° C.-4 months are shown in
FIG. 3 . - As a result, at both 4° C. and 25° C., MgCl2 was observed to have a stabilization effect. On the other hand, such an effect was hardly observed for trehalose, which is known to be a conventional protein stabilizer and which has been found to have a stabilization effect on IgM during lyophilizing as reported in Journal of Immunological Methods 1995, 181(1), 37-43 (
FIG. 3 ). - <Preparation>
- A large-scale dialysis of MABON-01 was carried out in “50 mM sodium citrate, 180 mM NaCl, pH 5.5, 5% sucrose” buffer, “50 mM sodium citrate, 180 mM ArgHCl, pH 5.5, 5% sucrose” buffer, or “50 mM sodium citrate, 180 mM MgCl2, pH 5.5, 5% sucrose” buffer. After dialysis, the solutions were concentrated by filter centrifugation. Centrifugation was carried out using VIVASPIN6 5000MWCO (VIVASCIENCE, VS061) on himac CF8DL (Hitachi, No. SZGEQ054) at 3,000 rpm. After concentrating and collecting the samples, their concentrations were determined based on UV absorption (ε=1.40). Next, the samples were diluted to 48.4 mg/mL using the buffers. 1% polysorbate80 solution was further added to each sample to prepare a 0.01% polysorbate formulation. 500 μL each of these samples was individually seeded into 5-mL glass vials, and 30 μL of each sample was placed in Multiply-Safecup 0.1 mL Biosph. (SARSTEDT). The 5-mL glass vials were lyophilized under the following conditions. 1 mg/mL MABON-01 was used as the initial sample, and was stored at 4° C. until analysis.
TABLE 2 Temperature [° C.] Time [hr] −50 24 −20 0.02 −20 18 23 2.5 23 28 30 0.25 30 10 Total 82.77
<Experimental Conditions> - “50 mM sodium citrate, 180 mM NaCl, pH 5.5, 5% sucrose, 0.01% polysorbate80”
- “50 mM sodium citrate, 180 mM ArgHCl, pH 5.5, 5% sucrose, 0.01% polysorbate80”
- “50 mM sodium citrate, 180 mM MgCl2, pH 5.5, 5% sucrose, 0.01% polysorbate80”
- MABON-01: 48.4 mg/mL
- Liquid/40° C.-8 days
- Liquid/40° C.-8 days+4° C.-3 days
- Lyophilized/40° C.-8 days
- Lyophilized/40° C.-8 days+4° C.-3 days (after reconstitution)
- Lyophilized/40° C.-8 days+4° C.-3 days (after reconstitution)+relyophilized/50° C.-8 days
<Analysis> - After incubation, 10 μL of solutions diluted to approximately 1 mg/mL (1/50 dilution solution: 4+296 μL) was analyzed by SEC. “50 mM sodium citrate, 500 mM KCl, pH 7.4” was used as the mobile phase (flow rate: 0.3 mL/min; detection at 280 nm or 220 nm) and SEC analysis was carried out using G4000SWXL (TOSOH) (
FIG. 4 ). - Stability of the lyophilized material was higher with ArgHCl or MgCl2 than with NaCl at the same concentration, even during incubation at 50° C.
- <Preparation>
- A large-scale dialysis of MABON-01 was carried out using “50 mM sodium citrate, 180 mM NaCl, pH 5.5, 5% sucrose” and “50 mM sodium citrate, 180 mM ArgHCl, pH 5.5, 5% sucrose” buffers. After dialysis, the solutions were concentrated by filter centrifugation. Centrifugation was carried out using VIVASPIN6 5000MWCO (VIVASCIENCE, VS061) on himac CF8DL (Hitachi, No. SZGEQ054) at 3,000 rpm. After concentrating and collecting the samples, their concentrations were determined based on UV absorption (ε=1.40). 1% polysorbate80 solution was further added to each sample to prepare a 0.01% polysorbate formulation. The samples were diluted to obtain 50 mg/mL formulations. 300 μL each of these samples was individually seeded into three 5-mL glass vials. The 5-mL glass vials were lyophilized under the following conditions. The lyophilized formulations were stored as the initial samples at 4° C. until analysis.
TABLE 3 Temperature [° C.] Time [hr] −50 24 −20 0.02 −20 18 23 2.5 23 28 30 0.25 30 10 Total 82.77
<Experimental Conditions> - “50 mM sodium citrate, 180 mM NaCl, pH 5.5, 5% sucrose, 0.01% polysorbate80”
- “50 mM sodium citrate, 180 mM ArgHCl, pH 5.5, 5% sucrose, 0.01% polysorbate80”
- MABON-01: 50 mg/mL
- Lyophilized/40° C.-2 months
<Analysis> - After incubation, 10 μL of solutions diluted to approximately 1 mg/mL (1/50 dilution solution: 4+296 μL) was analyzed by SEC. The sample obtained by using “50 mM sodium citrate, 500 mM KCl, pH 7.4” was used as the mobile phase (0.3 mL/min of flow rate; detection at 280 nm or 220 nm) and SEC analysis was carried out using G4000SWXL (TOSOH) (
FIG. 5 ). - Stabilization effect was higher with ArgHCl than with NaCl at the same concentration, even after a long-term accelerated study at 40° C.-2 months.
- A gene encoding the heavy chain of a human antibody that binds to ganglioside GM3 was amplified by RT-PCR using total RNAs extracted from human B cells transformed with Epstein-Barr virus (hereinafter, denoted as anti-ganglioside GM3 human antibody-expressing B cells).
- Total RNAs were extracted from 1×107 anti-ganglioside GM3 human antibody-expressing B cells using RNeasy Plant Mini Kit (QIAGEN). Two oligonucleotides (LMH-f3 and LMH-r3) were designed based on the nucleotide sequence of anti-ganglioside GM3 human antibody gene reported by Hoon et al. (Cancer Research 1993; 53: 5244-5250). LMH-f3 (SEQ ID NO: 7) was synthesized in the sense direction, and LMH-r3 (SEQ ID NO: 8) was synthesized in the antisense direction. Using 1 μg of total RNAs, gene fragments were amplified separately for the 5′ end and the 3′ end by SMART RACE cDNA Amplification Kit (CLONTECH). Synthetic oligonucleotides LMH-r3 and LMH-f3 were used for amplifying the 5′ and 3′ ends of the gene, respectively. Reverse transcription reaction was carried out at 42° C. for 1.5 hours.
- The composition of the PCR reaction solution (50 μL) is shown below:
-
-
- 5 μL of 10×
Advantage 2 PCR Buffer, - 5 μL of 10× Universal Primer A Mix,
- 0.2 mM dNTPs (dATP, dGTP, dCTP, and dTTP),
- 1 μL of
Advantage 2 Polymerase Mix,- (All the above were from CLONTECH)
- 2.5 μL of reverse transcription product, and
- 10 pmol of synthetic oligonucleotide LMH-f3 or LMH-r3.
The reaction was carried out under the conditions of: - 94° C. (initial temperature) for 30 seconds,
- 5 cycles of 94° C. for 5 seconds and 72° C. for 3 minutes,
- 5 cycles of 94° C. for 5 seconds, 70° C. for 10 seconds, and 72° C. for 3 minutes,
- 25 cycles of 94° C. for 5 seconds, 68° C. for 10 seconds, and 72° C. for 3 minutes, and finally 72° C. for 7 minutes.
- 5 μL of 10×
- The PCR products were purified from agarose gel using QIAquick Gel Extraction Kit (QIAGEN), and then cloned into pGEM-T Easy vector (Promega). After sequencing, an approximately 1.1 kbp fragment was obtained by digesting the vector comprising the 5′ end of the gene using restriction enzymes ApaI (Takara Shuzo) and SacII (Takara Shuzo), while an approximately 1.1 kbp fragment was obtained by digesting the vector comprising the 3′ end of the gene using restriction enzymes ApaI (Takara Shuzo) and NotI (Takara Shuzo). The fragments were then mixed, and cloned into pBluescript KS+ vector (TOYOBO) to obtain a full-length anti-ganglioside GM3 human antibody heavy chain gene.
- To clone into vectors for expression in animal cells, full-length gene fragments were amplified using synthetic oligonucleotides LMH-fxho and LMH-rsal. LMH-fxho (SEQ ID NO: 11) is a forward primer designed to hybridize to the 5′ end of the anti-ganglioside GM3 human antibody heavy chain gene, and to comprise an XhoI restriction enzyme recognition sequence and a Kozak sequence. LMH-rsal (SEQ ID NO: 12) is a reverse primer designed to hybridize to the 3′ end of the anti-ganglioside GM3 human antibody heavy chain gene, and to comprise a SalI restriction enzyme recognition sequence.
- The composition of the PCR reaction solution (50 μL) is shown below:
-
-
- 5 μL of 10× PCR Buffer,
- 1 mM MgSO4,
- 0.2 mM dNTPs (dATP, dGTP, dCTP, and dTTP),
- 1 unit of DNA polymerase KOD-Plus,
- (All the above were from TOYOBO),
- 10 ng of pBluescript KS+ vector comprising the full-length anti-ganglioside GM3 human antibody heavy chain gene, and
- 10 pmol of synthetic oligonucleotides LMH-fxho and LMH-rsal.
The reaction was carried out under conditions of: - 94° C. (initial temperature) for 2 minutes,
- 30 cycles of 94° C. for 15 seconds, 60° C. for 30 seconds, and 68° C. for 2 minutes, and finally 72° C. for 5 minutes.
- The amplified gene fragment was cloned by digesting with the XhoI restriction enzyme (Takara Shuzo) and the SalI restriction enzyme (Takara Shuzo), then purifying using QIAquick PCR Purification Kit (QIAGEN), and linking to the XhoI restriction enzyme site of pUCAG. This pUCAG vector is obtained by: linking the 2.6 kbp fragment obtained by digesting pCXN (Niwa et al., Gene 1991; 108: 193-200) using the BamHI restriction enzyme to the BamHI restriction enzyme site of pUC19 vector (TOYOBO). The obtained plasmid was named pUCAG/L612H. The nucleotide sequence and amino acid sequence of the anti-ganglioside GM3 human antibody heavy chain in this plasmid are shown in SEQ ID NOs: 1 and 2, respectively.
- A gene encoding the light chain of anti-ganglioside GM3 human antibody was amplified by RT-PCR using total RNAs extracted from the anti-ganglioside GM3 human antibody-expressing B cells. The total RNAs were extracted from the anti-ganglioside GM3 human antibody-expressing B cells in a manner similar to that mentioned above. Two oligonucleotides (LML-f1 and LML-r1) were designed based on the nucleotide sequence of anti-ganglioside GM3 human antibody gene reported by Hoon et al. (Cancer Research 1993; 53: 5244-5250). LML-f1 (SEQ ID NO: 9) and LML-r1 (SEQ ID NO: 10) were synthesized in the sense and antisense directions, respectively.
- Using 1 μg of total RNAs, gene fragments were amplified separately for the 5′ end and the 3′ end by the SMART RACE cDNA Amplification Kit (CLONTECH). Synthetic oligonucleotides LML-r1 and LML-f1 were used for amplifying the 5′ and 3′ ends of the gene, respectively. Reverse transcription reaction was carried out at 42° C. for 1.5 hours.
- The composition of the PCR reaction solution (50 μL) is shown below:
-
-
- 5 μL of 10×
Advantage 2 PCR Buffer, - 5 μL of 10× Universal Primer A Mix,
- 0.2 mM dNTPs (dATP, dGTP, dCTP, and dTTP),
- 1 μL of
Advantage 2 Polymerase Mix,- (All the above were from CLONTECH)
- 2.5 μL of reverse transcription product, and
- 10 pmol of synthetic oligonucleotide LML-f1 or LML-r1
The reaction was carried out under conditions of: - 94° C. (initial temperature) for 30 seconds,
- 5 cycles of 94° C. for 5 seconds and 72° C. for 3 minutes,
- 5 cycles of 94° C. for 5 seconds, 70° C. for 10 seconds, and 72° C. for 3 minutes,
- 25 cycles of 94° C. for 5 seconds, 68° C. for 10 seconds, and 72° C. for 3 minutes, and finally 72° C. for 7 minutes.
- 5 μL of 10×
- PCR product was purified from the agarose gel using QIAquick Gel Extraction Kit (QIAGEN), and then cloned into pGEM-T Easy vector (Promega). After sequencing, an approximately 0.7 kbp fragment was obtained by digesting the vector comprising the 5′ end of the gene using the EcoRI restriction enzyme (Takara Shuzo), while an approximately 0.9 kbp fragment was obtained by digesting the vector comprising the 3′ end of the gene using the EcoRI restriction enzyme (Takara Shuzo). The two fragments were mixed, and used to amplify the full-length gene fragment using synthetic oligonucleotides LML-feco and LML-rnot. LML-feco (SEQ ID NO: 13) is a forward primer, and was designed to hybridize to the 5′ end of the anti-ganglioside GM3 human antibody light chain gene, and to comprise an EcoRI restriction enzyme recognition sequence and a Kozak sequence. LML-rnot (SEQ ID NO: 14) is a reverse primer, and was designed to hybridize to the 3′ end of the anti-ganglioside GM3 human antibody light chain gene, and to comprise a NotI restriction enzyme recognition sequence.
- The composition of the PCR reaction solution (50 μL) is shown below:
-
-
- 5 μL of 10× PCR Buffer,
- 1 mM MgSO4,
- 0.2 mM dNTPs (dATP, dGTP, dCTP, and dTTP),
- 1 unit of DNA polymerase KOD-Plus,
- (All the above were from TOYOBO)
- 5′-end gene fragment,
- 3′-end gene fragment, and
- 10 pmol of synthetic oligonucleotides LML-feco and LML-rnot.
The reaction was carried out under conditions of: - 94° C. (initial temperature) for 2 minutes,
- 30 cycles of 94° C. for 15 seconds, 60° C. for 30 seconds, and 68° C. for 2 minutes, and finally 72° C. for 5 minutes.
- The amplified gene fragment was cloned by digesting with the EcoRI restriction enzyme (Takara Shuzo) and the NotI restriction enzyme (Takara Shuzo), then purifying using QIAquick PCR Purification Kit (QIAGEN) and linking to the EcoRI and NotI restriction enzyme cleavage sites of pCXND3.
- The pCXND3 vector was constructed as follows: DHFR-ΔE-rvH-PM1-f (see WO 92/19759) was digested at the EcoRI/SmaI restriction enzyme site to separate their antibody heavy chain gene and vector region. Only the vector portion was then collected, into which the EcoRI-NotI-BamHI adaptor (Takara Shuzo) was cloned. This vector was named pCHOI.
- A vector in which the DHFR gene expression site of pCHOI is cloned into the HindIII restriction enzyme site of pCXN (Niwa et al., Gene 1991; 108:193-200) was named pCXND3. Furthermore, the light-chain gene fragment was cloned into pCXND3 and the obtained plasmid was named pCXND3/L612L. The nucleotide sequence and amino acid sequence of anti-ganglioside GM3 human antibody light chain in this plasmid are shown in SEQ ID NOs: 3 and 4, respectively.
- To produce the anti-ganglioside GM3 human antibody expression vector, pUCAG/L612H was digested with the HindIII restriction enzyme (Takara Shuzo), and the resulting approximately 4.0 kbp fragment was linked to the HindIII restriction enzyme cleavage site of pCXND3/1612L. The obtained plasmid was named pCXND3/L612IgM. This plasmid expresses the neomycin-resistance gene, DHFR gene, and anti-ganglioside GM3 human antibody gene in animal cells.
- A gene encoding the J chain of anti-ganglioside GM3 human antibody was amplified by RT-PCR using total RNAs extracted from anti-ganglioside GM3 human antibody-expressing B cells. Total RNAs were extracted from anti-ganglioside GM3 human antibody-expressing B cells in a manner similar to that mentioned above. Two oligonucleotides (J-f1 and J-r1) were designed and synthesized based on the nucleotide sequence of the human antibody J chain gene registered in GenBank (GenBank accession number: M12759). J-f1 (SEQ ID NO: 15) hybridizes to human antibody J
chain gene Exon 3 in the sense direction, and J-r1 (SEQ ID NO: 16) hybridizes to the human antibody Jchain gene Exon 4 in the antisense direction. - Using 1 μg of total RNAs, gene fragments were amplified separately for the 5′ end and the 3′ end by the SMART RACE cDNA Amplification Kit (CLONTECH). Synthetic oligonucleotides J-r1 and J-f1 were used for amplifying the 5′ and 3′ ends of the gene, respectively. Reverse transcription reaction was carried out at 42° C. for 1.5 hours.
- The composition of the PCR reaction solution (50 μL) is shown below:
-
-
- 5 μL of 10×
Advantage 2 PCR Buffer, - 5 μL of 10× Universal Primer A Mix,
- 0.2 mM dNTPs (dATP, dGTP, dCTP, and dTTP),
- 1 μL of
Advantage 2 Polymerase Mix,- (All the above were all from CLONTECH)
- 2.5 μL of reverse transcription product, and
- 10 pmol of synthetic oligonucleotide J-f1 or J-r1
The reaction was carried out under conditions of: - 94° C. (initial temperature) for 30 seconds,
- 5 cycles of 94° C. for 5 seconds and 72° C. for 3 minutes,
- 5 cycles of 94° C. for 5 seconds, 70° C. for 10 seconds, and 72° C. for 3 minutes,
- 25 cycles of 94° C. for 5 seconds, 68° C. for 10 seconds, and 72° C. for 3 minutes, and finally 72° C. for 7 minutes.
- 5 μL of 10×
- PCR product was purified from the agarose gel using QIAquick Gel Extraction Kit (QIAGEN), and then cloned into pGEM-T Easy vector (Promega).
- After sequencing, an approximately 0.5 kbp fragment was obtained by digesting the vector comprising the 5′ end of the gene using the EcoRI restriction enzyme (Takara Shuzo), and an approximately 1.0 kbp fragment was obtained by digesting the vector comprising the 3′ end of the gene using the EcoRI restriction enzyme (Takara Shuzo). The two fragments were mixed, and used to amplify the full-length gene fragment using synthetic oligonucleotides J-feco and J-rxba.
- J-feco (SEQ ID NO: 17) is a forward primer designed to hybridize to the 5′ end of the anti-ganglioside GM3 human antibody J chain gene, and to comprise an EcoRI restriction enzyme recognition sequence and a Kozak sequence. J-rxba (SEQ ID NO: 18) is a reverse primer designed to hybridize to the 3′ end of the anti-ganglioside GM3 human antibody J chain gene, and to comprise an XbaI restriction enzyme recognition sequence.
- The composition of the PCR reaction solution (50 μL) is shown below:
-
-
- 5 μL of 10× PCR Buffer,
- 1 mM MgSO4,
- 0.2 mM dNTPs (dATP, dGTP, dCTP, and dTTP),
- 1 unit of DNA polymerase KOD-Plus
- (the above-mentioned ingredients were all from TOYOBO),
- 5′-end gene fragment,
- 3′-end gene fragment, and
- 10 pmol of synthetic oligonucleotides LML-feco and LML-rxba
The reaction was carried out under conditions of: - 94° C. (initial temperature) for 2 minutes,
- 30 cycles of 94° C. for 15 seconds, 60° C. for 30 seconds, and 68° C. for 2 minutes, and finally 72° C. for 5 minutes.
- The amplified gene fragment was cloned by digesting with the EcoRI restriction enzyme (Takara Shuzo) and the XbaI restriction enzyme (Takara Shuzo), then purifying using QIAquick PCR Purification Kit (QIAGEN), and linking to the EcoRI and XbaI restriction enzyme cleavage sites of pCOSII-Zeo.
- This pCOSII-Zeo vector is obtained by removing the DHFR gene expression site of pCHOI, and cloning the Zeocin-resistant gene expression site thereto. The obtained plasmid was named pCOSII-Zeo/J chain. The nucleotide sequence and amino acid sequence of anti-ganglioside GM3 human antibody J chain in this plasmid are shown in SEQ ID NOs: 5 and 6, respectively.
- Stable expression cell lines derived from CHO cells (DG44 line) were produced as described below.
- Genes were introduced by electroporation using Gene Pulser II (BioRad).
- Introduction of genes to obtain cell lines that do not express the J chain is described below. 0.75 mL of CHO cells suspended in PBS (1×107 cells/mL) was mixed with anti-ganglioside GM3 human antibody expression vector pCXND3/L612IgM (25 μg), cooled on ice for 10 minutes, transferred to a cuvette, and then pulsed at 1.5 kV and 25 μFD.
- After a recovery period of 10 minutes at room temperature, the electroporated cells were suspended in 40 mL of CHO—S—SFMII medium (Invitrogen) comprising 1× HT Supplement (Invitrogen). A 50-fold diluted solution was further prepared using the same medium, and then aliquoted at 100 μL/well into a 96-well culture plate. After incubation for 24 hours in a CO2 incubator (5% CO2), Geneticin (Invitrogen) was added to the wells at 0.5 mg/mL and cultured for 2 weeks.
- The IgM levels in the culture supernatants of wells in which colonies of Geneticin-resistant transformants were found were measured by the concentration assay described in Reference Example 1.6. Cell lines highly expressing the anti-ganglioside GM3 human antibodies were successively expanded to obtain anti-ganglioside GM3 human antibody-expressing stable cell lines CA02, CA15, CA19, CA20, and CA24.
- Introduction of genes to obtain cell lines expressing the J chain is described below. 0.75 mL of CHO cells suspended in PBS (1×107 cells/mL) was mixed with anti-ganglioside GM3 human antibody expression vector pCXND3/L612IgM (25 μg) and J chain expression vector pCOSII-Zeo/J chain (20 μg), cooled on ice for 10 minutes, transferred to a cuvette, and then pulsed at 1.5 kV and 25 μFD.
- After recovered for 10 minutes at room temperature, the electroporated cells were suspended in 40 mL of CHO—S—SFMII medium (Invitrogen) comprising 1× HT Supplement (Invitrogen).
- A 50-fold diluted solution was further prepared using the same medium and aliquoted at 100 μL/well into a 96-well culture plate. After incubation for 24 hours in a CO2 incubator (5% CO2), 0.5 mg/mL Geneticin (Invitrogen) and 0.6 mg/mL Zeocin (Invitrogen) were added to wells, and cultured for 2 weeks. The IgM levels in the culture supernatants of wells in which colonies of Geneticin- and Zeocin-resistant transformants were found were measured by the concentration assay described in Reference Example 1.6. Cell lines highly expressing the anti-ganglioside GM3 human antibodies were successively expanded to obtain anti-ganglioside GM3 human antibody-expressing stable cell lines (CJ15, CJ25, CJ38, CJ45, and CJ67).
- IgM concentration in the culture supernatants was measured as described below. Anti-Human IgM (BIOSOURCE) was diluted using a coating buffer (0.1 M NaHCO3 and 0.02% NaN3) to prepare a 1 μg/mL solution. The diluted solution was added to a 96-well ELISA plate at 100 μL/well, and then reacted at 4° C. for 24 hours or longer to coat the plate.
- After washing the wells with Rinse Buffer, blocking was carried out by adding 200 μL/well of Diluent Buffer and reacting at room temperature for 1 hour or longer. Compositions of the Rinse Buffer and Diluent Buffer are shown below.
- Rinse Buffer: PBS(−)
-
- 0.05% Tween20
- Diluent Buffer: 50 mM Tris,
-
- 1 mM MgCl2,
- 0.15 M NaCl,
- 0.05% Tween20,
- 0.02% NaN3,
- 1% BSA
- Next, culture supernatant suitably diluted with Diluent Buffer was added to the wells at 100 μL/well, and allowed to react at room temperature for 1 hour. After washing with Rinse Buffer, alkaline phosphatase-conjugated goat anti-human IgM (BIOSOURCE) diluted 4,000 times with Diluent Buffer was added at 100 μL/well, and reacted at room temperature for 1 hour. Finally, wells were washed with Rinse Buffer, and alkaline phosphatase substrate (SIGMA) was added thereto. The absorbance was determined at the 405 nm measurement wavelength and 655 nm reference wavelength using Benchmark Plus absorption spectrometer (BioRad). The concentration of IgM was calculated by comparing with a purified anti-ganglioside GM3 human antibody (Hoon et al., Cancer Research 1993; 53: 5244-5250).
- Each type of stable cell line expressing anti-ganglioside GM3 human antibodies was cultured in a 75 cm2-culture flask at an initial cell density of 2×105 cells/mL. The IgM concentration in the culture supernatants was measured by the method described above. The results are shown in Table 4. The amount of IgM produced was approximately 20 mg/L on the third day and approximately 50 mg/L on the seventh day. The productivity indicating the production ability of a single cell was 5 to 19 pg/cell/day. Since IgM is a type of immunoglobulin that forms multimers, expression level of IgM in recombinants is low, and therefore, its large-scale preparation was considered difficult. However, the present results showed that highly productive recombinant IgM-expressing cells can be produced from CHO cells.
TABLE 4 Production Production amount after amount after J-chain Cell culturing for culturing for Productivity expression lines 3 days (mg/L) 7 days (mg/L) (pg/cell/day) Absent CA02 24.1 36.9 14.1 CA15 11.8 39.7 4.9 CA19 27.1 62.3 13.1 CA20 20.2 35.4 10.5 CA24 25.0 41.5 10.7 Present CJ15 29.4 N.T. 19.4 CJ25 24.4 N.T. 18.1 CJ38 14.9 N.T. 12.4 CJ45 26.4 N.T. 18.7 CJ67 18.0 N.T. 12.8
N.T.: Not Tested
- Gel filtration chromatographic analysis of MABON-01 was carried out using the following buffers as mobile phase. In all analyses, a TSKgel G4000SWXL column was used, the flow rate was 0.3 mL/min, absorbance at 280 nm was detected, and the injection amount of samples was 10 μg.
- 1. 50 mM sodium phosphate, 500 mM KCl, pH 6.2
- 2. 50 mM sodium phosphate, 500 mM KCl, pH 6.5
- 3. 50 mM sodium phosphate, 500 mM KCl, pH 6.8
- 4. 50 mM sodium phosphate, 500 mM KCl, pH 7.1
- 5. 50 mM sodium phosphate, 500 mM KCl, pH 7.4
- 6. 50 mM sodium phosphate, 300 mM KCl, pH 6.5
- 7. 50 mM sodium phosphate, 300 mM KCl, pH 7.4
- 8. 50 mM sodium phosphate, 500 mM NaCl, pH 6.5
- 9. 50 mM sodium phosphate, 500 mM NaCl, pH 7.4
- 10. 50 mM sodium phosphate, 300 mM NaCl, pH 6.5
- 11. 50 mM sodium phosphate, 300 mM NaCl, pH 7.4
- The aggregate peak area and the monomer peak area values (peak assignments were carried out separately) based on the obtained chromatograms are shown in Table 5.
TABLE 5 KCl NaCl 500 mM 300 mM 500 mM 300 mM pH 6.2 3073386 — — Total peak area value 146342 2927044 — — — — Aggregate Monomer pH 6.5 3096904 2959509 3044989 2818198 155304 2941600 124880 2834629 127467 2917522 82928 2735270 pH 6.8 3074760 — — — 153682 2921078 — — — — — — pH 7.1 3033846 — — — 154085 2879761 — — — — — — pH 7.4 3074597 3098757 3130093 2948932 163747 2910850 157320 2941437 144630 2985463 112427 2836505
In each cell, the total peak area value is shown in the top row, the area value for the aggregate is shown in the bottom row on the left, and the area value for the monomer is shown in the bottom row on the right. - Table 6 shows the result of calculating the aggregate peak area rate and the monomer peak area rate relative to the total peak area value.
TABLE 6 KCl NaCl 500 mM 300 mM 500 mM 300 mM pH 6.2 4.8 95.2 — — — — Aggregate Monomer pH 6.5 5.0 95.0 4.2 95.8 4.2 95.8 2.9 97.1 pH 6.8 5.0 95.0 — — — — — — pH 7.1 5.1 94.9 — — — — — — pH 7.4 5.3 94.7 5.1 94.9 4.6 95.4 3.8 96.2
In each cell, the aggregate peak area rates are shown on the left, and the monomer peak area rates are shown on the right. - These results revealed that, for both the total peak area values and the aggregate peak area rates, the effects of salt type and salt concentration are suppressed when a pH 7.4 mobile phase buffer is used, compared to when mobile phase buffers of pH 6.2 to 7.1 are used. In addition, it was revealed that the effect of pH is suppressed when the salt concentration in the mobile phase buffer comprising either KCl or NaCl is 500 mM, compared to when the salt concentration is 300 mM. Furthermore, it was revealed that the effects of salt concentration and mobile phase pH are suppressed when a mobile phase buffer comprises KCl, compared to when a mobile phase buffer comprises NaCl. Therefore, conditions for the mobile phase for the gel filtration chromatographic analysis of MABON-01 were set at 50 mM sodium phosphate, 500 mM KCl, and pH 7.4.
- GPC-MALLS analysis of MABON-01 was carried out to determine the molecular weight of each peak. The mobile phase buffer was 50 mM sodium phosphate, 500 mM KCl, pH 7.4. TSKgel G4000SWXL column was used. The flow rate was 0.3 mL/min. Absorbance at 280 nm was detected. Injection amount of samples was 113 μg. According to the obtained results, the molecular weights were calculated by the Debye method.
- Resulting chromatogram and the calculated molecular weights are shown as a superimposed diagram in
FIG. 6 . The average molecular weights ofpeak 1 andpeak 2 in the figure, and the theoretical molecular weight of MABON-01 calculated from its amino acid sequence are shown in Table 7.TABLE 7 Average molecular weights Theoretical molecular weights M.W. M.W. [kDa] [kDa] Peak 11,109 Monomer 1,034 Peak 22,193 Dimer 2,068 - The average molecular weights were obtained by averaging the molecular weights corresponding to 21.5 to 22.0 minutes for
peak 2, and 24.5 to 25.0 minutes forpeak 1. The average molecular weight values forpeak 1 andpeak 2 are close to the theoretical molecular weight values for the monomer and dimer, respectively. In addition, the average molecular weight value forpeak 2 is approximately two times that ofpeak 1. Accordingly,peak 1 was found to comprise the MABON-01 monomer, andpeak 2 was found to comprise the dimer. - The present invention enabled stabilization of highly concentrated IgM in solutions. Since the present invention enables stable long-term storage of pharmaceutical formulations comprising IgM as an active ingredient, it can significantly contribute to particularly the preparation of antibody formulations.
Claims (35)
1. A solution wherein a high concentration of immunoglobulin is stabilized, and wherein the immunoglobulin is IgM.
2. The solution of claim 1 , comprising IgM at a concentration higher than 1 mg/mL.
3. The solution of claim 1 , which is an aqueous solution.
4. The solution of claim 1 , which is a pharmaceutical formulation.
5. The solution of claim 1 , comprising a polyvalent cationic ion.
6. The solution of claim 5 , comprising the polyvalent cationic ion at a concentration of 1 mM to 1,000 mM.
7. The solution of claim 5 , wherein the polyvalent cationic ion is a magnesium ion or an arginine ion.
8. The solution of claim 5 , further comprising sugars.
9. The solution of claim 1 , which is pH 5 to pH 8.
10. The solution of claim 1 , wherein the solution does not intrinsically comprise human-derived proteins other than IgM.
11. The solution of claim 1 , wherein the solution does not intrinsically comprise proteins other than IgM.
12. A pharmaceutical formulation obtained by freezing or lyophilizing the solution of claim 1 .
13. A method for stabilizing a solution comprising a high concentration of immunoglobulin, wherein the immunoglobulin is IgM and wherein the method comprises adding a polyvalent cationic ion to the solution.
14. The method of claim 13 , wherein the solution comprises IgM at a concentration higher than 1 mg/mL.
15. The method of claim 13 , wherein the solution is an aqueous solution.
16. The method of claim 13 , wherein the solution is a pharmaceutical formulation.
17. The method of claim 13 , which comprises adding a polyvalent cationic ion to the solution such that the solution comprises the polyvalent cationic ion at a concentration of 1 mM to 1,000 mM.
18. The method of claim 13 , wherein the polyvalent cationic ion is a magnesium ion or an arginine ion.
19. The method of claim 13 , further comprising addition of sugars.
20. The method of claim 13 , wherein the pH of the solution is 5 to 8.
21. The method of claim 13 , wherein the solution does not intrinsically comprise human-derived proteins other than IgM.
22. The method of claim 13 , wherein the solution does not intrinsically comprise proteins other than IgM.
23. A method for stabilizing a pharmaceutical formulation, which comprises the steps of:
(a) performing the method of claim 13; and
(b) freezing or lyophilizing the solution stabilized in step (a).
24. A method for producing a solution comprising a high concentration of stabilized immunoglobulin, wherein the immunoglobulin is IgM and wherein the method comprises the step of adding a polyvalent cationic ion to the solution.
25. The method of claim 24 , wherein the solution comprises IgM at a concentration higher than 1 mg/mL.
26. The method of claim 24 , wherein the solution is an aqueous solution.
27. The method of claim 24 , wherein the solution is a pharmaceutical formulation.
28. The method of claim 24 , which comprises the step of adding a polyvalent cationic ion to the solution such that the solution comprises the polyvalent cationic ion at a concentration of 1 mM to 1000 mM.
29. The method of claim 24 , wherein the polyvalent cationic ion is a magnesium ion or an arginine ion.
30. The method of claim 24 , which further comprises the step of adding sugars.
31. The method of claim 24 , wherein the pH of the solution is 5 to 8.
32. The method of claim 24 , wherein the solution essentially does not comprise human-derived proteins other than IgM.
33. The method of claim 24 , wherein the solution essentially does not comprise proteins other than IgM.
34. A solution which is produced by the method of claim 24 .
35. A method for producing a pharmaceutical formulation, wherein the method comprises the steps of:
(a) performing the method of claim 24; and
(b) freezing or lyophilizing the solution produced in step (a).
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2003351388 | 2003-10-09 | ||
JP2003-351388 | 2003-10-09 | ||
PCT/JP2004/014935 WO2005035574A1 (en) | 2003-10-09 | 2004-10-08 | IgM HIGH CONCENTRATION STABILIZED SOLUTION |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2004/014935 A-371-Of-International WO2005035574A1 (en) | 2003-10-09 | 2004-10-08 | IgM HIGH CONCENTRATION STABILIZED SOLUTION |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/509,075 Continuation US8920797B2 (en) | 2003-10-09 | 2009-07-24 | Highly concentrated stabilized IgM solution |
Publications (1)
Publication Number | Publication Date |
---|---|
US20070212346A1 true US20070212346A1 (en) | 2007-09-13 |
Family
ID=34431075
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/575,192 Abandoned US20070212346A1 (en) | 2003-10-09 | 2004-10-08 | Highly Concentrated Stabilized Igm Solution |
US12/509,075 Active 2028-01-29 US8920797B2 (en) | 2003-10-09 | 2009-07-24 | Highly concentrated stabilized IgM solution |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/509,075 Active 2028-01-29 US8920797B2 (en) | 2003-10-09 | 2009-07-24 | Highly concentrated stabilized IgM solution |
Country Status (5)
Country | Link |
---|---|
US (2) | US20070212346A1 (en) |
EP (1) | EP1688432B1 (en) |
JP (1) | JP4762717B2 (en) |
AT (1) | ATE518888T1 (en) |
WO (1) | WO2005035574A1 (en) |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070154469A1 (en) * | 2003-07-15 | 2007-07-05 | Reiko Irie | IGM production by transformed cells and methods of quantifying said IgM production |
US20070249812A1 (en) * | 2003-10-09 | 2007-10-25 | Chugai Seiyaku Kabushiki Kaisha | Methods for Stabilizing Protein Solutions |
US7390786B2 (en) | 2005-12-21 | 2008-06-24 | Wyeth | Protein formulations with reduced viscosity and uses thereof |
US20080292642A1 (en) * | 2007-03-29 | 2008-11-27 | Borhani David W | Crystalline anti-human IL-12 antibodies |
US20090285802A1 (en) * | 2003-10-09 | 2009-11-19 | Tomoyuki Igawa | Highly concentrated stabilized igm solution |
EP3378486B1 (en) | 2010-01-20 | 2021-03-03 | Chugai Seiyaku Kabushiki Kaisha | Stabilized antibody-containing liquid formulations |
Families Citing this family (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2668947C (en) * | 2006-12-05 | 2017-02-07 | Crucell Holland B.V. | Liquid anti-rabies antibody formulations |
CA2734919C (en) * | 2008-08-27 | 2016-08-16 | Schering Corporation | Lyophilized formulations of engineered anti-il-23p19 antibodies |
KR20120115383A (en) * | 2010-01-29 | 2012-10-17 | 가부시키가이샤 아크시스 | Pharmaceutical composition for treatment or prevention of osteoarthritis, and method of production therefor |
MX2012012743A (en) * | 2010-05-03 | 2012-11-23 | Genentech Inc | Compositions and methods useful for reducing the viscosity of protein-containing formulations. |
EP2471554A1 (en) | 2010-12-28 | 2012-07-04 | Hexal AG | Pharmaceutical formulation comprising a biopharmaceutical drug |
FR2994390B1 (en) | 2012-08-10 | 2014-08-15 | Adocia | METHOD FOR LOWERING THE VISCOSITY OF HIGH CONCENTRATION PROTEIN SOLUTIONS |
CN110913906A (en) | 2017-05-02 | 2020-03-24 | 默沙东公司 | Formulations of anti-LAG 3 antibodies and co-formulations of anti-LAG 3 antibodies with anti-PD-1 antibodies |
JOP20190260A1 (en) | 2017-05-02 | 2019-10-31 | Merck Sharp & Dohme | Stable formulations of programmed death receptor 1 (pd-1) antibodies and methods of use thereof |
EP4178978A1 (en) * | 2020-07-10 | 2023-05-17 | Grifols Worldwide Operations Limited | Method for obtaining a composition comprising human plasma-derived immunoglobulin m |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5157133A (en) * | 1989-12-01 | 1992-10-20 | Rhone-Poulenc Nutrition Animale | Dihydropyran derivatives, processes for preparation and use |
US5157113A (en) * | 1987-08-10 | 1992-10-20 | Miles Inc. | Removal of nucleic acids from monoclonal antibody preparations |
US5190752A (en) * | 1988-07-27 | 1993-03-02 | Biotest Pharma Gmbh | Intravenously administerable polyclonal immunoglobulin preparation containing igm and method of manufacture |
US5792838A (en) * | 1991-10-28 | 1998-08-11 | Glaxo Wellcome Inc. | Method for stabilizing immunoglobulin compositions |
US5908826A (en) * | 1991-03-08 | 1999-06-01 | Mitsui Toatsu Chemicals Inc. | Freeze-dried preparation containing monoclonal antibody |
US6136312A (en) * | 1996-10-14 | 2000-10-24 | Rotkreuzstifung Zentrallaboratorium Blutspendedienst Srk | Method for producing an IgM preparation for intravenous application |
US20070249812A1 (en) * | 2003-10-09 | 2007-10-25 | Chugai Seiyaku Kabushiki Kaisha | Methods for Stabilizing Protein Solutions |
Family Cites Families (36)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ES2045027T3 (en) * | 1987-08-10 | 1994-01-16 | Miles Inc | PURIFIED IGM. |
GB8719963D0 (en) | 1987-08-24 | 1987-09-30 | Cattaneo A | Recombinant dna products |
CA1312030C (en) | 1987-11-18 | 1992-12-29 | Brian Maiorella | Method to increase antibody titer |
US6238891B1 (en) * | 1987-11-18 | 2001-05-29 | Cetus Oncology Corporation | Method of increasing product expression through solute stress |
JP2501116B2 (en) | 1988-03-05 | 1996-05-29 | 日本電信電話株式会社 | Circuit-controlled circuit switching method |
GB9010944D0 (en) | 1990-05-16 | 1990-07-04 | Central Blood Lab Authority | Chemical compounds |
US5419904A (en) | 1990-11-05 | 1995-05-30 | The Regents Of The University Of California | Human B-lymphoblastoid cell line secreting anti-ganglioside antibody |
EP0492409A1 (en) | 1990-12-18 | 1992-07-01 | Ishihara Sangyo Kaisha, Ltd. | Human monoclonal antibody |
JPH05192181A (en) | 1990-12-18 | 1993-08-03 | Ishihara Sangyo Kaisha Ltd | Human monoclonal antibody |
US5547853A (en) * | 1991-03-12 | 1996-08-20 | Biogen, Inc. | CD2-binding domain of lymphocyte function associated antigen 3 |
JPH04360696A (en) | 1991-06-06 | 1992-12-14 | Sumitomo Chem Co Ltd | Recombinant human antibody produced with new human b lymphocyte strain as host |
US5830470A (en) * | 1992-09-07 | 1998-11-03 | Kyowa Hakko Kogyo Co., Ltd. | Humanized antibodies to ganglioside GM2 |
JP3565572B2 (en) | 1992-09-07 | 2004-09-15 | 協和醗酵工業株式会社 | Humanized antibodies |
JPH06178689A (en) | 1992-12-11 | 1994-06-28 | Mitsui Toatsu Chem Inc | Gene coding human antibody against type i pseudomonas aeruginosa as antigen |
JPH06189781A (en) | 1992-12-25 | 1994-07-12 | Mitsui Toatsu Chem Inc | Stabilization of bioactive protein in culture solution |
JPH09127112A (en) * | 1995-10-30 | 1997-05-16 | Dainabotsuto Kk | Modified igm immunological measuring reagent |
JPH09127114A (en) * | 1995-11-01 | 1997-05-16 | Dainabotsuto Kk | Stabilized igm reagent for immunoassay |
WO1998006749A2 (en) | 1996-08-16 | 1998-02-19 | President And Fellows Of Harvard College | Soluble monovalent and multivalent mhc class ii fusion proteins, and uses therefor |
JPH10324699A (en) | 1997-03-21 | 1998-12-08 | Sankyo Co Ltd | Anti-human fas humanized antibody |
US5994511A (en) | 1997-07-02 | 1999-11-30 | Genentech, Inc. | Anti-IgE antibodies and methods of improving polypeptides |
CA2318231A1 (en) | 1998-01-16 | 1999-07-22 | Iq Corporation | Use of radiolabeled monoclonal igm in therapy for cancer and autoimmune disease |
AU2444899A (en) | 1998-01-22 | 1999-08-09 | Astrazeneca Ab | Pharmaceutical formulation comprising an antibody and a citrate buffer |
JP2002504342A (en) | 1998-02-19 | 2002-02-12 | プレジデント アンド フェローズ オブ ハーバード カレッジ | Monovalent MHC binding domain fusion proteins and conjugates, multivalent MHC binding domain fusion proteins and conjugates, and multimeric MHC binding domain fusion proteins and conjugates, and uses therefor |
JP2000154149A (en) | 1998-09-18 | 2000-06-06 | Sankyo Co Ltd | Antirheumatic agent containing anti-human fas humanized antibody |
ATE327254T1 (en) | 1998-10-16 | 2006-06-15 | Biogen Idec Inc | INTERFERON-BETA FUSION PROTEINS AND THEIR USES |
CA2704600C (en) | 1999-04-09 | 2016-10-25 | Kyowa Hakko Kirin Co., Ltd. | A method for producing antibodies with increased adcc activity |
EP1174148A4 (en) | 1999-04-28 | 2005-05-04 | Yamanouchi Pharma Co Ltd | Parenteral medicinal composition containing humanized monoclonal antibody fragment and method for stabilizing the same |
AU7449200A (en) | 1999-09-30 | 2001-04-30 | Kyowa Hakko Kogyo Co. Ltd. | Derivatives of antibody against ganglioside gm2 |
AU1052701A (en) | 1999-11-05 | 2001-06-06 | Kyowa Hakko Kogyo Co. Ltd. | Transgenic non-human mammals for monitoring change in calcium ion concentration in cells |
KR20020093029A (en) | 2000-04-11 | 2002-12-12 | 제넨테크, 인크. | Multivalent Antibodies And Uses Therefor |
WO2002010371A1 (en) | 2000-08-01 | 2002-02-07 | Kyowa Hakko Kogyo Co., Ltd. | Novel physiologically active peptide and use thereof |
GB0113179D0 (en) * | 2001-05-31 | 2001-07-25 | Novartis Ag | Organic compounds |
JP2003128576A (en) | 2001-10-25 | 2003-05-08 | Taisho Pharmaceut Co Ltd | Therapeutic agent for endotoxin-inducing disease |
AU2002356749A1 (en) | 2001-11-28 | 2003-06-10 | Hermann Katinger | Process for the production of polypeptides in mammalian cell cultures |
CA2532966C (en) * | 2003-07-15 | 2012-12-04 | Chugai Seiyaku Kabushiki Kaisha | Igm production by transformed cells and methods for quantifying said igm production |
ATE518888T1 (en) | 2003-10-09 | 2011-08-15 | Chugai Pharmaceutical Co Ltd | STABILIZED SOLUTION WITH HIGH IGM CONCENTRATION |
-
2004
- 2004-10-08 AT AT04792204T patent/ATE518888T1/en not_active IP Right Cessation
- 2004-10-08 WO PCT/JP2004/014935 patent/WO2005035574A1/en active Application Filing
- 2004-10-08 US US10/575,192 patent/US20070212346A1/en not_active Abandoned
- 2004-10-08 JP JP2005514615A patent/JP4762717B2/en not_active Expired - Fee Related
- 2004-10-08 EP EP04792204A patent/EP1688432B1/en not_active Not-in-force
-
2009
- 2009-07-24 US US12/509,075 patent/US8920797B2/en active Active
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5157113A (en) * | 1987-08-10 | 1992-10-20 | Miles Inc. | Removal of nucleic acids from monoclonal antibody preparations |
US5190752A (en) * | 1988-07-27 | 1993-03-02 | Biotest Pharma Gmbh | Intravenously administerable polyclonal immunoglobulin preparation containing igm and method of manufacture |
US5157133A (en) * | 1989-12-01 | 1992-10-20 | Rhone-Poulenc Nutrition Animale | Dihydropyran derivatives, processes for preparation and use |
US5908826A (en) * | 1991-03-08 | 1999-06-01 | Mitsui Toatsu Chemicals Inc. | Freeze-dried preparation containing monoclonal antibody |
US5792838A (en) * | 1991-10-28 | 1998-08-11 | Glaxo Wellcome Inc. | Method for stabilizing immunoglobulin compositions |
US6136312A (en) * | 1996-10-14 | 2000-10-24 | Rotkreuzstifung Zentrallaboratorium Blutspendedienst Srk | Method for producing an IgM preparation for intravenous application |
US20070249812A1 (en) * | 2003-10-09 | 2007-10-25 | Chugai Seiyaku Kabushiki Kaisha | Methods for Stabilizing Protein Solutions |
Cited By (18)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070154469A1 (en) * | 2003-07-15 | 2007-07-05 | Reiko Irie | IGM production by transformed cells and methods of quantifying said IgM production |
US8257703B2 (en) | 2003-07-15 | 2012-09-04 | Chugai Seiyaku Kabushiki Kaisha | Anti-ganglioside antibodies and compositions |
US7709615B2 (en) | 2003-07-15 | 2010-05-04 | Chugai Seiyaku Kabushiki Kaisha | Polynucleotides encoding anti-ganglioside antibodies |
US20100172899A1 (en) * | 2003-07-15 | 2010-07-08 | Chugai Seiyaku Kabushiki Kaisha | IgM Production by Transformed Cell and Method of Quantifying the Same |
US20070249812A1 (en) * | 2003-10-09 | 2007-10-25 | Chugai Seiyaku Kabushiki Kaisha | Methods for Stabilizing Protein Solutions |
US8920797B2 (en) | 2003-10-09 | 2014-12-30 | Chugai Seiyaku Kabushiki Kaisha | Highly concentrated stabilized IgM solution |
US7803914B2 (en) | 2003-10-09 | 2010-09-28 | Chugai Seiyaku Kabushiki Kaisha | Methods for stabilizing protein solutions |
US20090285802A1 (en) * | 2003-10-09 | 2009-11-19 | Tomoyuki Igawa | Highly concentrated stabilized igm solution |
US7758860B2 (en) | 2005-12-21 | 2010-07-20 | Wyeth Llc | Protein formulations with reduced viscosity and uses thereof |
US20110027292A1 (en) * | 2005-12-21 | 2011-02-03 | Wyeth Llc | Protein Formulations with Reduced Viscosity and Uses Thereof |
US20080160014A1 (en) * | 2005-12-21 | 2008-07-03 | Wyeth | Protein Formulations With Reduced Viscosity and Uses Thereof |
US7390786B2 (en) | 2005-12-21 | 2008-06-24 | Wyeth | Protein formulations with reduced viscosity and uses thereof |
US20080292642A1 (en) * | 2007-03-29 | 2008-11-27 | Borhani David W | Crystalline anti-human IL-12 antibodies |
US8168760B2 (en) | 2007-03-29 | 2012-05-01 | Abbott Laboratories | Crystalline anti-human IL-12 antibodies |
US8404819B2 (en) | 2007-03-29 | 2013-03-26 | Abbvie Inc. | Crystalline anti-human IL-12 antibodies |
US8940873B2 (en) | 2007-03-29 | 2015-01-27 | Abbvie Inc. | Crystalline anti-human IL-12 antibodies |
EP3378486B1 (en) | 2010-01-20 | 2021-03-03 | Chugai Seiyaku Kabushiki Kaisha | Stabilized antibody-containing liquid formulations |
US11612562B2 (en) | 2010-01-20 | 2023-03-28 | Chugai Seiyaku Kabushiki Kaisha | Solution preparation containing stabilized antibody |
Also Published As
Publication number | Publication date |
---|---|
ATE518888T1 (en) | 2011-08-15 |
WO2005035574A1 (en) | 2005-04-21 |
US8920797B2 (en) | 2014-12-30 |
EP1688432A4 (en) | 2007-09-12 |
EP1688432A1 (en) | 2006-08-09 |
EP1688432B1 (en) | 2011-08-03 |
US20090285802A1 (en) | 2009-11-19 |
JP4762717B2 (en) | 2011-08-31 |
JPWO2005035574A1 (en) | 2008-02-14 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US8920797B2 (en) | Highly concentrated stabilized IgM solution | |
US10899841B2 (en) | Anti-BAFFR antibody formulations and methods of use thereof | |
US11612659B2 (en) | Anti-CD40 antibody formulation delivery device | |
EP2620450B1 (en) | Anti-CTLA-4 antibody compositions | |
EP2858671B1 (en) | Antibody formulation | |
US7803914B2 (en) | Methods for stabilizing protein solutions | |
WO2020072896A1 (en) | Anti-fgfr2 antibody formulations | |
WO2022184148A1 (en) | Il-21-anti-albumin single-domain antibody fusion protein pharmaceutical composition and use thereof | |
CN114793422A (en) | anti-CTLA-4 monoclonal antibody and preparation method and application thereof | |
CN117982437A (en) | Antibody preparation for targeting coronavirus and application thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: CHUGAI SEIYAKU KABUSHIKI KAISHA, JAPAN Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:IGAWA, TOMOYUKI;SEKIMORI, YASUO;REEL/FRAME:018821/0980;SIGNING DATES FROM 20060710 TO 20060711 |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |