EP2717904A1 - Préparation pharmaceutique - Google Patents
Préparation pharmaceutiqueInfo
- Publication number
- EP2717904A1 EP2717904A1 EP12730594.4A EP12730594A EP2717904A1 EP 2717904 A1 EP2717904 A1 EP 2717904A1 EP 12730594 A EP12730594 A EP 12730594A EP 2717904 A1 EP2717904 A1 EP 2717904A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- fsh
- stimulating hormone
- follicle stimulating
- sialylation
- glycan structures
- 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.)
- Withdrawn
Links
- 239000000825 pharmaceutical preparation Substances 0.000 title description 2
- 150000004676 glycans Chemical group 0.000 claims abstract description 102
- 238000002360 preparation method Methods 0.000 claims abstract description 47
- 102000012673 Follicle Stimulating Hormone Human genes 0.000 claims description 325
- 108010079345 Follicle Stimulating Hormone Proteins 0.000 claims description 325
- 229940028334 follicle stimulating hormone Drugs 0.000 claims description 322
- 230000009450 sialylation Effects 0.000 claims description 116
- 210000004027 cell Anatomy 0.000 claims description 76
- MBLBDJOUHNCFQT-UHFFFAOYSA-N N-acetyl-D-galactosamine Natural products CC(=O)NC(C=O)C(O)C(O)C(O)CO MBLBDJOUHNCFQT-UHFFFAOYSA-N 0.000 claims description 54
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 claims description 48
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 claims description 48
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 claims description 45
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 claims description 43
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 claims description 33
- 229950006780 n-acetylglucosamine Drugs 0.000 claims description 33
- 238000000034 method Methods 0.000 claims description 22
- 108090000623 proteins and genes Proteins 0.000 claims description 20
- 239000008194 pharmaceutical composition Substances 0.000 claims description 18
- 210000005260 human cell Anatomy 0.000 claims description 16
- 102000004169 proteins and genes Human genes 0.000 claims description 16
- OVRNDRQMDRJTHS-RTRLPJTCSA-N N-acetyl-D-glucosamine Chemical compound CC(=O)N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-RTRLPJTCSA-N 0.000 claims description 15
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 claims description 13
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 claims description 13
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 claims description 13
- 238000004519 manufacturing process Methods 0.000 claims description 12
- 230000036512 infertility Effects 0.000 claims description 10
- 208000000509 infertility Diseases 0.000 claims description 10
- 231100000535 infertility Toxicity 0.000 claims description 9
- 230000007935 neutral effect Effects 0.000 claims description 8
- 229940088597 hormone Drugs 0.000 claims description 7
- 239000005556 hormone Substances 0.000 claims description 7
- OVRNDRQMDRJTHS-CBQIKETKSA-N N-Acetyl-D-Galactosamine Chemical compound CC(=O)N[C@H]1[C@@H](O)O[C@H](CO)[C@H](O)[C@@H]1O OVRNDRQMDRJTHS-CBQIKETKSA-N 0.000 claims description 6
- 239000003814 drug Substances 0.000 claims description 4
- OVRNDRQMDRJTHS-KEWYIRBNSA-N N-acetyl-D-galactosamine Chemical compound CC(=O)N[C@H]1C(O)O[C@H](CO)[C@H](O)[C@@H]1O OVRNDRQMDRJTHS-KEWYIRBNSA-N 0.000 claims 7
- 230000004936 stimulating effect Effects 0.000 claims 1
- 108090000288 Glycoproteins Proteins 0.000 description 36
- 102000003886 Glycoproteins Human genes 0.000 description 36
- 239000000047 product Substances 0.000 description 30
- 239000000203 mixture Substances 0.000 description 25
- 102000003838 Sialyltransferases Human genes 0.000 description 21
- 108090000141 Sialyltransferases Proteins 0.000 description 21
- 125000005629 sialic acid group Chemical group 0.000 description 19
- 235000018102 proteins Nutrition 0.000 description 15
- 230000000694 effects Effects 0.000 description 13
- PXBFMLJZNCDSMP-UHFFFAOYSA-N 2-Aminobenzamide Chemical compound NC(=O)C1=CC=CC=C1N PXBFMLJZNCDSMP-UHFFFAOYSA-N 0.000 description 12
- 102000001708 Protein Isoforms Human genes 0.000 description 12
- 108010029485 Protein Isoforms Proteins 0.000 description 12
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 12
- 239000013604 expression vector Substances 0.000 description 10
- 229940057854 gonal f Drugs 0.000 description 9
- 108010018078 FE 999049 Proteins 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 8
- 238000009826 distribution Methods 0.000 description 8
- 239000000463 material Substances 0.000 description 8
- 230000036515 potency Effects 0.000 description 8
- 108091026890 Coding region Proteins 0.000 description 7
- 235000000346 sugar Nutrition 0.000 description 7
- 238000001890 transfection Methods 0.000 description 7
- 210000002700 urine Anatomy 0.000 description 7
- 102100031974 CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,3-sialyltransferase 4 Human genes 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 6
- 102000002287 alpha Subunit Glycoprotein Hormones Human genes 0.000 description 6
- 108010000732 alpha Subunit Glycoprotein Hormones Proteins 0.000 description 6
- 102000035824 beta Subunit Follicle Stimulating Hormone Human genes 0.000 description 6
- 108010081485 beta Subunit Follicle Stimulating Hormone Proteins 0.000 description 6
- 238000010276 construction Methods 0.000 description 6
- 108010006578 follitropin alfa Proteins 0.000 description 6
- 238000001727 in vivo Methods 0.000 description 6
- 230000001817 pituitary effect Effects 0.000 description 6
- 101000863864 Homo sapiens Beta-galactoside alpha-2,6-sialyltransferase 1 Proteins 0.000 description 5
- 108010082302 Human Follicle Stimulating Hormone Proteins 0.000 description 5
- 102000003864 Human Follicle Stimulating Hormone Human genes 0.000 description 5
- 102000000447 Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase Human genes 0.000 description 5
- 108010055817 Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase Proteins 0.000 description 5
- 150000002482 oligosaccharides Polymers 0.000 description 5
- 230000002485 urinary effect Effects 0.000 description 5
- 238000012935 Averaging Methods 0.000 description 4
- 102100029945 Beta-galactoside alpha-2,6-sialyltransferase 1 Human genes 0.000 description 4
- 102000011022 Chorionic Gonadotropin Human genes 0.000 description 4
- 108010062540 Chorionic Gonadotropin Proteins 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 4
- 101000703754 Homo sapiens CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,3-sialyltransferase 4 Proteins 0.000 description 4
- 102000005348 Neuraminidase Human genes 0.000 description 4
- 108010006232 Neuraminidase Proteins 0.000 description 4
- 230000002378 acidificating effect Effects 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 239000007972 injectable composition Substances 0.000 description 4
- 238000001155 isoelectric focusing Methods 0.000 description 4
- 238000002955 isolation Methods 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 239000002773 nucleotide Substances 0.000 description 4
- 125000003729 nucleotide group Chemical group 0.000 description 4
- 229920001542 oligosaccharide Polymers 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 229920001184 polypeptide Polymers 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 239000013598 vector Substances 0.000 description 4
- ZDRRIRUAESZNIH-BZGUUIOASA-N (2s)-1-[(4r,7s,10s,13s,16s,19r)-19-amino-7-(2-amino-2-oxoethyl)-13-[(2s)-butan-2-yl]-10-[(1r)-1-hydroxyethyl]-16-[(4-hydroxyphenyl)methyl]-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-pentazacycloicosane-4-carbonyl]-n-[(2s)-1-[(2-amino-2-oxoethyl)amino]- Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@H](C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CSSC[C@H](N)C(=O)N1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(C)C)C(=O)NCC(N)=O)[C@@H](C)O)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZDRRIRUAESZNIH-BZGUUIOASA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- 108010047196 Urofollitropin Proteins 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 229940024606 amino acid Drugs 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- -1 and the like) Substances 0.000 description 3
- 238000005349 anion exchange Methods 0.000 description 3
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 3
- 229940054242 bravelle Drugs 0.000 description 3
- 125000000837 carbohydrate group Chemical group 0.000 description 3
- 150000001720 carbohydrates Chemical class 0.000 description 3
- 235000014633 carbohydrates Nutrition 0.000 description 3
- 229940015047 chorionic gonadotropin Drugs 0.000 description 3
- 239000003937 drug carrier Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 230000013595 glycosylation Effects 0.000 description 3
- 238000006206 glycosylation reaction Methods 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 230000016087 ovulation Effects 0.000 description 3
- 230000000144 pharmacologic effect Effects 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 239000012679 serum free medium Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- HORYNJCIACQHGZ-BHVWUGLYSA-N 1-[(3R,4R,5S,6R)-3-amino-2,4-dihydroxy-6-(hydroxymethyl)-5-[(2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxan-2-yl]ethanone Chemical compound C(C)(=O)C1(O)[C@H](N)[C@@H](O)[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@@H](O)[C@H](O2)CO)[C@H](O1)CO HORYNJCIACQHGZ-BHVWUGLYSA-N 0.000 description 2
- VOXZDWNPVJITMN-ZBRFXRBCSA-N 17β-estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 VOXZDWNPVJITMN-ZBRFXRBCSA-N 0.000 description 2
- MSWZFWKMSRAUBD-GASJEMHNSA-N 2-amino-2-deoxy-D-galactopyranose Chemical group N[C@H]1C(O)O[C@H](CO)[C@H](O)[C@@H]1O MSWZFWKMSRAUBD-GASJEMHNSA-N 0.000 description 2
- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 description 2
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 2
- 101710083574 CMP-N-acetylneuraminate-beta-galactosamide-alpha-2,3-sialyltransferase 4 Proteins 0.000 description 2
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 2
- 102000008175 FSH Receptors Human genes 0.000 description 2
- 108010060374 FSH Receptors Proteins 0.000 description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 2
- 229930182816 L-glutamine Natural products 0.000 description 2
- 102000009151 Luteinizing Hormone Human genes 0.000 description 2
- 108010073521 Luteinizing Hormone Proteins 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical compound C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 102000011923 Thyrotropin Human genes 0.000 description 2
- 108010061174 Thyrotropin Proteins 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 229960001230 asparagine Drugs 0.000 description 2
- 235000009582 asparagine Nutrition 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 229920002988 biodegradable polymer Polymers 0.000 description 2
- 239000004621 biodegradable polymer Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- OSASVXMJTNOKOY-UHFFFAOYSA-N chlorobutanol Chemical compound CC(C)(O)C(Cl)(Cl)Cl OSASVXMJTNOKOY-UHFFFAOYSA-N 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 229930182833 estradiol Natural products 0.000 description 2
- 108010081934 follitropin beta Proteins 0.000 description 2
- 230000033581 fucosylation Effects 0.000 description 2
- 229960002442 glucosamine Drugs 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 108010067479 inhibin B Proteins 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- RLSSMJSEOOYNOY-UHFFFAOYSA-N m-cresol Chemical compound CC1=CC=CC(O)=C1 RLSSMJSEOOYNOY-UHFFFAOYSA-N 0.000 description 2
- 230000003285 pharmacodynamic effect Effects 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 230000003389 potentiating effect Effects 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 230000010473 stable expression Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 229960000874 thyrotropin Drugs 0.000 description 2
- 230000001748 thyrotropin Effects 0.000 description 2
- DQJCDTNMLBYVAY-ZXXIYAEKSA-N (2S,5R,10R,13R)-16-{[(2R,3S,4R,5R)-3-{[(2S,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}-5-(ethylamino)-6-hydroxy-2-(hydroxymethyl)oxan-4-yl]oxy}-5-(4-aminobutyl)-10-carbamoyl-2,13-dimethyl-4,7,12,15-tetraoxo-3,6,11,14-tetraazaheptadecan-1-oic acid Chemical compound NCCCC[C@H](C(=O)N[C@@H](C)C(O)=O)NC(=O)CC[C@H](C(N)=O)NC(=O)[C@@H](C)NC(=O)C(C)O[C@@H]1[C@@H](NCC)C(O)O[C@H](CO)[C@H]1O[C@H]1[C@H](NC(C)=O)[C@@H](O)[C@H](O)[C@@H](CO)O1 DQJCDTNMLBYVAY-ZXXIYAEKSA-N 0.000 description 1
- FJKROLUGYXJWQN-UHFFFAOYSA-N 4-hydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 1
- 108010002913 Asialoglycoproteins Proteins 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- LVGKNOAMLMIIKO-UHFFFAOYSA-N Elaidinsaeure-aethylester Natural products CCCCCCCCC=CCCCCCCCC(=O)OCC LVGKNOAMLMIIKO-UHFFFAOYSA-N 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108091006027 G proteins Proteins 0.000 description 1
- 102000030782 GTP binding Human genes 0.000 description 1
- 108091000058 GTP-Binding Proteins 0.000 description 1
- AEMRFAOFKBGASW-UHFFFAOYSA-N Glycolic acid Polymers OCC(O)=O AEMRFAOFKBGASW-UHFFFAOYSA-N 0.000 description 1
- 102000002068 Glycopeptides Human genes 0.000 description 1
- 108010015899 Glycopeptides Proteins 0.000 description 1
- 108700023372 Glycosyltransferases Proteins 0.000 description 1
- 239000000579 Gonadotropin-Releasing Hormone Substances 0.000 description 1
- 101000863891 Homo sapiens Beta-galactoside alpha-2,6-sialyltransferase 2 Proteins 0.000 description 1
- SQVRNKJHWKZAKO-LUWBGTNYSA-N N-acetylneuraminic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)CC(O)(C(O)=O)O[C@H]1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-LUWBGTNYSA-N 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 229920002732 Polyanhydride Polymers 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 229920000954 Polyglycolide Polymers 0.000 description 1
- 229920001710 Polyorthoester Polymers 0.000 description 1
- 102000048176 Prostaglandin-D synthases Human genes 0.000 description 1
- 108030003866 Prostaglandin-D synthases Proteins 0.000 description 1
- 108010001267 Protein Subunits Proteins 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 101000857870 Squalus acanthias Gonadoliberin Proteins 0.000 description 1
- 102000004357 Transferases Human genes 0.000 description 1
- 108090000992 Transferases Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 210000004198 anterior pituitary gland Anatomy 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 239000003429 antifungal agent Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000001593 cAMP accumulation Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 229960004926 chlorobutanol Drugs 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 239000006196 drop Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 230000007515 enzymatic degradation Effects 0.000 description 1
- 229960005309 estradiol Drugs 0.000 description 1
- BEFDCLMNVWHSGT-UHFFFAOYSA-N ethenylcyclopentane Chemical compound C=CC1CCCC1 BEFDCLMNVWHSGT-UHFFFAOYSA-N 0.000 description 1
- LVGKNOAMLMIIKO-QXMHVHEDSA-N ethyl oleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC LVGKNOAMLMIIKO-QXMHVHEDSA-N 0.000 description 1
- 229940093471 ethyl oleate Drugs 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000008217 follicular development Effects 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 102000045442 glycosyltransferase activity proteins Human genes 0.000 description 1
- 108700014210 glycosyltransferase activity proteins Proteins 0.000 description 1
- 230000002710 gonadal effect Effects 0.000 description 1
- XLXSAKCOAKORKW-AQJXLSMYSA-N gonadorelin Chemical compound C([C@@H](C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1[C@@H](CCC1)C(=O)NCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H]1NC(=O)CC1)C1=CC=C(O)C=C1 XLXSAKCOAKORKW-AQJXLSMYSA-N 0.000 description 1
- 229940035638 gonadotropin-releasing hormone Drugs 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 102000048059 human St6Gal2 Human genes 0.000 description 1
- 229940084986 human chorionic gonadotropin Drugs 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000009027 insemination Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 239000007951 isotonicity adjuster Substances 0.000 description 1
- 238000011545 laboratory measurement Methods 0.000 description 1
- 239000012669 liquid formulation Substances 0.000 description 1
- 239000006194 liquid suspension Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000001592 luteinising effect Effects 0.000 description 1
- 238000001906 matrix-assisted laser desorption--ionisation mass spectrometry Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000004530 micro-emulsion Substances 0.000 description 1
- 239000007922 nasal spray Substances 0.000 description 1
- 229940097496 nasal spray Drugs 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 229940060155 neuac Drugs 0.000 description 1
- CERZMXAJYMMUDR-UHFFFAOYSA-N neuraminic acid Natural products NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO CERZMXAJYMMUDR-UHFFFAOYSA-N 0.000 description 1
- 231100001079 no serious adverse effect Toxicity 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 210000000287 oocyte Anatomy 0.000 description 1
- 150000002895 organic esters Chemical class 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 230000027758 ovulation cycle Effects 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 239000008024 pharmaceutical diluent Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 239000008063 pharmaceutical solvent Substances 0.000 description 1
- 229960003742 phenol Drugs 0.000 description 1
- WVDDGKGOMKODPV-ZQBYOMGUSA-N phenyl(114C)methanol Chemical compound O[14CH2]C1=CC=CC=C1 WVDDGKGOMKODPV-ZQBYOMGUSA-N 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 230000004983 pleiotropic effect Effects 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
- 150000003077 polyols Chemical class 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 229940071643 prefilled syringe Drugs 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000013587 production medium Substances 0.000 description 1
- 239000000186 progesterone Substances 0.000 description 1
- 229960003387 progesterone Drugs 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 210000003370 receptor cell Anatomy 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000008247 solid mixture Substances 0.000 description 1
- 229940075582 sorbic acid Drugs 0.000 description 1
- 235000010199 sorbic acid Nutrition 0.000 description 1
- 239000004334 sorbic acid Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000003206 sterilizing agent Substances 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L sulfate group Chemical group S(=O)(=O)([O-])[O-] QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 229910021653 sulphate ion Inorganic materials 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002463 transducing effect Effects 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates to gonadotrophins for use in the treatment of infertility.
- gonadotrophins for use in the treatment of infertility.
- FSH follicle stimulating hormone
- the gonadotrophins are a group of heterodimeric glycoprotein hormones which regulate gonadal function in the male and female. They include follicle stimulating hormone (FSH), luteinising hormone (LH) and chorionic gonadotropin (CG).
- FSH follicle stimulating hormone
- LH luteinising hormone
- CG chorionic gonadotropin
- FSH is naturally secreted by the anterior pituitary gland and functions to support follicular development and ovulation.
- FSH comprises a 92 amino acid alpha sub-unit, also common to the other glycoprotein hormones LH and CG, and a 111 amino acid beta sub- unit unique to FSH that confers the biological specificity of the hormone (Pierce and Parsons, 1981).
- Each sub-unit is post translationally modified by the addition of complex carbohydrate residues. Both subunits carry 2 sites for N-linked glycan attachment, the alpha sub-unit at amino acids 52 and 78 and the beta sub-unit at amino acid residues 7 and 24 (Rathnam and Saxena, 1975, Saxena and Rathnam, 1976).
- FSH is thus glycosylated to about 30% by mass (Dias and Van Roey. 2001. Fox et al. 2001 ).
- FSH purified from post-menopausal human urine has been used for many years in infertility treatment; both to promote ovulation in natural reproduction and to provide oocytes for assisted reproduction technologies.
- Two recombinant versions of FSH, Gonal-F (Serono) and Puregon (Organon) became available in the mid-1990's. These are both expressed in Chinese hamster ovary (CHO) cells (Howies, 1996).
- FSH FSH
- Individual FSH isoforms exhibit identical amino acid sequences but differ in the extent to which they are post- translationally modified; particular isoforms are characterised by heterogeneity of the carbohydrate branch structures and differing amounts of sialic acid (a terminal sugar) incorporation, both of which appear to influence the specific isoform bioactivity.
- Glycosylation of natural FSH is highly complex.
- the glycans in naturally derived pituitary FSH can contain a wide range of structures that can include combinations of mono-, bi-, tri- and tetra-antennary glycans (Pierce and Parsons, 1981. Ryan ef a/., 1987. Baenziger and Green, 1988).
- the glycans can carry further modifications: core fucosylation, bisecting glucosamine, chains extended with acetyl lactosamine, partial or complete sialylation, sialylation with a2,3 and a2,6 linkages, and sulphated galactosamine substituted for galactose (Dalpathado ef al., 2006).
- glycan structures there are differences between the distributions of glycan structures at the individual glycosylation sites.
- a comparable level of glycan complexity has been found in FSH derived from the serum of individuals and from the urine of post-menopausal women (Wide et al., 2007).
- the glycosylation of recombinant FSH products reflects the range of glycosyl- transferases present in the host cell line.
- Commercially available rFSH products are derived from engineered Chinese hamster ovary cells (CHO cells). The range of glycan modifications in CHO cell derived rFSH are more limited than those found on the natural products.
- Examples of the reduced glycan heterogeneity found in CHO derived rFSH include a lack of bisecting glucosamine and a reduced content of core fucosylation and acetyl lactosamine extensions (Hard et al., 1990).
- CHO cells are only able to add sialic acid using the a2,3 linkage (Kagawa et al, 1988, Takeuchi et al, 1988, Svensson et al., 1990); CHO cell derived rFSH only includes a2,3-linked sialic acid and does not include a2,6-linked sialic acid. This is different from naturally produced FSH (e.g. human Pituitary/ serum/ urinary FSH) which contains glycans with a mixture of a2,3 and a2,6- linked sialic acid, with a predominance of the former.
- FSH e.g. human Pituitary/ serum/ urinary FSH
- a recombinant FSH preparation differs in the amounts of FSH with an isoelectric point (pi) of below 4 (considered the acidic isoforms) when compared to pituitary, serum or post-menopausal urine FSH (Ulloa-Aguirre et al. 1995).
- the amount of acidic isoforms in the urinary preparations was much higher as compared to the recombinant products, Gonal-f (Serono) and Puregon (Organon) (Andersen et al. 2004).
- the circulatory life-time of FSH has been documented for materials from a variety of sources. Some of these materials have been fractionated on the basis of overall molecular charge, as characterised by their pi, in which more acid equates to a higher negative charge.
- the major contributor to overall molecular charge is the total sialic content of each FSH molecule.
- rFSH Organon
- urine-derived FSH has a higher sialic acid content
- the corresponding plasma clearance rates in the rat are 0.34 and 0.14 ml/min (Ulloa-Aguirre et al. 2003).
- recombinant proteins expressed using the CHO system will differ from their natural counterparts in their type of terminal sialic acid linkages. This is an important consideration in the production of biologicals for pharmaceutical use since the carbohydrate moieties may contribute to the pharmacological attributes of the molecule.
- Recombinant FSH with a mixture of both a2,3 and a2,6-linked sialic acid was made by engineering a human cell line to express both rFSH and a2,3 sialyltransferase.
- the expressed product is highly acidic and carries a mix of both a2,3- and a2,6-linked sialic acids; the latter provided by the endogenous sialyl transferase activity.
- the rFSH product disclosed in International Patent Application No. PCT/GB2009/000978 contains branched glycan moieties.
- FSH comprises glycans (attached to the FSH glycoproteins) and these glycans may contain a wide variety of structures.
- branching can occur with the result that the glycan may have 1 , 2, 3, 4 or more terminal sugar residues or "antennae”; glycans with 1 , 2, 3 or 4 terminal sugar residues or "antennae” are referred to respectively as mono- antennary, di-antennary, tri-antennary or tetra-antennary structures.
- Glycans may have sialylation presence on mono-antennary and/or di-antennary and/or tri-antennary and/or tetra-antennary structures.
- a mono-sialylated glycan structure carries one sialic acid residue; a di-sialylated glycan structure carries two sialic acid residues; a tri-sialylated glycan structure carries three sialic acid residues; and a tetra-sialylated glycan structure carries four sialic acid residues.
- X% mono-sialylated refers to the number of glycan structures on FSH which are mono-, di, tri or tetra sialylated (respectively), expressed as a percentage (X%) of the total number of glycan structures on the FSH which are sialylated in any way (carry sialic acid).
- the phrase "20 - 36% tri-sialylated glycan structures” means that, of the total number of glycan structures on the FSH which carry sialic acid residues (that is, are sialylated), 20 to 36% of these glycan structures are tri sialylated (carry three sialic acid residues).
- follicle stimulating hormone including mono-, di-, tri- and tetra-sialylated glycan structures, wherein 15-24%, for example 17-23% of the sialylated glycan structures are tetrasialylated glycan structures (e.g. as shown by WAX analysis of charged glycans, as set out in the Examples below).
- the FSH includes cr2,3- and ct2,6- sialylation.
- the FSH (rFSH) according to the invention may have 1 % to 99% of the total sialylation being a2,3-sialylation.
- the FSH (rFSH) according to the invention may have 1% to 99% of the total sialylation being a2,6- sialylation.
- 50 to 70% for example 60 to 69%, for example about 65%, of the total sialylation is a2,3-sialylation.
- the FSH comprises glycans (attached to the FSH glycoproteins). It is well known that glycans in FSH may contain a wide variety of structures. These may include combinations of mono, bi, tri and tetra-antennary glycans.
- terminology such as "X% of the sialylated glycan structures are tetrasialylated glycan structures" refers to the number of glycan structures on the FSH which are tetra sialylated, i.e. carry four sialic acid residues, expressed as a percentage (X%) of the total number of glycan structures on the FSH which are sialylated in any way (carry sialic acid).
- the phrase "15-24% of the sialylated glycan structures are tetrasialylated glycan structures" means that, of the total number of glycan structures on FSH which carry sialic acid residues (that is, are sialylated), 15 to 24% of these glycan structures are tetra sialylated (carry four sialic acid residues).
- a follicle stimulating hormone preparation for example a recombinant follicle stimulating hormone preparation, comprising follicle stimulating hormone (FSH) including mono-, di-, tri- and tetra-sialylated glycan structures, wherein 15-24%, for example 17-23% of the sialylated glycan structures are tetrasialylated glycan structures.
- FSH follicle stimulating hormone
- the FSH includes a2,3- and a2,6- sialylation.
- the FSH (rFSH) preparation according to the invention may have 1% to 99% of the total sialylation being a2,3-sialylation.
- the FSH (rFSH) preparation according to the invention may have 1% to 99% of the total sialylation being a2,6-sialylation.
- 50 to 70% for example 60 to 69%, for example about 65%, of the total sialylation is a2,3- sialylation.
- the term "recombinant FSH preparation" includes a preparation for e.g. pharmaceutical use which includes recombinant FSH.
- the rFSH may be present as a single isoform or as a mixture of isoforms.
- the FSH is a recombinant FSH ("rFSH" or "recFSH”).
- FSH is a human cell line derived recombinant FSH.
- the rFSH may preferably include 27 - 33%, for example 30 - 32%, tri-sialylated glycan structures.
- the rFSH may preferably include 24 - 33%, for example 26 - 30%, di- sialylated glycan structures.
- the rFSH may preferably include 12 - 21 %, for example 15 - 17%, mono-sialylated glycan structures.
- the rFSH preferably includes mono-sialylated, di- sialylated, tri- sialylated and tetra- sialylated glycan structures with relative amounts as follows: 15 to 17% mono-sialylated; 26 - 30% di-sialylated; 27 - 33% (e.g. 29 to 32%, e.g 30-32%, e.g 30 to 31 %) tri-sialylated and 17 - 23 % tetra-sialylated (e.g. as shown by WAX analysis of charged glycans, as set out in the Examples).
- the rFSH may include from 0 to 7%, for example 3 to 6%, for example 5 to 6%, neutral sialylated structures.
- the FSH comprises glycans (attached to the FSH glycoproteins).
- terminology such as “X% mono-sialylated”, “X% di-sialylated”, “X% tri-sialylated” or “X% tetra-sialylated” refers to the number of glycan structures on FSH which are mono-, di, tri or tetra sialylated (respectively), expressed as a percentage (X%) of the total number of glycan structures on the FSH which are sialylated in any way (carry sialic acid).
- the phrase "27 - 33% tri-sialylated glycan structures” means that, of the total number of glycan structures on FSH which carry sialic acid residues (that is, are sialylated), 27 to 33% of these glycan structures are tri sialylated (carry three sialic acid residues).
- the rFSH (or rFSH preparation) according to the invention may have a sialic acid content [expressed in terms of a ratio of moles of sialic acid to moles of protein] of 6 mol/mol or greater, for example between 6 mol/mol and 15 mo!/mol, e.g between 8 mol/mol and 14 mol/mol, for example between 10 mol/mol and 14 mol/mol, e.g between 11 mol/mol and 14 mol/mol, e.g between 12 mol/mol and 14 mol/mol, e.g. between 12 mol/mol and 13 mol/mol.
- the rFSH of the invention may be produced or expressed in a human cell line.
- the rFSH (or rFSH preparation) according to the invention may have 10% or more of the total sialylation being a2,3-sia)ylation. For example, 20, 30, 40, 50, 60, 70, 80 or 90% or more of the total sialylation may be d2,3-sialylation.
- the rFSH (or rFSH preparation) may preferably include a2,3-sialylation in an amount which is from 50 to 70% of the total sialylation, for example from 60 to 69% of the total sialylation, for example from 63 to 67%, for example around 65% of the total sialylation.
- the rFSH (or rFSH preparation) of the invention may have 5% or more, for example 5% to 99%, of the total sialylation being a2,6- sialylation.
- the rFSH (or rFSH preparation) of the invention may have 50% or less of the total sialylation being a2,6-sialylation.
- the rFSH (or rFSH preparation) may preferably include a2,6-sialylation in an amount which is from 25 to 50% of the total sialylation, for example from 30 to 50% of the total sialylation, for example from 31 to 38%, for example around 35% of the total sialylation.
- sialylation it is meant the amount of sialic residues present on the FSH carbohydrate structures.
- a2,3-sialylation means sialylation at the 2,3 position (as is well known in the art) and a2,6 sialylation at the 2,6 position (also well known in the art).
- % of the total sialylation may be a 2,3 sialylation” refers to the % of the total number of sialic acid residues present in the FSH which are sialyiated in the 2,3 position.
- the term "% of the total sialylation being a2,6-sialylation” refers to the % of the total number of sialic acid residues present in the FSH which are sialyiated in the 2,6 position.
- the rFSH (or rFSH preparation) according to the invention may have a sialic acid content (amount of sialylation per FSH molecule) of (based on the mass of protein, rather than the mass of protein plus carbohydrate) of 6% or greater (e.g. between 6% and 15%, e.g. between 7% and 13%, e.g. between 8% and 12%, e.g. between 11 % and 15%, e.g. between 12% and 14%) by mass.
- sialic acid content amount of sialylation per FSH molecule
- the rFSH (or rFSH preparation) according to the invention may be FSH or a FSH preparation in which 16 % or fewer (e.g. 0.1 to 16%) of the glycans comprise (e.g. carry) bisecting N-acetylglucosamine (bisecting GlcNAc or bisGlcNAc).
- the rFSH (or rFSH preparation) according to the invention is an FSH or FSH preparation in which 8 to 14.5% of the glycans comprise (e.g. carry) a bisecting N-acetylglucosamine (bisecting GlcNAc or bisGlcNAc).
- FSH comprises glycans attached to the FSH glycoproteins. It will also be understood that 00% of the glycans refers to or means all of the glycans attached to the FSH glycoproteins.
- the terminology "8 to 14.5% of the glycans comprise (carry) bisecting N-acetylglucosamine” means that 8 to 14.5% of the total number of glycans attached to the FSH glycoproteins include/carry bisecting N- acetylglucosamine; "16% or fewer of the glycans comprise (carry) bisecting N- acetylglucosamine” means that 16 % or fewer of the total number of glycans attached to the FSH glycoproteins include/carry bisecting N-acetylglucosamine, and so on.
- recombinant FSH rFSH preparations; rFSH compositions
- 16% or fewer (e.g. 8 to 14.5%) of the glycans comprised in the FSH glycoproteins carry bisecting GlcNac may have advantageous pharmacokinetic properties. It is believed the advantageous properties may arise because the amount of glycans which carry bisecting GlcNac is similar to that in the human urinary derived product Bravelle, which is rather less than that of other recombinant FSH preparations such as those disclosed in WO2012/017058.
- the rFSH (or rFSH preparation) according to the invention may be an FSH or FSH preparation in which 20% or more of the glycans comprise (e.g. carry) N- Acety!galactosamine (GalNAc), for example in which 20% or more of the glycans comprise (e.g. carry) a terminal GalNAc.
- the rFSH (or rFSH preparation) according to the invention is an FSH or FSH preparation in which the 40 to 55%, for example 42% to 52%, of the glycans comprise (e.g. carry) GalNAc.
- the rFSH (or rFSH preparation) according to the invention is an FSH or FSH preparation in which the 40 to 55%, for example 42% to 52%, of the glycans comprise (e.g. carry) terminal GalNAc.
- FSH comprises glycans attached to the FSH glycoproteins. It will also be understood that 100% of the glycans refers to or means all of the glycans attached to the FSH glycoproteins.
- the terminology "wherein 20% or more of the glycans comprise (e.g. carry) GalNAc” means that 20% or more of the total number of glycans attached to the FSH glycoproteins include/carry N- Acetylgalactosamine (GalNAc); "40 to 55%, for example 42% to 52%, of the glycans comprise (e.g. carry) terminal GalNAc” means that 40 to 55 %, for example 42% to 52%, of the total number of glycans attached to the FSH glycoproteins include/carry terminal GalNAc, and so on.
- rFSH is distinguished over other approved products because of the sugar composition: it includes, or may include, a specific amount of GalNac. This may be linked to tetrasialylation and potency because the 2,6- sialylation is associated with GalNac.
- the present applicants have developed an rFSH product which includes specific characteristics (2,6- linker sites, GalNac) which provide rFSH with high degree of sialylation, which appears to lead to improved potency in vivo.
- the rFSH (or rFSH preparation) may have 16 to 24% of the glycans comprising (e.g. terminal) 1 fucose-lewis, for example 16.5 to 18% of the glycans comprising (e.g. terminal) 1 fucose-lewis.
- the rFSH (or rFSH preparation) may have 1.5 to 4.5%, for example 2 to 4%, for example 3.7%, of the glycans comprising (e.g. terminal) 2 fucose - lewis.
- the content of fucose-lewis may have an effect on potency.
- the rFSH of the invention may be produced or expressed in a human cell line, for example a Per.C6 cell line, a HEK293 cell line, a HT1080 cell line etc.. This may simplify (and render more efficient) the production method because manipulation and control of e.g. the cell growth medium to retain sialylation may be less critical than with known processes.
- the method may also be more efficient because there is little basic rFSH produced compared to production of known rFSH products; more acidic rFSH is produced and separation/removal of basic FSH is less problematic.
- the rFSH may be produced or expressed in a PER.C6® cell line, a PER.C6® derived cell line or a modified PER.C6® cell line.
- rFSH which is produced or expressed in a human cell line (e.g. PER.C6® cell line, HEK293 cell line, HT1080 cell line etc.) will include some a2,6-linked sialic acids ( ⁇ 2,6 sialylation) provided by endogenous sialyl transferase activity [of the cell line] and will include some a2,3-linked sialic acids (a2,3 sialylation) provided by endogenous sialyl transferase activity.
- the cell line may be modified using a2,3-sialyltransferase.
- the cell line may be modified using a2,6-sialyltransferase.
- the rFSH may include a2,6-linked sialic acids (a2,6 sialylation) provided by endogenous sialyl transferase activity [of the cell line].
- the rFSH may be produced using a2,3- and/or a2,6-sialyltransferase.
- rFSH is produced using a2,3- sialyltransferase.
- the rFSH may include Delinked sialic acids (a2,6 sialylation) provided by endogenous sialyl transferase activity.
- a method of production of rFSH and/or an rFSH preparation as described herein comprising the step of producing or expressing the rFSH in a human cell line, for example a PER.C6® cell line, a PER.C6® derived cell line or a modified PER.C6® cell line, for example a cell line which has been modified using a2,3-sialyltransferase.
- a human cell line for example a PER.C6® cell line, a PER.C6® derived cell line or a modified PER.C6® cell line, for example a cell line which has been modified using a2,3-sialyltransferase.
- rFSH produced (e.g. expressed) in a human cell line, the rFSH including including mono-, di-, tri- and tetra-sialylated glycan structures, wherein 15-24%, for example 17-23% of the sialylated glycan structures are tetrasialylated glycan structures (e.g. as shown by WAX analysis of charged glycans, as set out in the Examples below).
- the FSH includes ⁇ 2,3- and a2,6- sialylation.
- the FSH (rFSH) according to the invention may have 1% to 99% of the total sialylation being a2,3-sialylation.
- the FSH (rFSH) according to the invention may have 1% to 99% of the total sialylation being a2,6-sia!ylation.
- 50 to 70% for example 60 to 69%, for example about 65%
- of the total sialylation is a2,3- sialylation.
- 25 to 50%, for example 30 to 50 %, for example 31 to 38%, for example about 35%, of the total sialylation is ⁇ 2,6- sialylation.
- the rFSH may be produced or expressed in a PER.C6® cell line, a PER.C6® derived cell line or a modified PER.C6® cell line.
- the cell line may be modified using a2,3- sialyltransferase.
- the cell line may be modified using a2,6-sialyltransferase.
- the rFSH may include a2,6-linked sialic acids (a2,6 sialylation) provided by endogenous sialyl transferase activity [of the cell line].
- the rFSH (or rFSH preparation) may have 40% or more of the total sialylation being a2,3-sia)ylation, for example 50-70%, for example 60 to 69%, for example about 65%, of the total sialylation may be a2,3- sialylation.
- the rFSH of the invention may have 5% or more, for example 5% to 99%, of the total sialylation being d2,6-sialylation.
- the rFSH (or rFSH preparation) of the invention may have 50% or less of the total sialylation being a2,6-sialylation, for example 30 to 50 %, for example 31 to 38%, for example about 35%,of the total sialylation may be a2,6- sialylation.
- the rFSH may have a sialic acid content [expressed in terms of a ratio of moles of sialic acid to moles of protein] of 6 mol/mol or greater, for example between 6mol/mo! and 15 mol/mol.
- the rFSH (produced in the human cell line) may be an FSH comprising glycans wherein 16 % or fewer (e.g. 0.1 to 16%) of the glycans comprise (e.g. carry) bisecting N- acetylglucosamine (bisecting GlcNAc or bisGlcNAc).
- the rFSH is an FSH comprising glycans wherein 8 to 14.5% of the glycans comprise (e.g. carry) a bisecting N- acetylglucosamine (bisecting GicNAc or bisGlcNAc).
- FSH comprises glycans attached to the FSH glycoproteins. It will also be understood that 100% of the glycans refers to or means all of the glycans attached to the FSH glycoproteins.
- the terminology "8 to 14.5% of the glycans comprise (carry) bisecting N-acetylglucosamine” means that 8 to 14.5% of the total number of glycans attached to the FSH glycoproteins include/carry bisecting N- acetylglucosamine; "16% or fewer of the glycans comprise (carry) bisecting N- acetylglucosamine” means that 16 % or fewer of the total number of glycans attached to the FSH glycoproteins include/carry bisecting N-acetylglucosamine, and so on.
- recombinant FSH rFSH preparations; rFSH compositions
- 16% or fewer (e.g. 8 to 14.5%) of the glycans comprised in the FSH glycoproteins carry bisecting GlcNac may have advantageous pharmacokinetic properties.
- the rFSH (produced in the human cell line) may be an FSH in which 20% or more of the glycans comprise (e.g. carry) N-Acetylgalactosamine (GalNAc), for example in which 20% or more of the glycans comprise (e.g. carry) a terminal GalNAc.
- the rFSH is an FSH in which the 40 to 55%, for example 42% to 52%, of the glycans comprise (e.g. carry) GalNAc.
- the rFSH is an FSH in which the 40 to 55%, for example 42% to 52%, of the glycans comprise (e.g. carry) terminal GalNAc.
- FSH comprises glycans attached to the FSH glycoproteins. It will also be understood that 100% of the glycans refers to or means all of the glycans attached to the FSH glycoproteins.
- the terminology "wherein 20% or more of the glycans comprise (e.g. carry) GalNAc” means that 20% or more of the total number of glycans attached to the FSH glycoproteins include/carry N- Acetylgalactosamine (GalNAc); "40 to 55%, for example 42% to 52%, of the glycans comprise (e.g. carry) terminal GalNAc” means that 40 to 55 %, for example 42% to 52%, of the total number of glycans attached to the FSH glycoproteins include/carry terminal GalNAc, and so on.
- rFSH is distinguished over other approved products because of the sugar composition: it includes, or may include, a specific amount of GalNac. This may be linked to tetrasialylation and potency because the 2,6- sialylation is associated with GalNac.
- the present applicants have developed an rFSH product which includes specific characteristics (2,6- linker sites, GalNac) which provide rFSH with high degree of sialylation, which appears to lead to improved potency in vivo.
- the rFSH (produced in a human cell line) may have 16 to 24% of the glycans comprising (e.g. terminal) 1 fucose-lewis, for example 16.5 to 18% of the glycans comprising (e.g. terminal) 1 fucose-lewis.
- the rFSH (produced in a human cell line) may have 1.5 to 4.5%, for example 2 to 4%, for example 3.7%, of the glycans comprising (e.g. terminal) 2 fucose -lewis.
- a (recombinant) follicle stimulating hormone (FSH) wherein 16 % or fewer (e.g. 0.1 to 16%) of the glycans comprise (e.g. carry) bisecting N-acetylglucosamine (bisecting GlcNAc or bisGlcNAc).
- FSH follicle stimulating hormone
- the glycans comprise (e.g. carry) bisecting N-acetylglucosamine (bisecting GlcNAc or bisGlcNAc).
- a pharmaceutical composition or a preparation comprising a (recombinant) follicle stimulating hormone (FSH) wherein 16 % or fewer (e.g. 0.1 to 16%) of the glycans comprise (e.g. carry) bisecting N-acetylglucosamine (bisecting GlcNAc or bisGlcNAc).
- FSH follicle stimulating hormone
- the glycans comprise (e.g. carry) bisecting N-acetylglucosamine (bisecting GlcNAc or bisGlcNAc).
- FSH comprises glycans attached to the FSH glycoproteins. It will also be understood that 100% of the glycans refers to or means all of the glycans attached to the FSH glycoproteins.
- the terminology "8 to 1 .5% of the glycans comprise (carry) bisecting N-acetylglucosamine” means that 8 to 14.5% of the total number of glycans attached to the FSH glycoproteins include/carry bisecting N- acetylglucosamine; "16% or fewer of the glycans comprise (carry) bisecting N- acetylglucosamine” means that 16 % or fewer of the total number of glycans attached to the FSH glycoproteins include/carry bisecting N-acetylglucosamine, and so on.
- recombinant FSH rFSH preparations; rFSH compositions
- 6% or fewer (e.g. 8 to 14.5%) of the glycans comprised in the FSH glycoproteins carry bisecting GlcNac may have advantageous pharmacokinetic properties. It is believed the advantageous properties may arise because the amount of glycans which carry bisecting GlcNac is similar to that in the human urinary derived product Bravelle, and is rather less than that of other recombinant FSH preparations such as those disclosed in WO2012/017058.
- the FSH is a recombinant FSH.
- the recombinant FSH is produced or expressed in a human cell line, for example a PER.C6® cell line, a PER.C6® derived cell line or a modified PER.C6® cell line.
- the follicle stimulating hormone (FSH) may include a2,3- sialylation and a2,6- sialylation.
- the FSH (rFSH) may have 1% to 99% of the total sialylation being a2,3- sialylation.
- the FSH (rFSH) may have 1% to 99% of the total sialylation being a2,6- sialylation.
- Preferably 25 to 50%, for example 30 to 50%, of the total sialylation is a2, 6- sialylation.
- Preferably 50 to 70% of the total sialylation is ⁇ 2,3- sialylation.
- the follicle stimulating hormone may include mono-, di-, tri- and tetra- sialylated glycan structures, wherein 15-24%, for example 17-23% of the sialylated glycan structures are tetrasialylated glycan structures. Preferably 27 to 33% of the sialyated glycan structures are tri-sialylated glycan structures. Preferably, 24 to 33% of the sialyated glycan structures are di-sialylated glycan structures. Preferably, 12 to 21% of the sialyated glycan structures are mono-sialylated glycan structures.
- the FSH includes from 0.1 to 7% neutral glycan structures.
- the FSH has a sialic acid content [expressed in terms of a ratio of moles of sialic acid to moles of protein] of 6 mol/mol or greater, for example between 6mol/mol and 15 mol/mol.
- the rFSH may be an FSH in which 20% or more of the glycans comprise (e.g. carry) N-Acetylgalactosamine (GalNAc), for example in which 20% or more of the glycans comprise (e.g. carry) a terminal GalNAc.
- GalNAc N-Acetylgalactosamine
- the rFSH is an FSH in which the 40 to 55%, for example 42% to 52%, of the glycans comprise (e.g. carry) GalNAc.
- the rFSH is an FSH in which the 40 to 55%, for example 42% to 52%, of the glycans comprise
- GalNAc (e.g. carry) terminal GalNAc.
- the rFSH may have 6 to 24% of the glycans comprising (e.g. terminal) 1 fucose- lewis, for example 16.5 to 18% of the glycans comprising (e.g. terminal) 1 fucose-lewis.
- the rFSH may have 1.5 to 4.5%, for example 2 to 4%, for example 3.7%, of the glycans comprising (e.g. terminal) 2 fucose -lewis.
- a follicle stimulating hormone wherein 20% or more of the glycans comprise (e.g. carry)
- GalNAc a terminal GalNAc.
- GalNAc Preferably 40 to 55%, for example 42% to 52%, of the glycans comprise (e.g. carry) terminal GalNAc.
- a pharmaceutical composition or a preparation comprising a follicle stimulating hormone (FSH) wherein 20% or more of the glycans comprise (e.g. carry) GalNAc.
- FSH follicle stimulating hormone
- FSH follicle stimulating hormone
- glycans comprise (e.g. carry) GalNAc.
- glycans Preferably 40 to 55%, for example 42% to 52%, of the glycans comprise (e.g. carry) terminal GalNAc.
- FSH comprises glycans attached to the FSH glycoproteins. It will also be understood that 100% of the glycans refers to or means all of the glycans attached to the FSH glycoproteins.
- the terminology "wherein 20% or more of the glycans comprise (e.g. carry) GalNAc” means that 20% or more of the total number of glycans attached to the FSH glycoproteins include/carry GalNAc; "40 to 55%, for example 42% to 52%, of the glycans comprise (e.g. carry) terminal GalNAc” means that 40 to 55 %, for example 42% to 52%, of the total number of glycans attached to the FSH glycoproteins include/carry terminal GalNAc, and so on.
- the FSH is a recombinant FSH.
- the recombinant FSH is produced or expressed in a human cell line, for example a PER.C6® cell line, a PER.C6® derived cell line or a modified PER.C6® cell line.
- the follicle stimulating hormone (FSH) may include a2,3- sialylation and a2,6- sialylation.
- the FSH (rFSH) may have 1% to 99% of the total sialylation being a2,3- sialylation.
- the FSH (rFSH) may have 1% to 99% of the total sialylation being a2,6- sialylation.
- Preferably 25 to 50%, for example 30 to 50%, of the total sialylation is a2, 6- sialylation.
- Preferably 50 to 70% of the total sialylation is a 2,3- sialylation.
- rFSH is distinguished over other approved products because of the sugar composition: it includes, or may include, a specific amount of GalNac. This may be linked to tetrasialylation and potency because the 2,6- sialylation is associated with GalNac.
- the present applicants have developed an rFSH product which includes specific characteristics (2,6- linker sites, GalNac) which provide rFSH with high degree of sialylation, which appears to lead to improved potency in vivo.
- the follicle stimulating hormone may include mono-, di-, tri- and tetra- sialylated glycan structures, wherein 15-24%, for example 17-23% of the sialylated glycan structures are tetrasialylated glycan structures. Preferably 27 to 33% of the sialyated glycan structures are tri-sialylated glycan structures. Preferably, 24 to 33% of the sialyated glycan structures are di-sialylated glycan structures. Preferably, 12 to 21% of the sialyated glycan structures are mono-sialylated glycan structures.
- the FSH includes from 0.1 to 7% neutral glycan structures.
- the FSH has a sialic acid content [expressed in terms of a ratio of moles of sialic acid to moles of protein] of 6 mol/mol or greater, for example between 6mol/mol and 15 mol/mol.
- the rFSH (produced in the human cell line) may be an FSH comprising glycans wherein 16 % or fewer (e.g. 0.1 to 16%) of the glycans comprise (e.g. carry) bisecting N- acetylglucosamine (bisecting GlcNAc or bisGlcNAc).
- the rFSH is an FSH comprising glycans wherein 8 to 14.5% of the glycans comprise (e.g. carry) a bisecting N- acetylglucosamine (bisecting GlcNAc or bisGlcNAc).
- the rFSH may have 16 to 24% of the glycans comprising (e.g. terminal) 1 fucose- lewis, for example 16.5 to 18% of the glycans comprising (e.g. terminal) 1 fucose-lewis.
- the rFSH may have 1.5 to 4.5%, for example 2 to 4%, for example 3.7%, of the glycans comprising (e.g. terminal) 2 fucose -lewis.
- a recombinant follicle stimulating hormone wherein 16 to 24% of the glycans comprise (e.g. terminal) 1 fucose-lewis.
- the rFSH may have 15 to 23% of the glycans comprising (e.g. terminal) 1 fucose-lewis, for example 16.5 to 18% of the glycans comprising (e.g. terminal) 1 fucose-lewis.
- a recombinant follicle stimulating hormone wherein 1.5 to 4.5% of the glycans comprise (e.g. terminal) 2 fucose-lewis.
- the rFSH may have 1,5 to 4.5%, for example 2 to 4%, for example 3.7%, of the glycans comprising (e.g. terminal) 2 fucose - lewis.
- a pharmaceutical composition comprising a recombinant follicle stimulating hormone (FSH) wherein 16 to 24% of the glycans comprise (e.g. terminal) 1 fucose-lewis.
- FSH recombinant follicle stimulating hormone
- the rFSH may have 15 to 23% of the glycans comprising (e.g. terminal) 1 fucose-lewis, for example 16.5 to 18% of the glycans comprising (e.g. terminal) 1 fucose-lewis.
- a pharmaceutical composition comprising a recombinant follicle stimulating hormone (FSH) wherein 1.5 to 4.5% of the glycans comprise (e.g. terminal) 2 fucose-lewis.
- FSH follicle stimulating hormone
- the rFSH may have 1.5 to 4.5%, for example 2 to 4%, for example 3.7%, of the glycans comprising (e.g. terminal) 2 fucose -lewis.
- a pharmaceutical composition comprising (e.g. recombinant) follicle stimulating hormone (rFSH) including including mono-, di-, tri- and tetra-sialylated glycan structures, wherein 15- 24%, for example 17-23% of the sialylated glycan structures are tetrasialylated glycan structures (e.g. as shown by WAX analysis of charged glycans, as set out in the Examples below).
- rFSH follicle stimulating hormone
- the pharmaceutical composition may further comprise hCG and/or LH and/or LH activity.
- the rFSH may include a2,3- and a2,6-sialylation.
- the FSH (rFSH) may have 1% to 99% of the total sialylation being a2,3-sialylation.
- the FSH (rFSH) may have 1% to 99% of the total sialylation being a2,6-sialylation.
- the rFSH may be produced or expressed in a PER.C6® cell line, a PER.C6® derived cell line or a modified PER.C6® cell line.
- the cell line may be modified using a2,3-sialyltransf erase.
- the cell line may be modified using a2,6-sialyltransferase.
- the rFSH may include a2,6-linked sialic acids (a2,6 sialylation) provided by endogenous sialyl transferase activity [of the cell line].
- the rFSH (or rFSH preparation) may have 40% or more of the total sialylation being a2,3- sialylation, for example 50-70%, for example 60 to 69%, for example about 65%, of the total sialylation may be a2,3-sialylation.
- the rFSH of the invention may have 5% or more, for example 5% to 99%, of the total sialylation being a2,6-sialylation.
- the rFSH (or rFSH preparation) of the invention may have 50% or less of the total sialylation being a2,6- sialylation, for example 25-50%, for example 30 to 50 %, for example 31 to 38%, for example about 35%, of the total sialylation may be a2,6- sialylation.
- the rFSH may have a sialic acid content [expressed in terms of a ratio of moles of sialic acid to moles of protein] of 6 mol/mol or greater, for example between 6mol/mol and 15 mol/mol.
- 50 to 70% of the total sialylation is a2, 3-sialylation.
- 25 to 50 %, e.g. 30 to 50%, of the total sialylation is a2, 6- sialylation.
- the rFSH may be an FSH comprising glycans wherein 16 % or fewer (e.g. 0.1 to 16%) of the glycans comprise (e.g. carry) bisecting N-acetylglucosamine (bisecting GlcNAc or bisGlcNAc).
- the rFSH is an FSH comprising glycans wherein 8 to 14.5% of the glycans comprise (e.g. carry) a bisecting N-acetylglucosamine (bisecting GlcNAc or bisGlcNAc).
- the rFSH may be an FSH in which 20% or more of the glycans comprise (e.g. carry) N-Acetylgalactosamine (GalNAc), for example in which 20% or more of the glycans comprise (e.g. carry) a terminal GalNAc.
- GalNAc N-Acetylgalactosamine
- the rFSH is an FSH in which the 40 to 55%, for example 42% to 52%, of the glycans comprise (e.g. carry) GalNAc.
- the rFSH is an FSH in which the 40 to 55%, for example 42% to 52%, of the glycans comprise (e.g. carry) terminal GalNAc.
- a pharmaceutical composition comprising (e.g. recombinant) follicle stimulating hormone (rFSH) wherein 16 % or fewer (e.g. 0.1 to 16%) of the glycans comprise (e.g. carry) bisecting N-acetylglucosamine (bisecting GlcNAc or bisGlcNAc). Preferably 8 to 14.5% of the glycans comprise (e.g. carry) a bisecting N-acetylglucosamine (bisecting GlcNAc or bisGlcNAc).
- the pharmaceutical composition may further comprise hCG and/or LH and/or LH activity.
- a pharmaceutical composition e.g. recombinant follicle stimulating hormone (rFSH) wherein 20% or more of the glycans comprise (e.g. carry) GalNAc.
- 20% or more of the glycans comprise (e.g. carry) a terminal GalNAc.
- 40 to 55%, for example 42% to 52%, of the glycans comprise (e.g. carry) GalNAc.
- 40 to 55%, for example 42% to 52%, of the glycans comprise (e.g. carry) terminal GalNAc.
- the pharmaceutical composition may further comprise hCG and/or LH and/or LH activity.
- hCG can be obtained by any means known in the art.
- hCG as used herein includes humah-derived and recombinant hCG.
- Human-derived hCG can be purified from any appropriate source (e.g. urine, and placenta) by any method known in the art. Methods of expressing and purifying recombinant hCG are well known in the art.
- LH can be obtained by any means known in the art.
- LH as used herein, includes human-derived and recombinant LH.
- Human-derived LH can be purified from any appropriate source (e.g. urine) by any method known in the art. Methods of expressing and purifying recombinant LH are known in the art.
- the pharmaceutical composition may be for the treatment of infertility, e.g. for use in e.g. assisted reproductive technologies (ART), ovulation induction or intrauterine insemination (IUI).
- the pharmaceutical composition may be used, for example, in medical indications where known FSH preparations are used.
- the present invention also provides the use of rFSH and/or an rFSH preparation described herein (according to aspects of the invent/on) for, or in the manufacture of a medicament for, the treatment of infertility.
- the pharmaceutical compositions of the present invention can be formulated into well-known compositions for any route of drug administration, e.g. oral, rectal, parenteral, transdermal (e.g.
- a typical composition comprises a pharmaceutically acceptable carrier, such as aqueous solution, non toxic excipients, including salts and preservatives, buffers and the like, as described in Remington's Pharmaceutical Sciences fifteenth edition (Matt Publishing Company, 1975), at pages 1405 to 1412 and 1461 - 87, and the national formulary XIV fourteenth edition (American Pharmaceutical Association, 1975), among others.
- a pharmaceutically acceptable carrier such as aqueous solution, non toxic excipients, including salts and preservatives, buffers and the like, as described in Remington's Pharmaceutical Sciences fifteenth edition (Matt Publishing Company, 1975), at pages 1405 to 1412 and 1461 - 87, and the national formulary XIV fourteenth edition (American Pharmaceutical Association, 1975), among others.
- aqueous and non-aqueous pharmaceutical carriers, diluents, solvents or vehicles examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectible organic esters such as ethyl oleate.
- compositions of the present invention also can contain additives such as but not limited to preservatives, wetting agents, emulsifying agents, and dispersing agents.
- Antibacterial and antifungal agents can be included to prevent growth of microbes and includes, for example, m-cresol, benzyl alcohol, paraben, chlorobutanol, phenol, sorbic acid, and the like.
- isotonic agents such as sugars, sodium chloride, and the like.
- FSH FSH
- other active ingredients if present
- the rate of absorption of FSH then depends upon its rate of dissolution which, in turn, can depend upon crystal size and crystalline form.
- delayed absorption of a parenteraliy administered FSH combination form is accomplished by dissolving or suspending the FSH combination in an oil vehicle.
- injectable depot forms can be made by forming microencapsule matrices of the FSH (and other agents, if present) in biodegradable polymers such as polylactide-polyglycolide.
- the rate of FSH release can be controlled.
- biodegradable polymers include polyvinylpyrrolidone, poly(orthoesters), poly(anhydrides) etc.
- Depot injectable formulations are also prepared by entrapping the FSH in liposomes or microemulsions which are compatible with body tissues.
- Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.
- Injectable formulations can be supplied in any suitable container, e.g. vial, pre-filled syringe, injection cartridges, and the like.
- Formulations may be supplied as a product having pharmaceutical compositions containing FSH (optionally with hCG, LH etc.) If there is more than one active ingredient (i.e. FSH and e.g. hCG or LH) these may be suitable for administration separately or together. If administered separately, administration can be sequential.
- the product can be supplied in any appropriate package.
- a product can contain a number of pre-filled syringes or vials containing either FSH, hCG, or a combination of both FSH and hCG.
- the syringes or vials may be packaged in a blister package or other means to maintain sterility.
- a product can optionally contain instructions for using the FSH and hCG formulations.
- compositions of the invention are supplied as compositions for parenteral administration.
- General methods for the preparation of the parenteral formulations are known in the art and are described in REMINGTON; THE SCIENCE AND PRACTICE OF PHARMACY, supra, at pages 780-820.
- the parenteral compositions can be supplied in liquid formulation or as a solid which will be mixed with a sterile injectable medium just prior to administration.
- the parenteral compositions are supplied in dosage unit form for ease of administration and uniformity of dosage.
- Figure 1 shows a plasmid map of the pFSHalpha/beta expression vector
- Figure 2 shows the a2,3-sialyltransf erase (ST3GAL4) expression vector
- Figure 3 shows the a2,6-sialyltransferase (ST6GAL1) expression vector
- Figure 4 shows % abundance sialic acid distribution of examples of recombinant FSH produced by PER.C6® cells stably expressing FSH after engineering with 02,3- sialyltransferase
- Figure 5 shows % abundance of glycan charge distribution of examples of recombinant FSH produced by PER.C6® cells stably expressing FSH after engineering with a2,3- sialyltransferase;
- Figure 6 shows a comparison of concentration of inhibin-B following administration of 225IU Gonal f (bottom line, dotted line) and 225 IU of the Example (top line, full line) of Invention.
- the coding region of the gene for the FSH alpha polypeptide was used to according to Fiddes and Goodman. (1981). The sequence is banked as AH007338 and at the time of construction there were no other variants of this protein sequence. The sequence is referred herein as SEQ ID NO:1.
- the coding region of the gene for FSH beta polypeptide was used according to Keene et al (1989).
- the sequence is banked as N _000510 and at the time of construction there were no other variants of this protein sequence.
- the sequence is referred herein as SEQ ID NO:2.
- a2,6-Sialyltransferase The coding region of the gene for beta-galactosamide alpha-2,6-sialyltransferase 1 (a2,6-sialyltransferase, ST6GAL1) was used according to Grundmann et al. (1990). The sequence is banked as N _003032 and referred herein as SEQ ID NO:4.
- FSH alpha polypeptide AH007338, SEQ ID NO:1
- FSH beta polypeptide NM_003032, SEQ ID NO:2
- FSHa-fw and FSHa-rev and FSHb-fw and FSHb-rec were amplified by PCR using the primer combinations FSHa-fw and FSHa-rev and FSHb-fw and FSHb-rec respectively.
- FSHa-fw 5'-CCAGGATCCGCCACCATGGATTACTACAGAAAAATATGC-3' FSHa-rev 5'-GGATGGCTAGCTTAAGATTTGTGATAATAAC-3' (SEQ ID NO.10)
- FSHb-fw 5'-CCAGGCGCGCCACCATGAAGACACTCCAGTTTTTC-3' FSHb-rev S'-CCGGGTTAACTTATTATTCTTTCATTTCACCAAAGG-S' (SEQ ID NO: 12)
- the resulting amplified FSH beta DNA was digested with the restriction enzymes Ascl and Hpal and inserted into the Ascl and Hpal sites on the CMV driven mammalian expression vector carrying a neomycin selection marker.
- the FSH alpha DNA was digested with BamHI and Nhe ⁇ and inserted into the sites BamYW and Nhe) on the expression vector already containing the FSH beta polypeptide DNA.
- the vector DNA was used to transform the DH5a strain of E.coli. Colonies were picked for amplification. Colonies containing the vector containing both FSH alpha and beta were selected for sequencing and all contained the correct sequences according to SEQ ID NO.1 and SEQ ID NO:2. Plasmid pFSH A+B#17 was selected for transfection ( Figure 1).
- beta-galactoside alpha-2,3-sialyltransf erase 4 (ST3, L23767, SEQ ID NO:3) was amplified by PCR using the primer combination 2,3STfw and 2,3STrev.
- the coding sequence of beta-galactosamide alpha-2,6-sialyltransferase 1 was amplified by PCR using the primer combination 2,6STfw and 2,6STrev.
- Example 4 Stable expression of pFSH ⁇ + ⁇ in PER.C6® cells. Transfection isolation and screening of clones.
- PER.C6®clones producing FSH were generated by expressing both polypeptide chains of FSH from a single plasmid (see Example 1).
- a liposome based transfection agent with the pFSH ⁇ + ⁇ construct Stable clones were selected in VPRO supplemented with 10% FCS and containing G418. Three weeks after transfection G418 resistant clones grew out. Clones were selected for isolation. The isolated clones were cultured in selection medium until 70- 80% confluent. Supernatants were assayed for FSH protein content using an FSH selective ELISA and pharmacological activity at the FSH receptor in cloned cell line, using a cAMP accumulation assay. Clones expressing functional protein were progressed for culture expansion to 24 well, 6 well and T80 flasks.
- Example 5 Level of sialylation is increased in cells that over express a2,3- sialyltransferase. Stable expression of pST3 in FSH expressing PER.C6® cells; Transfection isolation and screening of clones.
- PER.C6® clones producing highly sialylated FSH were generated by expressing a2,3 sialyltransferase from separate plasmids (Example 2) in PER.C6® cells already expressing both polypeptide chains of FSH (from Example 4). Clones produced from PER.C6® cells as set out in Example 4 were selected for their characteristics including productivity, good growth profile, production of functional protein, and produced FSH which included some sialylation. Stable clones were generated as previously described in Example 4. Clones were isolated, expanded and assayed. The a2,3-sialyltransferase clones were adapted to serum free media and suspension conditions.
- FSH from a2,3- clone (Example 5) was prepared using a using a modification of the method described by Lowry ef al. (1976).
- PER.C6®-FSH For the production of PER.C6®-FSH, the cell lines were adapted to a serum- free medium, i.e., Excell 525 (JRH Biosciences). The cells were first cultured to form a 70%- 90% confluent monolayer in a T80 culture flask. On passage the cells were re-suspended in the serum free medium, Excell 525 + 4 m L-Glutamine, to a cell density of 0.3x10 6 cells/ml. A 25 ml cell suspension was put in a 250 ml shaker flask and shaken at 100 rpm at 37°C at 5% C0 2 .
- Excell 525 JRH Biosciences
- the cells were sub- cultured to a cell density of 0.2 or 0.3x10 6 cells/ml and further cultured in shaker flasks at 37°C, 5% C0 2 and 00 rpm.
- VPRO serum-free production medium
- VPRO JRH Biosciences
- PER.C6®cells very high cell densities (usually > 10 7 cells/ml in a batch culture).
- the cells were first cultured to > 1x10 s cells/ml in Excell 525, then spun down for 5 min at 1000 rpm and subsequently suspended in VPRO medium + 6 mM L-glutamine to a density of 1x10 6 cells/ml. The cells were then cultured in a shaker flask for 7-10 days at 37°C, 5% C0 2 and 100 rpm.
- the cells grew to a density of > 10 7 cells/ml.
- the culture medium was harvested after the cell viability started to decline.
- the cells were spun down for 5 min at 1000 rpm and the supernatant was used for the quantification and purification of FSH.
- the concentration of FSH was determined using ELISA (DRG EIA 1288). Thereafter, purification of FSH was carried out using a modification of the method described by Lowry et al. (1976). Purification using charge selective chromatography was carried out to enrich the highly sialylated forms by methods well known in the art.
- N-Glycans were released from the samples using PNGase F under denaturative conditions and then labelled with 2-aminobenzamide. Released glycan forms were then separated and analysed by Weak Anion Exchange (WAX) column for determination of charge distribution. Labelled glycans treated with 2,3,6,8 sialidase for determination of total sialic acid and 2,3 sialidase for determination of 2,3 sialic acid, were further analyzed by wax column.
- WAX Weak Anion Exchange
- the relative percentages of the charged glycans were calculated from structures present in the undigested and digested glycan pools and are shown in Figure 4 (for 8 samples). These were found to be in the ranges 50% - 70% (e.g. about 60% or 65%) for a2,3 sialylation and 28 to 50%, generally 30 to 35% (e.g. about 31% or 35%), for ⁇ 2,6 sialylation.
- N Glycans were released from the samples using PNGase F under denaturative conditions and then were labeled with 2-aminobenzamide.
- Glycans were released from the samples using PNGase F under denaturative conditions and then labeled with 2- aminobenzamide. Released glycan forms were then separated and analysed by Weak Anion Exchange (WAX) column for determination of sialylation distribution. The relative amounts of neutral, mono-sialylated, di-sialylated, tri-sialylated and tetra-sialylated structures are shown in Figure 5 (for the 8 samples shown in Fig 4).
- WAX Weak Anion Exchange
- the rFSH includes neutral , mono-sialylated, di- sialylated, tri- sialylated and tetra- sialylated glycan structures with relative amounts as follows: neutral 5-6 %; 5- 7% mono- sialylated; 26-30% di-sialylated; 30-32% tri-sialylated and 17-23 % tetra-sialylated.
- N-Glycans were released from the samples using PNGase F under denaturative conditions and then labelled with 2-aminobenzamide. Released glycan forms were then separated and analysed by Weak Anion Exchange (WAX) column for determination of charge distribution. Labelled glycans treated with 2,3,6,8 sialidase for determination of total sialic acid and 2,3 sialidase for determination of 2,3 sialic acid, were further analyzed by wax column (see Example 8). The analysis allows calculation of a2,6 sialic acid.
- WAX Weak Anion Exchange
- the relative percentages of the charged glycans were calculated from structures present in the undigested and digested glycan pools and are shown in the following Table. These were found to be in the ranges 25 to 50%, generally 30 to 35% for a2,6 sialylation.
- the glycans were analyzed by MALDI-MS The relative amounts of alpha 2,6-sialic acid and the terminal residues are shown in the following table, together with those for Gonal F (CHO cell derived recombinant FSH) and Bravelle (human urinary FSH).
- Gonal F CHO cell derived recombinant FSH
- Bravelle human urinary FSH
- the amount of bisecting GlcNac in the FSH of the invention varies between 8.7 and 13.9%, averaging approximately at 10.9%.
- the amount of 1 Fucose Lewis in the FSH of the invention varies between 16.1 and 23.3%, averaging approximately at 19%.
- Example 9 A multiple dose study investigating the safety, tolerability, pharmacokinetics, pharmacodynamics, and immunogenicity of FE 999049 in comparison to GONAL-F. Study population
- the composition has relative percentage amounts of mono, di, tri and tetra sialylated structures as set out in Example 8 [neutral 5-6 %; 15-17% mono-sialylated; 26-30% di-sialylated; 30-32% tri-sialylated and 17-23 % tetra— sialylated.]
- the amount of GalNAc is approximately 50%, the amount of bisGlcNac about 9%, the amount of 1 Fucose Lewis is about 20 % and the amount of 2 Fucose Lewis about 3%.
- FE 999049 and GONAL-F were safe and generally well tolerated as assessed by Adverse Events (AEs), vital signs, ECG, clinical laboratory measurements, and physical examination. No serious adverse event or death occurred during the study.
- AEs Adverse Events
- vital signs vital signs
- ECG ECG
- clinical laboratory measurements ECG
- physical examination No serious adverse event or death occurred during the study.
- concentrations of inhibin-B (see figure 6), oestradiol, and progesterone all increased subsequent to administration of FE 999049 and GONAL-F, however to a greater extent following administration of FE 999049 compared to GONAL-F. Both number and size distribution of follicles showed a greater response to FE 999049 compared to GONAL-F.
- Example 9 demonstrates that FSH having a specific amount (17-23%) of tetra-sialylated glycan structures and e.g. specific amounts of a2,3 sialylation and a2,6 sialylation is markedly more potent then recombinant FSH products which are currently on the market.
- Timossi CM Barrios-de-Tomasi J, GonzaJez-Suarez R, Arranz MC, Padmanabhan V, Conn PM, and Ulloa-Aguirre A. (2000). Differential effects of the charge variants of human follicle-stimulating hormone. J Endocrinol. 165(2), 93-205.
- Protein sequence of FSH alpha (SEQ ID NO:5) 1 MDYYRKYAAI FLVTLSVFLH VLHSAPDVQD CPECTLQENP FFSQPGAPIL QC GCCFSRA
- Beta-galactosamide alpha-2,6-sialyltransferase 1 Accession number NM 003032 Nucleotide sequence of ST6GAL1
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Reproductive Health (AREA)
- Endocrinology (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pregnancy & Childbirth (AREA)
- Pharmacology & Pharmacy (AREA)
- Engineering & Computer Science (AREA)
- Animal Behavior & Ethology (AREA)
- Biophysics (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- General Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Gynecology & Obstetrics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
L'invention concerne des préparations incluant de la FSH (hormone folliculostimulante), par exemple de la FSH recombinante, 17% à 23% de structures de type glycane sialylé étant des structures de type glycane tétrasialylé.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP12730594.4A EP2717904A1 (fr) | 2011-06-06 | 2012-06-06 | Préparation pharmaceutique |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP11168816 | 2011-06-06 | ||
EP12730594.4A EP2717904A1 (fr) | 2011-06-06 | 2012-06-06 | Préparation pharmaceutique |
PCT/GB2012/000491 WO2012168680A1 (fr) | 2011-06-06 | 2012-06-06 | Préparation pharmaceutique |
Publications (1)
Publication Number | Publication Date |
---|---|
EP2717904A1 true EP2717904A1 (fr) | 2014-04-16 |
Family
ID=46397316
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP12730594.4A Withdrawn EP2717904A1 (fr) | 2011-06-06 | 2012-06-06 | Préparation pharmaceutique |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP2717904A1 (fr) |
AR (1) | AR090095A1 (fr) |
WO (1) | WO2012168680A1 (fr) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
TWI488640B (zh) | 2008-04-16 | 2015-06-21 | Ferring Int Ct Sa | 藥學製劑 |
US20180264086A1 (en) * | 2015-09-17 | 2018-09-20 | Glycotope Gmbh | Mammalian follicle-stimulating hormone composition with increased stability |
IL272763B2 (en) | 2017-09-01 | 2024-02-01 | Ferring Bv | The composition for controlled stimulation of the ovary |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
MEP38608A (en) * | 2001-10-22 | 2011-02-10 | Merck Serono Sa | Gonadotrophins for folliculogenesis |
US8609370B2 (en) * | 2004-02-13 | 2013-12-17 | Glycotope Gmbh | Highly active glycoproteins-process conditions and an efficient method for their production |
TWI488640B (zh) | 2008-04-16 | 2015-06-21 | Ferring Int Ct Sa | 藥學製劑 |
TWI532495B (zh) * | 2009-10-05 | 2016-05-11 | 菲瑞茵國際中心股份有限公司 | 藥學製劑 |
WO2012016576A1 (fr) | 2010-08-04 | 2012-02-09 | Glycotope Gmbh | Hormone folliculostimulante humaine recombinante améliorée |
-
2012
- 2012-06-06 AR ARP120101991A patent/AR090095A1/es unknown
- 2012-06-06 WO PCT/GB2012/000491 patent/WO2012168680A1/fr active Application Filing
- 2012-06-06 EP EP12730594.4A patent/EP2717904A1/fr not_active Withdrawn
Non-Patent Citations (1)
Title |
---|
See references of WO2012168680A1 * |
Also Published As
Publication number | Publication date |
---|---|
AR090095A1 (es) | 2014-10-22 |
WO2012168680A1 (fr) | 2012-12-13 |
WO2012168680A8 (fr) | 2014-01-03 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11952407B2 (en) | Pharmaceutical preparation | |
JP6718951B2 (ja) | 調節卵巣刺激のための組成物 | |
WO2012168680A1 (fr) | Préparation pharmaceutique | |
TW201302783A (zh) | 藥學製備物 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 20131125 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR |
|
DAX | Request for extension of the european patent (deleted) | ||
17Q | First examination report despatched |
Effective date: 20150813 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN |
|
18W | Application withdrawn |
Effective date: 20160905 |