US20180186832A1 - Method for the reduction of host cell proteins in affinity chromatography - Google Patents
Method for the reduction of host cell proteins in affinity chromatography Download PDFInfo
- Publication number
- US20180186832A1 US20180186832A1 US15/900,461 US201815900461A US2018186832A1 US 20180186832 A1 US20180186832 A1 US 20180186832A1 US 201815900461 A US201815900461 A US 201815900461A US 2018186832 A1 US2018186832 A1 US 2018186832A1
- Authority
- US
- United States
- Prior art keywords
- antibody
- aqueous solution
- antibody against
- protein
- conductivity aqueous
- 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
- 238000000034 method Methods 0.000 title claims abstract description 204
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 162
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 162
- 238000001042 affinity chromatography Methods 0.000 title claims abstract description 138
- 230000009467 reduction Effects 0.000 title description 3
- 239000007864 aqueous solution Substances 0.000 claims abstract description 235
- 238000005406 washing Methods 0.000 claims abstract description 84
- 230000001603 reducing effect Effects 0.000 claims abstract description 11
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 198
- 239000007983 Tris buffer Substances 0.000 claims description 195
- 239000000463 material Substances 0.000 claims description 79
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 60
- 238000004587 chromatography analysis Methods 0.000 claims description 57
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 claims description 32
- 102000009524 Vascular Endothelial Growth Factor A Human genes 0.000 claims description 32
- 229910000160 potassium phosphate Inorganic materials 0.000 claims description 29
- 235000011009 potassium phosphates Nutrition 0.000 claims description 29
- 102000013455 Amyloid beta-Peptides Human genes 0.000 claims description 25
- 108010090849 Amyloid beta-Peptides Proteins 0.000 claims description 25
- 102000015439 Phospholipases Human genes 0.000 claims description 25
- 108010064785 Phospholipases Proteins 0.000 claims description 25
- ZIIUUSVHCHPIQD-UHFFFAOYSA-N 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide Chemical compound CC1=CC(C)=CC(C)=C1S(=O)(=O)NC1=CC=CC(C(F)(F)F)=C1 ZIIUUSVHCHPIQD-UHFFFAOYSA-N 0.000 claims description 24
- 102000003780 Clusterin Human genes 0.000 claims description 24
- 108090000197 Clusterin Proteins 0.000 claims description 24
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 24
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 claims description 22
- 102100025475 Carcinoembryonic antigen-related cell adhesion molecule 5 Human genes 0.000 claims description 22
- 108010035766 P-Selectin Proteins 0.000 claims description 22
- 102100034608 Angiopoietin-2 Human genes 0.000 claims description 16
- 108010048049 Factor IXa Proteins 0.000 claims description 16
- 108010014173 Factor X Proteins 0.000 claims description 16
- 238000004519 manufacturing process Methods 0.000 claims description 15
- 108090000176 Interleukin-13 Proteins 0.000 claims description 14
- 102000003816 Interleukin-13 Human genes 0.000 claims description 14
- 102000039446 nucleic acids Human genes 0.000 claims description 14
- 108020004707 nucleic acids Proteins 0.000 claims description 14
- 150000007523 nucleic acids Chemical class 0.000 claims description 14
- 102100038080 B-cell receptor CD22 Human genes 0.000 claims description 13
- 101150029707 ERBB2 gene Proteins 0.000 claims description 13
- 101000924533 Homo sapiens Angiopoietin-2 Proteins 0.000 claims description 13
- 101000884305 Homo sapiens B-cell receptor CD22 Proteins 0.000 claims description 13
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 claims description 13
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 claims description 13
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 claims description 13
- 102100029986 Receptor tyrosine-protein kinase erbB-3 Human genes 0.000 claims description 12
- 101710100969 Receptor tyrosine-protein kinase erbB-3 Proteins 0.000 claims description 12
- 208000037798 influenza B Diseases 0.000 claims description 12
- 102000008212 P-Selectin Human genes 0.000 claims 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 166
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 108
- 210000004027 cell Anatomy 0.000 description 100
- 239000011780 sodium chloride Substances 0.000 description 83
- 238000010828 elution Methods 0.000 description 43
- 239000002609 medium Substances 0.000 description 43
- 238000011067 equilibration Methods 0.000 description 40
- 239000000243 solution Substances 0.000 description 30
- 238000000746 purification Methods 0.000 description 28
- 102100023472 P-selectin Human genes 0.000 description 20
- 230000027455 binding Effects 0.000 description 16
- 239000008367 deionised water Substances 0.000 description 13
- 229910021641 deionized water Inorganic materials 0.000 description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 13
- 239000012515 MabSelect SuRe Substances 0.000 description 12
- 239000003446 ligand Substances 0.000 description 12
- 239000011347 resin Substances 0.000 description 10
- 229920005989 resin Polymers 0.000 description 10
- -1 sulfopropyl Chemical group 0.000 description 10
- 239000000427 antigen Substances 0.000 description 9
- 239000000872 buffer Substances 0.000 description 9
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 8
- 238000005571 anion exchange chromatography Methods 0.000 description 8
- 102000036639 antigens Human genes 0.000 description 8
- 108091007433 antigens Proteins 0.000 description 8
- 238000003556 assay Methods 0.000 description 8
- 239000006167 equilibration buffer Substances 0.000 description 8
- 239000011534 wash buffer Substances 0.000 description 8
- 238000005277 cation exchange chromatography Methods 0.000 description 7
- 229920001184 polypeptide Polymers 0.000 description 7
- 102000004196 processed proteins & peptides Human genes 0.000 description 7
- 108090000765 processed proteins & peptides Proteins 0.000 description 7
- 241000699802 Cricetulus griseus Species 0.000 description 6
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 6
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 6
- 101710120037 Toxin CcdB Proteins 0.000 description 6
- 150000001413 amino acids Chemical class 0.000 description 6
- 230000013595 glycosylation Effects 0.000 description 6
- 238000006206 glycosylation reaction Methods 0.000 description 6
- 239000012561 harvest cell culture fluid Substances 0.000 description 6
- 238000004255 ion exchange chromatography Methods 0.000 description 6
- 239000012516 mab select resin Substances 0.000 description 6
- 230000035772 mutation Effects 0.000 description 6
- 210000001672 ovary Anatomy 0.000 description 6
- 102000005962 receptors Human genes 0.000 description 6
- 108020003175 receptors Proteins 0.000 description 6
- 239000007790 solid phase Substances 0.000 description 6
- NFGXHKASABOEEW-UHFFFAOYSA-N 1-methylethyl 11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate Chemical compound COC(C)(C)CCCC(C)CC=CC(C)=CC(=O)OC(C)C NFGXHKASABOEEW-UHFFFAOYSA-N 0.000 description 5
- 108060003951 Immunoglobulin Proteins 0.000 description 5
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 5
- 108010090804 Streptavidin Proteins 0.000 description 5
- 238000004191 hydrophobic interaction chromatography Methods 0.000 description 5
- 102000018358 immunoglobulin Human genes 0.000 description 5
- 239000011859 microparticle Substances 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 238000001542 size-exclusion chromatography Methods 0.000 description 5
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 4
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 4
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 4
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 4
- 229960002685 biotin Drugs 0.000 description 4
- 235000020958 biotin Nutrition 0.000 description 4
- 239000011616 biotin Substances 0.000 description 4
- 210000004899 c-terminal region Anatomy 0.000 description 4
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 4
- 238000004440 column chromatography Methods 0.000 description 4
- 238000011984 electrochemiluminescence immunoassay Methods 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 230000000063 preceeding effect Effects 0.000 description 4
- 108010048036 Angiopoietin-2 Proteins 0.000 description 3
- 102000011720 Lysophospholipase Human genes 0.000 description 3
- 108020002496 Lysophospholipase Proteins 0.000 description 3
- 108091007491 NSP3 Papain-like protease domains Proteins 0.000 description 3
- 102000011420 Phospholipase D Human genes 0.000 description 3
- 108090000553 Phospholipase D Proteins 0.000 description 3
- 101000942681 Rattus norvegicus Clusterin Proteins 0.000 description 3
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 3
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 102000014384 Type C Phospholipases Human genes 0.000 description 3
- 108010079194 Type C Phospholipases Proteins 0.000 description 3
- 238000011210 chromatographic step Methods 0.000 description 3
- 229950002508 gantenerumab Drugs 0.000 description 3
- 230000002209 hydrophobic effect Effects 0.000 description 3
- 229940072221 immunoglobulins Drugs 0.000 description 3
- 229950009230 inclacumab Drugs 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 241000894007 species Species 0.000 description 3
- CHEANNSDVJOIBS-MHZLTWQESA-N (3s)-3-cyclopropyl-3-[3-[[3-(5,5-dimethylcyclopenten-1-yl)-4-(2-fluoro-5-methoxyphenyl)phenyl]methoxy]phenyl]propanoic acid Chemical compound COC1=CC=C(F)C(C=2C(=CC(COC=3C=C(C=CC=3)[C@@H](CC(O)=O)C3CC3)=CC=2)C=2C(CCC=2)(C)C)=C1 CHEANNSDVJOIBS-MHZLTWQESA-N 0.000 description 2
- 125000000022 2-aminoethyl group Chemical group [H]C([*])([H])C([H])([H])N([H])[H] 0.000 description 2
- 101000878605 Homo sapiens Low affinity immunoglobulin epsilon Fc receptor Proteins 0.000 description 2
- 101000845170 Homo sapiens Thymic stromal lymphopoietin Proteins 0.000 description 2
- 102100038007 Low affinity immunoglobulin epsilon Fc receptor Human genes 0.000 description 2
- 102000043136 MAP kinase family Human genes 0.000 description 2
- 108091054455 MAP kinase family Proteins 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 239000012327 Ruthenium complex Substances 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 102100031294 Thymic stromal lymphopoietin Human genes 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 238000005377 adsorption chromatography Methods 0.000 description 2
- 238000005349 anion exchange Methods 0.000 description 2
- 125000003118 aryl group Chemical group 0.000 description 2
- 229960000397 bevacizumab Drugs 0.000 description 2
- 238000005251 capillar electrophoresis Methods 0.000 description 2
- 125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 description 2
- 238000005341 cation exchange Methods 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000012930 cell culture fluid Substances 0.000 description 2
- 239000013522 chelant Substances 0.000 description 2
- 239000012504 chromatography matrix Substances 0.000 description 2
- 239000000356 contaminant Substances 0.000 description 2
- 229950001954 crenezumab Drugs 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 229950002183 lebrikizumab Drugs 0.000 description 2
- JMZFEHDNIAQMNB-UHFFFAOYSA-N m-aminophenylboronic acid Chemical compound NC1=CC=CC(B(O)O)=C1 JMZFEHDNIAQMNB-UHFFFAOYSA-N 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 239000012562 protein A resin Substances 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 229960000575 trastuzumab Drugs 0.000 description 2
- 229950000449 vanucizumab Drugs 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- 101100067974 Arabidopsis thaliana POP2 gene Proteins 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 description 1
- 102100024167 C-C chemokine receptor type 3 Human genes 0.000 description 1
- 101710149862 C-C chemokine receptor type 3 Proteins 0.000 description 1
- 101710149863 C-C chemokine receptor type 4 Proteins 0.000 description 1
- 102100032976 CCR4-NOT transcription complex subunit 6 Human genes 0.000 description 1
- 101150013553 CD40 gene Proteins 0.000 description 1
- 108010016626 Dipeptides Proteins 0.000 description 1
- 102000001301 EGF receptor Human genes 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 102100032031 Epidermal growth factor-like protein 7 Human genes 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 description 1
- 101100118549 Homo sapiens EGFR gene Proteins 0.000 description 1
- 101000921195 Homo sapiens Epidermal growth factor-like protein 7 Proteins 0.000 description 1
- 101001046686 Homo sapiens Integrin alpha-M Proteins 0.000 description 1
- 101000935040 Homo sapiens Integrin beta-2 Proteins 0.000 description 1
- 101001019600 Homo sapiens Interleukin-17 receptor B Proteins 0.000 description 1
- 101000998146 Homo sapiens Interleukin-17A Proteins 0.000 description 1
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 description 1
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 1
- 102000006496 Immunoglobulin Heavy Chains Human genes 0.000 description 1
- 108010019476 Immunoglobulin Heavy Chains Proteins 0.000 description 1
- 102100022338 Integrin alpha-M Human genes 0.000 description 1
- 102100022297 Integrin alpha-X Human genes 0.000 description 1
- 108010008212 Integrin alpha4beta1 Proteins 0.000 description 1
- 102100025390 Integrin beta-2 Human genes 0.000 description 1
- 102100033016 Integrin beta-7 Human genes 0.000 description 1
- 102000007482 Interleukin-13 Receptor alpha2 Subunit Human genes 0.000 description 1
- 108010085418 Interleukin-13 Receptor alpha2 Subunit Proteins 0.000 description 1
- 102000013691 Interleukin-17 Human genes 0.000 description 1
- 108050003558 Interleukin-17 Proteins 0.000 description 1
- 102100035014 Interleukin-17 receptor B Human genes 0.000 description 1
- 102100033461 Interleukin-17A Human genes 0.000 description 1
- 102000010787 Interleukin-4 Receptors Human genes 0.000 description 1
- 108010038486 Interleukin-4 Receptors Proteins 0.000 description 1
- 102000010786 Interleukin-5 Receptors Human genes 0.000 description 1
- 108010038484 Interleukin-5 Receptors Proteins 0.000 description 1
- 102100037792 Interleukin-6 receptor subunit alpha Human genes 0.000 description 1
- 102000010682 Interleukin-9 Receptors Human genes 0.000 description 1
- 108010038414 Interleukin-9 Receptors Proteins 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 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
- 108090000772 Neuropilin-1 Proteins 0.000 description 1
- 108010042215 OX40 Ligand Proteins 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 108050000258 Prostaglandin D receptors Proteins 0.000 description 1
- 102100024218 Prostaglandin D2 receptor 2 Human genes 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100023068 Protein kinase C-binding protein NELL1 Human genes 0.000 description 1
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 description 1
- 102100029981 Receptor tyrosine-protein kinase erbB-4 Human genes 0.000 description 1
- 101710100963 Receptor tyrosine-protein kinase erbB-4 Proteins 0.000 description 1
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 1
- KJTLSVCANCCWHF-UHFFFAOYSA-N Ruthenium Chemical compound [Ru] KJTLSVCANCCWHF-UHFFFAOYSA-N 0.000 description 1
- 101100123851 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) HER1 gene Proteins 0.000 description 1
- 102100022563 Tubulin polymerization-promoting protein Human genes 0.000 description 1
- 102100026890 Tumor necrosis factor ligand superfamily member 4 Human genes 0.000 description 1
- 101710165473 Tumor necrosis factor receptor superfamily member 4 Proteins 0.000 description 1
- 102100022153 Tumor necrosis factor receptor superfamily member 4 Human genes 0.000 description 1
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 230000002494 anti-cea effect Effects 0.000 description 1
- 229940046836 anti-estrogen Drugs 0.000 description 1
- 230000001833 anti-estrogenic effect Effects 0.000 description 1
- 239000003418 antiprogestin Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000006172 buffering agent Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 239000000032 diagnostic agent Substances 0.000 description 1
- 229940039227 diagnostic agent Drugs 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229950006432 duligotuzumab Drugs 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 239000000328 estrogen antagonist Substances 0.000 description 1
- 229950004912 etrolizumab Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 108010015792 glycyllysine Proteins 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000000099 in vitro assay Methods 0.000 description 1
- 108010021315 integrin beta7 Proteins 0.000 description 1
- 108040006858 interleukin-6 receptor activity proteins Proteins 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229950000482 lampalizumab Drugs 0.000 description 1
- 108010032674 lampalizumab Proteins 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 229960003347 obinutuzumab Drugs 0.000 description 1
- 229950005751 ocrelizumab Drugs 0.000 description 1
- 229960000470 omalizumab Drugs 0.000 description 1
- 229950000846 onartuzumab Drugs 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 229950004260 parsatuzumab Drugs 0.000 description 1
- 229960002087 pertuzumab Drugs 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000011176 pooling Methods 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000003623 progesteronic effect Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 229950003033 quilizumab Drugs 0.000 description 1
- 229960003876 ranibizumab Drugs 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 229960004641 rituximab Drugs 0.000 description 1
- 229950010316 rontalizumab Drugs 0.000 description 1
- 229910052707 ruthenium Inorganic materials 0.000 description 1
- 238000003118 sandwich ELISA Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000011146 sterile filtration Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 238000003146 transient transfection Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1018—Orthomyxoviridae, e.g. influenza virus
-
- 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
-
- 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
- C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
-
- 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
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
- C07K16/244—Interleukins [IL]
-
- 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
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
-
- 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
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
-
- 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
- C07K16/2851—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
- C07K16/2854—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72 against selectins, e.g. CD62
-
- 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
- C07K16/3007—Carcino-embryonic Antigens
-
- 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
- C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
-
- 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
- C07K16/36—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
- C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
Definitions
- the present invention relates to the field of purification of polypeptides.
- the present invention in particular relates to the reduction of host cell proteins like phospholipase B-like 2 (PLBL2) or Clusterin in solutions containing antibodies.
- PLBL2 phospholipase B-like 2
- Clusterin in solutions containing antibodies.
- Proteins and especially immunoglobulins play an important role in today's medical portfolio. For human application every therapeutic protein has to meet distinct criteria. To ensure the safety of biopharmaceutical agents to humans by-products accumulating during the production process have to be removed especially. To fulfill the regulatory specifications one or more purification steps have to follow the manufacturing process. Among other things, purity, throughput, and yield play an important role in determining an appropriate purification process.
- affinity chromatography e.g. protein A or protein G affinity chromatography, single chain Fv ligand affinity chromatography
- ion exchange chromatography e.g. cation exchange (sulfopropyl or carboxymethyl resins), anion exchange (amino ethyl resins) and mixed-mode ion exchange
- thiophilic adsorption e.g. with beta-mercaptoethanol and other SH ligands
- hydrophobic interaction or aromatic adsorption chromatography e.g.
- an affinity chromatography step like protein A affinity chromatography is followed by one or more additional separation steps.
- high conductivity buffers are described to be employed in wash steps of affinity chromatrography methods.
- a method for purifying a protein including one or more chromatographic processes, in which an amino acid; or a dipeptide, an oligopeptide, or a polyamino acid thereof is included in a buffer solution used in at least one chromatographic process (equilibration buffer, wash buffer, and elution buffer), thereby purifying a high-purity protein with a very small quantity of the impurity (e.g., polymers or host cell proteins) is reported in EP2583973.
- a buffer solution used in at least one chromatographic process equilibration buffer, wash buffer, and elution buffer
- the method of the current invention which uses a low conductivity aqueous solution in a wash step of an affinity chromatography prior to the recovery of an antibody from the chromatographic material, that the content of certain host cell proteins in a solution comprising the antibody can be reduced. Accordingly, it has been found that the content of phospholipases (in particular phospholipase B-like 2 (PLBL2)) can be reduced. It has been found that the PLBL2 content can be reduced 100-fold or more if the antibody is of the IgG4 isotype.
- phospholipases in particular phospholipase B-like 2 (PLBL2)
- One aspect as reported herein is the use of a low conductivity aqueous solution in a wash step of a protein A chromatography for reducing the content of a host cell protein wherein the protein A chromatography is used to purify a human IgG1 or a human IgG4 isotype antibody.
- the human IgG4 isotype antibody is an antibody against P-selectin, or an bispecific antibody against factor IXa and factor X, or an antibody against IL-13, or an antibody against amyloid beta.
- the human IgG1 isotype antibody is an antibody against Influenza B, or an antibody against VEGF-A, or an antibody against CD22, or a bispecific antibody against HER3 and EGFR, or an antibody against amyloid beta, or an antibody against Her2, or a bispecific antibody against Ang2 and VEGF-A, or a bispecific antibody against carcinoembryonic antigen (CEA) and CD3.
- the low conductivity aqueous solution has a conductivity value of about 0.5 mS/cm or less.
- the host cell protein is phospholipase B-like 2 (PLBL2) or Clusterin.
- the low conductivity aqueous solution comprises about 0.1 mM to about 8 mM Tris.
- the low conductivity aqueous solution comprises about 0.05 mM to about 2 mM potassium phosphate.
- the low conductivity aqueous solution has a pH of about 7 or higher.
- the low conductivity aqueous solution wash step is preceded or succeeded by a high conductivity aqueous solution wash step.
- the high conductivity aqueous solution has a conductivity value of about 20 mS/cm or higher.
- an intermediate wash step is performed with a medium conductivity aqueous solution between the low conductivity aqueous solution wash step and the high conductivity aqueous solution wash step.
- the medium conductivity aqueous solution has a conductivity value of from more than 0.5 mS/cm to less than 20 mS/cm.
- the high (or medium) conductivity aqueous solution comprises Histidine.
- One aspect as reported herein is a method for producing a human IgG4 or IgG1 isotype antibody comprising the steps of
- One aspect as reported herein is method for purifying a human IgG4 or IgG1 isotype antibody from a sample comprising the steps of
- the human IgG4 isotype antibody is an antibody against P-selectin or a bispecific antibody against factor IXa and factor X or an antibody against IL-13 or an antibody against amyloid beta.
- the human IgG1 isotype antibody is an antibody against Influenza B or an antibody against VEGF-A or an antibody against CD22 or a bispecific antibody against HER3 and EGFR or an antibody against amyloid beta or an antibody against Her2 or a bispecific antibody against Ang2 and VEGF-A, or a bispecific antibody against carcinoembryonic antigen (CEA) and CD3.
- the low conductivity aqueous solution has a conductivity value of about 0.5 mS/cm or less.
- the content of a host cell protein is reduced and the (specific) host cell protein is phospholipase B-like 2 (PLBL2) or Clusterin.
- PLBL2 phospholipase B-like 2
- Clusterin phospholipase B-like 2
- the low conductivity aqueous solution comprises about 0.1 mM to about 8 mM Tris.
- the low conductivity aqueous solution comprises about 0.05 mM to about 2 mM potassium phosphate.
- the low conductivity aqueous solution has a pH of about 7 or higher.
- the method additionally comprises washing the affinity chromatography material with a high conductivity aqueous solution and/or with a medium conductivity aqueous solution before or after washing the protein A chromatography material with low conductivity aqueous solution.
- the high conductivity aqueous solution has a conductivity value of about 20 mS/cm or higher.
- the medium conductivity aqueous solution has a conductivity value of from more than 0.5 mS/cm to less than 20 mS/cm.
- the high or medium conductivity aqueous solution comprises Histidine.
- a wash step with a low conductivity aqueous solution when this wash step is used in an affinity chromatography step, e.g. a protein A chromatrography step.
- the affinity chromatography step is used in a purification or production method for antibodies.
- the low conductivity aqueous solution wash step is particularly effective to reduce the content of phospholipase B-like 2 (PLBL2).
- One aspect as reported herein is the use of a low conductivity aqueous solution in a wash step of an affinity chromatography for reducing the content of a (specific) host cell protein.
- One aspect as reported herein is a method for producing a human IgG isotype antibody comprising
- One aspect as reported herein is a method for purifying a human IgG isotype antibody from a sample comprising the steps of
- Recombinant polypeptides produced in CHO cells may be purified according to the methods described herein to remove or reduce levels of a host cell proteins.
- Exemplary recombinant polypeptides include therapeutic antibodies and immunoadhesins, including, without limitation, antibodies, including antibody fragments, to one or more of the following antigens: HER1 (EGFR), HER2 (e.g., trastuzumab, pertuzumab), HER3, HER4, VEGF (e.g., bevacizumab, ranibizumab), MET (e.g., onartuzumab), CD20 (e.g., rituximab, obinutuzumab, ocrelizumab), CD22, CD11a, CD11b, CD11c, CD18, an ICAM, VLA-4, VCAM, IL-17A and/or F, IgE (e.g., omalizumab), DRS, CD40, Apo2L/TRAIL, EGFL7 (e.g., parsatuzumab), NRP1, integrin beta7 (e.g., etro
- exemplary antibodies include those selected from, and without limitation, antiestrogen receptor antibody, anti-progesterone receptor antibody, anti-p53 antibody, anticathepsin D antibody, antiBcl-2 antibody, anti-E-cadherin antibody, anti-CA125 antibody, anti-CA15-3 antibody, antiCA19-9 antibody, anti-c-erbB-2 antibody, anti-P-glycoprotein antibody, anti-CEA antibody, Ki-67 antibody, anti-PCNA antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8 antibody, anti-CD9/p24 antibody, anti-CD10 antibody, anti-CD11c antibody, anti-CD13 antibody, anti-CD14 antibody, anti-CD15 antibody, anti-CD19 antibody, anti-CD23 antibody, anti-CD30 antibody, anti-CD31 antibody, anti-CD33 antibody, anti-CD34 antibody, anti-CD35 antibody, anti-CD38 antibody, anti-CD41 antibody, antiLCA/CD45 antibody, anti-CD45RO antibody
- exemplary antibodies include antibodies to Abeta, antibodies to IL17 A/F and antibodies to CMV.
- Exemplary anti-Abeta antibodies and methods of producing such antibodies have been described previously, for example, in WO2008011348, WO2007068429, WO2001062801, and WO2004071408.
- Exemplary anti-IL17 A/F antibodies and methods of producing such antibodies have been described previously, for example, in WO 2009136286 and U.S. Pat. No. 8,715,669.
- Exemplary anti-CMV antibodies, including anti-CMV-MSL, and methods of producing such antibodies have been described previously, for example, in WO 2012047732.
- the affinity chromatography is used to purify a human IgG isotype antibody. In some embodiments the affinity chromatography is used to purify an IgG4 antibody. In one embodiment the IgG4 isotype antibody is an antibody against P-selectin or a (bispecific) antibody against factor IXa and factor X or an antibody against IL-13 or an antibody against amyloid beta. In some embodiments the affinity chromatography is used to purify an IgG1 isotype antibody.
- the IgG1 isotype antibody is an antibody against Influenza B or an antibody against VEGF-A or an antibody against CD22 or an (bispecific) antibody against HER3 and EGFR or an antibody against amyloid beta or an antibody against Her2 or a bispecific antibody against Ang2 and VEGF-A or a bispecific antibody against carcinoembryonic antigen (CEA) and CD3.
- One aspect as reported herein is a method for producing a human IgG4 isotype antibody (containing solution) comprising
- One aspect as reported herein is a method for producing an IgG4 isotype antibody (containing solution) comprising
- One aspect as reported herein is a method for purifying a human IgG4 isotype antibody from a sample comprising the steps of
- One aspect as reported herein is a method for purifying an IgG4 isotype antibody from a sample comprising the steps of
- the content of a host cell protein can be reduced if the conductivity of the aqueous solution used in the wash step is low i.e a low conductivity aqueous solution is used for washing.
- the low conductivity aqueous solution has a conductivity value of about 1 mS/cm or less. In one preferred embodiment of all aspects the low conductivity aqueous solution has a conductivity value of about 0.5 mS/cm or less. In one embodiment the low conductivity aqueous solution has a conductivity value of from about 0.03 ⁇ S/cm to about 0.5 mS/cm.
- the low conductivity aqueous solution has a conductivity value of from about 0.05 ⁇ S/cm to about 0.35 mS/cm. In one embodiment of all aspects the low conductivity aqueous solution is deionized water. For some applications deionized water is not suitable to be used in a wash step. In some embodiments the low conductivity aqueous solution is not deionized water.
- the affinity chromatography is a protein A affinity chromatography.
- the protein A affinity chromatography is selected from the group comprising MabSelectSure affinity chromatography, ProSep vA affinity chromatography, Poros Mab Capture A affinity chromatography, ProSep Ultra Plus affinity chromatography, MabSelect SuRe LX, MabSelect, Eshmuno A, Toyopearl AF-rProtein A-650F; Toyopearl AF-rProtein A HC-650HF).
- the affinity chromatography is a protein G affinity chromatography.
- the affinity chromatography is an affinity chromatography that uses a recombinant protein as a ligand, that means that the affinity chromatography is a recombinant protein ligand affinity chromatography.
- the affinity chromatography is an affinity chromatography that uses a single chain Fv as a ligand, that means that the affinity chromatography is a single chain Fv ligand affinity chromatography.
- the affinity chromatography comprises a mutated Protein A coupled to a chromatography matrix or a fragment of Protein A coupled to a chromatography matrix.
- the content of (specific) host cell proteins can be reduced. It has been found that especially the content of phospholipase B-like 2 (PLBL2) can be reduced.
- the (specific) host cell protein is a Chinese hamster ovary (CHO) host cell protein.
- the (specific) host cell protein is phospholipase B-like 2 (PLBL2) or Clusterin.
- the (specific) host cell protein is phospholipase B-like 2 (PLBL2).
- low conductivity aqueous solution may comprise certain buffering substances e.g. Tris or potassium phosphate in low amounts.
- the low conductivity aqueous solution contains tris(hydroxymethyl)aminomethane (Tris).
- Tris tris(hydroxymethyl)aminomethane
- the low conductivity aqueous solution comprises about 0.1 mM to about 10 mM Tris.
- the low conductivity aqueous solution comprises about 0.5 mM to about 6.5 mM Tris.
- the low conductivity aqueous solution comprises about 2 mM Tris.
- the low conductivity aqueous solution contains potassium phosphate.
- the low conductivity aqueous solution comprises about 0.05 mM to about 5 mM potassium phosphate.
- the low conductivity aqueous solution comprises about 0.05 mM to about 2 mM potassium phosphate.
- the low conductivity aqueous solution comprises about 0.5 mM potassium phosphate.
- the low conductivity aqueous solution has a certain pH.
- the low conductivity aqueous solution has a pH of about 7 or higher.
- the low conductivity aqueous solution has a pH of about 7.5 or higher.
- the low conductivity aqueous solution has a pH of from about 7 to about 9.5.
- the low conductivity aqueous solution has a pH of from about 7.5 to about 8.5.
- the low conductivity aqueous solution has a pH of about 8.
- the low conductivity aqueous solution has a pH of about 9.
- the effect of reducing the content of a host cell protein can also be achieved if the pH of the low conductivity aqueous solution is about 8.5 or higher and the low conductivity aqueous solution has a conductivity value of about 1.2 mS/cm or less.
- the low conductivity aqueous solution has a pH of about 8.5 or higher and the low conductivity aqueous solution has a conductivity value of about 1.2 mS/cm or less.
- the low conductivity aqueous solution has a pH of about 8.5 or higher and the low conductivity aqueous solution has a conductivity value of about 1 mS/cm or less.
- low conductivity aqueous solution has a pH of about 8.5 or higher and the low conductivity aqueous solution comprises about 55 mM Tris or less. In one embodiment low conductivity aqueous solution has a pH of about 8.5 or higher and the low conductivity aqueous solution comprises about 30 mM Tris or less.
- the low conductivity aqueous solution is in the pH range of from pH 7 to less than pH 8.5 and has a conductivity value of about 0.5 mS/cm or less and at a pH value of 8.5 or more a conductivity value of about 1.2 mS/cm or less.
- the content of host cell proteins like PLBL2 can be reduced to a certain level, e.g. when compared to the load amount of PLBL2 prior to a purification step like an affinity chromatography step.
- the content of PLBL2 is reduced at least 20-fold.
- the content of PLBL2 is reduced at least 40-fold.
- the content of PLBL2 is reduced at least 50-fold.
- the content of PLBL2 is reduced at least 90-fold.
- the content of PLBL2 is reduced at least 100-fold. In some cases the level of reduction is even higher.
- the content of PLBL2 is reduced at least 200-fold.
- the content of PLBL2 is reduced at least 250-fold. In some embodiments the content of PLBL2 is reduced at least 300-fold. In some embodiments the content of PLBL2 is reduced at least 400-fold. In some embodiments the content of PLBL2 is reduced at least1000-fold. In one embodiment the content of PLBL2 is reduced at least by 50%. In one embodiment the content of PLBL2 is reduced at least by 66%. In one embodiment the content of PLBL2 is reduced at least by 80%. In one embodiment the content of PLBL2 is reduced at least by 90%. In one embodiment the content of PLBL2 is reduced at least by 95%. In some embodiments the content of PLBL2 is reduced to below 10 ng per mg of antibody. In some embodiments the content of PLBL2 is reduced to below 5 ng per mg of antibody. In some embodiments the content of PLBL2 is reduced to below 2 ng per mg of antibody.
- wash steps can be employed with medium and/or high conductivity aqueous solutions.
- the low conductivity aqueous solution wash step is preceded or succeeded by a high conductivity aqueous solution wash step.
- the high conductivity aqueous solution has a conductivity value of about 20 mS/cm or higher.
- the high conductivity aqueous solution has a conductivity value of from about 20 mS/cm to about 100 mS/cm.
- an intermediate wash step is performed with a medium conductivity aqueous solution between the low conductivity aqueous solution wash step and the high conductivity aqueous solution wash step.
- the medium conductivity aqueous solution has a conductivity value of from more than 0.5 mS/cm to less than 20 mS/cm.
- the host cell protein reducing effect can be improved when the high or medium conductivity aqueous solution further comprises an amino acid.
- the high or medium conductivity aqueous solution comprises an amino acid.
- the high or medium conductivity aqueous solution comprises Histidine or Arginine.
- the high or medium conductivity aqueous solution comprises Histidine.
- the high or medium conductivity aqueous solution comprises Histidine and Tris.
- the methods and the uses as reported herein may include one or more further chromatography steps.
- at least one additional chromatography method/step is performed.
- an additional ion exchange chromatography method/step is performed.
- an additional anion exchange chromatography method/step is performed.
- an additional anion exchange chromatography method/step and an additional cation exchange chromatography method/step are performed.
- hydrophobic interaction chromatography step may be omitted.
- the use or the methods is without an hydrophobic interaction chromatography method/step.
- One aspect as reported herein is the use of a low conductivity aqueous solution in a wash step of a protein A chromatography for reducing the content of PLBL2 or Clusterin wherein the protein A chromatography is used to purify an IgG4 or IgG1 isotype, e.g., a human IgG4 or IgG1, antibody and wherein the low conductivity aqueous solution has a conductivity value of about 0.5 mS/cm or less and a pH of about 7 or higher.
- One aspect is the use of a low conductivity aqueous solution in a wash step of a protein A chromatography for reducing the content of PLBL2 or Clusterin wherein the protein A chromatography is used to purify a human IgG4 or IgG1 isotype antibody and wherein the low conductivity aqueous solution has a conductivity value of about 0.5 mS/cm or less and a pH of about 7 or higher.
- the antibody is an IgG4 isotype antibody, e.g., an antibody against P-selectin, or a bispecific antibody against factor IXa and factor X, or an antibody against IL-13, or an antibody against amyloid beta.
- the antibody is a IgG1 isotype antibody, e.g., an antibody against Influenza B, or an antibody against VEGF-A, or an antibody against CD22, or a bispecific antibody against HER3 and EGFR, or an antibody against amyloid beta, or an antibody against Her2, or a bispecific antibody against Ang2 and VEGF-A, or a bispecific antibody against carcinoembryonic antigen (CEA) and CD3.
- IgG1 isotype antibody e.g., an antibody against Influenza B, or an antibody against VEGF-A, or an antibody against CD22, or a bispecific antibody against HER3 and EGFR, or an antibody against amyloid beta, or an antibody against Her2, or a bispecific antibody against Ang2 and VEGF-A, or a bispecific antibody against carcinoembryonic antigen (CEA) and CD3.
- IgG1 isotype antibody e.g., an antibody against Influenza B, or an antibody against VEGF-A
- the present disclosure provides a method for producing a human IgG4 or IgG1 isotype antibody comprising
- the present disclosure provides a method for producing a human IgG4 or IgG1 isotype antibody comprising
- the present disclosure provides a method for purifying a human IgG4 or IgG1 isotype antibody from a sample comprising the steps of
- the present disclosure provides a method for purifying a human IgG4 or IgG1 isotype antibody from a sample comprising the steps of
- the present disclosure provides a method for producing a human IgG4 isotype antibody comprising
- the present disclosure provides a method for purifying a human IgG4 isotype antibody from a sample comprising the steps of
- anti-P-selectin antibody and “an antibody that binds to P-selectin” or “antibody against P-selectin”refer to an antibody that is capable of binding P-selectin with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting P-selectin.
- the extent of binding of an anti-P-selectin antibody to an unrelated, non-P-selectin protein is less than about 10% of the binding of the antibody to P-selectin as measured, e.g., by ELISA or surface plasmon resonance.
- an anti-P-selectin antibody binds to an epitope of P-selectin that is conserved among P-selectin from different species.
- antibody against factor IXa and factor X or “antibody against IL-13” or “antibody against amyloid beta” or the like.
- the antibody against P-selectin is inclacumab (IgG4 isotype) as described in WO 2005/100402 or SEQ ID NO: 07 to 12.
- the antibody is a bispecific antibody against factor IXa and factor X, e.g., anti-FIXa/X antibody (IgG4 isotype) as described in WO 2012/067176.
- the antibody is an antibody against Her2, e.g., trastuzumab (IgG1 isotype) as described in WO 1992/022653.
- the antibody is a bispecific antibody against angiopoietin 2 (Ang2) and vascular endothelial growth factor A (VEGF-A), e.g., vanucizumab (IgG1 isotype) as described in WO 2011/117329 or SEQ ID NO: 01 to 04.
- the antibody is an antibody against amyloid beta, e.g., gantenerumab (IgG1 isotype) as described in WO 2003/070760 or SEQ ID NO: 05 to 06, or crenezumab (IgG4 isotype).
- the antibody is an antibody against CD22, an antibody against IL13 (e.g., lebrikizumab), a bispecific antibody against Her3 and EGFR (e.g., duligotuzumab), an antibody against VEGF-A (e.g., bevacizumab), and an antibody against Influenza B.
- IL13 e.g., lebrikizumab
- a bispecific antibody against Her3 and EGFR e.g., duligotuzumab
- an antibody against VEGF-A e.g., bevacizumab
- Influenza B e.g., bevacizumab
- VEGF or VEGF-A can be used interchangeably herein.
- binding refers to the binding of the antibody to an epitope of the antigen in an in-vitro assay, preferably in a surface plasmon resonance assay (SPR, BIAcore, GE-Healthcare Uppsala, Sweden).
- the affinity of the binding is defined by the terms ka (rate constant for the association of the antibody from the antibody/antigen complex), k d (dissociation constant), and K D (k d /k a ).
- Binding or specifically binding means a binding affinity (K D ) of 10 ⁇ 7 mol/L or less.
- antibody herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.
- antibody fragment refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
- antibody fragments include but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′) 2 ; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.
- a Fab fragment is an antibody fragment obtained by a papain digestion of a (full length/complete) antibody.
- Bispecific antibodies are antibodies which have two different antigen-binding specificities.
- the term “bispecific” antibody as used herein denotes an antibody that has at least two binding sites each of which bind to different epitopes.
- chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.
- the “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain.
- the heavy chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
- human IgG isotype antibody denotes an antibody that comprises a constant region that is derived from a human wild-type IgG isotype, i.e. for example it may comprise a constant region derived from a human IgG isotype with a mutation, e.g. an P329G mutation (numbering according to Kabat).
- human IgG4 isotype antibody denotes an antibody that comprises a constant region that is derived from a human wild-type IgG4 isotype, i.e. for example it may comprise a constant region derived from a human IgG4 isotype with a mutation, e.g. an an P329G mutation and/or S228P, L235E mutation (numbering according to Kabat).
- Fc-region herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
- the term includes native sequence Fc-regions and variant Fc-regions.
- a human IgG heavy chain Fc-region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain.
- the C-terminal lysine (Lys447) or the C-terminal glycyl-lysine dipeptide (Gly446Lys447) of the Fc-region may or may not be present.
- EU numbering system also called the EU index, as described in Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991), NIH Publication 91-3242.
- “Framework” or “FR” refers to variable domain residues other than hypervariable region (HVR) residues.
- the FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
- host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
- Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
- the term “cell” includes cells which are used for the expression of nucleic acids.
- the host cell is a CHO cell (e.g.
- the cell is a CHO cell, or a BHK cell, or a PER.C6® cell.
- the expression “cell” includes the subject cell and its progeny.
- washing denotes the applying of a solution to an affinity chromatography material in order to remove non specifically bound polypeptides and non-polypeptide compounds from the chromatography material, especially to remove host cell protein and host cell DNA.
- the term “washing” does not encompass the elution of bound material from an affinity chromatography material.
- affinity chromatography with microbial proteins e.g. protein A or protein G affinity chromatography
- affinity chromatographie with a recombinant protein as ligand e.g. single chain Fv as ligand, e.g. Kappa select
- ligand e.g. single chain Fv as ligand, e.g. Kappa select
- ion exchange chromatography e.g. cation exchange (carboxymethyl resins), anion exchange (amino ethyl resins) and mixed-mode exchange
- thiophilic adsorption e.g. with beta-mercaptoethanol and other SH ligands
- hydrophobic interaction or aromatic adsorption chromatography e.g.
- protein A denotes a protein A polypeptide either obtained from a natural source or produced synthetically.
- protein A chromatography material denotes an inert solid phase to which a protein A is covalently linked.
- the protein A chromatography material is selected from MabSelectSure, ProSep vA, Mab Capture A, ProSep Ultra Plus, Mab Select, Mab Select Xtra, Poros A, or ProSep A.
- high conductivity aquaeous solution denotes an aquaeous solution with a high conductivity value.
- the conductivity value may be about 20 mS/cm or higher.
- medium conductivity aquaeous solution denotes an aquaeous solution with a medium conductivity value.
- the conductivity value may be more than 0.5 mS/cm to less than 20 mS/cm.
- low conductivity aquaeous solution denotes an aquaeous solution with a low conductivity value.
- the conductivity value may be about 0.5 mS/cm or less.
- the conductivity value may be about 1.2 mS/cm or less, if the pH is about 8.5 or higher.
- the conductivity values can be determined by standard methods known to the person skilled in the art.
- variable heavy chain domain VH of ⁇ VEGF> SEQ ID NO: 02 variable light chain domain VL of ⁇ VEGF>
- 07 variable heavy chain domain VH1 of anti-P-selectin antibody SEQ ID NO: 08 variable heavy chain domain VH2 of anti-P-selectin antibody
- the current invention is exemplified using a number of exemplary antibodies, including: an antibody against P-selectin (anti-P-selectin antibody; inclacumab; IgG4 isotype) as described in WO 2005/100402 or SEQ ID NO: 07 to SEQ ID NO: 12; a bispecific antibody against factor IXa and factor X (anti-FIXa/X antibody; IgG4 isotype) as described in WO 2012/067176; with an antibody against Her2; a bispecific antibody against Ang2 and VEGF-A (anti-Ang2/VEGF-A antibody; vanucizumab; IgG1 isotype) as described in WO 2011/117329 or SEQ ID NO: 01 to SEQ ID NO: 04; an antibody against amyloid beta (anti-amyloid beta antibody; gantenerumab; IgG1 isotype) as described in WO 2003/070760 or SEQ ID NO: 05 to SEQ ID NO: 06. Also included herein
- HCP Host Cell Protein
- PLBL2 Phospholipase B-like 2 Protein
- the residual CHO HCP content in process samples is determined by an electrochemiluminescence immunoassay (ECLIA) on cobas e 411 immunoassay analyzer (Roche Diagnostics).
- ECLIA electrochemiluminescence immunoassay
- the assay is based on a sandwich principle using polyclonal anti-CHO HCP antibody from sheep.
- CHO HCP Chinese hamster ovary host cell protein
- Second incubation After addition of polyclonal CHO HCP-specific antibody labeled with ruthenium complex (Tris(2,2′-bipyridyl)ruthenium(II)-complex) a ternary sandwich complex is formed on the microparticles.
- ruthenium complex Tris(2,2′-bipyridyl)ruthenium(II)-complex
- the reaction mixture is aspirated into the measuring cell where the microparticles are magnetically captured onto the surface of the electrode. Unbound substances are then removed in a washing step. Application of a voltage to the electrode then induces chemiluminescent emission which is measured by a photomultiplier.
- the concentration of CHO HCP in the test sample is finally calculated from a CHO
- the residual Chinese hamster ovary (CHO) Phospholipase B-like 2 protein (PLBL2) content in process samples is determined by an electrochemiluminescence immunoassay (ECLIA) on cobas e 411 immunoassay analyzer (Roche Diagnostics).
- ELIA electrochemiluminescence immunoassay
- the assay is based on a sandwich principle using monoclonal anti-CHO PLBL2 antibody from mouse.
- CHO PLBL2 from 30 ⁇ L sample (neat and/or diluted), biotin labeled monoclonal CHO PLBL2-specific antibody, and a monoclonal CHO PLBL2-specific antibody labeled with a ruthenium complex (Tris(2,2′-bipyridyl)ruthenium(II)-complex) form a sandwich complex.
- a ruthenium complex Tris(2,2′-bipyridyl)ruthenium(II)-complex
- the ternary complex becomes bound to the solid phase via interaction of biotin and streptavidin.
- the reaction mixture is aspirated into the measuring cell where the microparticles are magnetically captured onto the surface of the electrode. Unbound substances are then removed in a washing step. Application of a voltage to the electrode then induces chemiluminescence, which is measured by a photomultiplier.
- the concentration of CHO PLBL2 in the test sample is finally calculated from a CHO PLBL2 standard curve of known concentration.
- the residual Clusterin content in process samples is determined by a commercial assay from Merck Millipore (GyroMark HT Kit GYRCLU-37K) which was used according to the manufacturer's instructions.
- this assay is a Sandwich ELISA based, sequentially, on:
- Antibody Anti-P-Selectin
- a solution containing an anti-P-Selectin antibody was applied to a Protein A affinity column after equilibration (step 1) of the column.
- Initial load of PLBL2 determined in solution containing an anti-P-Selectin antibody: 335 ng PLBL2/mg of antibody.
- Initial load of Clusterin determined in solution containing an anti-P-Selectin antibody: 2874.8 ng Clusterin/mg of antibody.
- Initial load of CHOP determined in solution containing an anti-P-Selectin antibody 100971 ng CHOP/mg of antibody.
- Step 2 Load of antibody containing solution
- Step 1 Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 3 Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 4 Wash II: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 7 Elution: 50 mM acetic acid, pH 4.0 b) low conductivity wash (with Tris buffer only)
- Step 1 Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 3 Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 4 Wash II: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 5 Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 6 Wash IV: 2 mM Tris, pH 8.0
- Step 7 Elution: 50 mM acetic acid, pH 4.0 c) low conductivity wash (with potassium phosphate (KP) only)
- Step 1 Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 3 Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 4 Wash II: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 5 Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 6 Wash IV: 0.5 mM potassium phosphate, pH 8.0
- Step 7 Elution: 50 mM acetic acid, pH 4.0 d) high conductivity wash (with Tris buffer only)
- Step 1 Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 3 Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 4 Wash II: 700 mM Tris, pH 7.2
- Step 5 Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 7 Elution: 50 mM acetic acid, pH 4.0 e) low conductivity wash (with Tris buffer only; pH 6.0)
- Step 1 Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 3 Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 4 Wash II: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 5 Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 6 Wash IV: 2 mM Tris, pH 6.0
- Step 7 Elution: 50 mM acetic acid, pH 4.0 f) high conductivity wash (with Histidine (His)/Tris buffer only)
- Step 1 Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 3 Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 4 Wash II: 200 mM His/1000 mM Tris, pH 7.0
- Step 5 Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 7 Elution: 50 mM acetic acid, pH 4.0 g) low conductivity Tris +high conductivity Histidine (His)/Tris
- Step 1 Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 3 Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 4 Wash II: 200 mM His/1000 mM Tris, pH 7.0
- Step 5 Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 6 Wash IV: 2 mM Tris, pH 8.0
- Step 7 Elution: 50 mM acetic acid, pH 4.0 h) low conductivity potassium phosphate (KP)+high conductivity Histidine (His)/Tris
- Step 1 Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 3 Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 4 Wash II: 200 mM His/1000 mM Tris, pH 7.0
- Step 5 Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 6 Wash IV: 0.5 mM potassium phosphate, pH 8.0
- Step 7 Elution: 50 mM acetic acid, pH 4.0 i) low conductivity Tris+high conductivity Tris
- Step 1 Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 3 Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 4 Wash II: 700 mM Tris, pH 7.2
- Step 5 Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 6 Wash IV: 2 mM Tris, pH 8.0
- Step 7 Elution: 50 mM acetic acid, pH 4.0 j) low conductivity Tris; pH 6.0+high conductivity Tris
- Step 1 Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 3 Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 4 Wash II: 700 mM Tris, pH 7.2
- Step 5 Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 6 Wash IV: 2 mM Tris, pH 6.0
- Step 7 Elution: 50 mM acetic acid, pH 4.0
- Antibody anti-amyloid beta.
- Step 1 Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 3 Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 4 Wash II: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 7 Elution: 50 mM acetic acid, pH 4.0 b) low conductivity wash (with Tris buffer only)
- Step 1 Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 3 Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 4 Wash II: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 5 Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 6 Wash IV: 2 mM Tris, pH 8.0
- Step 7 Elution: 50 mM acetic acid, pH 4.0 c) high conductivity wash (with Tris buffer only)
- Step 1 Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 3 Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 4 Wash II: 700 mM Tris, pH 7.2
- Step 5 Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 7 Elution: 50 mM acetic acid, pH 4.0 d) low conductivity Tris+high conductivity Histidine (His)/Tris
- Step 1 Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 3 Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 4 Wash II: 200 mM His/1000 mM Tris, pH 7.0
- Step 5 Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 6 Wash IV: 2 mM Tris, pH 8.0
- Step 7 Elution: 50 mM acetic acid, pH 4.0
- Antibody anti-Her2
- Step 1 Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 3 Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 4 Wash II: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 7 Elution: 50 mM acetic acid, pH 4.0 b) low conductivity wash (with Tris buffer only)
- Step 1 Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 3 Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 4 Wash II: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 5 Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 6 Wash IV: 2 mM Tris, pH 8.0
- Step 7 Elution: 50 mM acetic acid, pH 4.0 c) high conductivity wash (with Tris buffer only)
- Step 1 Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 3 Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 4 Wash II: 700 mM Tris, pH 7.2
- Step 5 Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 7 Elution: 50 mM acetic acid, pH 4.0 d) low conductivity Tris+high conductivity Histidine (His)/Tris
- Step 1 Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 3 Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 4 Wash II: 200 mM His/1000 mM Tris, pH 7.0
- Step 5 Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 6 Wash IV: 2 mM Tris, pH 8.0
- Step 7 Elution: 50 mM acetic acid, pH 4.0
- Antibody anti-Ang2/VEGF-A
- Step 1 Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 3 Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 4 Wash II: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 7 Elution: 50 mM acetic acid, pH 4.0 b) low conductivity wash (with Tris buffer only)
- Step 1 Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 3 Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 4 Wash II: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 5 Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 6 Wash IV: 2 mM Tris, pH 8.0
- Step 7 Elution: 50 mM acetic acid, pH 4.0 c) high conductivity wash (with Tris buffer only)
- Step 1 Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 3 Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 4 Wash II: 700 mM Tris, pH 7.2
- Step 5 Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 7 Elution: 50 mM acetic acid, pH 4.0 d) low conductivity Tris+high conductivity Histidine (His)/Tris
- Step 1 Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 3 Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 4 Wash II: 200 mM His/1000 mM Tris, pH 7.0
- Step 5 Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 6 Wash IV: 2 mM Tris, pH 8.0
- Step 7 Elution: 50 mM acetic acid, pH 4.0
- HCP total PLBL2 Clusterin Yield Run [ng/mg] [ng/mg] [ng/mg] [%] a 3035 1.0 n.d. 85.0 b 1707 0.8 n.d. 79.8 c 655 0.7 n.d. 52 d 1050 0.8 n.d. 92.3
- Antibody anti-FIXa/X
- Step 1 Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 3 Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 4 Wash II: 700 mM Tris, pH 7.2
- Step 5 Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 7 Elution: 50 mM acetic acid, pH 4.0 b) low conductivity Tris+high conductivity Histidine (His)/Tris
- Step 1 Equilibration: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 3 Wash I: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 4 Wash II: 200 mM His/1000 mM Tris, pH 7.0
- Step 5 Wash III: 25 mM Tris, 25 mM NaCl, pH 7.0
- Step 6 Wash IV: 2 mM Tris, pH 8.0
- Step 7 Elution: 50 mM acetic acid, pH 4.0
- a solution containing an anti-FIXa/X antibody was applied to a Protein A affinity column after equilibration (step 1) of the column.
- Step 2 Load of antibody containing solution
- Step 1 Equilibration: 20 mM NaPO4, pH 7.5
- Step 3 Wash I: 450 mM NaSO4, 20 mM NaAc, pH 4.8
- Step 4 Wash II: 20 mM NaPO4, pH 7.5
- Step 6 Elution: 35 mM acetic acid, pH 4.0 b) low conductivity wash (Tris 1 mM)+high conductivity wash (with NaSO4)
- Step 1 Equilibration: 20 mM NaPO4, pH 7.5
- Step 3 Wash I: 450 mM NaSO4, 20 mM NaAc, pH 4.8
- Step 4 Wash II: 20 mM NaPO4, pH 7.5
- Step 5 Wash III: 1 mM Tris, pH 8.0
- Step 6 Elution: 50 mM acetic acid, pH 4.0 c) low conductivity wash (Tris 2 mM)+high conductivity wash (with NaSO4)
- Step 1 Equilibration: 20 mM NaPO4, pH 7.5
- Step 3 Wash I: 450 mM NaSO4, 20 mM NaAc, pH 4.8
- Step 4 Wash II: 20 mM NaPO4, pH 7.5
- Step 5 Wash III: 2 mM Tris, pH 8.0
- Step 6 Elution: 35 mM acetic acid, pH 4.0 d) low conductivity wash (Tris 4 mM)+high conductivity wash (with NaSO4)
- Step 1 Equilibration: 20 mM NaPO4, pH 7.5
- Step 3 Wash I: 450 mM NaSO4, 20 mM NaAc, pH 4.8
- Step 4 Wash II: 20 mM NaPO4, pH 7.5
- Step 5 Wash III: 4 mM Tris, pH 8.0
- Step 6 Elution: 50 mM acetic acid, pH 4.0 e) low conductivity wash (Tris 6 mM)+high conductivity wash (with NaSO4)
- Step 1 Equilibration: 20 mM NaPO4, pH 7.5
- Step 3 Wash I: 450 mM NaSO4, 20 mM NaAc, pH 4.8
- Step 4 Wash II: 20 mM NaPO4, pH 7.5
- Step 5 Wash III: 6 mM Tris, pH 8.0
- Step 6 Elution: 50 mM acetic acid, pH 4.0 f) low conductivity wash (Tris 4 mM, pH 7.8)+high conductivity wash (with NaSO4)
- Step 1 Equilibration: 20 mM NaPO4, pH 7.5
- Step 3 Wash I: 450 mM NaSO4, 20 mM NaAc, pH 4.8
- Step 4 Wash II: 20 mM NaPO4, pH 7.5
- Step 5 Wash III: 4 mM Tris, pH 7.8
- Step 6 Elution: 50 mM acetic acid, pH 4.0 g) low conductivity wash (Tris 4 mM, pH 8.2)+high conductivity wash (with NaSO4)
- Step 1 Equilibration: 20 mM NaPO4, pH 7.5
- Step 3 Wash I: 450 mM NaSO4, 20 mM NaAc, pH 4.8
- Step 4 Wash II: 20 mM NaPO4, pH 7.5
- Step 5 Wash III: 4 mM Tris, pH 8.2
- Step 6 Elution: 50 mM acetic acid, pH 4.0 h) low conductivity wash (Tris 2 mM)+high conductivity wash (with Histidine (His)/Tris 1 M)
- Step 1 Equilibration: 20 mM NaPO4, pH 7.5
- Step 3 Wash I: 200 mM His/1000 mM Tris, pH 7.0
- Step 4 Wash II: 20 mM NaPO4, pH 7.5
- Step 5 Wash III: 2 mM Tris, pH 8.0
- Step 6 Elution: 35 mM acetic acid, pH 4.0 i) low conductivity wash (Tris 2 mM)+high conductivity wash (Histidine (His)/Tris 0.85 M)
- Step 1 Equilibration: 20 mM NaPO4, pH 7.5
- Step 3 Wash I: 200 mM His/850 mM Tris, pH 7.0
- Step 4 Wash II: 20 mM NaPO4, pH 7.5
- Step 5 Wash III: 2 mM Tris, pH 8.0
- Step 6 Elution: 50 mM acetic acid, pH 4.0 j) low conductivity wash (Tris 2 mM)+high conductivity wash (Histidine (His)/Tris 0.7 M)
- Step 1 Equilibration: 20 mM NaPO4, pH 7.5
- Step 3 Wash I: 200 mM His/700 mM Tris, pH 7.0
- Step 4 Wash II: 20 mM NaPO4, pH 7.5
- Step 5 Wash III: 2 mM Tris, pH 8.0
- Step 6 Elution: 50 mM acetic acid, pH 4.0 k) low conductivity wash (Tris 2 mM)+high conductivity wash (Histidine (His)/Tris 0.55 M)
- Step 1 Equilibration: 20 mM NaPO4, pH 7.5
- Step 3 Wash I: 200 mM His/550 mM Tris, pH 7.0
- Step 4 Wash II: 20 mM NaPO4, pH 7.5
- Step 5 Wash III: 2 mM Tris, pH 8.0
- Step 6 Elution: 50 mM acetic acid, pH 4.0
- Null harvested cell culture fluid was produced using non-transfected CHO-DP12 cells cultured in serum-free media. Fermentation was performed at the 2 L-scale using a representative cell culture process. At the end of 14 days of fermentation, cell culture fluid was harvested via centrifugation and sterile filtration. This harvested cell culture fluid (HCCF) was then stored at ⁇ 70° C. until experimentation.
- HCCF harvested cell culture fluid
- Recombinant CHO PLBL2 with a C-terminal hexahistidine-tag was expressed in 35 L-scale transient transfections and purified from harvested cell culture fluid as previously described (Vanderlaan et al., 2015). Purified PLBL2 was then formulated in a PBS solution and stored at ⁇ 70° C. until experimentation.
- Recombinant humanized antibodies were expressed in CHO cells and purified using column chromatography to ensure PLBL2 concentration was below 20 ng/mg. Prior to beginning each study, each antibody was buffer-exchanged into PBS using PD-10 desalting columns (GE Healthcare).
- purified antibodies were diluted to the same concentration with PBS and spiked into HCCF from a non-producing cell line to give a final antibody titer of 5 g/L.
- a control was also prepared wherein PBS was added instead of the purified antibody to evaluate non-specific host cell protein binding to the Protein A resin in the absence of antibody.
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Oncology (AREA)
- Hematology (AREA)
- Analytical Chemistry (AREA)
- Virology (AREA)
- Cell Biology (AREA)
- Communicable Diseases (AREA)
- Pulmonology (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US15/900,461 US20180186832A1 (en) | 2015-08-21 | 2018-02-20 | Method for the reduction of host cell proteins in affinity chromatography |
US17/328,408 US20220169675A1 (en) | 2015-08-21 | 2021-05-24 | Method for the reduction of host cell proteins in affinity chromatography |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201562208523P | 2015-08-21 | 2015-08-21 | |
PCT/EP2016/069604 WO2017032686A1 (en) | 2015-08-21 | 2016-08-18 | Method for the reduction of host cell proteins in affinity chromatography |
US15/900,461 US20180186832A1 (en) | 2015-08-21 | 2018-02-20 | Method for the reduction of host cell proteins in affinity chromatography |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP2016/069604 Continuation WO2017032686A1 (en) | 2015-08-21 | 2016-08-18 | Method for the reduction of host cell proteins in affinity chromatography |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US17/328,408 Continuation US20220169675A1 (en) | 2015-08-21 | 2021-05-24 | Method for the reduction of host cell proteins in affinity chromatography |
Publications (1)
Publication Number | Publication Date |
---|---|
US20180186832A1 true US20180186832A1 (en) | 2018-07-05 |
Family
ID=56802471
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/900,461 Abandoned US20180186832A1 (en) | 2015-08-21 | 2018-02-20 | Method for the reduction of host cell proteins in affinity chromatography |
US17/328,408 Pending US20220169675A1 (en) | 2015-08-21 | 2021-05-24 | Method for the reduction of host cell proteins in affinity chromatography |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US17/328,408 Pending US20220169675A1 (en) | 2015-08-21 | 2021-05-24 | Method for the reduction of host cell proteins in affinity chromatography |
Country Status (16)
Country | Link |
---|---|
US (2) | US20180186832A1 (ja) |
EP (1) | EP3337812B1 (ja) |
JP (3) | JP6968055B2 (ja) |
KR (1) | KR20180034500A (ja) |
CN (2) | CN116063375A (ja) |
AU (1) | AU2016312909B2 (ja) |
BR (1) | BR112018001511A2 (ja) |
CA (1) | CA2992420A1 (ja) |
ES (1) | ES2877532T3 (ja) |
HK (1) | HK1251584A1 (ja) |
HR (1) | HRP20211026T1 (ja) |
IL (1) | IL257146A (ja) |
MX (1) | MX2018001536A (ja) |
PL (1) | PL3337812T3 (ja) |
SI (1) | SI3337812T1 (ja) |
WO (1) | WO2017032686A1 (ja) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2021061504A1 (en) * | 2019-09-23 | 2021-04-01 | Merck Sharp & Dohme Corp. | Methods and compositions comprising an anti-ctla4 monoclonal antibody with reduced host cell proteins and increased polysorbate-80 stability |
WO2023137143A1 (en) * | 2022-01-14 | 2023-07-20 | Shattuck Labs, Inc. | Methods of contaminant removal from protein isolates |
Families Citing this family (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2959817A1 (en) | 2014-09-10 | 2016-03-17 | The Procter & Gamble Company | Nonwoven web |
EP3337818A1 (en) * | 2015-08-21 | 2018-06-27 | H. Hoffnabb-La Roche Ag | Affinity chromatography purification with low conductivity wash buffer |
EP3337812B1 (en) | 2015-08-21 | 2021-04-28 | F. Hoffmann-La Roche AG | Method for the reduction of host cell proteins in affinity chromatography |
JP7073253B2 (ja) | 2015-08-21 | 2022-05-23 | エフ.ホフマン-ラ ロシュ アーゲー | アフィニティークロマトグラフィーにおける宿主細胞タンパク質の低減方法 |
SG11202009499VA (en) * | 2018-05-04 | 2020-11-27 | Sigma Aldrich Co Llc | Producing recombinant proteins with reduced levels of host cell proteins |
EP3826743A4 (en) * | 2018-07-25 | 2022-09-14 | Merck Sharp & Dohme Corp. | METHODS FOR SEPARATION OF HOST CELL LIPASES FROM A PRODUCTION PROTEIN IN CHROMATOGRAPHIC METHODS |
WO2023284073A1 (zh) * | 2021-07-13 | 2023-01-19 | 江苏荃信生物医药股份有限公司 | 降低单克隆抗体生产中宿主细胞蛋白含量的亲和纯化方法、抗人ifnar1单克隆抗体浓缩溶液的制备方法以及液体制剂 |
Family Cites Families (15)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ES2626268T3 (es) | 2002-09-11 | 2017-07-24 | Chugai Seiyaku Kabushiki Kaisha | Método de purificación de proteínas |
TWI306862B (en) | 2005-01-03 | 2009-03-01 | Hoffmann La Roche | Antibodies against il-13 receptor alpha 1 and uses thereof |
PE20100748A1 (es) * | 2005-12-12 | 2010-11-12 | Hoffmann La Roche | Anticuerpo anti beta-4-amiloide que contiene asparagina glicosilada en la region variable de vh |
US8263750B2 (en) | 2006-03-16 | 2012-09-11 | Amgen Inc. | Method for purifying a protein using protein-A affinity chromatography using an intermediate wash step |
WO2011038894A1 (en) * | 2009-10-01 | 2011-04-07 | F. Hoffmann-La Roche Ag | Protein a chromatography |
SG10201408384PA (en) | 2009-12-18 | 2015-01-29 | Novartis Ag | Wash solution and method for affinity chromatography |
ES2671347T3 (es) * | 2010-06-21 | 2018-06-06 | Kyowa Hakko Kirin Co., Ltd. | Procedimiento para purificar una proteína que utiliza un aminoácido |
WO2014186350A1 (en) | 2013-05-15 | 2014-11-20 | Medimmune Limited | Purification of recombinantly produced polypeptides |
TWI596107B (zh) * | 2013-06-25 | 2017-08-21 | 卡地拉保健有限公司 | 單株抗體之新穎純化方法 |
US10144774B2 (en) * | 2013-07-01 | 2018-12-04 | Csl Behring Ag | Method for purifying IgG |
CN105473612A (zh) * | 2013-08-19 | 2016-04-06 | 豪夫迈·罗氏有限公司 | 用羟基磷灰石层析分离双特异性抗体和双特异性抗体生产副产物 |
CA3184564A1 (en) * | 2013-09-13 | 2015-03-19 | Genentech, Inc. | Methods and compositions comprising an anti-il13 antibody and residual hamster phospholipase b-like 2 |
CN103497248B (zh) * | 2013-09-22 | 2016-08-10 | 中国抗体制药有限公司 | 一种从细胞培养上清中分离纯化抗体的方法 |
JP7073253B2 (ja) | 2015-08-21 | 2022-05-23 | エフ.ホフマン-ラ ロシュ アーゲー | アフィニティークロマトグラフィーにおける宿主細胞タンパク質の低減方法 |
EP3337812B1 (en) | 2015-08-21 | 2021-04-28 | F. Hoffmann-La Roche AG | Method for the reduction of host cell proteins in affinity chromatography |
-
2016
- 2016-08-18 EP EP16757608.1A patent/EP3337812B1/en active Active
- 2016-08-18 AU AU2016312909A patent/AU2016312909B2/en active Active
- 2016-08-18 SI SI201631245T patent/SI3337812T1/sl unknown
- 2016-08-18 CN CN202211094785.4A patent/CN116063375A/zh active Pending
- 2016-08-18 CN CN201680042079.1A patent/CN107849087B/zh active Active
- 2016-08-18 PL PL16757608T patent/PL3337812T3/pl unknown
- 2016-08-18 CA CA2992420A patent/CA2992420A1/en active Pending
- 2016-08-18 JP JP2018509846A patent/JP6968055B2/ja active Active
- 2016-08-18 WO PCT/EP2016/069604 patent/WO2017032686A1/en active Application Filing
- 2016-08-18 ES ES16757608T patent/ES2877532T3/es active Active
- 2016-08-18 MX MX2018001536A patent/MX2018001536A/es unknown
- 2016-08-18 KR KR1020187004868A patent/KR20180034500A/ko active IP Right Grant
- 2016-08-18 BR BR112018001511A patent/BR112018001511A2/pt active Search and Examination
-
2018
- 2018-01-25 IL IL257146A patent/IL257146A/en unknown
- 2018-02-20 US US15/900,461 patent/US20180186832A1/en not_active Abandoned
- 2018-08-23 HK HK18110896.4A patent/HK1251584A1/zh unknown
-
2021
- 2021-05-24 US US17/328,408 patent/US20220169675A1/en active Pending
- 2021-06-29 HR HRP20211026TT patent/HRP20211026T1/hr unknown
- 2021-07-30 JP JP2021124951A patent/JP7258964B2/ja active Active
-
2023
- 2023-04-05 JP JP2023061111A patent/JP2023076617A/ja active Pending
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2021061504A1 (en) * | 2019-09-23 | 2021-04-01 | Merck Sharp & Dohme Corp. | Methods and compositions comprising an anti-ctla4 monoclonal antibody with reduced host cell proteins and increased polysorbate-80 stability |
WO2023137143A1 (en) * | 2022-01-14 | 2023-07-20 | Shattuck Labs, Inc. | Methods of contaminant removal from protein isolates |
Also Published As
Publication number | Publication date |
---|---|
KR20180034500A (ko) | 2018-04-04 |
PL3337812T3 (pl) | 2021-10-11 |
JP2023076617A (ja) | 2023-06-01 |
ES2877532T3 (es) | 2021-11-17 |
HK1251584A1 (zh) | 2019-02-01 |
EP3337812A1 (en) | 2018-06-27 |
MX2018001536A (es) | 2018-04-24 |
CN116063375A (zh) | 2023-05-05 |
CN107849087A (zh) | 2018-03-27 |
CN107849087B (zh) | 2022-09-06 |
EP3337812B1 (en) | 2021-04-28 |
SI3337812T1 (sl) | 2021-08-31 |
JP7258964B2 (ja) | 2023-04-17 |
HRP20211026T1 (hr) | 2021-10-01 |
JP2021176900A (ja) | 2021-11-11 |
JP2018531895A (ja) | 2018-11-01 |
AU2016312909B2 (en) | 2022-12-08 |
WO2017032686A1 (en) | 2017-03-02 |
CA2992420A1 (en) | 2017-03-02 |
IL257146A (en) | 2018-03-29 |
JP6968055B2 (ja) | 2021-11-17 |
AU2016312909A1 (en) | 2018-02-22 |
US20220169675A1 (en) | 2022-06-02 |
BR112018001511A2 (pt) | 2018-09-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP7258964B2 (ja) | アフィニティークロマトグラフィーにおける宿主細胞タンパク質の低減方法 | |
JP6435193B2 (ja) | 抗体を精製する方法 | |
CA2921999C (en) | Methods and compositions comprising an anti-il13 antibody and residual hamster phospholipase b-like 2 | |
US20220119500A1 (en) | Affinity chromatography purification with low conductivity wash buffer | |
US20220119499A1 (en) | Method for the reduction of host cell proteins in affinity chromatography | |
US20230273216A1 (en) | Methods for identifying free thiols in proteins | |
US20170029495A1 (en) | Human Antibodies that Bind Human TNF-Alpha and Methods of Preparing the Same | |
CA3179460A1 (en) | A high-throughput and mass-spectrometry-based method for quantitating antibodies |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: FINAL REJECTION MAILED |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |
|
AS | Assignment |
Owner name: HOFFMANN-LA ROCHE INC., NEW JERSEY Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:F. HOFFMANN-LA ROCHE AG;REEL/FRAME:061681/0016 Effective date: 20160721 Owner name: F. HOFFMANN-LA ROCHE AG, SWITZERLAND Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:ROCHE DIAGNOSTICS GMBH;REEL/FRAME:061680/0947 Effective date: 20160628 Owner name: ROCHE DIAGNOSTICS GMBH, GERMANY Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:FALKENSTEIN, ROBERTO;KLEINJANS, ANNIKA;KOEHNLEIN, WOLFGANG;AND OTHERS;SIGNING DATES FROM 20160415 TO 20160513;REEL/FRAME:061680/0818 Owner name: GENENTECH, INC., CALIFORNIA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:TRAN, BENJAMIN;ST. JOHN, RICHARD;MCDONALD, PAUL;AND OTHERS;SIGNING DATES FROM 20160728 TO 20160921;REEL/FRAME:061679/0974 |