WO1999018130A1 - Procede de purification d'immunoglobulines - Google Patents
Procede de purification d'immunoglobulines Download PDFInfo
- Publication number
- WO1999018130A1 WO1999018130A1 PCT/IL1998/000483 IL9800483W WO9918130A1 WO 1999018130 A1 WO1999018130 A1 WO 1999018130A1 IL 9800483 W IL9800483 W IL 9800483W WO 9918130 A1 WO9918130 A1 WO 9918130A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- column
- solution
- immunoglobulins
- cation exchange
- purification
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 54
- 108060003951 Immunoglobulin Proteins 0.000 title claims abstract description 49
- 102000018358 immunoglobulin Human genes 0.000 title claims abstract description 49
- 229940072221 immunoglobulins Drugs 0.000 title claims abstract description 42
- 238000000746 purification Methods 0.000 title claims abstract description 27
- 239000003729 cation exchange resin Substances 0.000 claims abstract description 25
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims abstract description 18
- 239000000243 solution Substances 0.000 claims description 47
- 239000003599 detergent Substances 0.000 claims description 25
- 230000002209 hydrophobic effect Effects 0.000 claims description 16
- 150000003839 salts Chemical class 0.000 claims description 16
- 108010044316 Cohn fraction III Proteins 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 238000005341 cation exchange Methods 0.000 claims description 10
- 229940023913 cation exchange resins Drugs 0.000 claims description 8
- 239000003960 organic solvent Substances 0.000 claims description 7
- 239000003929 acidic solution Substances 0.000 claims description 5
- 239000011347 resin Substances 0.000 description 28
- 229920005989 resin Polymers 0.000 description 28
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 27
- 102000004169 proteins and genes Human genes 0.000 description 27
- 108090000623 proteins and genes Proteins 0.000 description 27
- 239000002904 solvent Substances 0.000 description 17
- 229940008228 intravenous immunoglobulins Drugs 0.000 description 16
- 125000002091 cationic group Chemical group 0.000 description 14
- 239000000047 product Substances 0.000 description 14
- 238000011026 diafiltration Methods 0.000 description 12
- 239000013504 Triton X-100 Substances 0.000 description 11
- 229920004890 Triton X-100 Polymers 0.000 description 11
- 238000004519 manufacturing process Methods 0.000 description 11
- 239000000203 mixture Substances 0.000 description 10
- 238000002360 preparation method Methods 0.000 description 10
- STCOOQWBFONSKY-UHFFFAOYSA-N tributyl phosphate Chemical compound CCCCOP(=O)(OCCCC)OCCCC STCOOQWBFONSKY-UHFFFAOYSA-N 0.000 description 10
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 10
- 239000003153 chemical reaction reagent Substances 0.000 description 9
- 241000700605 Viruses Species 0.000 description 8
- 239000003957 anion exchange resin Substances 0.000 description 8
- 210000002381 plasma Anatomy 0.000 description 8
- 238000004587 chromatography analysis Methods 0.000 description 7
- 230000008030 elimination Effects 0.000 description 7
- 238000003379 elimination reaction Methods 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 238000010828 elution Methods 0.000 description 6
- 150000002632 lipids Chemical class 0.000 description 6
- 238000011084 recovery Methods 0.000 description 6
- 102000004895 Lipoproteins Human genes 0.000 description 5
- 108090001030 Lipoproteins Proteins 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- -1 carboxy, sulfo Chemical group 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 230000002779 inactivation Effects 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 4
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 150000001768 cations Chemical class 0.000 description 4
- 230000001143 conditioned effect Effects 0.000 description 4
- 239000000706 filtrate Substances 0.000 description 4
- 239000013020 final formulation Substances 0.000 description 4
- 238000011068 loading method Methods 0.000 description 4
- 239000011159 matrix material Substances 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 3
- 150000001450 anions Chemical class 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 229940027941 immunoglobulin g Drugs 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 239000003456 ion exchange resin Substances 0.000 description 3
- 229920003303 ion-exchange polymer Polymers 0.000 description 3
- 238000012454 limulus amebocyte lysate test Methods 0.000 description 3
- 230000007935 neutral effect Effects 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 229910052708 sodium Inorganic materials 0.000 description 3
- 208000030507 AIDS Diseases 0.000 description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 239000012190 activator Substances 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- 230000002391 anti-complement effect Effects 0.000 description 2
- 108010008730 anticomplement Proteins 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 238000011210 chromatographic step Methods 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 230000003750 conditioning effect Effects 0.000 description 2
- 239000012149 elution buffer Substances 0.000 description 2
- 238000011067 equilibration Methods 0.000 description 2
- 238000012869 ethanol precipitation Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 238000005194 fractionation Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000005342 ion exchange Methods 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002480 mineral oil Substances 0.000 description 2
- 235000010446 mineral oil Nutrition 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 2
- 229920000053 polysorbate 80 Polymers 0.000 description 2
- 238000002203 pretreatment Methods 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- LBCZOTMMGHGTPH-UHFFFAOYSA-N 2-[2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethoxy]ethanol Chemical compound CC(C)(C)CC(C)(C)C1=CC=C(OCCOCCO)C=C1 LBCZOTMMGHGTPH-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 108010032597 Cohn fraction II Proteins 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 101150096839 Fcmr gene Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 208000005176 Hepatitis C Diseases 0.000 description 1
- 238000012369 In process control Methods 0.000 description 1
- 101100018618 Mus musculus Igll1 gene Proteins 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 102000005686 Serum Globulins Human genes 0.000 description 1
- 108010045362 Serum Globulins Proteins 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000010306 acid treatment Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 238000005349 anion exchange Methods 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 230000003171 anti-complementary effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000013065 commercial product Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 125000004093 cyano group Chemical group *C#N 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000006862 enzymatic digestion Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 108010074605 gamma-Globulins Proteins 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000000951 immunodiffusion Effects 0.000 description 1
- 238000010965 in-process control Methods 0.000 description 1
- 238000010949 in-process test method Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 239000003014 ion exchange membrane Substances 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 238000010979 pH adjustment Methods 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 238000001050 pharmacotherapy Methods 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- NRHMKIHPTBHXPF-TUJRSCDTSA-M sodium cholate Chemical compound [Na+].C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 NRHMKIHPTBHXPF-TUJRSCDTSA-M 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000011146 sterile filtration Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 125000004964 sulfoalkyl group Chemical group 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000041 toxicology testing Toxicity 0.000 description 1
- RXPQRKFMDQNODS-UHFFFAOYSA-N tripropyl phosphate Chemical compound CCCOP(=O)(OCCC)OCCC RXPQRKFMDQNODS-UHFFFAOYSA-N 0.000 description 1
- MGMXGCZJYUCMGY-UHFFFAOYSA-N tris(4-nonylphenyl) phosphite Chemical compound C1=CC(CCCCCCCCC)=CC=C1OP(OC=1C=CC(CCCCCCCCC)=CC=1)OC1=CC=C(CCCCCCCCC)C=C1 MGMXGCZJYUCMGY-UHFFFAOYSA-N 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 230000003253 viricidal effect Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
- C07K16/065—Purification, fragmentation
-
- 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
- C07K1/18—Ion-exchange chromatography
-
- 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
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
Definitions
- the present invention is generally in the field of purification of immunoglobulins from a source solution and more specifically concerns purification of immunoglobulins utilizing ion-exchange resins.
- ISG immune serum globulins
- the final pH ranges from 4.2-5.5.
- acid treatment Uemura, Y., J. Exp. Med, 141(3 ):337-349 (1983)
- trypsin trypsin in low pH. All these modifications were proved to be inferior to natural intact Immunoglobulins.
- ISG products both IMG and IVIG
- ISG products have been considered generally safe
- ISG products do not transmit active viruses such as those associated with hepatitis or, HIV which is associated with Acquired Immune Deficiency Syndrome (AIDS).
- AIDS Acquired Immune Deficiency Syndrome
- Cohn fraction II the main source of Immunoglobulins used in the 80s was contaminated with Hepatitis C.
- a new method for virus inactivation was developed by the New York Blood Center. The method is based on the virucidal effect of an organic solvent combined with an nonionic detergent (US 4,481,189 and US 4,315,919).
- the high amount of both the detergent and the organic solvent is toxic, therefore both have to be removed from the final formulation.
- the purification by anion exchange resins has the disadvantages of loss of IgG sub-classes 3 and 4, as compared to the source solution.
- purification by anion exchange resins causes the purified gamma-globulins to aggregate, which aggregates have to be eventually disassociated by digestion with trypsin and pepsin, resulting in an Ig molecule of reduced efficacy.
- the yield of purification by anion exchange resins is usually in the range of 75 to 85% since a large amount of the immunoglobulins are not absorbed on the anion exchange resin and are thus discarded.
- the present invention is based on the surprising finding that cation exchange resins, preferably pre-conditioned with a pH lower than 4.5, can leave a retinue of relatively large quantities of immunoglobulins as compared to anionic resins. According to the finding of the invention, it was discovered that by using cation-exchange resins, essentially all the solvent/detergent was eliminated. A further finding was that the elution at higher salt concentration and pH above 7.5 releases (eluted) all the immunoglobulins G heavy chain subclasses, notably IgG subclass 3 and 4, so that the purified immunoglobulins have a sub-class ratio similar to that of the source solution.
- the present invention provides a method for the purification of immunoglobulins from a source solution comprising: contacting the source solution with a cation exchange resin; and - eluting the immunoglobulins bound to said cation exchange resin.
- immunoglobulin refers to immunoglobulins in particularly IgG of all sub-classes.
- the purification may be from any source solution such as: Cohn Fraction Paste II, PEG precipitated plasma supernatant, DEAE purified plasma effluent, ammonium sulfate precipitate of plasma or serum.
- the purification is from a Cohn Fraction III, i.e. from a fraction obtained from blood plasma by a cold ethanol precipitation technique as specified in Technical Procedure I, hereinafter.
- the method comprises contacting the source solution with a cation exchange resin.
- cation exchange resin refers to a highly polymerized synthetic organic compound consisting of a large, non-diffusable anion and a simple, dif ⁇ usable cation, which latter can be exchanged for a cation in the medium in which the resin is placed.
- Examples of cation exchange resins are any kind of matrix fiinctionalized with a carboxy, sulfo, sulfoalkyl, cyano and phenyl groups.
- the cation exchange compound may be bound to any type of solid support including any matrix resins.
- the cation exchange resin is constructed in the form of a chromatographic column and the method includes passing the source solution through the cation exchange chromatographic column which leads to binding of the immunoglobulins to the cation exchange resins.
- the resin is pre-treated with an acidic solution.
- an acidic solution it was surprisingly found that such pre-treatment facilitates binding of the immunoglobulins to the resins, increases the capacity of the cation exchange resin, and the yield of the whole process, from about 30 mg per ml of resin to 50 mg of SP resin.
- the concept of utilizing pof the ion-exchange resin by acidic solution is contrary to the state of the art concept of using neutral ion exchange membranes. Ion exchange resins are usually cleaned by acid followed by base or vice versa.
- the immunoglobulins are removed or "eluted" from the cation exchange resin, by rinsing it with a solution having a high salt concentration, usually in the range of 0.3 molar 1.5 molar, preferably 0.45 to 0.65 molar, most preferably 0.5 molar and having a pH in the range of 6.0-9.0, preferably 6.5 to 7.5, most preferably a pH of 7.0.
- a solution having a high salt concentration usually in the range of 0.3 molar 1.5 molar, preferably 0.45 to 0.65 molar, most preferably 0.5 molar and having a pH in the range of 6.0-9.0, preferably 6.5 to 7.5, most preferably a pH of 7.0.
- it is preferable to treat the source solution, prior to contact with a cation exchange resin, with an nonionic detergent and an organic solvent, in order to inactivate viruses present in the source solution as specified in U.S. 4,481,189, and U.S.
- the source solution should be incubated with a solvent/detergent in an acidic pH, at a pH in the range of 5.1 to 5.4, preferably a pH of 5.2 for several o hours at low temperatures such as 4°C.
- the cationic exchange chromatography serves to remove virtually all the solvent/detergent reagents. However, if it is desired to remove also various hydrophobic lipids, or lipoprotein impurities which are present in the source solution, it is possible also to pass the immunoglobulins eluted from the 5 cation exchange resins through a hydrophobic column, preferably a hydrophobic column having a substantially smaller volume than the volume of the cation exchange column, for example, 7 to 12 times smaller preferably about 10 times smaller.
- the present invention provides a method for the 0 purification of immunoglobulins from Cohn Fraction III solution comprising: (i) diafiltratring the Cohn Fraction III solution against water; (ii) adjusting the pH of the diafiltrated solution to a pH of 5.1 to 5.4; (iii) contacting the solution obtained in (ii) with an organic solvent and a detergent; 5 (iv) passing the solution obtained in (iii) through a cation exchange chromatographic column; (v) eluting the immunoglobulins absorbed on the column by passing a solution having a salt concentration of 0.3 M - 1.5 M and a pH of 6.0-7.0.
- step (vi) diafiltrating the elute of step (v) against water: and optionally (vii) passing the eluted immunoglobulin obtained in step (vi) through a hydrophobic column which is 7 to 12 times smaller than the cation exchange column.
- Fig. 1 shows a schematic flow chart for the preparation of Cohn Fraction III.
- Fig. 1 shows a schematic representation of the preparation of Cohn Fraction III.
- Example 1 The effect of various buffers used for conditioning and elution on the concentration of IgG purified on an SP column
- the combined mixture was loaded on a pre-conditioned FPLC (Pharmacia, Sweden) mounted with a 16 mm column loaded with 74 ml Fractogel SP (TosoHaas, Japan) resin, which is a hydrophilic methacrylate copolymer matrix functionalized with a sulphopropyl (-CH2CH 2 CH 2 SO3) group.
- This 470x16 column as the same bed height as the large scale column.
- the Fractogel SP column was pre-conditioned with 2 column volumes of various buffers as specified in Table 2 below, the Immunoglobulin (concentrated Cohn Fraction III prepared according to Fig. 1) mixture with the solvent detergent reagent was loaded at a rate of 1 ml/min. followed by running various elusion buffers as specified in Table 2 below. After the elusion of the SD from the mixture the protein was eluted from the SP column by various elution buffers (see Table 2).
- Example 2 The effect on recovery and elution volume of Immunoglobulin G using three types of cationic exchange resins A. Technical procedure
- SP0611- SP-Toyopearl 650 (M) - from Tosoh corporation Tokyo, Japan - CH 2 CH 2 CH 2 SO 3 (sulphopropyl).
- the ionic strength of the cationic residue has a significant effect on the gel capacity.
- the operating mechanism of ion exchange chromatography is by reversible bidning of charged molecules. Binding strength is governed by the degree of charge on the substrate - the pKa of the ion exchange matrix, and the aqueous solution properties pH and ionic strength.
- the pKa of SP resins is 2.3 and for all the CM resin is 4.2.
- the ion exchange capacity is 0.08-0.12 meq/ml resin for CM resin and 0.13-0.17 meq/ml resin for the SP resin.
- the subclasses IgG distribution of the immunoglobulins in the preparation purified on the SP resin is the same as in the starting material indicating that the purification method of the invention, utilizing cation exchange resins, does not result in loss of IgG subclasses 3 and 4. This being in contrast to a significant IgG subclass 3 and 4 loss evident in purification procedures utilizing anion-exchange resins.
- a major factor in choosing purification techniques is the ability of the technique to completely remove essentially all of the detergent, (such as X-100), which was added in order to eliminate viruses.
- Intravenous immunoglobulin solution was prepared from frozen human plasma by means of Cohn method Fraction III followed by additional purification and virus inactivation steps (McCue et al, supra). Fraction III which contains about 3-5 mg/ml protein and 17% ethanol was concentrated by Diafiltration to 60-70 mg/ml and the salt and the ethanol were removed by 6 volumes of Diafiltration against water. The pH was adjusted to 5.2 and solution was treated by solvent / detergent (S/D) solution comprising: 0.3% Tri (n-butyl) phosphate, 1% Triton X-100 for 6 hours at 4°C.
- S/D solvent / detergent
- the S/D reagents were removed by cationic exchange chromatography on a 200 ml SP Toyopearl 650 resin (Tosohaas Corporation, Tokyo, Japan (-CH 2 CH 2 CH 2 SO3) sulphopropyl functional group) at low pH (4.0). At this pH, the positively charged IVIg is bound to the column, allowing the removal of the >97 of the Triton X-100 in the column spend.
- SP Toyopearl 650 resin Tosohaas Corporation, Tokyo, Japan (-CH 2 CH 2 CH 2 SO3) sulphopropyl functional group
- the IVIg was removed from the cationic resin by increasing the salt concentration to 0.35 molar sodium chloride and increasing the pH to 7.0. Removal of salt and concentration of protein to about 70 mg/ml was than followed by Diafiltration against water. In order to remove the lipids or lipoproteins from the IVIg solution the solution was passed through a an hydrophobic column (C-18 resin) which is 10 times smaller than the cationic column or with a ratio of 500 mg protein per ml of resin. The IVIg protein was then incubated for 22 hours at 37°C at pH 4.0 in the presence of 10% maltose in order to reduce the aggregates of Immunoglobulins. This preparation was than subjected to an addition Diafiltration against water followed by final formulation of the balk material. The final formulation consists of 10% maltose and 50 mg/ml protein at pH 5.2 . The sterile filtered bulk product is then filled in 50 ml aliquots which was stored at room temperature.
- the cationic exchange chromatography step is highly efficient in removing the detergent reagents that are added for the purpose of viral inactivation. More than 95% Triton levels were reduced by the cationic exchange chromatography.
- the following step of Diafiltration brought the levels below the limits specified by the toxicological studies which are > than 50 PPM of Triton X-100 and 30 PPM of TnBP in the final product of 5 mg/ml. so that even without purification on a hydrophobic column Triton levels were reduced to a medically acceptable level. Therefore it can be concluded that the hydrophobic chromatography on the C18 column is not essential but rather optional and was performed solely for the elimination of hydrophobic lipid or lipoproteins, and not for elimination of the solvent or detergent.
- the source material for IVIg was Cohn Fraction III.
- the fraction which contains about 3-5 mg/ml protein and 17% ethanol was concentrated by Diafiltration to 60-70 mg/ml.
- the salt and the ethanol were removed by the ration of 6 volumes of Diafiltration against water.
- the S/D reagents were removed by cationic exchange chromatography. At low pH (4.0) the positively charged IVIg is bound to the column. The IVIg is removed from the cationic resin by increasing the salt concentration and increasing the pH to 7.0. Concentration of the protein to 70 mg/ml and removal of salt is done by Diafiltration against water.
- the solution is passed through a hydrophobic column which is 10 times smaller than the cationic column.
- a sample from the S/D treated product was collected half an hour after the administration of the mixture.
- the product sampled was eluted in the final step of column A.
- a sample from the product which passed diafiltration was then collected and mixed well for 10 mins. before collection.
- a sample from the effluent of the column B was collected while noting the total weight of the vessel. Initially the effluent from the column loading was collected while continuing to collect until the absorbency reaches 5%. The volume of the solution collected was determined, the sample was mixed four times and 10 ml samples were obtained for TNPP and Triton. The samples were frozen at -18°C or colder until ready for assaying. Table 7
- the cationic exchange chromatography step is highly efflciient in removing the solvent Detergent reagents that are added at the virus inactivation step.
- both the TNBP and Triton levels were reduced by greater than 99.8% to levels below the specified limits in the final product of 5 Vml and the tables of the SD elimination show consistency of the purification steps from batch to batch.
- the hydrophobic column has only a marginal role in the solvent detergent removal and therefore it serves the sole purpose of elimination of lipids and lipoprotein from the product.
- a batch of purified immunoglobulins prepared as above was transferred to Tel Ha Shomer Laboratory (Israel) for analysis.
- Purified immunoglobulins prepared as described above were stored for 12 months at 4°C and at room temperature. Parameters tested remained stable during the year's storage at both temperatures.
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU94568/98A AU9456898A (en) | 1997-10-07 | 1998-10-02 | A method for the purification of immunoglobulins |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
IL121900 | 1997-10-07 | ||
IL12190097A IL121900A (en) | 1997-10-07 | 1997-10-07 | A method for the purification of immunoglobulins |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1999018130A1 true WO1999018130A1 (fr) | 1999-04-15 |
Family
ID=11070714
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/IL1998/000483 WO1999018130A1 (fr) | 1997-10-07 | 1998-10-02 | Procede de purification d'immunoglobulines |
Country Status (3)
Country | Link |
---|---|
AU (1) | AU9456898A (fr) |
IL (1) | IL121900A (fr) |
WO (1) | WO1999018130A1 (fr) |
Cited By (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001072844A2 (fr) * | 2000-03-30 | 2001-10-04 | Amersham Biosciences Ab | Procédé de production d'igg |
EP1225180A2 (fr) * | 2001-01-17 | 2002-07-24 | Probitas Pharma, S.A. | Procédé de production de gammaglobuline G humaine à virus inactivé |
WO2007067856A2 (fr) * | 2005-12-06 | 2007-06-14 | Advantek Serum Laboratories Limited | Procédé pour l'inactivation et la suppression du virus de la dengue dans des échantillons biologiques |
JP2008540462A (ja) * | 2005-05-10 | 2008-11-20 | マレー ゴールバーン コーオペラティブ コー リミテッド | 免疫グロブリン分画およびそのためのプロセス |
WO2010048183A1 (fr) * | 2008-10-20 | 2010-04-29 | Abbott Laboratories | Anticorps se liant à il-18 et procédés de purification de ceux-ci |
WO2010064241A1 (fr) | 2008-12-04 | 2010-06-10 | Omrix Biopharmaceuticals Ltd. | Administration sous-cutanée d'anticorps anti-hépatite b |
US8354249B2 (en) | 2005-08-11 | 2013-01-15 | Omrix Biopharmaceuticals Ltd. | Intravenous immunoglobulin composition |
US8895709B2 (en) | 2008-10-20 | 2014-11-25 | Abbvie Inc. | Isolation and purification of antibodies using protein A affinity chromatography |
US9023994B2 (en) | 2011-05-16 | 2015-05-05 | Omrix Biopharmaceuticals, Ltd. | Immunoglobulin reduced in thrombogenic agents and preparation thereof |
US9109010B2 (en) | 2008-10-20 | 2015-08-18 | Abbvie Inc. | Viral inactivation during purification of antibodies cross reference to related applications |
US9631007B2 (en) | 2005-05-25 | 2017-04-25 | Hoffmann-La-Roche Inc. | Method for the purification of antibodies |
EP2061803B1 (fr) | 2006-08-28 | 2019-08-14 | Ares Trading S.A. | Procédé de purification de protéines contenant fc |
CN112574296A (zh) * | 2020-12-30 | 2021-03-30 | 中国医学科学院输血研究所 | 一种模拟IVIg的多人份混合人血浆IgG样品的分离纯化方法 |
WO2022161889A1 (fr) | 2021-01-27 | 2022-08-04 | Centre National De La Recherche Scientifique | Procede de fabrication d'un detecteur des flux d'un premier et d'un deuxieme rayonnements ionisants |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4877866A (en) * | 1986-11-27 | 1989-10-31 | Biotest Pharma Gmbh | Method of producing a virus safe, storage-stable, and intravenously tolerable immunoglobulin-G preparation |
EP0440483A2 (fr) * | 1990-02-01 | 1991-08-07 | Baxter International Inc. | Procédé de préparation d'immunoglobulines de sérum |
EP0525502A1 (fr) * | 1991-08-02 | 1993-02-03 | Octapharma Ag | Procédé pour la fabrication de solutions contenant des immunoglobulines et des viruses inactivés |
EP0530447A2 (fr) * | 1991-06-08 | 1993-03-10 | Biotest Pharma Gmbh | Procédé de purification d'anticorps monoclonaux du type IgG et leur application |
US5429746A (en) * | 1994-02-22 | 1995-07-04 | Smith Kline Beecham Corporation | Antibody purification |
-
1997
- 1997-10-07 IL IL12190097A patent/IL121900A/en not_active IP Right Cessation
-
1998
- 1998-10-02 AU AU94568/98A patent/AU9456898A/en not_active Abandoned
- 1998-10-02 WO PCT/IL1998/000483 patent/WO1999018130A1/fr active Application Filing
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4877866A (en) * | 1986-11-27 | 1989-10-31 | Biotest Pharma Gmbh | Method of producing a virus safe, storage-stable, and intravenously tolerable immunoglobulin-G preparation |
EP0440483A2 (fr) * | 1990-02-01 | 1991-08-07 | Baxter International Inc. | Procédé de préparation d'immunoglobulines de sérum |
EP0530447A2 (fr) * | 1991-06-08 | 1993-03-10 | Biotest Pharma Gmbh | Procédé de purification d'anticorps monoclonaux du type IgG et leur application |
EP0525502A1 (fr) * | 1991-08-02 | 1993-02-03 | Octapharma Ag | Procédé pour la fabrication de solutions contenant des immunoglobulines et des viruses inactivés |
US5429746A (en) * | 1994-02-22 | 1995-07-04 | Smith Kline Beecham Corporation | Antibody purification |
Cited By (29)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2001072844A3 (fr) * | 2000-03-30 | 2002-04-11 | Amersham Biosciences Ab | Procédé de production d'igg |
US6835379B2 (en) | 2000-03-30 | 2004-12-28 | Amersham Biosciences Ab | Method of producing IgG |
WO2001072844A2 (fr) * | 2000-03-30 | 2001-10-04 | Amersham Biosciences Ab | Procédé de production d'igg |
EP1225180A2 (fr) * | 2001-01-17 | 2002-07-24 | Probitas Pharma, S.A. | Procédé de production de gammaglobuline G humaine à virus inactivé |
EP1225180A3 (fr) * | 2001-01-17 | 2004-01-28 | Probitas Pharma, S.A. | Procédé de production de gammaglobuline G humaine à virus inactivé |
US6875848B2 (en) | 2001-01-17 | 2005-04-05 | Probitas Pharma, S.A. | Process for the production of virus-inactivated human gammaglobulin G |
JP2008540462A (ja) * | 2005-05-10 | 2008-11-20 | マレー ゴールバーン コーオペラティブ コー リミテッド | 免疫グロブリン分画およびそのためのプロセス |
US9631007B2 (en) | 2005-05-25 | 2017-04-25 | Hoffmann-La-Roche Inc. | Method for the purification of antibodies |
US11034754B2 (en) | 2005-05-25 | 2021-06-15 | Hoffmann-La Roche Inc. | Method for the purification of antibodies |
US8354249B2 (en) | 2005-08-11 | 2013-01-15 | Omrix Biopharmaceuticals Ltd. | Intravenous immunoglobulin composition |
US9365635B2 (en) | 2005-08-11 | 2016-06-14 | Omrix Biopharmaceuticals Ltd. | Intravenous immunoglobulin composition |
WO2007067856A2 (fr) * | 2005-12-06 | 2007-06-14 | Advantek Serum Laboratories Limited | Procédé pour l'inactivation et la suppression du virus de la dengue dans des échantillons biologiques |
WO2007067856A3 (fr) * | 2005-12-06 | 2008-01-24 | Advantek Serum Lab Ltd | Procédé pour l'inactivation et la suppression du virus de la dengue dans des échantillons biologiques |
EP2061803B1 (fr) | 2006-08-28 | 2019-08-14 | Ares Trading S.A. | Procédé de purification de protéines contenant fc |
EP2061803B2 (fr) † | 2006-08-28 | 2022-11-16 | Ares Trading S.A. | Procédé de purification de protéines contenant fc |
AU2009307728B2 (en) * | 2008-10-20 | 2014-12-11 | Abbvie Inc. | Antibodies that bind to IL-18 and methods of purifying the same |
WO2010048183A1 (fr) * | 2008-10-20 | 2010-04-29 | Abbott Laboratories | Anticorps se liant à il-18 et procédés de purification de ceux-ci |
US9109010B2 (en) | 2008-10-20 | 2015-08-18 | Abbvie Inc. | Viral inactivation during purification of antibodies cross reference to related applications |
US9018361B2 (en) | 2008-10-20 | 2015-04-28 | Abbvie Inc. | Isolation and purification of antibodies using protein a affinity chromatography |
US8895709B2 (en) | 2008-10-20 | 2014-11-25 | Abbvie Inc. | Isolation and purification of antibodies using protein A affinity chromatography |
US8795671B2 (en) | 2008-12-04 | 2014-08-05 | Omrix Biopharmaceuticals Ltd. | Subcutaneous administration of anti-hepatitis B antibodies |
WO2010064241A1 (fr) | 2008-12-04 | 2010-06-10 | Omrix Biopharmaceuticals Ltd. | Administration sous-cutanée d'anticorps anti-hépatite b |
US9657087B2 (en) | 2008-12-04 | 2017-05-23 | Omrix Biopharmaceuticals Ltd. | Subcutaneous administration of anti-hepatitis B antibodies |
US9796770B2 (en) | 2011-05-16 | 2017-10-24 | Omrix Biopharmaceuticals Ltd. | Immunoglobulin reduced in thrombogenic agents and preparation thereof |
US9023994B2 (en) | 2011-05-16 | 2015-05-05 | Omrix Biopharmaceuticals, Ltd. | Immunoglobulin reduced in thrombogenic agents and preparation thereof |
EP3572419A1 (fr) | 2011-05-16 | 2019-11-27 | Omrix Biopharmaceuticals Ltd. | Immunoglobuline à teneur réduite en agents thrombogéniques et sa préparation |
CN112574296A (zh) * | 2020-12-30 | 2021-03-30 | 中国医学科学院输血研究所 | 一种模拟IVIg的多人份混合人血浆IgG样品的分离纯化方法 |
CN112574296B (zh) * | 2020-12-30 | 2023-05-19 | 中国医学科学院输血研究所 | 一种模拟IVIg的多人份混合人血浆IgG样品的分离纯化方法 |
WO2022161889A1 (fr) | 2021-01-27 | 2022-08-04 | Centre National De La Recherche Scientifique | Procede de fabrication d'un detecteur des flux d'un premier et d'un deuxieme rayonnements ionisants |
Also Published As
Publication number | Publication date |
---|---|
AU9456898A (en) | 1999-04-27 |
IL121900A (en) | 2001-12-23 |
IL121900A0 (en) | 1998-03-10 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP4359347B2 (ja) | 抗体の高収率精製およびウイルス不活性化のためのクロマトグラフイー法 | |
US6069236A (en) | Immunoglobulin G concentrate for therapeutic use and process for producing said concentrate | |
JP4445202B2 (ja) | 治療的使用のためのヒト免疫グロブリン濃縮物の調製方法 | |
JP3148222B2 (ja) | 非変性静脈内投与可能なIgM―及び/又は/IgA―含有免疫グロブリン製剤及びその製造方法 | |
AU2001273907B2 (en) | A method of producing IgG | |
Curling et al. | A chromatographic procedure for the purification of human plasma albumin | |
WO1999018130A1 (fr) | Procede de purification d'immunoglobulines | |
US6955917B2 (en) | Chromatographic method for high yield purification and viral inactivation of antibodies | |
EP2822965B1 (fr) | Procédé d'enrichissement d'iga | |
US4075193A (en) | Process for producing intravenous immune globulin | |
AU2001273907A1 (en) | A method of producing IgG | |
US9663553B2 (en) | Integrated process for the production of therapeutics (human albumin, immunoglobulins, clotting factor VIII and clotting factor IX) from human plasma | |
JPH06107561A (ja) | 静脈内相容性免疫グロブリン− g− 製剤の製造方法 | |
CN106349387B (zh) | 一种从Cohn组分Ⅳ沉淀中纯化α1-抗胰蛋白酶的方法 | |
US5378365A (en) | Process for the isolation of highly purified factors IX, X and II from prothrombin complex or human plasma | |
EP2102335B1 (fr) | Purification de facteur xi | |
US7041798B1 (en) | Method for the chromatographic fractionation of plasma or serum, preparations, so obtained, and their use | |
US20090281282A1 (en) | Method for the isolation of haptoglobin | |
JPH09504532A (ja) | クロマトグラフィー法による第8因子を含むウィルス不活性化分画の製造方法 | |
JPH01258700A (ja) | 血液凝固第4因子の精製方法 | |
JP2721961B2 (ja) | 血液製剤 | |
JPS5810522A (ja) | 不活化トロンビンゲルによるアンチトロンビン3の精製法 | |
dans la Region | Ulllted States Patent [19][11] Patent Number: 6,069,236 | |
CZ2000983A3 (cs) | Způsob čištění antithrombinu III |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
NENP | Non-entry into the national phase |
Ref country code: KR |
|
WWE | Wipo information: entry into national phase |
Ref document number: 09529016 Country of ref document: US |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
122 | Ep: pct application non-entry in european phase | ||
NENP | Non-entry into the national phase |
Ref country code: CA |