CN113880908A - Method for purifying fusion protein of recombinant human serum albumin - Google Patents
Method for purifying fusion protein of recombinant human serum albumin Download PDFInfo
- Publication number
- CN113880908A CN113880908A CN202110983902.1A CN202110983902A CN113880908A CN 113880908 A CN113880908 A CN 113880908A CN 202110983902 A CN202110983902 A CN 202110983902A CN 113880908 A CN113880908 A CN 113880908A
- Authority
- CN
- China
- Prior art keywords
- solution
- chromatography
- fusion protein
- protein
- exchange chromatography
- 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.)
- Granted
Links
- 238000000034 method Methods 0.000 title claims abstract description 45
- 108020001507 fusion proteins Proteins 0.000 title claims abstract description 43
- 102000037865 fusion proteins Human genes 0.000 title claims abstract description 43
- 102000008100 Human Serum Albumin Human genes 0.000 title claims abstract description 21
- 108091006905 Human Serum Albumin Proteins 0.000 title claims abstract description 21
- 239000006228 supernatant Substances 0.000 claims abstract description 34
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 21
- 230000002209 hydrophobic effect Effects 0.000 claims abstract description 20
- 238000005571 anion exchange chromatography Methods 0.000 claims abstract description 11
- 238000005277 cation exchange chromatography Methods 0.000 claims abstract description 11
- 238000001556 precipitation Methods 0.000 claims abstract description 9
- 230000020477 pH reduction Effects 0.000 claims abstract description 8
- 239000000243 solution Substances 0.000 claims description 55
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 28
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 24
- 239000012149 elution buffer Substances 0.000 claims description 21
- 239000012501 chromatography medium Substances 0.000 claims description 16
- 239000003480 eluent Substances 0.000 claims description 16
- 239000006167 equilibration buffer Substances 0.000 claims description 16
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 15
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 14
- 239000003929 acidic solution Substances 0.000 claims description 12
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 12
- 102000000588 Interleukin-2 Human genes 0.000 claims description 11
- 108010002350 Interleukin-2 Proteins 0.000 claims description 11
- 239000007983 Tris buffer Substances 0.000 claims description 11
- 241000700605 Viruses Species 0.000 claims description 11
- 230000003472 neutralizing effect Effects 0.000 claims description 11
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 11
- 230000002779 inactivation Effects 0.000 claims description 10
- 239000012228 culture supernatant Substances 0.000 claims description 9
- 238000001914 filtration Methods 0.000 claims description 9
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 9
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 8
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 8
- 239000002609 medium Substances 0.000 claims description 8
- 239000001632 sodium acetate Substances 0.000 claims description 8
- 235000017281 sodium acetate Nutrition 0.000 claims description 8
- 102000004127 Cytokines Human genes 0.000 claims description 7
- 108090000695 Cytokines Proteins 0.000 claims description 7
- 239000003381 stabilizer Substances 0.000 claims description 7
- 239000003446 ligand Substances 0.000 claims description 6
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical group [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 5
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 4
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 claims description 4
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 claims description 4
- 229930006000 Sucrose Natural products 0.000 claims description 4
- 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 claims description 4
- 239000012670 alkaline solution Substances 0.000 claims description 4
- 229910001424 calcium ion Inorganic materials 0.000 claims description 4
- 159000000007 calcium salts Chemical class 0.000 claims description 4
- 239000000600 sorbitol Substances 0.000 claims description 4
- 239000005720 sucrose Substances 0.000 claims description 4
- 150000005846 sugar alcohols Chemical class 0.000 claims description 4
- 150000001413 amino acids Chemical class 0.000 claims description 3
- 238000004113 cell culture Methods 0.000 claims description 3
- 230000007935 neutral effect Effects 0.000 claims description 3
- 239000008188 pellet Substances 0.000 claims description 3
- 230000003612 virological effect Effects 0.000 claims description 3
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 claims description 2
- 230000002378 acidificating effect Effects 0.000 claims description 2
- 239000003637 basic solution Substances 0.000 claims description 2
- 239000001110 calcium chloride Substances 0.000 claims description 2
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 2
- 125000001453 quaternary ammonium group Chemical group 0.000 claims description 2
- -1 sulfopropyl Chemical group 0.000 claims description 2
- 239000002265 redox agent Substances 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 abstract description 63
- 102000004169 proteins and genes Human genes 0.000 abstract description 62
- 238000000746 purification Methods 0.000 abstract description 30
- 239000012535 impurity Substances 0.000 abstract description 22
- 239000000047 product Substances 0.000 abstract description 11
- 230000008569 process Effects 0.000 abstract description 8
- 238000011160 research Methods 0.000 abstract description 6
- 238000011031 large-scale manufacturing process Methods 0.000 abstract description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 44
- 238000011068 loading method Methods 0.000 description 30
- 238000005406 washing Methods 0.000 description 30
- 239000011780 sodium chloride Substances 0.000 description 22
- 239000012460 protein solution Substances 0.000 description 18
- 239000000523 sample Substances 0.000 description 16
- 239000000872 buffer Substances 0.000 description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 12
- 238000010828 elution Methods 0.000 description 11
- 239000007788 liquid Substances 0.000 description 11
- 210000004027 cell Anatomy 0.000 description 10
- 230000000694 effects Effects 0.000 description 9
- 239000000945 filler Substances 0.000 description 9
- 230000000052 comparative effect Effects 0.000 description 8
- 238000004128 high performance liquid chromatography Methods 0.000 description 8
- 230000035515 penetration Effects 0.000 description 8
- 239000008213 purified water Substances 0.000 description 8
- 238000002305 strong-anion-exchange chromatography Methods 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 239000007853 buffer solution Substances 0.000 description 7
- 238000011067 equilibration Methods 0.000 description 6
- 238000012423 maintenance Methods 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 239000003550 marker Substances 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 239000012521 purified sample Substances 0.000 description 6
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 6
- 238000000825 ultraviolet detection Methods 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 5
- 230000014759 maintenance of location Effects 0.000 description 5
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 description 4
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 4
- 229910019142 PO4 Inorganic materials 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 210000004962 mammalian cell Anatomy 0.000 description 4
- 239000010452 phosphate Substances 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 4
- 238000012437 strong cation exchange chromatography Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 3
- 239000008380 degradant Substances 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 239000002502 liposome Substances 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 239000001488 sodium phosphate Substances 0.000 description 3
- 229910000162 sodium phosphate Inorganic materials 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 210000001744 T-lymphocyte Anatomy 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000005349 anion exchange Methods 0.000 description 2
- 150000001450 anions Chemical class 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 229960000074 biopharmaceutical Drugs 0.000 description 2
- 238000005341 cation exchange Methods 0.000 description 2
- 150000001768 cations Chemical class 0.000 description 2
- 238000011097 chromatography purification Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000005538 encapsulation Methods 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000006386 neutralization reaction Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000000149 penetrating effect Effects 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- 208000008839 Kidney Neoplasms Diseases 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 206010038389 Renal cancer Diseases 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 229940125644 antibody drug Drugs 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000013019 capto adhere Substances 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 238000011210 chromatographic step Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000000091 immunopotentiator Effects 0.000 description 1
- 201000010982 kidney cancer Diseases 0.000 description 1
- 239000012092 media component Substances 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 230000001394 metastastic effect Effects 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 108010043655 penetratin Proteins 0.000 description 1
- MCYTYTUNNNZWOK-LCLOTLQISA-N penetratin Chemical compound C([C@H](NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CCCNC(N)=N)[C@@H](C)CC)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(N)=O)C1=CC=CC=C1 MCYTYTUNNNZWOK-LCLOTLQISA-N 0.000 description 1
- 230000036470 plasma concentration Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 239000012474 protein marker Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000012807 shake-flask culturing Methods 0.000 description 1
- 238000003998 size exclusion chromatography high performance liquid chromatography Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
Images
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
- C07K1/30—Extraction; Separation; Purification by precipitation
-
- 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
-
- 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
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/20—Partition-, reverse-phase or hydrophobic interaction 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
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Water Supply & Treatment (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a method for purifying fusion protein of recombinant human serum albumin, which comprises the following steps: the fusion protein supernatant is subjected to fine purification by acidification precipitation, cation exchange chromatography, anion exchange chromatography and hydrophobic chromatography in sequence, process-related impurities and product-related impurities in the fusion protein are basically removed, a high-quality product conforming to pharmaceutical research can be obtained, and the problems of low protein purity and low yield in large-scale production are effectively solved.
Description
Technical Field
The invention belongs to the field of biological medicine purification methods, and particularly relates to a method for purifying fusion protein of mammalian cell expression recombinant Human Serum Albumin (HSA).
Background
Human Serum Albumin (HSA), which accounts for half and more of the total serum protein, is a very important natural protein in blood circulation. The molecular weight is 66KDa, the compound can not be filtered by glomeruli under normal conditions, the half-life period in serum reaches about 3 weeks, the internal environment compatibility is good, and the immunogenicity is low. The research shows that the human serum albumin is used as a carrier, the fusion protein expressed by the fusion of the therapeutic protein or polypeptide and the human serum albumin not only keeps the bioactivity of the original protein or polypeptide, but also obviously reduces the clearance rate of the medicine in vivo, thereby prolonging the half-life period of the medicine, greatly reducing the administration frequency of patients and improving the compliance of the patients.
Interleukin 2(IL-2) is a cytokine of the chemokine family. Is mainly generated by T lymphocytes, can activate T cells, promote B cell differentiation and secrete antibodies, has important effects on immune response of organisms, tumor resistance, virus infection resistance and the like, and is a natural immunopotentiator. Currently, interleukin 2, which is clinically approved, is mainly used for the treatment of cancer (metastatic renal cancer, melanoma) and autoimmune diseases. However, no substantial and breakthrough progress has been made to date, the main reason being that the half-life of autologous plasma is short, only a few minutes in vivo, and it is difficult to achieve a stable effective plasma concentration, i.e. the desired immune effect is not achieved.
Thus, to date, there are three main approaches to study cytokine depuration: PEG modification, liposome encapsulation and fusion protein technology. The PEG modified protein has low technical yield and low binding activity of the protein and a receptor. Liposome encapsulation technology is complex, and high concentration enrichment can not occur when the liposome reaches a target region or a receptor in a body, so that the curative effect is influenced. The HSA fusion protein technology is to fuse and express a cytokine-HSA fragment through gene engineering and protein engineering technologies, and the fragment is combined with a receptor to form a compound which is not easy to degrade in vivo. The fusion technology has the advantages of prolonged half-life period, high yield, good curative effect and low immunogenicity, and is the direction of long-acting development of therapeutic cytokines.
However, most of the current purification methods for HSA fusion protein are complex, the product purity is not high, the protein yield is low (20%), the host cell protein content is high, the cost is high, and the amplification is not easy, which is a difficult problem for the purification of the fusion protein. In particular, when the HSA-secreted fusion protein is purified by mammalian Cell (CHO) culture, it was found that the fusion protein may produce some degradants and aggregates to different extents during the culture process, which are more obvious in large-scale culture than in shake flask culture, and their presence may adversely affect the activity of the fusion protein, and due to the fact that the physicochemical properties are very similar to those of the fusion protein, it is difficult to remove the fusion protein by some conventional chromatographic methods, especially the host cell protein, and this may present a great challenge to the purification process.
In view of biopharmaceutical safety, regulatory agencies apply stringent purification standards and quality attributes to proteins for human administration, which standards require therapeutic protein products to be substantially free of impurities (aggregates, degradants, variants, DNA, host cell proteins, media components, viral contaminants, endotoxins, etc.), where the quality of the purification process directly impacts product quality, cost of the product, which is critical to biopharmaceutical control costs. The invention establishes a brand new purification method, which not only improves the product quality and the protein yield and reduces the production cost, but also is a stable, reliable and enlargeable purification method.
Disclosure of Invention
The invention mainly aims to provide a method for purifying mammal Cell (CHO) expression HSA fusion protein, effectively removing various impurities, improving the protein purity and yield, reducing the production cost and ensuring that the quality of the final product conforms to the pharmaceutical research and application.
In order to achieve the purpose, the invention is mainly realized by the following technical scheme: and purifying the cell culture supernatant containing the fusion protein by sequentially carrying out acidification precipitation, cation exchange chromatography, anion exchange chromatography and hydrophobic chromatography. Wherein, the acidification precipitation step can remove most of Host Cell Protein (HCP) and DNA, the cation exchange chromatography step is used for capturing and separating out the fusion protein, the anion exchange chromatography step is used for further removing impurities such as host protein, polymers and the like, and the hydrophobic chromatography step is used for further improving the purity of the target protein, and simultaneously, the content of trace impurities is continuously reduced. Preferably, the method of the present invention further comprises steps of virus inactivation and filtration to effectively control the virus content in the final product.
Specifically, the method for purifying the fusion protein of the recombinant human serum albumin comprises the following steps:
adjusting the pH of the cell culture supernatant containing the fusion protein to acidity by using an acidic solution, carrying out acidification and precipitation, and filtering to obtain an acidified supernatant; subjecting the acidified supernatant obtained to cation exchange chromatography to obtain a first eluate; after virus inactivation, carrying out anion exchange chromatography on the first eluent to obtain a second eluent; and carrying out hydrophobic chromatography on the second eluent to obtain a solution containing the fusion protein.
In some embodiments, the fusion protein further comprises a cytokine; preferably, the cytokine is interleukin 2.
In some embodiments, prior to acidifying the pellet, calcium ions are added to the cell supernatant, which can result in more complete pellet; preferably, the calcium ions are in the form of calcium salts; more preferably, the calcium salt is calcium chloride.
In some embodiments, the acidic solution selected from one or more of phosphoric acid, hydrochloric acid, acetic acid and citric acid in the acidified precipitate of the purification method of the present invention has good effect of removing impurities such as aggregates, degradants, DNA and host cell proteins; preferably, the acidic solution is phosphoric acid; preferably, the supernatant is acidified by adjusting the pH of the supernatant to 4.5-6.0, preferably pH5.0-5.5, and the acidified supernatant is obtained by filtration.
In some embodiments, the purification method of the invention, the cation exchange chromatography is high resolution strong cation exchange chromatography, and the chromatography medium is a medium containing a sulfopropyl ligand; the ligand with positive charge interacts with the amino acid with negative charge on the surface of the protein to achieve the purpose of adsorption. Preferably, the chromatographic medium is selected from one of SP HP, NanoGel-50SP, Capto MMC, Toyopearl SP-650M, SP Bestarose HP, and the like, and the medium has the characteristics of high loading capacity, high resolution, high protein yield and the like, and more preferably, the chromatographic medium is SP HP.
In some embodiments, the purification method of the invention of cation exchange chromatography, equilibrium buffer can be selected from phosphate, acetate, citrate, 2- (N-morpholino) ethanesulfonic acid (MES) and so on, working pH 5.0-6.0, preferably pH 5.0-5.5; preferably, the equilibrium buffer is a sodium acetate solution, and the pH value is 5.0-6.0; more preferably, the equilibration buffer contains a 10-150mM sodium chloride in acetic acid. In the cation exchange chromatography, the elution buffer solution is phosphate, acetate, citrate, 2- (N-morpholino) ethanesulfonic acid (MES) and the like containing sodium chloride with a certain concentration, the pH value is 5.0-6.0, and preferably the pH value is 5.0-5.5; preferably, the elution buffer is a solution containing 20-100mM sodium acetate and 50-500mM sodium chloride, pH 5.0-6.0. The column retention time is 6-8 min. The loading capacity is 20-30g/L filler. During chromatography, the acidified supernatant is filtered and directly loaded without any adjustment.
In some embodiments, virus inactivation in the purification methods of the invention is adjusting the pH of the first eluate to acidic using an acidic solution in the presence of a stabilizing agent, and then neutralizing to neutral with a basic solution. Adding certain stabilizing substances such as sugar, amino acid, sugar alcohol, redox substance, etc. into the eluate, preferably sugar and sugar alcohol, more preferably sucrose and sorbitol; the concentration of the stabilizer is 2% -10%, preferably 5%. In low pH viral inactivation, the acidic solution may be selected from one or more of phosphoric acid, hydrochloric acid, acetic acid and citric acid, preferably phosphoric acid; the concentration of the acid solution is 0.5M-1.5M; the pH is adjusted to 3.5-3.8, preferably pH 3.6. The alkaline solution used when neutralizing the acidic solution may be selected from one or more of sodium hydroxide, Tris and sodium acetate, preferably Tris-HCl solution; the concentration of the alkaline solution is 1M-3M.
In some embodiments, the purification method of the invention, the anion exchange chromatography is high resolution strong anion exchange chromatography, and the chromatography medium is a medium containing a quaternary ammonium moiety; preferably, the chromatography medium is selected from one of Q HP, UniGel-30Q, Toyopearl SuperQ-650M and Q Bestarose HP, and the media have the characteristics of high loading capacity, high resolution, low counter pressure and the like; more preferably, the chromatography medium is Q HP.
In some embodiments, the equilibration buffer in the anion exchange chromatography in the purification method of the invention may be selected from one or more of phosphoric acid, Tris base, ethanolamine, pH 6.0-8.0, preferably pH 6.5-7.5; preferably, the equilibration buffer is a solution of 20-100mM Tris and 10-150mM sodium chloride, pH 6.0-8.0. The elution buffer solution in the anion exchange chromatography can be one or more selected from phosphoric acid, Tris alkali and ethanolamine, contains sodium chloride with certain concentration, and has pH of 6.0-8.0, preferably pH of 7.0-7.5. Preferably, the elution buffer is a solution containing 20-100mM Tris and not more than 500mM sodium chloride, pH 6.0-8.0. The column retention time is 7-9 min. The loading capacity is 15-25g/L filler.
In some embodiments, the chromatography medium in the hydrophobic chromatography of the purification method of the present invention is a medium containing a Phenyl ligand, preferably said chromatography medium is selected from one or more of Capto Phenyl (HS), Phenyl Bestarose HP, Toyopearl Phenyl-650M, Phenyl HP; the media have the characteristics of high flow rate, high carrying capacity and the like; preferably Capto Phenyl (HS).
In some embodiments, in the hydrophobic chromatography of the purification method of the present invention, the equilibration buffer may be selected from phosphate, Tris, citrate, MES, etc., at a pH of 6.0 to 8.0, preferably at a pH of 7.0 to 7.5; preferably, the equilibration buffer is a solution of 10-100mM sodium phosphate and 500-2500mM sodium chloride, pH 6.0-8.0. In the hydrophobic chromatography, the elution buffer solution and the equilibrium buffer solution can be the same or different; preferably, the elution buffer is a solution of 10-100mM sodium phosphate and 500-2500mM sodium chloride, pH 6.0-8.0. The column retention time is 3-5 min. The loading capacity is 50-70g/L of filler.
In some embodiments, the method of purifying a fusion protein of recombinant human serum albumin of the present invention comprises the steps of:
obtaining cell supernatant containing the fusion protein from upstream, adding calcium chloride solution, adjusting the pH of the supernatant to acidity by using acid solution, carrying out acidification precipitation at room temperature, and filtering to obtain acidified supernatant; subjecting the obtained acidified supernatant to strong cation exchange chromatography to obtain a first eluate containing the fusion protein; adding a stabilizer into the obtained first eluent, adjusting the pH value by using an acidic solution to inactivate the low-pH viruses, and then neutralizing the low-pH viruses to be neutral by using an alkaline solution; carrying out strong anion exchange chromatography on the obtained neutralized eluent to obtain a second eluent containing the fusion protein; and carrying out strong hydrophobic chromatography on the obtained second eluent to obtain a solution containing the fusion protein.
The purification method has the following beneficial effects:
(1) after the large-scale culture product of the CHO cells is sequentially subjected to acidification precipitation and fractional purification of a high-resolution high-capacity strong cation exchange chromatography medium, a high-resolution high-capacity strong anion exchange chromatography medium and a high-capacity hydrophobic chromatography medium, process-related impurities and product-related impurities in the fusion protein are basically removed, and finally a high-quality product conforming to pharmaceutical research is obtained, so that the problems of low protein purity and low yield in large-scale production are effectively solved.
(2) The medium used in the invention has good repeatability, is easy to be produced in an enlarged way, is more beneficial to large-scale operation compared with certain composite chromatographic media, has low price, is easy to obtain, has long service cycle, and further reduces the production cost.
Drawings
The invention will be further explained by the following figures and examples, wherein M in electrophoresis chart represents protein Marker, and arrows in chromatogram and SEC-HPLC chart represent target protein peak.
FIG. 1 is a HPLC purity determination map of the culture supernatant in example 1 of the present invention.
FIG. 2 is a SP cation exchange chromatogram of example 1 of the present invention.
FIG. 3 is the HPLC purity determination map of the SP elution protein solution in example 1 of the present invention.
FIG. 4 is a Q anion exchange chromatogram of example 1 of the present invention.
FIG. 5 is the HPLC purity determination map of the Q-eluting protein solution in example 1 of the present invention.
FIG. 6 is a Capto Pheny hydrophobic chromatogram of example 1 of the present invention.
FIG. 7 is a HPLC purity determination map of Capto Pheny penetratin liquid in example 1 of the present invention.
FIG. 8 is an electrophoretic (SDS-PAGE) pattern of a purified sample in example 1 of the present invention; m is Marker, 1 is culture supernatant, 2 is acidified supernatant, 3 is SP eluent, 4 is Q eluent, and 5 is Capto Pheny penetration liquid.
FIG. 9 is a SP cation exchange chromatogram of example 2 of the present invention.
FIG. 10 is a Q anion exchange chromatogram of example 2 of the present invention.
FIG. 11 is a Capto Pheny hydrophobic chromatogram of example 2 of the present invention.
FIG. 12 is an electrophoretic (SDS-PAGE) pattern of a purified sample in example 2 of the present invention; m is Marker, 1 is culture supernatant, 2 is acidified supernatant, 3 is SP eluent, 4 is Q eluent, and 5 is Capto Pheny penetration liquid.
FIG. 13 is an electrophoretic (SDS-PAGE) contrast map of purified samples according to examples 1 and 2 of the present invention; m is Marker, 1 is the Capto Pheny penetration liquid 1 of example 1, and 2 is the Capto Pheny penetration liquid 2 of example 2.
Detailed Description
The technical solutions of the present invention will be described in more detail with reference to the accompanying drawings and specific embodiments, which are only a part of the embodiments of the present invention and are provided for the purpose of better understanding, and the present invention will fully convey the disclosure to those skilled in the art.
Example 1: method for purifying fusion protein of recombinant Human Serum Albumin (HSA) and interleukin 2(IL-2)
The method of the embodiment mainly comprises the following steps:
the HPLC purity determination map of the fusion protein culture supernatant of recombinant Human Serum Albumin (HSA) and interleukin 2(IL-2) is shown in FIG. 1.
1. Pretreatment of supernatant fluid: obtaining cell supernatant from upstream, adding calcium chloride solution to make the final concentration of the supernatant to be 10mM, adjusting the pH of the supernatant to 5.0 with 1M phosphoric acid, standing at room temperature for about 1 hour, and removing precipitate and partial impurities by deep filtration to obtain acidified supernatant.
2. Capturing and purifying the acidified supernate obtained in the step 1 by using an SP HP high-resolution strong cation chromatographic column to obtain a large amount of target protein and remove most impurities; the specific operation steps are as follows:
2.1 preparation of the solution
And (3) an equilibrium buffer: 50mM sodium acetate +100mM sodium chloride, pH 5.0.
Elution buffer: 50mM sodium acetate +300mM sodium chloride, pH 5.0.
2.2 Balancing
The column was washed with equilibration buffer for 3-5 column volumes until the conductivity and pH of the buffer exiting the column was the same as before equilibration, at which point the UV detection was zeroed.
2.3 Loading
Directly loading the acidified supernatant obtained in the step 1 with 20g/L of filler, wherein the loading time of the sample is 6-8 min.
2.4 washing
After the sample loading is finished, washing the chromatographic column by 5-7 column volumes by using an equilibrium buffer solution, so that the components which are not combined or are relatively weakly combined are sufficiently washed off, and washing until the effluent liquid ultraviolet absorption value shows that the baseline is flat and stable.
2.5 elution
After washing, continuously washing the chromatographic column by using an elution buffer solution to fully elute the combined target protein, and collecting a main peak at the ultraviolet position of 280nm, wherein the peak collection parameters are as follows: the UV collection was started at 100mAU and ended at 100 mAU.
2.6 maintenance
Washing the chromatographic column with 2M sodium chloride solution, 0.5M sodium hydroxide solution and purified water for 3 column volumes, preserving the chromatographic column with 2 column volumes of 20% ethanol, and storing at 4 deg.C.
As shown in FIG. 2, the arrow marks the target protein.
The HPLC purity profile of SP eluted protein solution is shown in FIG. 3.
3. And (3) performing low-pH virus inactivation on the crude pure target protein solution obtained in the step (2), and removing part of process-related impurities, wherein the specific operation process is as follows:
respectively adding a sorbitol solution with a final concentration of 5% and a sucrose solution with a final concentration of 5% into the protein solution obtained from the crude elution, uniformly mixing at room temperature, adjusting the pH of the protein solution to 3.6 by using 1M phosphoric acid, standing for 1.5 hours, adjusting the pH of the protein solution to 7.4 by using a 2M Tris-HCl neutralizing solution with a pH of 10.0, and then obtaining a protein neutralizing solution through deep filtration.
4. And (3) further carrying out chromatographic purification on the protein neutralization solution in the step (3) by using a Q HP high-resolution strong anion chromatographic column to remove degraded substances and part of other impurities, wherein the specific operation steps are as follows:
4.1 solution preparation
And (3) an equilibrium buffer: 25mM Tris +30mM sodium chloride, pH 7.4.
Elution buffer E: 25mM Tris +150mM sodium chloride, pH 7.4.
4.2 Balancing
The column was washed with equilibration buffer for 3-5 column volumes until the conductivity and pH of the buffer exiting the column was the same as before equilibration, at which point the UV detection was zeroed.
4.3 sample application
And (3) loading the protein neutralizing solution obtained in the step (3), wherein the lowest loading capacity of the loaded protein neutralizing solution is 15g/L of filler, and the loading retention time is 7-9 min.
4.4 washing
After the sample loading is finished, washing the chromatographic column by 5-7 column volumes by using an equilibrium buffer solution, so that the components which are not combined or are relatively weak to be combined are sufficiently washed, and washing until the ultraviolet absorption value shows that the baseline is flat and stable.
4.5 elution
After washing, continuously washing the chromatographic column by using an elution buffer solution to fully elute the combined target protein, and collecting a main peak at the ultraviolet position of 280nm, wherein the peak collection parameters are as follows: the UV collection was started at 100mAU and ended at 100 mAU.
4.6 maintenance
Washing the chromatographic column with 2M sodium chloride solution, 0.5M sodium hydroxide solution and purified water for 3 column volumes, preserving the chromatographic column with 2 column volumes of 20% ethanol, and storing at 4 deg.C.
As shown in FIG. 4, the arrow marks the target protein.
The HPLC purity map of the Q-eluted protein solution is shown in FIG. 5.
5. And (3) further finely purifying the eluted protein solution obtained in the step (4) by using a Capto Phenyl (HS) hydrophobic chromatographic column, and removing part of refractory impurities in a penetration mode to obtain a high-quality fusion protein solution, wherein the specific operation steps are as follows:
5.1 solution preparation
And (3) an equilibrium buffer: 20mM phosphate +1000mM sodium chloride, pH 7.0.
The elution buffer was the same as the equilibration buffer.
5.2 Balancing
The column was washed with equilibration buffer for 3-5 column volumes until the conductivity and pH of the buffer exiting the column was the same as before equilibration, at which point the UV detection was zeroed.
5.3 sample application
And (4) adjusting the conductivity and pH of the eluted protein solution obtained in the step (4) to be consistent with those of the equilibrium solution, loading, collecting a penetrating target protein peak, starting to collect filler with ultraviolet of 100mAU and loading capacity of 50-70g/L, and keeping the loading for 3-5 min.
5.4 elution
After the sample loading is finished, the chromatography is continuously washed by the balance buffer solution, the ultraviolet is reduced to 100mAU, and the collection is stopped.
5.5 elution
After washing, continuously washing the chromatographic column by using an elution buffer solution to fully elute the combined target protein, and collecting a main peak at the ultraviolet position of 280nm, wherein the peak collection parameters are as follows: the UV collection is 150mAU at the beginning, and the UV collection is 150mAU at the end, and the collected sample is the final chromatography target protein sample.
5.6 maintenance
Washing the chromatographic column with purified water, 0.5M sodium hydroxide solution and purified water for 3 column volumes, preserving the chromatographic column with 2 column volumes of 20% ethanol, and storing at 4 deg.C.
As shown in FIG. 6, the arrow marks the target protein.
The HPLC purity determination map of the Capto Pheny penetration protein solution is shown in FIG. 7.
The electrophoresis (SDS-PAGE) result of the purified sample in example 1 of the present invention is shown in FIG. 8, wherein M is Marker, 1 is culture supernatant, 2 is acidified supernatant, 3 is SP eluate, 4 is Q eluate, and 5 is Capto Pheny permeant.
Example 2: method for purifying fusion protein of recombinant Human Serum Albumin (HSA) and interleukin 2(IL-2)
The method of the embodiment mainly comprises the following steps:
1. pretreatment of supernatant fluid: obtaining cell supernatant from upstream, adding calcium chloride solution to make the final concentration of the supernatant to be 10mM, adjusting the pH of the supernatant to 5.5 with 1M phosphoric acid, standing at room temperature for about 1 hour, and removing precipitate and partial impurities by deep filtration to obtain acidified supernatant.
2. And (2) capturing and purifying the acidified supernatant obtained in the step (1) by using an SP HP high-resolution strong cation chromatographic column to obtain a large amount of target protein and remove part of impurities, wherein the specific operation steps are as follows:
2.1 preparation of the solution
And (3) an equilibrium buffer: 25mM sodium acetate +50mM sodium chloride, pH 5.5.
Elution buffer: 25mM sodium acetate +200mM sodium chloride, pH 5.5.
2.2 Balancing
The column was washed with equilibration buffer for 3-5 column volumes until the conductivity and pH of the buffer exiting the column was the same as before equilibration, at which point the UV detection was zeroed.
2.3 Loading
Directly loading the acidified supernatant obtained in the step 1 with 30g/L filler, wherein the loading time of the sample is 6-8 min.
2.4 washing
After the sample loading is finished, washing the chromatographic column by 5-7 column volumes by using an equilibrium buffer solution, so that the components which are not combined or are relatively weakly combined are sufficiently washed off, and washing until the effluent liquid ultraviolet absorption value shows that the baseline is flat and stable.
2.5 elution
After washing, continuously washing the chromatographic column by using an elution buffer solution to fully elute the combined target protein, and collecting a main peak at the ultraviolet position of 280nm, wherein the peak collection parameters are as follows: the UV collection was started at 120mAU and ended at 120 mAU.
2.6 maintenance
Washing the chromatographic column with 2M sodium chloride solution, 0.5M sodium hydroxide solution and purified water for 3 column volumes, preserving the chromatographic column with 2 column volumes of 20% ethanol, and storing at 4 deg.C.
As shown in FIG. 9, the arrow marks the target protein.
3. And (3) performing low-pH virus inactivation on the crude pure target protein solution obtained in the step (2), and removing part of impurities, wherein the specific operation process is as follows:
respectively adding a sorbitol solution with a final concentration of 5% and a sucrose solution with a final concentration of 5% into the protein solution obtained from the crude elution, uniformly mixing at room temperature, adjusting the pH of the protein solution to 3.6 by using 1M phosphoric acid, standing for 1.5 hours, adjusting the pH of the protein solution to 7.4 by using a 2M Tris-HCl neutralizing solution with a pH of 10.0, and then obtaining a protein neutralizing solution through deep filtration.
4. And (3) further carrying out chromatographic purification on the protein neutralization solution in the step (3) by using a Q HP high-resolution strong anion chromatographic column to remove degraded substances and part of other impurities, wherein the specific operation steps are as follows:
4.1 solution preparation
And (3) an equilibrium buffer: 50mM Tris +30mM sodium chloride, pH 6.5.
Elution buffer: 50mM Tris +130mM sodium chloride, pH 6.5.
4.2 Balancing
The column was washed with equilibration buffer for 3-5 column volumes until the conductivity and pH of the buffer exiting the column was the same as before equilibration, at which point the UV detection was zeroed.
4.3 sample application
And (3) loading the protein neutralizing solution obtained in the step (3), wherein the loading capacity of the loading is 25g/L of filler, and the loading retention time is 7-9 min.
4.4 washing
After the sample loading is finished, washing the chromatographic column by 5-7 column volumes by using an equilibrium buffer solution, so that the components which are not combined or are relatively weak to be combined are sufficiently washed, and washing until the ultraviolet absorption value shows that the baseline is flat and stable.
4.5 elution
After washing, continuously washing the chromatographic column by using an elution buffer solution to fully elute the combined target protein, and collecting a main peak at the ultraviolet position of 280nm, wherein the peak collection parameters are as follows: the UV collection was 150mAU at the beginning and 150mAU at the end.
4.6 maintenance
Washing the chromatographic column with 2M sodium chloride solution, 0.5M sodium hydroxide solution and purified water for 3 column volumes, preserving the chromatographic column with 2 column volumes of 20% ethanol, and storing at 4 deg.C.
As shown in FIG. 10, the arrow marks the target protein.
5. And (3) further finely purifying the eluted protein liquid obtained in the step (4) by using a Capto Phenyl (HS) hydrophobic chromatographic column, and removing part of refractory impurities in a penetration mode to obtain a high-quality fusion protein solution, wherein the specific operation steps are as follows:
5.1 solution preparation
And (3) an equilibrium buffer: 100mM sodium phosphate +1000mM sodium chloride, pH 7.0.
The elution buffer was the same as the equilibration buffer.
5.2 Balancing
The column was washed with equilibration buffer for 3-5 column volumes until the conductivity and pH of the buffer exiting the column was the same as before equilibration, at which point the UV detection was zeroed.
5.3 sample application
And (4) adjusting the conductivity and pH of the eluted protein solution obtained in the step (4) to be consistent with those of the equilibrium solution, loading, collecting a penetrating target protein peak, starting to collect filler with ultraviolet of 100mAU and loading capacity of 50-70g/L, and keeping the loading for 3-5 min.
5.4 elution
After the sample loading is finished, the chromatography is continuously washed by the balance buffer solution, the ultraviolet is reduced to 100mAU, and the collection is stopped.
5.5 elution
After washing, continuously washing the chromatographic column by using an elution buffer solution to fully elute the combined target protein, and collecting a main peak at the ultraviolet position of 280nm, wherein the peak collection parameters are as follows: the UV collection is 150mAU at the beginning, and the UV collection is 150mAU at the end, and the collected sample is the final chromatography target protein sample.
5.6 maintenance
Washing the chromatographic column with purified water, 0.5M sodium hydroxide solution and purified water for 3 column volumes, preserving the chromatographic column with 2 column volumes of 20% ethanol, and storing at 4 deg.C.
As shown in FIG. 11, the arrow marks the target protein.
An electrophoresis (SDS-PAGE) map of a purified sample in the embodiment 2 of the invention is shown in a figure 12, wherein M is Marker, 1 is culture supernatant, 2 is acidified supernatant, 3 is SP eluent, 4 is Q eluent and 5 is Capto Pheny penetration liquid.
The electrophoresis (SDS-PAGE) contrast of the purified samples in examples 1 and 2 of the invention is shown in FIG. 13, wherein M is Marker, 1 is the Capto Pheny permeant liquid 1 of example 1, and 2 is the Capto Pheny permeant liquid 2 of example 2.
From the above examples, it can be seen that the purification method of the present invention sequentially performs acidification-SP-Q-Capto Phenyl (HS) purification on the CHO mammalian cell expressed fusion protein supernatant to obtain high quality target protein, the purity is above 99.5%, the protein yield is above 45%, and the method is in accordance with pharmaceutical research and application.
Experimental example 1:
the removal of impurities is an important part of the development of biotechnological drug purification processes, in particular the removal of host proteins. There is no clear international regulation on host protein residues, and the typical host protein residue content for antibody drugs is 100ng/mg protein. The invention particularly compares the removal effect of different purification processes on host proteins in the development process of the purification process.
The purification processes of comparative example 1 and comparative example 2 are different from those of example 1, and the host protein (HCP) removal effect is significantly different, and the results are shown in table 1.
TABLE 1 different purification procedures and host protein removal efficiency
| Experiment number | Purification process | Protein concentration | HCP content |
| Example 1 | SP HP→Q HP→Capto Pheny | 3.95mg/mL | 65ng/mg |
| Comparative example 1 | SP HP→Q HP | 3.20mg/mL | 347ng/mg |
| Comparative example 2 | SP HP→Q HP→Capto adhere | 2.28mg/mL | 333ng/mg |
And (4) conclusion: compared with the example 1, the step of hydrophobic chromatography is reduced in the comparative example 1, compared with the example 1, the hydrophobic chromatography in the comparative example 2 adopts different chromatography media, after the fusion protein is purified, the HCP residual content in the comparative example 1 and the comparative example 2 is higher, while the purification effect on the fusion protein in the example 1 is good, and most host proteins are basically removed.
Discussion:
the specific embodiment shows the same purification effect for removing process-related impurities and product-related impurities in the fusion protein, and confirms the feasibility and stability of the technical scheme of the invention. Particularly, after the supernatant fluid of the fusion protein expressed by the CHO mammalian cells is subjected to fine purification by acidification precipitation, strong cation exchange chromatography, virus inactivation, strong anion exchange chromatography and hydrophobic chromatography in sequence, the high-quality target protein can be obtained, the purity is over 99.5 percent, the protein yield is over 45 percent, and the protein meets the pharmaceutical research and application standards.
From the economic consideration, the technical scheme of the invention can greatly improve the yield and purity of the protein, shorten the production period, reduce the production cost and improve the working efficiency, and the most direct economic benefit is easy amplification production and good stability.
The illustrated examples are only preferred technical solutions of the present invention, and other chromatography mediums mentioned in the present invention can achieve the objects of the present invention.
The scope of the present invention described above is not limited to the above-described embodiments, and any modification and variation may be made within the scope of the present invention.
Claims (10)
1. A method for purifying a fusion protein of recombinant human serum albumin, comprising the steps of:
adjusting the pH of the cell culture supernatant containing the fusion protein to acidity by using an acidic solution, carrying out acidification and precipitation, and filtering to obtain an acidified supernatant; subjecting the acidified supernatant obtained to cation exchange chromatography to obtain a first eluate; after virus inactivation, carrying out anion exchange chromatography on the first eluent to obtain a second eluent; and carrying out hydrophobic chromatography on the second eluent to obtain a solution containing the fusion protein.
2. The method of claim 1, wherein the fusion protein further comprises a cytokine; preferably, the cytokine is interleukin 2.
3. The method of claim 1, wherein calcium ions are added to the cell supernatant prior to acidifying the pellet; preferably, the calcium ions are in the form of calcium salts; more preferably, the calcium salt is calcium chloride.
4. The method of claim 1, wherein in the acidified precipitation, the acidic solution is selected from one or more of phosphoric acid, hydrochloric acid, acetic acid, and citric acid;
preferably, the acidic solution is phosphoric acid.
5. The method of claim 1, wherein the chromatography medium of cation exchange chromatography is a medium containing sulfopropyl ligands;
preferably, the pH of the equilibration buffer in the cation exchange chromatography is 5.0-6.0;
preferably, the pH of the elution buffer in the cation exchange chromatography is 5.0-6.0.
6. The method of claim 1, wherein the viral inactivation is adjusting the pH of the first eluate to acidic using an acidic solution in the presence of a stabilizing agent, and then neutralizing to neutral with a basic solution.
7. The method of claim 6, wherein the stabilizer is selected from one or more of a sugar, an amino acid, a sugar alcohol, and a redox agent; preferably, the stabilizer is a sugar or sugar alcohol; more preferably, the stabilizers are sucrose and sorbitol.
8. The method according to claim 6, wherein the acidic solution in the virus inactivation is selected from one or more of phosphoric acid, hydrochloric acid, acetic acid and citric acid, preferably phosphoric acid; and/or the alkaline solution is selected from one or more of sodium hydroxide, Tris and sodium acetate, preferably a Tris-HCl solution.
9. The method of claim 1, wherein the chromatography medium of anion exchange chromatography is a medium containing a quaternary ammonium-based ligand;
preferably, the pH of the equilibration buffer in the anion exchange chromatography is 6.0-8.0;
preferably, the pH of the elution buffer in the anion exchange chromatography is 6.0-8.0.
10. The method of claim 1, wherein the chromatography medium of hydrophobic chromatography is a medium containing a phenyl ligand,
preferably, the pH of the equilibration buffer in hydrophobic chromatography is 6.0-8.0;
preferably, the pH of the elution buffer in the hydrophobic chromatography is 6.0-8.0.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110983902.1A CN113880908B (en) | 2021-08-25 | 2021-08-25 | Method for purifying fusion protein of recombinant human serum albumin |
| PCT/CN2021/121807 WO2023024214A1 (en) | 2021-08-25 | 2021-09-29 | Method for purifying fusion protein of recombinant human serum albumin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110983902.1A CN113880908B (en) | 2021-08-25 | 2021-08-25 | Method for purifying fusion protein of recombinant human serum albumin |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113880908A true CN113880908A (en) | 2022-01-04 |
| CN113880908B CN113880908B (en) | 2024-05-14 |
Family
ID=79011496
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110983902.1A Active CN113880908B (en) | 2021-08-25 | 2021-08-25 | Method for purifying fusion protein of recombinant human serum albumin |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN113880908B (en) |
| WO (1) | WO2023024214A1 (en) |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5440018A (en) * | 1992-05-20 | 1995-08-08 | The Green Cross Corporation | Recombinant human serum albumin, process for producing the same and pharmaceutical preparation containing the same |
| CN101240033A (en) * | 2008-03-04 | 2008-08-13 | 无锡和邦生物科技有限公司 | Fusion protein of insulin secretion accelerating peptide and human serum albumin, and preparation method thereof |
| CN101260145A (en) * | 2008-04-11 | 2008-09-10 | 北京未名福源基因药物研究中心有限公司 | Technique for separating and purifying recombination human serum albumin and fusion protein thereof |
| CN102911250A (en) * | 2012-09-29 | 2013-02-06 | 浙江海正药业股份有限公司 | Method for purifying acidic recombinant protein medicament |
| CN104974251A (en) * | 2008-10-20 | 2015-10-14 | Abbvie公司 | Viral inactivation during purification of antibodies |
| US20180030106A1 (en) * | 2014-10-30 | 2018-02-01 | Soonchunhyang University Industry Academy Cooperation Foundation | Interleukin-2 expression construct using human serium albumin |
| CN110526982A (en) * | 2018-05-23 | 2019-12-03 | 信立泰(成都)生物技术有限公司 | A kind of purification process of human glucagon-like-peptide-1 analog fusion |
| CN110770251A (en) * | 2017-04-20 | 2020-02-07 | 诺和诺德股份有限公司 | Method for purifying albumin fusion proteins |
| CN111484557A (en) * | 2019-01-25 | 2020-08-04 | 武汉禾元生物科技股份有限公司 | Method for separating and purifying recombinant human serum albumin-epidermal growth factor fusion protein from genetically engineered rice seeds |
| CN112724259A (en) * | 2020-11-16 | 2021-04-30 | 天津林达生物科技有限公司 | Fusion protein of human serum albumin and interleukin 2 and application thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SE526227C2 (en) * | 2002-05-15 | 2005-08-02 | North China Pharmaceutical Group | Method of purifying recombinant human serum albumin |
| CN112210002B (en) * | 2020-10-15 | 2022-06-10 | 楚天源创生物技术(长沙)有限公司 | Purification method of recombinant human serum albumin |
-
2021
- 2021-08-25 CN CN202110983902.1A patent/CN113880908B/en active Active
- 2021-09-29 WO PCT/CN2021/121807 patent/WO2023024214A1/en unknown
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5440018A (en) * | 1992-05-20 | 1995-08-08 | The Green Cross Corporation | Recombinant human serum albumin, process for producing the same and pharmaceutical preparation containing the same |
| CN101240033A (en) * | 2008-03-04 | 2008-08-13 | 无锡和邦生物科技有限公司 | Fusion protein of insulin secretion accelerating peptide and human serum albumin, and preparation method thereof |
| CN101260145A (en) * | 2008-04-11 | 2008-09-10 | 北京未名福源基因药物研究中心有限公司 | Technique for separating and purifying recombination human serum albumin and fusion protein thereof |
| CN104974251A (en) * | 2008-10-20 | 2015-10-14 | Abbvie公司 | Viral inactivation during purification of antibodies |
| CN102911250A (en) * | 2012-09-29 | 2013-02-06 | 浙江海正药业股份有限公司 | Method for purifying acidic recombinant protein medicament |
| US20180030106A1 (en) * | 2014-10-30 | 2018-02-01 | Soonchunhyang University Industry Academy Cooperation Foundation | Interleukin-2 expression construct using human serium albumin |
| CN110770251A (en) * | 2017-04-20 | 2020-02-07 | 诺和诺德股份有限公司 | Method for purifying albumin fusion proteins |
| CN110526982A (en) * | 2018-05-23 | 2019-12-03 | 信立泰(成都)生物技术有限公司 | A kind of purification process of human glucagon-like-peptide-1 analog fusion |
| CN111484557A (en) * | 2019-01-25 | 2020-08-04 | 武汉禾元生物科技股份有限公司 | Method for separating and purifying recombinant human serum albumin-epidermal growth factor fusion protein from genetically engineered rice seeds |
| CN112724259A (en) * | 2020-11-16 | 2021-04-30 | 天津林达生物科技有限公司 | Fusion protein of human serum albumin and interleukin 2 and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| ANDRAS KOVACS: "Fractionation of the human plasma proteome for monoclonal antibody proteomics-based biomarker discovery", ELECTROPHORESIS, pages 1916 * |
| 路秀玲, 苏志国: "人血清白蛋白纯化技术研究进展", 生物工程学报, no. 06, pages 761 - 766 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113880908B (en) | 2024-05-14 |
| WO2023024214A1 (en) | 2023-03-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5948343B2 (en) | Method for purifying human serum albumin from seeds of transgenic rice | |
| CA2816050C (en) | Method for purifying human granulocyte-colony stimulating factor from recombinant e. coli | |
| US20170152298A1 (en) | Process for the purification of tnfr:fc fusion protein | |
| CN102911250B (en) | Method for purifying acidic recombinant protein medicament | |
| RU2614119C9 (en) | Method of producing human immunoglobulin | |
| CN101260145B (en) | Technique for separating and purifying recombination human serum albumin and fusion protein thereof | |
| JP2017526649A (en) | New process for purification of rHu-GCSF | |
| CN109627322A (en) | A method of human serum albumins is isolated and purified from V supernatant of Cohn component | |
| WO2020078254A1 (en) | Method for isolating recombinant human growth hormone from genetically engineered rice seeds and purifying same | |
| CN113880908A (en) | Method for purifying fusion protein of recombinant human serum albumin | |
| CN109929038B (en) | Purification method of VEGF (vascular endothelial growth factor) capture agent fusion protein | |
| CN113121637A (en) | Separation and purification method of recombinant protein | |
| CN110590931B (en) | Method for removing and/or inactivating virus in recombinant human thrombopoietin stock solution | |
| CN114885608A (en) | Method for purifying recombinant proteins | |
| CN110724204A (en) | Method for purifying Fc fusion protein | |
| CN116199768B (en) | Preparation method and application of high-purity plant source recombinant human serum albumin | |
| CN105541994B (en) | method for purifying thrombopoietin or variant or derivative thereof | |
| CN113121638B (en) | Method for purifying recombinant protein | |
| EP0446850B1 (en) | Process for purifying recombinant human beta-interferon | |
| WO2011015919A1 (en) | A highly efficient process of purification and production of recombinant infliximab | |
| WO2011015921A1 (en) | A highly efficient process of purification and production of recombinant cetuximab | |
| CN114891061A (en) | Purification method of glycosylated recombinant protein | |
| CN112979806A (en) | Preparation method of anti-Her 2 antibody | |
| WO2021262041A1 (en) | Method of producing a highly purified recombinant inhibitor of human c1 esterase | |
| JPS6317898A (en) | Purification of lymphotoxin compound |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |