WO2014081225A1 - Method for preparing novel long-acting human growth hormone monomer - Google Patents
Method for preparing novel long-acting human growth hormone monomer Download PDFInfo
- Publication number
- WO2014081225A1 WO2014081225A1 PCT/KR2013/010630 KR2013010630W WO2014081225A1 WO 2014081225 A1 WO2014081225 A1 WO 2014081225A1 KR 2013010630 W KR2013010630 W KR 2013010630W WO 2014081225 A1 WO2014081225 A1 WO 2014081225A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- growth hormone
- human growth
- nexp
- hgh
- protein
- Prior art date
Links
- 102000002265 Human Growth Hormone Human genes 0.000 title claims abstract description 121
- 108010000521 Human Growth Hormone Proteins 0.000 title claims abstract description 121
- 239000000854 Human Growth Hormone Substances 0.000 title claims abstract description 121
- 239000000178 monomer Substances 0.000 title claims abstract description 53
- 238000000034 method Methods 0.000 title claims abstract description 37
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 68
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 67
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 27
- 239000003957 anion exchange resin Substances 0.000 claims abstract description 26
- 230000002459 sustained effect Effects 0.000 claims description 60
- 102000015395 alpha 1-Antitrypsin Human genes 0.000 claims description 50
- 229940024142 alpha 1-antitrypsin Drugs 0.000 claims description 46
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 44
- 108010050122 alpha 1-Antitrypsin Proteins 0.000 claims description 42
- 150000003839 salts Chemical class 0.000 claims description 29
- 239000011780 sodium chloride Substances 0.000 claims description 22
- 239000011347 resin Substances 0.000 claims description 20
- 229920005989 resin Polymers 0.000 claims description 20
- 239000000203 mixture Substances 0.000 claims description 12
- 239000012064 sodium phosphate buffer Substances 0.000 claims description 12
- 239000007983 Tris buffer Substances 0.000 claims description 10
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 10
- 102000008394 Immunoglobulin Fragments Human genes 0.000 claims description 8
- 108010021625 Immunoglobulin Fragments Proteins 0.000 claims description 8
- 238000001042 affinity chromatography Methods 0.000 claims description 7
- 238000005571 anion exchange chromatography Methods 0.000 claims description 7
- 230000002209 hydrophobic effect Effects 0.000 claims description 7
- 239000012149 elution buffer Substances 0.000 claims description 5
- 210000004102 animal cell Anatomy 0.000 claims description 4
- 238000011067 equilibration Methods 0.000 claims description 4
- 125000000524 functional group Chemical group 0.000 claims description 4
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 3
- 150000001412 amines Chemical group 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 239000008057 potassium phosphate buffer Substances 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims 2
- 238000002347 injection Methods 0.000 claims 2
- 239000007924 injection Substances 0.000 claims 2
- -1 amino ethynyl Chemical group 0.000 claims 1
- 235000018102 proteins Nutrition 0.000 description 52
- 229920000642 polymer Polymers 0.000 description 34
- 239000000243 solution Substances 0.000 description 16
- 150000001413 amino acids Chemical class 0.000 description 13
- 235000001014 amino acid Nutrition 0.000 description 12
- 229940024606 amino acid Drugs 0.000 description 11
- 238000004519 manufacturing process Methods 0.000 description 11
- 238000000746 purification Methods 0.000 description 9
- 239000012614 Q-Sepharose Substances 0.000 description 8
- 238000011026 diafiltration Methods 0.000 description 8
- 101000823116 Homo sapiens Alpha-1-antitrypsin Proteins 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 7
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 7
- 102000018997 Growth Hormone Human genes 0.000 description 6
- 108010051696 Growth Hormone Proteins 0.000 description 6
- 229920002684 Sepharose Polymers 0.000 description 6
- 239000000122 growth hormone Substances 0.000 description 6
- 239000013598 vector Substances 0.000 description 6
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 5
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 5
- 229960001230 asparagine Drugs 0.000 description 5
- 235000009582 asparagine Nutrition 0.000 description 5
- 230000008901 benefit Effects 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 4
- 239000012535 impurity Substances 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- 238000001542 size-exclusion chromatography Methods 0.000 description 4
- 239000001488 sodium phosphate Substances 0.000 description 4
- 229910000162 sodium phosphate Inorganic materials 0.000 description 4
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 3
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 3
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 3
- MZVQCMJNVPIDEA-UHFFFAOYSA-N [CH2]CN(CC)CC Chemical compound [CH2]CN(CC)CC MZVQCMJNVPIDEA-UHFFFAOYSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 210000000845 cartilage Anatomy 0.000 description 3
- 230000024245 cell differentiation Effects 0.000 description 3
- 238000010353 genetic engineering Methods 0.000 description 3
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 3
- 210000004349 growth plate Anatomy 0.000 description 3
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 3
- 230000002688 persistence Effects 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000003998 size exclusion chromatography high performance liquid chromatography Methods 0.000 description 3
- 230000004936 stimulating effect Effects 0.000 description 3
- 108700028369 Alleles Proteins 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 2
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 239000012471 diafiltration solution Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 230000013595 glycosylation Effects 0.000 description 2
- 238000006206 glycosylation reaction Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 238000001155 isoelectric focusing Methods 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 230000037081 physical activity Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 229940099982 prolastin Drugs 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 230000011664 signaling Effects 0.000 description 2
- 238000002741 site-directed mutagenesis Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000012609 strong anion exchange resin Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000013268 sustained release Methods 0.000 description 2
- 239000012730 sustained-release form Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 1
- 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 description 1
- 125000000022 2-aminoethyl group Chemical group [H]C([*])([H])C([H])([H])N([H])[H] 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 101150074155 DHFR gene Proteins 0.000 description 1
- 206010014561 Emphysema Diseases 0.000 description 1
- 206010062767 Hypophysitis Diseases 0.000 description 1
- 208000001953 Hypotension Diseases 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 230000004988 N-glycosylation Effects 0.000 description 1
- 101710098761 Protein alpha-1 Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 239000012062 aqueous buffer Substances 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000037182 bone density Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000003729 cation exchange resin Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 108010005905 delta-hGHR Proteins 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 239000002532 enzyme inhibitor Substances 0.000 description 1
- 229940125532 enzyme inhibitor Drugs 0.000 description 1
- 239000006167 equilibration buffer Substances 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 238000009578 growth hormone therapy Methods 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- 208000021822 hypotensive Diseases 0.000 description 1
- 230000001077 hypotensive effect Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229940063149 nutropin Drugs 0.000 description 1
- 239000000813 peptide hormone Substances 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 210000003635 pituitary gland Anatomy 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 238000013094 purity test Methods 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 159000000000 sodium salts Chemical group 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 239000002753 trypsin inhibitor Substances 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- 239000012610 weak anion exchange resin Substances 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- 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
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/61—Growth hormone [GH], i.e. somatotropin
Definitions
- the present invention relates to a method for producing a high purity monomer of NexP-hGH protein, a sustained human growth hormone using anion exchange chromatography, and a monomer of the NexP-hGH protein prepared by the above method.
- Human growth hormone is a protein hormone with a molecular weight of approximately 21,500 Da consisting of 191 amino acids, and is responsible for promoting growth by stimulating cell differentiation of cartilage growth plates in the body. If there is a deficiency in the synthesis and secretion of human growth hormone in the body, it may cause hypotensive, increased risk of cardiovascular disease, muscle and bone density decrease.
- the present inventors also endeavored to develop a sustained human growth hormone with increased half-life in the body by maintaining the persistence in the body, and as a result developed a sustained human growth hormone NexP-hGH.
- the preparation method and protein sequence thereof are disclosed in Korean Patent Registration No. 10-1183262.
- the long-acting human growth hormone NexP-hGH is an N-terminus or C-terminus of human growth hormone that replaces NexP with specific amino acids to eliminate the intrinsic activity of the protein alpha-1 antitrypsin (A1AT) and increase its half-life. It is a protein fused by genetic recombination.
- NexP-hGH has the advantage of improved half-life in the body compared to first-generation human growth hormone, and it is made to be glycosylated when expressed in animal cells, CHO cells, and it is different from most first-generation human growth hormone drugs manufactured in Escherichia coli.
- hGH fused with NexP still retains the structural and chemical properties of hGH itself.
- hGH is known as a well-formed protein, and hGH in polymer form is known to not only induce antigenicity in the body but also to hinder the binding between hGH and its receptor, thereby reducing hGH signaling.
- Such polymers may arise from proteins that do not form the appropriate tertiary structure when expressed in the target protein from the host, or may result from reduced stability of the target protein in the process.
- such polymers are classified as abnormal peptides that are product-derived impurities, and their amount is regulated in the purity test. Accordingly, many attempts have been made to remove polymers during the production process, and size-exclusion chromatography (SEC) is generally used to separate polymers and monomers by molecular weight.
- SEC size-exclusion chromatography
- the target protein since the volume of the column loaded is limited (typically within 10% of the bed volume), the target protein must be concentrated at a high concentration. There is a problem.
- the present inventors confirmed that the polymer was formed during the purification process of the sustained human growth hormone NexP-hGH, thereby separating the polymer and the monomer of the new continuous human growth hormone, which was difficult to purify by the existing method, to maintain high purity Efforts have been made to develop a method for producing monomers of the type human growth hormone NexP-hGH. As a result, anion exchange resins are used to separate the polymers and monomers of the type of sustained human growth hormone NexP-hGH from each other. It was confirmed that the monomer of hGH can be prepared and completed the present invention.
- One object of the present invention is to provide a method for preparing monomers of NexP-hGH protein, which is a sustained human growth hormone fused with human growth hormone and alpha-1 antitrypsin variant, using anion exchange resin chromatography.
- Another object of the present invention is to provide a monomer of NexP-hGH protein, which is a sustained human growth hormone fused to human growth hormone and alpha-1 antitrypsin variant prepared by the above method.
- the method of the present invention has the advantage that only monomers can be obtained with a high purity of 99% or more from a mixture comprising a polymer and a monomer of the sustained human growth hormone NexP-hGH using an anion exchange resin.
- the high purity purification method provided by the method of the present invention is applied to a production process, it is possible to purify high concentration and high purity sustained human growth hormone in large quantities, thereby increasing bioactivity and reducing antigenicity.
- Sustained human growth hormone NexP-hGH protein can be prepared, the present invention can be usefully used for the industrial production of sustained human growth hormone.
- FIG. 1 is a diagram showing a chromatographic separation of polymers and monomers of the sustained human growth hormone NexP-hGH using anion exchange resin chromatography.
- FIGS. 2a and b are diagrams showing the results of measuring the purity of the sustained human growth hormone monomer by fraction in anion exchange resin chromatography by size exclusion-HPLC (SE-HPLC).
- Figure 2a shows a high purity sustained human growth hormone monomer obtained at a salt concentration of 100mM to 250mM in anion exchange resin chromatography
- Figure 2b is a human obtained at a salt concentration of 250mM to 350mM in anion exchange resin chromatography Purity of growth hormone monomer is shown.
- the present invention uses anion exchange resin chromatography to prepare monomers of NexP-hGH protein, which is a sustained human growth hormone fused with human growth hormone (hGH) and alpha-1 antitrypsin variant (NexP). Provide a method.
- NexP-hGH protein which is a sustained human growth hormone fused with human growth hormone (hGH) and alpha-1 antitrypsin variant (NexP).
- the method preferably comprises the steps of: a) injecting a sample comprising a mixture of sustained human growth hormone NexP-hGH protein into a pre-equilibration anion exchange chromatography column; And b) eluting a monomer of NexP-hGH protein, which is a sustained human growth hormone, at a salt concentration gradient of 0M to 0.4M using an elution buffer having a pH of 6.0 to 9.0.
- the sustained human growth hormone NexP-hGH protein When the sustained human growth hormone NexP-hGH protein is produced from a host cell, in addition to the monomeric sustained human growth hormone NexP-hGH protein which binds to the human growth hormone receptor and exhibits biological activity, it does not form an appropriate tertiary structure. Due to the poor stability of NexP-hGH protein or NexP-hGH protein, there is a problem in that NexP-hGH protein is produced in the form of a polymer. Therefore, when producing NexP-hGH protein from a host cell, there is a need to separate the NexP-hGH protein in high purity from the mixture containing both polymer and monomer of NexP-hGH protein, which can be used as protein medicine. It was.
- the method of the present invention can be prepared by separating the monomer of NexP-hGH protein from the mixture of NexP-hGH protein with high purity by using anion exchange resin chromatography, the separation and purification of NexP-hGH protein in monomer form It can be useful.
- human growth hormone is a peptide hormone, which means a hormone that can lead to human growth, cell reproduction or regeneration.
- the human growth hormone includes any protein that can promote growth by stimulating cell differentiation of cartilage growth plates in the body.
- the human growth hormone includes both growth hormone produced naturally and growth hormone produced using genetic engineering technology, preferably growth hormone produced using genetic engineering technology, but is not limited thereto.
- information on the human growth hormone can be obtained from a known database such as the National Institutes of Health (NCBI) GenBank, for example, human growth hormone having an Accession Number of AAA98618, but is not limited thereto.
- the human growth hormone can promote growth by stimulating cell differentiation of cartilage growth plates in the body, it can be usefully used as a therapeutic protein in individuals having problems in synthesis or secretion in the body.
- alpha-1 antitrypsin variant (NexP) having an increased half-life in the body by maintaining its persistence in the body can be fused to human growth hormone, thereby being used as a form of a fusion protein (NexP-hGH) having an increased half-life.
- NexP-hGH is a fusion protein of human growth hormone (hGH) and alpha-1 antitrypsin variant (NexP), and may be used in combination with "persistent human growth hormone” in the present invention.
- the NexP is a variant of alpha-1 antitrypsin (A1AT), which is a protein in the body whose half-life is increased by maintaining the persistence in the body developed by the present inventors, and is a term named by the present inventors.
- A1AT alpha-1 antitrypsin
- Protein sequence of alpha-1 antitrypsin, which has been deactivated by a protease inhibitor, and a manufacturing method thereof are disclosed in Korean Patent Registration No. 10-1183262 (Publication No.
- alpha-1 antitrypsin variant that can be applied to the sustained human growth hormone NexP-hGH of the present invention is not limited to the variant disclosed in the Republic of Korea Patent Registration No. 10-1183262, the protein degradation of alpha-1 antitrypsin All mutations of certain amino acids are included for the purpose of eliminating intrinsic body activity as an enzyme inhibitor and increasing half-life.
- the long-acting human growth hormone NexP-hGH is a protein in which the mutant of alpha-1 antitrypsin (A1AT) is fused to the N-terminus or C-terminus of human growth hormone by a gene recombinant method, compared to the first generation human growth hormone It is a substance with improved half-life in the body.
- Most of the first generation human growth hormone drugs are manufactured in Escherichia coli, whereas the sustained human growth hormone NexP-hGH is expressed in animal cells such as CHO cells to be glycosylated. Such glycosylation has the advantage of reducing the risk of causing antigenicity in the human body by producing human growth hormone similar to the natural form.
- the alpha-1 antitrypsin is one of proteins in mammalian blood having a molecular weight of about 50,000 Da, one of the main blood proteins having a blood concentration of about 2 mg / ml, and an alpha-1 protease inhibitor (alpha- 1 protease inhibitor).
- Alpha-1 antitrypsin extracted from blood is marketed as emphysema under the name Prolastin under FDA approval.
- Prolastin is commonly administered to the human body by intravenous injection at weekly intervals at a dose of 60 mg / kg and is a protein that has been demonstrated to be safe and harmful in humans.
- the role and structure of alpha-1 antitrypsin as a protease inhibitor is well known (Elliott, P.
- alpha-1 antitrypsin has more than 100 alleles in nature, and phenotypes are classified from A to Z according to the type of isoelectric focusing (IEF) (Stoller et al., The Lancet, 365, 2225-2236, 2005). Most of the M alleles are normal and are subdivided into various subtypes such as M1 (Val 213 ), M2, and M3 by amino acid sequence variation. Thus, alpha-1 antitrypsin used in the present invention is a specific subtype present in nature, and the same effect can be obtained with other subtypes.
- IEF isoelectric focusing
- alpha-1 antitrypsin protein may be obtained from a known database such as the National Institutes of Health (NCBI) GenBank, and examples thereof include an alpha-1 antitrypsin protein having an Accession Number of ABV21360 and CAJ15161, but is not limited thereto. It doesn't work.
- NCBI National Institutes of Health
- Such alpha-1 antitrypsin can modify one or more amino acid residues using site-directed mutagenesis to eliminate intrinsic body activity and increase half-life.
- Variation of one or more amino acids in the alpha-1 antitrypsin variant is characterized in that the 357th amino acid Proline (P), which is the P2 position, is mutated, and more specifically, it is mutated to asparagine (N). It is done.
- the variation of one or more amino acids in the alpha-1 antitrypsin variant includes a mutation of proline, the 357th amino acid, which is the P2 position, into an asparagine, and a variation of one or more amino acids in another position.
- the mutation of one or more amino acids in the other position is a ninth amino acid glutamine (Glutamine, Q) to asparagine, the 232rd amino acid Cysteine (C) to Serine (Serine, S) Or the 359 th serine (Srine, S) includes all of the mutations (Threonine, T).
- the human growth hormone NexP-hGH of the present invention which is a human growth hormone of which the half-life is increased by fusion of the alpha-1 antitrypsin variant, is obtained by introducing and expressing an expression vector containing a polynucleotide encoding the same into an animal cell. can do.
- the obtained product containing NexP-hGH protein which is a sustained human growth hormone, may not form an appropriate tertiary structure during expression or may form a polymer in addition to the monomer due to deterioration in stability during the purification process.
- the sustained human growth hormone in the form of a polymer may not be able to bring proper signaling in vivo or may be recognized as an antigen, and thus, it is necessary to separate monomers from the sample containing both the polymer and the monomer in high purity. In the case of using the production method of the present invention, the fraction in which the monomers are contained in high purity can be separated.
- Step a) is a step of injecting a sample containing a mixture of the sustained human growth hormone NexP-hGH protein in a pre-equilibration anion exchange chromatography column.
- anion exchange resin it is possible to remove other components other than the sustained human growth hormone in the sample, not only to concentrate the sustained human growth hormone monomer, but also to remove the polymer of the sustained human growth hormone. can do.
- anion exchange chromatography refers to the separation of molecules according to their charge by binding a negatively charged (or acidic) molecule to a positively charged support.
- the homologues of the molecules (acidic, basic and neutral) can be easily separated by this technique.
- strong anion exchange resin and weak anion exchange resin can be used without limitation, for example, Sephadex, Sepharose, Sauce, Mono, Mini (trade name, GE healthcare), etc.
- a resin in which the functional group of the resin is Q (Quaternary amine), DEAE (DiEthylAminoEthyl), or QAE (Quaternary Amino Ethyl) may be used.
- the functional group of the resin may be Q or DEAE, and most preferably Q-sepharose, which is a strong anion exchange resin, may be used.
- a BPG column filled with a Q-Sepharose resin having a functional group of Q was used (Example 3).
- the anion exchange resin used in the anion exchange resin chromatography of the present invention may be pre-equilibration with an aqueous buffer before injecting a sample comprising a mixture of sustained human growth hormone NexP-hGH protein.
- sample comprising a mixture of sustained human growth hormone NexP-hGH protein refers to a cell culture supernant or a cell lysate of the cells that produces the sustained human growth hormone NexP-hGH protein. cell extract), but is not limited thereto.
- the term "partially purified” refers to a state in which a polymer or the like exists in addition to a monomer of a desired NexP-hGH protein, although at least one fractionation procedure such as chromatography is performed.
- the partial purification process is not particularly limited in kind, and may be, for example, hydrophobic chromatography, anion exchange resin chromatography, affinity chromatography using a resin to which an antibody fragment is attached, and / or diafiltration. It is not limited.
- the partial purification is preferably purified using at least one method selected from the group consisting of hydrophobic chromatography, anion exchange resin chromatography and affinity chromatography with a resin to which the antibody fragment is attached, more preferably a A) applying a sample comprising NexP-hGH to anion exchange resin chromatography, b) applying the eluate generated in step a) to hydrophobic resin chromatography; And c) applying the eluate generated in step b) to affinity chromatography filled with a resin to which an anti alpha-1 antitrypsin antibody fragment is attached, but is not limited thereto.
- the solution purified by the affinity chromatography may be applied to anion exchange resin chromatography for separating the monomer of the present invention without diafiltration, but is not limited thereto, and then subjected to diafiltration and then applied to the production method of the present invention. It is possible.
- the culture solution obtained from the transformed CHO cells is diafiltered and purified sequentially using anion exchange resin chromatography using Q-sepharose resin and hydrophobic chromatography using phenyl sepharose. Diafiltration was again performed and partially purified by an affinity chromatography process using a resin to which an anti-alpha1 antitrypsin antibody fragment was attached (Examples 1 and 2).
- the partially purified eluate may be a sample having a final salt concentration of 0-50 mM NaCl or 0-50 mM magnesium chloride (MgCl 2 ) diluted in 0-20 mM Tris buffer at pH 6.0-9.0 without diafiltration step.
- the present invention is not limited thereto.
- the sample is used after dilution with NaCl of 50 mM or less or MgCl 2 or less of magnesium chloride (MgCl 2 ) without a filtration step, it has the advantage of reducing polymer loss and protein loss due to diafiltration.
- Step b) is a step of eluting the monomer of NexP-hGH protein, a sustained human growth hormone, at a salt concentration gradient of 0M to 0.4M using an elution buffer of pH 6.0 to 9.0.
- step b) the column into which the sample is injected is washed with a washing buffer, and eluted with a salt gradient using an elution buffer having a pH of 6.0 to 9.0.
- the salt gradient may be a continuous salt gradient or a stepwise salt gradient, but is not limited thereto.
- the continuous salt gradient or step salt gradient may be any one as long as the monomer of the sustained human growth hormone can be separated with high purity.
- monomers of the sustained human growth hormone were eluted with high purity in a continuous salt gradient (Example 3).
- a salt concentration capable of separating the monomer of the NexP-hGH protein, which is a sustained human growth hormone of the present invention may be a salt concentration of 0M to 0.4M, preferably a salt concentration of 50mM to 300mM, and more preferably 100mM. Salt concentration may be from 250 mM, but is not limited thereto.
- the salt is preferably a sodium salt, more preferably sodium chloride, but is not limited thereto.
- the type of buffer used in the washing and eluting step is not particularly limited, and may be, for example, sodium phosphate buffer, potassium phosphate buffer or Tris buffer, but It is not limited. However, since the polymer increases when the storage time of NexP-hGH in Tris buffer containing MgCl 2 increases, it is preferable to remove MgCl 2 in the washing and eluting steps.
- a culture medium containing the sustained human growth hormone NexP-hGH protein was obtained from transformed CHO cells, diafiltered, and then anion exchange resin chromatography and hydrophobic chromatography were sequentially Purification was carried out. It was then diafiltered again and purified by affinity chromatography using a resin with anti-alpha1-antitrypsin antibody fragment attached.
- An anion chromatography column (BPG column, 7 cm in diameter) using a Q-Sepharose resin was used to prepare a sample containing monomers of the sustained human growth hormone NexP-hGH protein having a purity of about 90% on SE-HPLC.
- the 20 mM Tris buffer solution (pH 7.4) and 20 mM sodium phosphate elution buffer solution (pH 7.4) were sequentially flowed in 3 column volumes at a flow rate of 40 mL / min to remove impurities. Washed. Then, 20 mM sodium phosphate eluting solvent containing 0.4 M NaCl (pH 7.4) was used to elute the continuous human growth hormone by flowing a continuous salt concentration gradient by 10 column capacity at a flow rate of 40 ml / min.
- the sustained human growth hormone was isolated with high purity at a NaCl concentration of 100 mM to 250 mM, while the sustained human growth hormone containing 60% or more polymers was separated at the NaCl concentration of 250 mM to 350 mM ( 1 and 2).
- the high-purity sustained human growth hormone from which the polymer was separated in this manner did not increase the polymer by more than 1% even when concentrated at a high concentration of 12 mg / ml or more.
- the present invention is a method capable of efficiently separating only long-acting human growth hormone NexP-hGH protein in monomer form in high purity by efficiently removing the easy-to-separate polymer form of long-acting human growth hormone NexP-hGH.
- it can be used to separately prepare NexP-hGH with a purity of 95 to 100%.
- the present invention provides a monomer of NexP-hGH protein, which is a sustained human growth hormone fused to human growth hormone (hGH) and alpha-1 antitrypsin variant (NexP) prepared by the above method.
- hGH human growth hormone
- NexP alpha-1 antitrypsin variant
- the human growth hormone, alpha-1 antitrypsin variant and NexP-hGH are as described above.
- proline (P) 357 was first reacted with site directed mutagenesis.
- An alpha-1 antitrypsin variant (A1AT (P357N)) substituted with (Asparagine, N) was prepared. Then, it was cloned with pG001-derived PCR product hGH and vector pAV1 having Xho1 and BamH1 restriction enzyme sites to prepare pT107.
- pT107 is a P357N variant wherein hGH and A1AT are bound without a linker.
- Clones made of the vector of ⁇ 1-1> were transduced into Chinese hamster ovary cells (CHO) and the expression of human growth hormone / alpha-1 antitrypsin mutant (107N) was confirmed.
- Human growth hormone / alpha-1 antitrypsin fusion (107N) expressing cells were cultured in CD Opti-CHO medium at 8% CO 2 and 37 ° C. temperature, and incubated in suspension at 0.5 ⁇ 10 6 cells / ml concentration. .
- a phenyl-sepharose loading solution was prepared by adding 4M NaCl / 20 mM sodium phosphate buffer (pH 8.0) to the Q-sepharose column eluate to give about 2.5 M NaCl / 20 mM sodium phosphate (pH 8.0). .
- phenyl-sepharose (GE Healthcare) resin was charged to an XK-50 column (GE Healthcare), and then the column was equilibrated with sufficient flow of 20 mM sodium phosphate buffer (pH 7.5) containing 2.5 M NaCl. .
- 20 mM sodium phosphate buffer (pH 8.0) containing 2.5 M NaCl was added at about 3 CV (column volume). Wash the column.
- a solution containing the sustained human growth hormone NexP-hGH was recovered by flowing 20 mM sodium phosphate buffer (pH 8.0) at a flow rate of 20 ml / min at about 4 CV.
- Resin GE Healthcare, hereinafter referred to as 'AIAT'
- A1AT anti alpha1-antitrypsin
- 'AIAT' XK-50 column
- the column was equilibrated with sufficient flow of Tris buffer (pH 7.4). After about 1 L of the diafiltration solution was flowed to the prepared A1AT column at a flow rate of 20 mL / min, the column was washed again by adding Tris buffer solution (pH 7.4) containing 150 mM NaCl (pH 7.4) to about 3 CV (column volume).
- Sustained human growth hormone NexP-hGH purified by the above method was present in more than 10% of the polymer to monomer ratio.
- Example 3 Isolation and Purification of Sustained Human Growth Hormone NexP-hGH Polymer and Monomer by Anion Exchange Resin Chromatography
- the purified long-acting human growth hormone NexP-hGH of Example 2 has a ratio of 10% or more of the polymer to the monomer, and it is necessary to specifically separate and purify only the monomer from the mixture in which both the monomer and the polymer are present.
- the present inventors separated and purified only the monomer from the mixture of the polymer and monomer in the following manner.
- NexP-hGH eluted in Example 2 was diluted 7-fold with Q column equilibration buffer, and loaded onto a BPG column (7 cm in diameter) filled with Q-Sepharose resin at a flow rate of 40 ml / min.
- 20 mM sodium phosphate buffer (pH 7.4) was used in a three column capacity at a flow rate of 40 ml / min to wash off impurities that did not adhere to the column.
- a sustained human growth hormone having a monomer concentration of about 98% or more is isolated at a salt concentration of about 100 mM to 250 mM, and a sustained human growth hormone comprising at least 60% of a polymer compared to the monomer at a salt concentration of 250 mM to 350 mM is isolated. It confirmed that it becomes (FIGS. 1-2).
- high-purity sustained human growth hormone in which the polymer was separated it was confirmed that the polymer was not increased by more than 1% even when concentrated at a high concentration of 12 mg / mL or more.
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Endocrinology (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Analytical Chemistry (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to: a method for preparing a high-purity monomer of a NexP-hGH protein, which is a long-acting human growth hormone, using anion-exchange resin chromatography; and a monomer of a NexP-hGH protein prepared by the method.
Description
본 발명은 음이온 교환 수지 크로마토그래피(anion exchange chromatography)를 이용하여 지속형 인간 성장호르몬인 NexP-hGH 단백질의 단량체를 고순도로 제조하는 방법 및 상기 방법으로 제조된 NexP-hGH 단백질의 단량체에 관한 것이다. The present invention relates to a method for producing a high purity monomer of NexP-hGH protein, a sustained human growth hormone using anion exchange chromatography, and a monomer of the NexP-hGH protein prepared by the above method.
인간 성장호르몬(human growth hormone, hGH)은 191개의 아미노산으로 구성된 분자량 약 21,500 Da의 단백질 호르몬으로 체내에서 연골 성장판의 세포 분화를 자극하여 성장을 촉진시키는 기능을 담당한다. 이러한 인간 성장호르몬의 체내 합성 및 분비 결핍이 있을 경우, 저 신장증, 심혈관 질환의 위험도 증가, 근육 및 골밀도 감소 등을 유발할 수 있다.Human growth hormone (hGH) is a protein hormone with a molecular weight of approximately 21,500 Da consisting of 191 amino acids, and is responsible for promoting growth by stimulating cell differentiation of cartilage growth plates in the body. If there is a deficiency in the synthesis and secretion of human growth hormone in the body, it may cause hypotensive, increased risk of cardiovascular disease, muscle and bone density decrease.
이를 치료하기 위한 하나의 방편으로, 인간은 1950년대 후반부터 인간의 뇌하수체에서 유래한 성장호르몬의 형태로 성장호르몬 치료(growth hormone therapy)를 받게 되었다. 유전공학기술의 발달로 1985년부터 성장호르몬은 대장균이나 효모에서 대량 생산이 가능해져 이를 의약품으로 개발하여 왔으며, 1990년대 후반부터 제약사들은 환자의 투여 편의성을 개선 및 증대시키기 위하여 투여횟수를 크게 줄일 수 있는 지속형 인간 성장호르몬 개발 및 출시하고 있다. 구체적으로, 1999년 제넨텍 사는 봉합체(encapsulation)로 만든 서방형 제품인 뉴트로핀 디팟 (nutropin depot)을 최초로 출시하였으며, 국내에서는 LG 생명과학에서 히알루론산을 매트릭스로 한 서방형 제품인 디클라제를 2007년 국내에 출시하였다.As a way to treat this, humans have received growth hormone therapy in the form of growth hormone derived from the human pituitary gland since the late 1950s. With the development of genetic engineering technology, growth hormone can be produced in large quantities in E. coli or yeast since 1985, and it has been developed as a medicine. Since the late 1990s, pharmaceutical companies have been able to significantly reduce the frequency of administration to improve and increase the convenience of patients. Is developing and releasing sustainable human growth hormone. Specifically, in 1999, Genentech released the first release of the nutropin depot, a sustained-release product made from encapsulation, and in Korea, LG Life Sciences released the de-laze, a sustained-release product based on hyaluronic acid as a matrix, in 2007. Released.
본 발명자들 역시 체내 지속성을 유지함으로 체내 반감기가 증가된 지속형 인간 성장호르몬을 개발하기 위해 노력하였고, 그 결과 지속형 인간 성장호르몬 NexP-hGH를 개발하였다. 이의 제작방법 및 단백질 서열 등은 대한민국 등록특허 제10-1183262호에 개시되었다. 지속형 인간 성장호르몬 NexP-hGH는 체내 단백질인 알파-1 안티 트립신(A1AT)의 고유한 체내 활성을 없애고 반감기를 증가시키기 위하여 특정 아미노산을 변형한 NexP를 인간 성장호르몬의 N-말단 또는 C-말단에 유전자 재조합 방식으로 융합한 단백질이다. NexP-hGH는 1세대 인간 성장호르몬에 비해 체내 반감기가 개선된 장점을 지니며, 동물세포인 CHO 세포에서 발현되는 경우 당화가 되도록 만들어져 있어 대장균에서 제조되는 대다수의 1세대 인간 성장호르몬 의약품과 차별점을 가진다.The present inventors also endeavored to develop a sustained human growth hormone with increased half-life in the body by maintaining the persistence in the body, and as a result developed a sustained human growth hormone NexP-hGH. The preparation method and protein sequence thereof are disclosed in Korean Patent Registration No. 10-1183262. The long-acting human growth hormone NexP-hGH is an N-terminus or C-terminus of human growth hormone that replaces NexP with specific amino acids to eliminate the intrinsic activity of the protein alpha-1 antitrypsin (A1AT) and increase its half-life. It is a protein fused by genetic recombination. NexP-hGH has the advantage of improved half-life in the body compared to first-generation human growth hormone, and it is made to be glycosylated when expressed in animal cells, CHO cells, and it is different from most first-generation human growth hormone drugs manufactured in Escherichia coli. Have
또한, NexP와 융합된 hGH는 hGH 자체의 구조 및 화학적 특성을 여전히 보유하고 있다. hGH는 중합체를 잘 이루는 단백질로 알려져 있는데, 중합체 형태의 hGH는 체내에서 항원성을 유발할 수 있을 뿐만 아니라, hGH와 이의 수용체 간의 결합을 방해하여 hGH 신호전달을 감소시키는 것으로 알려져 있다. In addition, hGH fused with NexP still retains the structural and chemical properties of hGH itself. hGH is known as a well-formed protein, and hGH in polymer form is known to not only induce antigenicity in the body but also to hinder the binding between hGH and its receptor, thereby reducing hGH signaling.
이러한 중합체는 숙주로부터 목적 단백질 발현시킬 때, 적절한 3차 구조가 형성되지 못한 단백질로부터 발생하거나, 공정 중 목적 단백질의 안정성 저하로 생성될 수 있다. 상업적 생산에서 상기와 같은 중합체는 제품 유래 불순물인 이상 펩타이드로 분류되어 순도시험에서 그 양이 규제되고 있다. 따라서, 생산 공정 중 중합체를 제거하기 위하여 많은 시도가 있었으며, 일반적으로 중합체와 단량체를 분자량으로 분리하는 크기배제 크로마토그래피(size-exclusion chromatography, SEC)가 일반적으로 이용되고 있다. 그러나 크기배제 크로마토그래피의 경우, 컬럼에 로딩하는 부피가 한정적(일반적으로 bed volume의 10% 이내)이기 때문에 목적 단백질을 고농도로 농축해야 하므로, 고농도 농축이 어려운 단백질에 적용할 경우 수율이 저하되는 등의 문제점이 있다. Such polymers may arise from proteins that do not form the appropriate tertiary structure when expressed in the target protein from the host, or may result from reduced stability of the target protein in the process. In commercial production, such polymers are classified as abnormal peptides that are product-derived impurities, and their amount is regulated in the purity test. Accordingly, many attempts have been made to remove polymers during the production process, and size-exclusion chromatography (SEC) is generally used to separate polymers and monomers by molecular weight. However, in size exclusion chromatography, since the volume of the column loaded is limited (typically within 10% of the bed volume), the target protein must be concentrated at a high concentration. There is a problem.
이러한 문제를 극복하기 위하여 근래에는 크기배제 크로마토그래피 외에 컬럼을 이용한 중합체 분리가 시도되고 있다. 양이온 교환수지를 이용하여 면역 글로불린 중합체와 단량체를 서로 분리하는 방법(일본 공개특허 H7-285885) 및 음이온 교환수지를 이용하여 항체 및 우혈청 알부민 중합체를 단량체와 분리하는 방법(유럽 공개특허 EP 1084136)이 개시된 바 있다. 그러나, 목적 단백질의 단량체를 분리하는 방법은 목적 단백질의 종류 등에 따라 조건이 달라질 수 있으며, 기존에 알려진 방법을 다른 단백질의 분리 및 제조에 적용하는 경우 큰 효과를 나타내지 않는 경우가 빈번하게 존재한다. 따라서, 원하는 목적 단백질에 따라 이의 단량체를 고순도 및 고농도로 분리할 수 있는 적절한 조건을 규명해야 할 필요가 있으며, 이러한 조건을 규명하는 것은 단백질 의약품과 같은 단백질 제조 시장에 있어 매우 중요한 문제이다.In order to overcome this problem, polymer separation using a column has been attempted in addition to size exclusion chromatography. A method for separating an immunoglobulin polymer and a monomer from each other using a cation exchange resin (Japanese Patent Laid-Open No. H7-285885) and a method for separating an antibody and a bovine serum albumin polymer from a monomer using an anion exchange resin (European Patent EP 1084136) This has been disclosed. However, the method of separating the monomer of the target protein may vary depending on the type of the target protein and the like, and when the known method is applied to the separation and production of other proteins, there are frequently cases that do not show a great effect. Therefore, there is a need to identify suitable conditions for separating the monomers of high purity and high concentration according to the desired protein of interest, which is a very important problem in the protein manufacturing market such as protein pharmaceuticals.
이에 본 발명자들은 지속형 인간 성장호르몬 NexP-hGH의 정제 공정 과정에서 중합체가 형성됨을 확인하여, 기존의 방법으로 정제가 어려웠던 신규한 지속형 인간 성장호르몬의 중합체와 단량체를 서로 분리함으로써, 고순도의 지속형 인간 성장호르몬 NexP-hGH의 단량체를 제조하는 방법을 개발하기 위하여 예의 노력한 결과, 음이온 교환 수지를 이용함으로써 지속형 인간 성장호르몬 NexP-hGH의 중합체와 단량체를 서로 분리하여 지속형 인간 성장호르몬 NexP-hGH의 단량체를 제조할 수 있음을 확인하고 본 발명을 완성하였다.Accordingly, the present inventors confirmed that the polymer was formed during the purification process of the sustained human growth hormone NexP-hGH, thereby separating the polymer and the monomer of the new continuous human growth hormone, which was difficult to purify by the existing method, to maintain high purity Efforts have been made to develop a method for producing monomers of the type human growth hormone NexP-hGH. As a result, anion exchange resins are used to separate the polymers and monomers of the type of sustained human growth hormone NexP-hGH from each other. It was confirmed that the monomer of hGH can be prepared and completed the present invention.
본 발명의 하나의 목적은 음이온 교환 수지 크로마토그래피를 이용하여 인간 성장호르몬 및 알파-1 안티트립신 변이체가 융합된 지속형 인간 성장호르몬인 NexP-hGH 단백질의 단량체를 제조하는 방법을 제공하는 것이다.One object of the present invention is to provide a method for preparing monomers of NexP-hGH protein, which is a sustained human growth hormone fused with human growth hormone and alpha-1 antitrypsin variant, using anion exchange resin chromatography.
본 발명의 다른 목적은 상기 방법으로 제조된, 인간 성장호르몬 및 알파-1 안티트립신 변이체가 융합된 지속형 인간 성장호르몬인 NexP-hGH 단백질의 단량체를 제공하는 것이다. Another object of the present invention is to provide a monomer of NexP-hGH protein, which is a sustained human growth hormone fused to human growth hormone and alpha-1 antitrypsin variant prepared by the above method.
본 발명의 방법은 음이온 교환 수지를 이용하여 지속형 인간 성장호르몬인 NexP-hGH의 중합체와 단량체를 포함하는 혼합물로부터 단량체만을 99% 이상의 높은 순도로 얻을 수 있는 장점을 지닌다. 본 발명의 방법에서 제공하는 고순도의 정제 방법을 생산 공정에 적용할 경우, 대량으로 고농도 및 고순도의 지속형 인간 성장호르몬을 정제할 수 있으며, 이로 인해 생활성(bioactivity)를 높이고 항원성을 감소시킬 수 있는 지속형 인간 성장호르몬 NexP-hGH 단백질을 제조할 수 있어, 본 발명은 지속형 인간 성장호르몬의 산업적 생산에 유용하게 이용될 수 있다.The method of the present invention has the advantage that only monomers can be obtained with a high purity of 99% or more from a mixture comprising a polymer and a monomer of the sustained human growth hormone NexP-hGH using an anion exchange resin. When the high purity purification method provided by the method of the present invention is applied to a production process, it is possible to purify high concentration and high purity sustained human growth hormone in large quantities, thereby increasing bioactivity and reducing antigenicity. Sustained human growth hormone NexP-hGH protein can be prepared, the present invention can be usefully used for the industrial production of sustained human growth hormone.
도 1은, 음이온 교환 수지 크로마토그래피를 이용하여 지속형 인간 성장호르몬 NexP-hGH의 중합체 및 단량체를 분리한 크로마토그래피를 나타낸 도이다.1 is a diagram showing a chromatographic separation of polymers and monomers of the sustained human growth hormone NexP-hGH using anion exchange resin chromatography.
도 2a 및 b는, 음이온 교환 수지 크로마토그래피에서 분획(fraction)별 지속형 인간 성장호르몬 단량체의 순도를 SE-HPLC(Size exclusion-HPLC)로 측정한 결과를 나타내는 도이다. 도 2a는, 음이온 교환 수지 크로마토그래피에서 100mM 내지 250mM의 염 농도에서 수득한 고순도의 지속형 인간 성장호르몬 단량체를 나타내며, 도 2b는, 음이온 교환 수지 크로마토그래피에서 250mM 내지 350mM의 염 농도에서 수득한 인간성장호르몬 단량체의 순도를 나타낸다.2a and b are diagrams showing the results of measuring the purity of the sustained human growth hormone monomer by fraction in anion exchange resin chromatography by size exclusion-HPLC (SE-HPLC). Figure 2a shows a high purity sustained human growth hormone monomer obtained at a salt concentration of 100mM to 250mM in anion exchange resin chromatography, Figure 2b is a human obtained at a salt concentration of 250mM to 350mM in anion exchange resin chromatography Purity of growth hormone monomer is shown.
하나의 양태로서, 본 발명은 음이온 교환 수지 크로마토그래피를 이용하여 인간 성장호르몬(hGH) 및 알파-1 안티트립신 변이체(NexP)가 융합된 지속형 인간 성장호르몬인 NexP-hGH 단백질의 단량체를 제조하는 방법을 제공한다.In one embodiment, the present invention uses anion exchange resin chromatography to prepare monomers of NexP-hGH protein, which is a sustained human growth hormone fused with human growth hormone (hGH) and alpha-1 antitrypsin variant (NexP). Provide a method.
상기 방법은 바람직하게는 a) 전-평형화(pre-equilibration)된 음이온 교환 수지 크로마토그래피(anion exchange chromatography) 컬럼에 지속형 인간 성장호르몬 NexP-hGH 단백질의 혼합액을 포함하는 시료를 주입하는 단계; 및 b) pH 6.0 내지 9.0인 용출 완충용액을 이용하여 0M 내지 0.4M의 염 농도 구배에서 지속형 인간 성장호르몬인 NexP-hGH 단백질의 단량체를 용출시키는 단계를 포함할 수 있다. The method preferably comprises the steps of: a) injecting a sample comprising a mixture of sustained human growth hormone NexP-hGH protein into a pre-equilibration anion exchange chromatography column; And b) eluting a monomer of NexP-hGH protein, which is a sustained human growth hormone, at a salt concentration gradient of 0M to 0.4M using an elution buffer having a pH of 6.0 to 9.0.
상기 지속형 인간 성장호르몬 NexP-hGH 단백질을 숙주세포로부터 생산하는 경우, 인간 성장호르몬 수용체에 결합하여 생물학적 활성을 나타내는 단량체 형태의 지속형 인간 성장호르몬 NexP-hGH 단백질 외에도, 적절한 3차 구조를 형성하지 못한 NexP-hGH 단백질 또는 NexP-hGH 단백질의 안정성 저하로 인하여 형성된 중합체 형태의 NexP-hGH 단백질이 생산되는 문제점이 있다. 따라서, NexP-hGH 단백질을 숙주 세포로부터 생산하는 경우, NexP-hGH 단백질의 중합체 및 단량체가 모두 포함되어 있는 혼합물로부터 단백질 의약품으로 사용할 수 있는 단량체 형태의 NexP-hGH 단백질을 고순도로 분리해낼 필요성이 존재하였다. 본 발명의 방법은 음이온 교환 수지 크로마토그래피를 이용하여 NexP-hGH 단백질의 혼합액으로부터 NexP-hGH 단백질의 단량체를 고순도로 분리하여 제조할 수 있는 방법으로, 단량체 형태의 NexP-hGH 단백질의 분리 및 정제에 유용하게 사용할 수 있다.When the sustained human growth hormone NexP-hGH protein is produced from a host cell, in addition to the monomeric sustained human growth hormone NexP-hGH protein which binds to the human growth hormone receptor and exhibits biological activity, it does not form an appropriate tertiary structure. Due to the poor stability of NexP-hGH protein or NexP-hGH protein, there is a problem in that NexP-hGH protein is produced in the form of a polymer. Therefore, when producing NexP-hGH protein from a host cell, there is a need to separate the NexP-hGH protein in high purity from the mixture containing both polymer and monomer of NexP-hGH protein, which can be used as protein medicine. It was. The method of the present invention can be prepared by separating the monomer of NexP-hGH protein from the mixture of NexP-hGH protein with high purity by using anion exchange resin chromatography, the separation and purification of NexP-hGH protein in monomer form It can be useful.
본 발명에서 용어, "인간 성장호르몬(human growth hormone, hGH)"은 펩타이드 호르몬으로서, 인간의 성장, 세포 생산(cell reproduction) 또는 재생(regeneration)을 가져올 수 있는 호르몬을 의미한다. 본 발명의 목적상 상기 인간 성장호르몬은 체내에서 연골 성장판의 세포 분화를 자극하여 성장을 촉진시킬 수 있는 단백질이라면 제한 없이 포함한다. 상기 인간 성장호르몬은 자연적으로 생산된 성장호르몬 및 유전공학기술을 이용하여 생산된 성장호르몬을 모두 포함하며, 바람직하게는 유전공학기술을 이용하여 생산된 성장호르몬이나, 이에 제한되지 않는다. 또한, 상기 인간 성장호르몬에 대한 정보는 미국 국립보건원(NCBI) GenBank와 같은 공지의 데이터베이스로부터 얻을 수 있으며, 그 예로 Accession Number가 AAA98618인 인간 성장호르몬 등이 있으나, 이에 제한되지 않는다. In the present invention, the term "human growth hormone (hGH)" is a peptide hormone, which means a hormone that can lead to human growth, cell reproduction or regeneration. For the purpose of the present invention, the human growth hormone includes any protein that can promote growth by stimulating cell differentiation of cartilage growth plates in the body. The human growth hormone includes both growth hormone produced naturally and growth hormone produced using genetic engineering technology, preferably growth hormone produced using genetic engineering technology, but is not limited thereto. In addition, information on the human growth hormone can be obtained from a known database such as the National Institutes of Health (NCBI) GenBank, for example, human growth hormone having an Accession Number of AAA98618, but is not limited thereto.
상기 인간 성장호르몬은 체내에서 연골 성장판의 세포 분화를 자극함으로써 성장을 촉진시킬 수 있으므로, 체내 합성 또는 분비에 있어 문제가 있는 개체에 있어 치료용 단백질로서 유용하게 사용될 수 있다. 다만, 환자의 투여 편의성 및 치료 효율을 위하여 반감기를 증대시킬 필요성이 있다. 이를 위하여 체내 지속성을 유지함으로써 체내 반감기가 증대된 알파-1 안티트립신 변이체(NexP)를 인간 성장호르몬에 융합시킴으로써, 반감기를 증대시킨 융합 단백질의 형태(NexP-hGH)로서 이용할 수 있다.Since the human growth hormone can promote growth by stimulating cell differentiation of cartilage growth plates in the body, it can be usefully used as a therapeutic protein in individuals having problems in synthesis or secretion in the body. However, there is a need to increase the half-life for the convenience of administration and treatment efficiency. To this end, alpha-1 antitrypsin variant (NexP) having an increased half-life in the body by maintaining its persistence in the body can be fused to human growth hormone, thereby being used as a form of a fusion protein (NexP-hGH) having an increased half-life.
본 발명에서 용어, "NexP-hGH"은 인간 성장호르몬(hGH) 및 알파-1 안티트립신 변이체(NexP)가 융합된 형태의 단백질로서, 본 발명에서 "지속형 인간 성장호르몬"과 혼용되어 사용될 수 있다. 상기 NexP는 본 발명자들에 의해 개발된 체내 지속성을 유지함으로 체내 반감기가 증가된 체내 단백질인 알파-1 안티 트립신(alpha 1-Antitrypsin, A1AT)의 변이체로, 본 발명자들에 의해 명명된 용어이다. 단백질 분해 효소 억제제의 활성을 없앤 알파-1 안티 트립신의 단백질 서열, 제작 방법 등은 대한민국 등록특허 제10-1183262호 (공개번호: KR 제10-2010-0116558호)에 개시되어 있으며, 대한민국 등록특허 제10-1183262호 명세서 전체는 본 발명의 참고자료로 포함된다. 다만, 본 발명의 지속형 인간 성장호르몬 NexP-hGH에 적용될 수 있는 알파-1 안티트립신 변이체는 상기 대한민국 등록특허 제10-1183262호에 개시된 변이체에 한정되지 않고, 알파-1 안티 트립신이 지닌 단백질 분해 효소 억제제로서의 고유한 체내 활성을 없애고 반감기를 증가시킬 목적으로 일정 아미노산을 돌연변이시킨 것을 모두 포함한다. 상기 지속형 인간 성장호르몬 NexP-hGH은 이러한 알파-1 안티 트립신(A1AT)의 변이체를 인간 성장호르몬의 N-말단 또는 C-말단에 유전자 재조합 방식으로 융합한 단백질이며, 1세대 인간 성장호르몬에 비해 체내 반감기가 개선된 물질이다. 대다수의 1세대 인간 성장호르몬 의약품은 대장균에서 제조되는 반면, 지속형 인간 성장호르몬 NexP-hGH는 CHO 세포 등 동물 세포에서 발현되어 당화가 되도록 만들어졌다. 이와 같은 당화는 천연형과 유사한 인간 성장호르몬을 생산하여 인체 내에서 항원성을 유발시킬 위험을 줄일 수 있는 장점이 있다. As used herein, the term "NexP-hGH" is a fusion protein of human growth hormone (hGH) and alpha-1 antitrypsin variant (NexP), and may be used in combination with "persistent human growth hormone" in the present invention. have. The NexP is a variant of alpha-1 antitrypsin (A1AT), which is a protein in the body whose half-life is increased by maintaining the persistence in the body developed by the present inventors, and is a term named by the present inventors. Protein sequence of alpha-1 antitrypsin, which has been deactivated by a protease inhibitor, and a manufacturing method thereof are disclosed in Korean Patent Registration No. 10-1183262 (Publication No. KR 10-2010-0116558) and Korean Patent Registration The entirety of No. 10-1183262 is hereby incorporated by reference. However, the alpha-1 antitrypsin variant that can be applied to the sustained human growth hormone NexP-hGH of the present invention is not limited to the variant disclosed in the Republic of Korea Patent Registration No. 10-1183262, the protein degradation of alpha-1 antitrypsin All mutations of certain amino acids are included for the purpose of eliminating intrinsic body activity as an enzyme inhibitor and increasing half-life. The long-acting human growth hormone NexP-hGH is a protein in which the mutant of alpha-1 antitrypsin (A1AT) is fused to the N-terminus or C-terminus of human growth hormone by a gene recombinant method, compared to the first generation human growth hormone It is a substance with improved half-life in the body. Most of the first generation human growth hormone drugs are manufactured in Escherichia coli, whereas the sustained human growth hormone NexP-hGH is expressed in animal cells such as CHO cells to be glycosylated. Such glycosylation has the advantage of reducing the risk of causing antigenicity in the human body by producing human growth hormone similar to the natural form.
상기 알파-1 안티트립신은 분자량이 약 50,000Da의 포유류의 혈액 내에 존재하는 단백질 중의 하나로서, 혈액 내 농도는 약 2㎎/㎖에 달하는 주 혈액 단백질 중의 하나이며, 알파-1 프로테아제 억제제(alpha-1 protease inhibitor)라고도 불리운다. 혈액 내에서 추출한 알파-1 안티트립신은 FDA의 허가를 거쳐서 프로라스틴(Prolastin)이라는 상품명으로 폐기종(emphysema) 치료제로 판매되고 있다. 프로라스틴은 통상적으로 60㎎/㎏의 용량으로 1주 간격으로 정맥 주사로 인체에 투여되며, 인체에서의 안전성 및 유해성이 입증이 된 단백질이다. 또한, 알파-1 안티 트립신의 프로테아제 억제제로의 역할 및 구조 등은 이미 잘 알려져 있다(Elliott, P. et al., JMB 275, 419-425, 1998). 상기 알파-1 안티트립신은 자연계에 100여 종 이상의 대립유전자(allele)가 존재하고, 표현형(phenotype)은 IEF(isoelectric focusing) 유형에 따라 A에서 Z로 구분한다(Stoller et al., The Lancet, 365, 2225-2236, 2005). 이중 가장 많은 M 대립 유전자는 정상형으로 아미노산 서열 변이에 의해 다시 M1(Val213), M2, M3와 같이 여러 가지 아형(subtype)으로 구분된다. 따라서, 본 발명에 사용된 알파-1 안티트립신은 자연계에 존재하는 특정 아형이며, 다른 아형에 대하여도 동일한 효과를 얻을 수 있다. 상기 알파-1 안티트립신 단백질에 대한 기본적인 정보는 미국 국립보건원(NCBI) GenBank와 같은 공지의 데이터베이스로부터 얻을 수 있으며, 그 예로 Accession Number가 ABV21360, CAJ15161인 알파-1 안티트립신 단백질 등이 있으나, 이에 제한되지 않는다.The alpha-1 antitrypsin is one of proteins in mammalian blood having a molecular weight of about 50,000 Da, one of the main blood proteins having a blood concentration of about 2 mg / ml, and an alpha-1 protease inhibitor (alpha- 1 protease inhibitor). Alpha-1 antitrypsin extracted from blood is marketed as emphysema under the name Prolastin under FDA approval. Prolastin is commonly administered to the human body by intravenous injection at weekly intervals at a dose of 60 mg / kg and is a protein that has been demonstrated to be safe and harmful in humans. In addition, the role and structure of alpha-1 antitrypsin as a protease inhibitor is well known (Elliott, P. et al., JMB 275, 419-425, 1998). The alpha-1 antitrypsin has more than 100 alleles in nature, and phenotypes are classified from A to Z according to the type of isoelectric focusing (IEF) (Stoller et al., The Lancet, 365, 2225-2236, 2005). Most of the M alleles are normal and are subdivided into various subtypes such as M1 (Val 213 ), M2, and M3 by amino acid sequence variation. Thus, alpha-1 antitrypsin used in the present invention is a specific subtype present in nature, and the same effect can be obtained with other subtypes. Basic information about the alpha-1 antitrypsin protein may be obtained from a known database such as the National Institutes of Health (NCBI) GenBank, and examples thereof include an alpha-1 antitrypsin protein having an Accession Number of ABV21360 and CAJ15161, but is not limited thereto. It doesn't work.
이러한 알파-1 안티트립신은 특정부위 돌연변이 유발(site-directed mutagenesis) 방법을 이용하여 하나 이상의 아미노산 잔기를 변형시켜 고유한 체내 활성을 없애고 반감기를 증가시킬 수 있다. 이러한 알파-1 안티트립신 변이체에서 하나 이상의 아미노산의 변이는 P2 위치인 357번째 아미노산인 프롤린(Proline, P)이 변이된 것을 특징으로 하며, 보다 구체적으로 아스파라진(Asparagine, N)으로 변이시킨 것을 특징으로 한다. 또한, 알파-1 안티트립신 변이체에서 하나 이상의 아미노산의 변이는 P2 위치인 357번째 아미노산인 프롤린이 아스파라진으로 변이된 것을 포함하고 다른 위치에 있는 하나 이상의 아미노산을 변이시킨 것도 포함한다. 아울러, 상기 다른 위치에 있는 하나 이상의 아미노산의 변이는 9번째 아미노산인 글루타민(Glutamine, Q)이 아스파라진으로의 변이, 232번째 아미노산인 시스테인(Cysteine, C)이 세린(Serine, S)으로의 변이 또는 359번째 세린(Serine, S)이 트레오닌(Threonine, T)으로 변이된 것을 모두 포함한다. 이러한 알파-1 안티트립신 변이체는 357Asn-358X-359Ser 또는 357Asn-358X-359Thr의 새로운 N-당화 부위가 생성되어 알파-1 안티트립신의 프로테아제 억제제의 활성을 중화시키는 동시에 체내 주입시에 아미노산 치환에 의한 면역원성 가능성을 최소화할 수 있으며, 유리 시스테인기에 의한 이중체 형성 등을 제거할 수 있는 장점을 지닌다. Such alpha-1 antitrypsin can modify one or more amino acid residues using site-directed mutagenesis to eliminate intrinsic body activity and increase half-life. Variation of one or more amino acids in the alpha-1 antitrypsin variant is characterized in that the 357th amino acid Proline (P), which is the P2 position, is mutated, and more specifically, it is mutated to asparagine (N). It is done. In addition, the variation of one or more amino acids in the alpha-1 antitrypsin variant includes a mutation of proline, the 357th amino acid, which is the P2 position, into an asparagine, and a variation of one or more amino acids in another position. In addition, the mutation of one or more amino acids in the other position is a ninth amino acid glutamine (Glutamine, Q) to asparagine, the 232rd amino acid Cysteine (C) to Serine (Serine, S) Or the 359 th serine (Srine, S) includes all of the mutations (Threonine, T). These alpha-1 antitrypsin variants generate a new N-glycosylation site of 357 Asn- 358 X- 359 Ser or 357 Asn- 358 X- 359 Thr to neutralize the activity of the alpha-1 antitrypsin protease inhibitors while simultaneously injecting into the body. The possibility of immunogenicity by amino acid substitution at the time can be minimized, and it has the advantage of eliminating duplex formation by free cysteine group.
상기 알파-1 안티트립신 변이체를 융합하여 반감기가 증대된 인간 성장호르몬인 본 발명의 지속형 인간 성장호르몬인 NexP-hGH은 이를 코딩하는 폴리뉴클레오티드를 포함하는 발현벡터를 동물세포에 도입하여 발현함으로써 수득할 수 있다. 상기 지속형 인간 성장호르몬인 NexP-hGH 단백질을 포함하는 수득물은 발현시 적절한 3차 구조를 형성하지 못하거나 정제 과정을 거치는 동안 안정성 저하 등으로 단량체 외에도 중합체를 형성할 수 있다. 이러한 중합체 형태의 지속형 인간 성장호르몬은 생체 내에서 적절한 신호전달을 가져오지 못하거나 항원으로 인식될 수 있어 중합체 및 단량체가 모두 포함되어 있는 시료로부터 단량체를 고순도로 분리해낼 필요가 있다. 본 발명의 제조 방법을 이용하는 경우에는 단량체가 고순도로 포함되어 있는 분획을 분리해낼 수 있다.The human growth hormone NexP-hGH of the present invention, which is a human growth hormone of which the half-life is increased by fusion of the alpha-1 antitrypsin variant, is obtained by introducing and expressing an expression vector containing a polynucleotide encoding the same into an animal cell. can do. The obtained product containing NexP-hGH protein, which is a sustained human growth hormone, may not form an appropriate tertiary structure during expression or may form a polymer in addition to the monomer due to deterioration in stability during the purification process. The sustained human growth hormone in the form of a polymer may not be able to bring proper signaling in vivo or may be recognized as an antigen, and thus, it is necessary to separate monomers from the sample containing both the polymer and the monomer in high purity. In the case of using the production method of the present invention, the fraction in which the monomers are contained in high purity can be separated.
상기 제조 방법의 각 단계를 구체적으로 설명하면 다음과 같다.Hereinafter, each step of the manufacturing method will be described in detail.
상기 a) 단계는 전-평형화(pre-equilibration)된 음이온 교환 수지 크로마토그래피(anion exchange chromatography) 컬럼에 지속형 인간 성장호르몬 NexP-hGH 단백질의 혼합액을 포함하는 시료를 주입하는 단계이다. 이와 같은 음이온 교환 수지를 이용하는 경우, 시료 내에 지속형 인간 성장호르몬 외의 다른 성분 등을 제거하는 것이 가능하고, 지속형 인간 성장 호르몬 단량체를 농축할 수 있을 뿐만 아니라, 지속형 인간 성장호르몬의 중합체를 제거할 수 있다.Step a) is a step of injecting a sample containing a mixture of the sustained human growth hormone NexP-hGH protein in a pre-equilibration anion exchange chromatography column. In the case of using such anion exchange resin, it is possible to remove other components other than the sustained human growth hormone in the sample, not only to concentrate the sustained human growth hormone monomer, but also to remove the polymer of the sustained human growth hormone. can do.
본 발명에서 용어, "음이온 교환 수지 크로마토그래피(anion exchange chromatography)"는 양으로 하전된 지지체에 음으로 하전된(또는 산성) 분자를 결합시키는 것에 의해 분자들을 이들의 전하에 따라 분리할 수 있는 것으로서, 분자들의 동족체(산성, 염기성 및 중성)는 이 기법에 의해 쉽게 분리할 수 있다. 본 발명의 음이온교환크로마토그래피에 사용될 수 있는 수지로는 강음이온 교환 수지와 약음이온 교환 수지를 제한 없이 사용할 수 있으며, 그 예로 세파덱스, 세파로즈, 소스, 모노, 미니(상품명, GE healthcare) 등일 수 있으며, 이에 제한되는 것은 아니나 상기 수지의 작용기가 Q(Quaternary amine), DEAE(DiEthylAminoEthyl) 또는 QAE(Quaternary Amino Ethyl) 등인 수지를 사용할 수 있다. 바람직하게는 상기 수지의 작용기가 Q 또는 DEAE 일 수 있으며, 가장 바람직하게는 강음이온 교환 수지인 Q-세파로즈를 사용할 수 있다. 본 발명의 일 실시예에서는 수지의 작용기가 Q인 Q-세파로즈 수지가 충진된 BPG 컬럼을 사용하였다(실시예 3). As used herein, the term "anion exchange chromatography" refers to the separation of molecules according to their charge by binding a negatively charged (or acidic) molecule to a positively charged support. The homologues of the molecules (acidic, basic and neutral) can be easily separated by this technique. As the resin that can be used in the anion exchange chromatography of the present invention, strong anion exchange resin and weak anion exchange resin can be used without limitation, for example, Sephadex, Sepharose, Sauce, Mono, Mini (trade name, GE healthcare), etc. Although not limited thereto, a resin in which the functional group of the resin is Q (Quaternary amine), DEAE (DiEthylAminoEthyl), or QAE (Quaternary Amino Ethyl) may be used. Preferably, the functional group of the resin may be Q or DEAE, and most preferably Q-sepharose, which is a strong anion exchange resin, may be used. In one embodiment of the present invention, a BPG column filled with a Q-Sepharose resin having a functional group of Q was used (Example 3).
본 발명의 음이온 교환 수지 크로마토그래피에 사용되는 음이온 교환 수지는 지속형 인간 성장호르몬 NexP-hGH 단백질의 혼합액을 포함하는 시료를 주입시키기 전에 수성 완충용액으로 전-평형화(pre-equilibration)시킬 수 있다.The anion exchange resin used in the anion exchange resin chromatography of the present invention may be pre-equilibration with an aqueous buffer before injecting a sample comprising a mixture of sustained human growth hormone NexP-hGH protein.
본 발명에서 용어, "지속형 인간 성장호르몬 NexP-hGH 단백질의 혼합액을 포함하는 시료"는 지속형 인간 성장호르몬 NexP-hGH 단백질을 생산하는 세포의 배양 상등액(cell culture supernant) 또는 상기 세포의 파쇄물(cell extract)로부터 부분 정제된 형태일 수 있으나, 이에 제한되지 않는다.As used herein, the term "sample comprising a mixture of sustained human growth hormone NexP-hGH protein" refers to a cell culture supernant or a cell lysate of the cells that produces the sustained human growth hormone NexP-hGH protein. cell extract), but is not limited thereto.
본 발명에서 용어, "부분 정제(partially purified)"는 크로마토그래피와 같은 분류 과정(fractionation procedure)을 하나 이상 수행하였으나 목적하는 NexP-hGH 단백질의 단량체 외에도 중합체 등이 존재하는 상태를 의미한다. 상기 부분 정제 과정은 특별히 그 종류가 제한되지 않으며, 그 예로 소수성 크로마토그래피, 음이온 교환 수지 크로마토그래피, 항체 단편이 부착된 수지를 이용한 친화성 크로마토그래피 또는/및 정용여과(diafiltration)일 수 있으나, 이에 제한되지 않는다. 상기 부분 정제는 바람직하게는 소수성 크로마토그래피, 음이온 교환 수지 크로마토그래피 및 항체 단편이 부착된 수지를 이용한 친화성 크로마토그래피로 이루어진 군으로부터 선택되는 하나 이상의 방법을 이용하여 정제하는 것이며, 더욱 바람직하게는 a) NexP-hGH를 포함하는 시료를 음이온 교환 수지 크로마토그래피에 적용하는 단계, b) 단계 a)에서 생성된 용출액을 소수성 수지 크로마토그래피에 적용하는 단계; 및 c) 단계 b)에서 생성된 용출액을 안티 알파-1 안티트립신 항체 단편이 부착된 수지로 충진된 친화성 크로마토그래피에 적용하는 단계를 포함하는 부분 정제 방법일 수 있으나, 이에 제한되지 않는다. 상기 친화성 크로마토그래피로 정제한 용액은 정용 여과 없이 본 발명의 단량체를 분리하기 위한 음이온 교환 수지 크로마토그래피에 적용될 수 있으나, 이에 국한되지 않으며 정용여과를 거친 다음 본 발명의 제조 방법에 적용하는 것 또한 가능하다.As used herein, the term "partially purified" refers to a state in which a polymer or the like exists in addition to a monomer of a desired NexP-hGH protein, although at least one fractionation procedure such as chromatography is performed. The partial purification process is not particularly limited in kind, and may be, for example, hydrophobic chromatography, anion exchange resin chromatography, affinity chromatography using a resin to which an antibody fragment is attached, and / or diafiltration. It is not limited. The partial purification is preferably purified using at least one method selected from the group consisting of hydrophobic chromatography, anion exchange resin chromatography and affinity chromatography with a resin to which the antibody fragment is attached, more preferably a A) applying a sample comprising NexP-hGH to anion exchange resin chromatography, b) applying the eluate generated in step a) to hydrophobic resin chromatography; And c) applying the eluate generated in step b) to affinity chromatography filled with a resin to which an anti alpha-1 antitrypsin antibody fragment is attached, but is not limited thereto. The solution purified by the affinity chromatography may be applied to anion exchange resin chromatography for separating the monomer of the present invention without diafiltration, but is not limited thereto, and then subjected to diafiltration and then applied to the production method of the present invention. It is possible.
본 발명의 일 실시예에서는 형질전환된 CHO 세포로부터 수득한 배양액을 정용여과하고 이를 Q-세파로즈 수지를 이용한 음이온 교환 수지 크로마토그래피, 페닐 세파로즈를 이용한 소수성 크로마토그래피를 이용하여 순차적으로 정제한 다음, 다시 정용여과를 수행하고, 안티-알파1 안티트립신 항체 단편이 부착된 수지를 이용한 친화성 크로마토그래피 공정으로 부분 정제하였다(실시예 1 및 2).In an embodiment of the present invention, the culture solution obtained from the transformed CHO cells is diafiltered and purified sequentially using anion exchange resin chromatography using Q-sepharose resin and hydrophobic chromatography using phenyl sepharose. Diafiltration was again performed and partially purified by an affinity chromatography process using a resin to which an anti-alpha1 antitrypsin antibody fragment was attached (Examples 1 and 2).
또한, 부분 정제된 용출액은 정용여과 단계 없이 pH 6.0 내지 9.0인 0 내지 20mM 트리스(Tris) 완충용액으로 희석한 최종 염 농도가 0 내지 50mM NaCl 또는 0 내지 50mM 염화마그네슘(MgCl2)인 시료일 수 있으나, 이에 제한되지 않는다. 상기 시료를 여과단계 없이 최종 염 농도가 50mM 이하의 NaCl 또는 50mM 이하 염화마그네슘(MgCl2)으로 희석시켜 사용하는 경우, 정용여과로 인한 중합체의 증가 및 단백질의 손실을 줄일 수 있는 이점을 지닌다.In addition, the partially purified eluate may be a sample having a final salt concentration of 0-50 mM NaCl or 0-50 mM magnesium chloride (MgCl 2 ) diluted in 0-20 mM Tris buffer at pH 6.0-9.0 without diafiltration step. However, the present invention is not limited thereto. When the sample is used after dilution with NaCl of 50 mM or less or MgCl 2 or less of magnesium chloride (MgCl 2 ) without a filtration step, it has the advantage of reducing polymer loss and protein loss due to diafiltration.
상기 b) 단계는 pH 6.0 내지 9.0인 용출 완충용액을 이용하여 0M 내지 0.4M의 염 농도 구배에서 지속형 인간 성장호르몬인 NexP-hGH 단백질의 단량체를 용출시키는 단계이다.Step b) is a step of eluting the monomer of NexP-hGH protein, a sustained human growth hormone, at a salt concentration gradient of 0M to 0.4M using an elution buffer of pH 6.0 to 9.0.
구체적으로, b) 단계에서는 시료를 주입한 컬럼을 세척 완충액으로 세척하고, pH 6.0 내지 9.0인 용출 완충용액을 이용하여 염 구배(salt gradient)로 용출시킨다. 상기 염 구배는 연속식 염 구배(continuous salt gradient) 또는 단계식 염 구배(stepwise salt gradient)일 수 있으나, 이에 제한되지 않는다. 상기 연속식 염 구배 또는 단계식 염 구배는 지속형 인간 성장호르몬의 단량체를 고순도로 분리할 수만 있다면 어느것이든 무방하다. 본 발명의 일 실시예에서는 연속식 염 구배로 지속형 인간 성장호르몬의 단량체를 고순도로 용출하였다(실시예 3).Specifically, in step b), the column into which the sample is injected is washed with a washing buffer, and eluted with a salt gradient using an elution buffer having a pH of 6.0 to 9.0. The salt gradient may be a continuous salt gradient or a stepwise salt gradient, but is not limited thereto. The continuous salt gradient or step salt gradient may be any one as long as the monomer of the sustained human growth hormone can be separated with high purity. In one embodiment of the present invention, monomers of the sustained human growth hormone were eluted with high purity in a continuous salt gradient (Example 3).
본 발명의 지속형 인간 성장호르몬인 NexP-hGH 단백질의 단량체를 분리할 수 있는 염 농도는 0M 내지 0.4M의 염 농도일 수 있으며, 바람직하게는 50mM 내지 300mM의 염 농도이며, 더욱 바람직하게는 100mM 내지 250mM의 염 농도일 수 있으나, 이에 제한되지 않는다. 또한, 상기 염은 바람직하게는 나트륨 염이며, 더욱 바람직하게는 염화나트륨이나, 이에 제한되지 않는다. A salt concentration capable of separating the monomer of the NexP-hGH protein, which is a sustained human growth hormone of the present invention, may be a salt concentration of 0M to 0.4M, preferably a salt concentration of 50mM to 300mM, and more preferably 100mM. Salt concentration may be from 250 mM, but is not limited thereto. In addition, the salt is preferably a sodium salt, more preferably sodium chloride, but is not limited thereto.
상기 세척 및 용출 단계에서 사용되는 완충용액은 그 종류가 특별히 제한되지 않으며, 그 예로 인산나트륨(Sodium phosphate) 완충용액, 인산칼륨(Potassium phosphate) 완충용액 또는 트리스(Tris) 완충용액일 수 있으나, 이에 제한되지 않는다. 단, MgCl2 가 포함된 트리스(Tris) 완충용액에서 NexP-hGH의 보관시간이 길어지면 중합체가 증가하므로 세척 및 용출단계에는 MgCl2를 제거하는 것이 바람직하다.The type of buffer used in the washing and eluting step is not particularly limited, and may be, for example, sodium phosphate buffer, potassium phosphate buffer or Tris buffer, but It is not limited. However, since the polymer increases when the storage time of NexP-hGH in Tris buffer containing MgCl 2 increases, it is preferable to remove MgCl 2 in the washing and eluting steps.
본 발명의 일 실시예에 따르면, 지속형 인간 성장호르몬 NexP-hGH 단백질을 포함하는 배양액을 형질전환된 CHO 세포로부터 수득하였고, 이를 정용여과한 다음, 음이온 교환 수지 크로마토그래피 및 소수성 크로마토그래피를 순차적으로 수행하여 정제하였다. 그 다음, 다시 정용여과하고 항-알파1-안티트립신 항체 단편이 부착된 수지를 이용한 친화성 크로마토그래피로 정제하였다. 이와 같은 과정으로 부분정제한 SE-HPLC 상 순도 약 90%인 지속형 인간 성장호르몬 NexP-hGH 단백질의 단량체를 포함하는 시료를 Q-세파로즈 수지를 사용한 음이온 크로마토그래피 컬럼(BPG 컬럼, 직경 7cm)에 40㎖/min의 유속으로 주입하여 결합시킨 후, 20mM 트리스 완충용액(pH 7.4) 및 20mM 인산나트륨 용출 완충용액(pH 7.4)를 순차적으로 40㎖/min의 유속으로 3 컬럼 용량을 흘려 불순물을 세척하였다. 그 다음, 0.4M NaCl이 포함된 20mM 소듐 포스페이트 용출 용매(pH 7.4)를 사용하여 40㎖/min의 유속으로 10 컬럼 용량만큼 연속식 염 농도구배로 흘려 지속형 인간 성장호르몬을 용출하였다. 그 결과, 100mM 내지 250mM의 NaCl 농도에서 지속형 인간 성장호르몬을 고순도로 분리해냈으며, 반면, 250mM 내지 350mM의 NaCl 농도에서는 단량체에 비하여 60% 이상의 중합체를 포함하는 지속형 인간 성장호르몬이 분리되었다(도 1 및 2). 또한 이와 같은 방법으로 중합체가 분리된 고순도의 지속형 인간 성장호르몬은 12mg/㎖ 이상의 고농도로 농축하여도 중합체가 1% 이상 증가되지 않음을 확인하였다. According to one embodiment of the present invention, a culture medium containing the sustained human growth hormone NexP-hGH protein was obtained from transformed CHO cells, diafiltered, and then anion exchange resin chromatography and hydrophobic chromatography were sequentially Purification was carried out. It was then diafiltered again and purified by affinity chromatography using a resin with anti-alpha1-antitrypsin antibody fragment attached. An anion chromatography column (BPG column, 7 cm in diameter) using a Q-Sepharose resin was used to prepare a sample containing monomers of the sustained human growth hormone NexP-hGH protein having a purity of about 90% on SE-HPLC. After injecting at 40 mL / min at the flow rate, the 20 mM Tris buffer solution (pH 7.4) and 20 mM sodium phosphate elution buffer solution (pH 7.4) were sequentially flowed in 3 column volumes at a flow rate of 40 mL / min to remove impurities. Washed. Then, 20 mM sodium phosphate eluting solvent containing 0.4 M NaCl (pH 7.4) was used to elute the continuous human growth hormone by flowing a continuous salt concentration gradient by 10 column capacity at a flow rate of 40 ml / min. As a result, the sustained human growth hormone was isolated with high purity at a NaCl concentration of 100 mM to 250 mM, while the sustained human growth hormone containing 60% or more polymers was separated at the NaCl concentration of 250 mM to 350 mM ( 1 and 2). In addition, it was confirmed that the high-purity sustained human growth hormone from which the polymer was separated in this manner did not increase the polymer by more than 1% even when concentrated at a high concentration of 12 mg / ml or more.
따라서, 본 발명은 분리가 용이하지 않은 중합체 형태의 지속형 인간 성장호르몬 NexP-hGH 단백질을 효율적으로 제거하여 단량체 형태의 지속형 인간 성장호르몬 NexP-hGH 단백질만을 고순도로 분리할 수 있는 방법으로, 바람직하게는 95 내지 100%의 순도로 NexP-hGH를 분리하여 제조하는데 이용할 수 있다.Therefore, the present invention is a method capable of efficiently separating only long-acting human growth hormone NexP-hGH protein in monomer form in high purity by efficiently removing the easy-to-separate polymer form of long-acting human growth hormone NexP-hGH. Preferably, it can be used to separately prepare NexP-hGH with a purity of 95 to 100%.
또 다른 양태로서, 본 발명은 상기 방법으로 제조된 인간 성장호르몬(hGH) 및 알파-1 안티트립신 변이체(NexP)가 융합된 지속형 인간 성장호르몬인 NexP-hGH 단백질의 단량체를 제공한다.In another embodiment, the present invention provides a monomer of NexP-hGH protein, which is a sustained human growth hormone fused to human growth hormone (hGH) and alpha-1 antitrypsin variant (NexP) prepared by the above method.
상기 인간 성장호르몬, 알파-1 안티트립신 변이체 및 NexP-hGH에 대해서는 상기에서 설명한 바와 같다. The human growth hormone, alpha-1 antitrypsin variant and NexP-hGH are as described above.
이하, 본 발명을 실시예를 통하여 보다 상세하게 설명한다. 그러나 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.
실시예 1: 지속형 인간 성장호르몬 NexP-hGH의 제조Example 1 Preparation of Sustained Human Growth Hormone NexP-hGH
<1-1> 인간 성장호르몬/알파-1 안티트립신 변이융합체를 발현하는 벡터의 제조[hGH/A1 AT(Q9N,P357N)]<1-1> Preparation of a vector expressing a human growth hormone / alpha-1 antitrypsin mutant [hGH / A1 AT (Q9N, P357N)]
인간 성장호르몬을 알파-1 안티트립신 변이융합체의 N 말단에 융합시킨 단백질을 발현하는 벡터를 제조하기 위하여 우선, 부위 특이적 돌연변이(site directed mutagenesis)를 통하여 357번 프롤린(Proline, P)을 아스파라진(Asparagine, N)으로 치환시킨 알파-1 안티트립신 변이체(A1AT(P357N))를 제조하였다. 그 다음, 이를 Xho1과 BamH1 제한효소 부위를 가지는 pG001 유래 PCR product hGH 및 벡터 pAV1과 클로닝하여 pT107을 제조하였다. pT107은 hGH와 A1AT가 링커 없이 결합되어 있는 P357N 변이체이다. 당화 증가를 위하여 이 클론의 A1AT(P357N)를 9번 글루타민을 아스파라진으로 점 돌연변이(point mutation)시켜서 이중 변이체(A1AT(Q9N, P357N))을 만들고 DHFR 유전자 및 Kozac 서열 등을 추가 삽입하여 벡터를 제조하였다. 상기 벡터에서 발현되는 인간 성장호르몬/알파-1 안티트립신 변이융합체를 107N으로 명명하였다.To prepare a vector expressing a protein in which human growth hormone was fused to the N-terminus of an alpha-1 antitrypsin mutant, proline (P) 357 was first reacted with site directed mutagenesis. An alpha-1 antitrypsin variant (A1AT (P357N)) substituted with (Asparagine, N) was prepared. Then, it was cloned with pG001-derived PCR product hGH and vector pAV1 having Xho1 and BamH1 restriction enzyme sites to prepare pT107. pT107 is a P357N variant wherein hGH and A1AT are bound without a linker. To increase glycosylation, point clones of the clone's A1AT (P357N) and Glutamine No. 9 with asparagine were used to create double variants (A1AT (Q9N, P357N)), and the vector was inserted by additional DHFR gene and Kozac sequence. Prepared. Human growth hormone / alpha-1 antitrypsin mutant expressed in the vector was named 107N.
<1-2> 지속형 인간성장호르몬 NexP-hGH인 인간 성장호르몬/알파-1 안티트립신 변이융합체 107N의 발현<1-2> Expression of Human Growth Hormone / alpha-1 Antitrypsin Mutant 107N, Sustained Human Growth Hormone NexP-hGH
상기 <1-1>의 벡터로 만들어진 클론을 차이니즈 햄스터 난소세포(CHO)에 형질도입시키고 인간 성장호르몬/알파-1 안티트립신 변이융합체(107N)의 발현을 확인하였다. 인간 성장호르몬/알파-1 안티트립신 변이융합체(107N) 발현 세포는 8% CO2및 37℃ 온도 조건에서, CD Opti-CHO 배지에서 배양하였고, 0.5x106 cells/㎖ 농도로 접종하여 현탁 배양하였다.Clones made of the vector of <1-1> were transduced into Chinese hamster ovary cells (CHO) and the expression of human growth hormone / alpha-1 antitrypsin mutant (107N) was confirmed. Human growth hormone / alpha-1 antitrypsin fusion (107N) expressing cells were cultured in CD Opti-CHO medium at 8% CO 2 and 37 ° C. temperature, and incubated in suspension at 0.5 × 10 6 cells / ml concentration. .
실시예 2: 음이온 교환 수지, 소수성 수지 크로마토그래피 및 안티 알파1-안티트립신 항체 단편이 부착된 수지를 이용한 지속형 인간 성장호르몬 NexP-hGH의 정제Example 2 Purification of Sustained Human Growth Hormone NexP-hGH Using Anion Exchange Resin, Hydrophobic Resin Chromatography and Resin Attached with Anti-alpha-Antitrypsin Antibody Fragment
상기에서 형질 전환된 CHO 세포로부터 제조한 지속형 인간 성장호르몬 NexP-hGH을 발현하여 얻은 배양액 약 2ℓ를 20mM 소듐 포스페이트 완충용액(pH 8.0)을 이용하여 한외 여과 시스템(분자량 컷 오프 30,000)을 이용하여 정용여과 (diafiltration)하였다. About 2 L of the culture medium obtained by expressing the sustained human growth hormone NexP-hGH prepared from the transformed CHO cells was subjected to an ultrafiltration system (molecular weight cutoff 30,000) using 20 mM sodium phosphate buffer (pH 8.0). Diafiltration was performed.
약 250㎖ Q-sepharose(GE Healthcare사) 수지를 XK-50컬럼(GE Healthcare사)에 충진 한 다음, 20mM 소듐 포스페이트 완충용액(pH 8.0)을 충분히 흘려 컬럼을 평형화시켰다. 준비된 Q-sepharose 컬럼에 상기 정용여과액 약 1ℓ를 20㎖/min의 유속으로 흘린 후, 다시 20mM 소듐 포스페이트 완충용액(pH 8.0)을 약 3CV(column volume)흘려 컬럼을 세척하였다. 그 다음, 100mM NaCl이 포함된 20mM 소듐 포스페이트 완충용액(pH 8.0)을 20㎖/min의 유속으로 약 3CV정도 흘려 불순 단백질들을 제거한 후, 180mM NaCl이 포함된 20mM 소듐 포스페이트 완충용액(pH 8.0)을 20㎖/min의 유속으로 약 3CV정도 흘려 지속형 인간 성장호르몬 NexP-hGH가 포함된 용액을 회수하였다.About 250 mL Q-sepharose (GE Healthcare) resin was charged into an XK-50 column (GE Healthcare), and then the column was equilibrated with sufficient flow of 20 mM sodium phosphate buffer (pH 8.0). The prepared Q-sepharose column was flowed about 1 L of the diafiltration solution at a flow rate of 20 mL / min, and then washed again with 20 mM sodium phosphate buffer (pH 8.0) by about 3 CV (column volume). Then, 20 mM sodium phosphate buffer solution containing 100 mM NaCl (pH 8.0) was flowed at about 3 CV at a flow rate of 20 ml / min to remove impurities, and then 20 mM sodium phosphate buffer solution (pH 8.0) containing 180 mM NaCl was removed. The solution containing the sustained human growth hormone NexP-hGH was recovered by flowing about 3 CV at a flow rate of 20 ml / min.
Q-sepharose 컬럼 용출액에 4M NaCl/20mM 소듐 포스페이트 완충용액(pH 8.0)을 첨가하여 약 2.5M NaCl/20mM 소듐 포스페이트(pH 8.0)가 되도록 하여 페닐-세파로즈(Phenyl-sepharose) 로딩액을 준비하였다.A phenyl-sepharose loading solution was prepared by adding 4M NaCl / 20 mM sodium phosphate buffer (pH 8.0) to the Q-sepharose column eluate to give about 2.5 M NaCl / 20 mM sodium phosphate (pH 8.0). .
약 250㎖ 페닐-세파로즈(GE Healthcare사) 수지를 XK-50컬럼(GE Healthcare사)에 충진한 다음, 2.5M NaCl이 포함된 20mM 소듐 포스페이트 완충용액(pH 7.5)을 충분히 흘려 컬럼을 평형화시켰다. 준비된 페닐-세파로즈 컬럼에 상기 페닐-세파로즈 로딩액 약 1.2ℓ를 20㎖/min의 유속으로 흘린 후, 다시 2.5M NaCl이 포함된 20mM 소듐 포스페이트 완충용액(pH 8.0)을 약 3CV(column volume)흘려 컬럼을 세척하였다.Approximately 250 ml phenyl-sepharose (GE Healthcare) resin was charged to an XK-50 column (GE Healthcare), and then the column was equilibrated with sufficient flow of 20 mM sodium phosphate buffer (pH 7.5) containing 2.5 M NaCl. . About 1.2 liters of the phenyl-sepharose loading solution was flowed into the prepared phenyl-sepharose column at a flow rate of 20 ml / min, and then 20 mM sodium phosphate buffer solution (pH 8.0) containing 2.5 M NaCl was added at about 3 CV (column volume). Wash the column.
20mM 소듐 포스페이트 완충용액(pH 8.0)을 20㎖/min의 유속으로 약 4CV정도 흘려 지속형 인간 성장호르몬 NexP-hGH가 포함된 용액을 회수하였다.A solution containing the sustained human growth hormone NexP-hGH was recovered by flowing 20 mM sodium phosphate buffer (pH 8.0) at a flow rate of 20 ml / min at about 4 CV.
그 다음, 상기에서 얻은 지속형 인간 성장호르몬 NexP-hGH가 포함된 용액 약 1ℓ를 150mM NaCl이 포함된 트리스(Tris) 완충용액(pH 7.4)을 이용하여 한외 여과 시스템(분자량 컷 오프 30,000)으로 정용여과(diafiltration)하였다.Then, about 1 L of the solution containing the sustained human growth hormone NexP-hGH obtained above was defined using an ultrafiltration system (molecular weight cutoff 30,000) using Tris buffer solution (pH 7.4) containing 150 mM NaCl. Diafiltration was performed.
약 250㎖ 안티 알파1-안티트립신(A1AT) 항체 단편이 부착된 수지(GE Healthcare사, 이하 'AIAT'로 명명)를 XK-50컬럼(GE Healthcare사)에 충진한 다음, 150mM NaCl이 포함된 트리스(Tris) 완충용액(pH 7.4)을 충분히 흘려 컬럼을 평형화시켰다. 준비된 A1AT 컬럼에 상기 정용여과액 약 1ℓ를 20㎖/min의 유속으로 흘린 후, 다시 150mM NaCl이 포함된 트리스(Tris) 완충용액(pH 7.4)을 약 3CV(column volume)흘려 컬럼을 세척하였다. 이후 50mM MgCl2가 포함된 트리스(Tris) 완충용액 (pH 7.4)으로 컬럼을 세척한 후, 180mM MgCl2가 포함된 트리스(Tris) 완충용액(pH 7.4)을 흘려 지속형 인간 성장호르몬 NexP-hGH가 포함된 분획을 용출하였다.Resin (GE Healthcare, hereinafter referred to as 'AIAT') to which about 250 ml anti alpha1-antitrypsin (A1AT) antibody fragment was attached was filled in an XK-50 column (GE Healthcare), followed by 150 mM NaCl. The column was equilibrated with sufficient flow of Tris buffer (pH 7.4). After about 1 L of the diafiltration solution was flowed to the prepared A1AT column at a flow rate of 20 mL / min, the column was washed again by adding Tris buffer solution (pH 7.4) containing 150 mM NaCl (pH 7.4) to about 3 CV (column volume). Thereafter, the column was washed with Tris buffer solution containing 50 mM MgCl 2 (pH 7.4), and then tris buffer solution (pH 7.4) containing 180 mM MgCl 2 was flowed to sustain human growth hormone NexP-hGH. Fractions containing were eluted.
상기의 방법으로 정제한 지속형 인간 성장호르몬 NexP-hGH은 단량체 대비 중합체의 비율이 10% 이상 존재하였다.Sustained human growth hormone NexP-hGH purified by the above method was present in more than 10% of the polymer to monomer ratio.
실시예 3: 음이온 교환 수지 크로마토그래피를 이용한 지속형 인간 성장호르몬 NexP-hGH 중합체와 단량체의 분리 및 정제Example 3: Isolation and Purification of Sustained Human Growth Hormone NexP-hGH Polymer and Monomer by Anion Exchange Resin Chromatography
상기 실시예 2의 정제된 지속형 인간 성장호르몬 NexP-hGH는 단량체 대비 중합체의 비율이 10% 이상으로, 단량체 및 중합체가 모두 존재하는 혼합물로부터 단량체만을 특이적으로 분리정제할 필요성이 있다. 이에 본 발명자들은 하기와 같은 방법으로 중합체 및 단량체의 혼합액으로부터 단량체만을 분리정제하였다.The purified long-acting human growth hormone NexP-hGH of Example 2 has a ratio of 10% or more of the polymer to the monomer, and it is necessary to specifically separate and purify only the monomer from the mixture in which both the monomer and the polymer are present. The present inventors separated and purified only the monomer from the mixture of the polymer and monomer in the following manner.
상기 실시예 2에서 용출한 NexP-hGH를 Q컬럼 평형 완충용액으로 7배 희석한 다음, Q-세파로즈 수지가 충진된 BPG 컬럼(직경 7cm)에 40㎖/min의 유속으로 부하하여 결합시켰다. 그 다음, 20mM 소듐 포스페이트 완충용액(pH 7.4)을 40㎖/min의 유속으로 3 컬럼 용량으로 사용하여 컬럼에 붙지 못한 불순물을 세척하였다. 지속형 인간 성장 호르몬 NexP-hGH 단량체를 고순도로 분리정제하기 위하여, 0.4M NaCl이 포함된 20mM 소듐 포스페이트 용출 용매(pH 7.4)를 40㎖/min 유속으로 10 컬럼 용량만큼 연속식 염 농도 구배로 흘려 NexP-hGH를 용출시켰다. NexP-hGH eluted in Example 2 was diluted 7-fold with Q column equilibration buffer, and loaded onto a BPG column (7 cm in diameter) filled with Q-Sepharose resin at a flow rate of 40 ml / min. Next, 20 mM sodium phosphate buffer (pH 7.4) was used in a three column capacity at a flow rate of 40 ml / min to wash off impurities that did not adhere to the column. In order to isolate and purify the sustained human growth hormone NexP-hGH monomer with high purity, 20 mM sodium phosphate eluting solvent (pH 7.4) containing 0.4 M NaCl was flowed in a continuous salt concentration gradient by 10 column capacity at a flow rate of 40 ml / min. NexP-hGH was eluted.
그 결과, 약 100mM 내지 250 mM의 염 농도에서 단량체가 약 98% 이상인 지속형 인간 성장호르몬이 분리되며, 250mM 내지 350mM의 염 농도에서 단량체 대비 60% 이상의 중합체를 포함하는 지속형 인간 성장호르몬이 분리되는 것을 확인하였다(도 1 내지 2). 또한, 중합체가 분리된 고순도의 지속형 인간 성장호르몬의 경우, 12mg/mL 이상의 고농도로 농축하여도 중합체가 1% 이상 증가되지 않음을 확인하였다.As a result, a sustained human growth hormone having a monomer concentration of about 98% or more is isolated at a salt concentration of about 100 mM to 250 mM, and a sustained human growth hormone comprising at least 60% of a polymer compared to the monomer at a salt concentration of 250 mM to 350 mM is isolated. It confirmed that it becomes (FIGS. 1-2). In addition, in the case of high-purity sustained human growth hormone in which the polymer was separated, it was confirmed that the polymer was not increased by more than 1% even when concentrated at a high concentration of 12 mg / mL or more.
상기와 같은 결과는 음이온 교환 수지 크로마토그래피를 이용한 본 발명의 방법이 단량체 형태의 NexP-hGH 단백질을 고순도로 분리하는데 매우 유용하게 사용될 수 있음을 나타내는 것으로, 특히 100mM 내지 250mM의 염 농도에서 단량체 형태의 NexP-hGH 단백질을 고순도로 분리할 수 있음을 나타내는 결과이다. The above results indicate that the method of the present invention using anion exchange resin chromatography can be very useful for high purity separation of monomeric NexP-hGH protein, especially at the salt concentration of 100 mM to 250 mM. The results show that the NexP-hGH protein can be separated with high purity.
이상의 설명으로부터, 본 발명이 속하는 기술 분야의 당업자는 본 발명이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 실시 예들은 모든 면에서 예시적인 것이며 한정적인 것이 아닌 것으로서 이해해야만 한다. 본 발명의 범위는 상기 상세한 설명보다는 후술하는 특허 청구범위의 의미 및 범위 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, the embodiments described above are to be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.
Claims (11)
- 음이온 교환 수지 크로마토그래피를 이용하여 인간 성장호르몬(hGH) 및 알파-1 안티트립신 변이체(NexP)가 융합된 지속형 인간 성장호르몬인 NexP-hGH 단백질의 단량체를 제조하는 방법.A method for preparing monomers of NexP-hGH protein, which is a sustained human growth hormone fused with human growth hormone (hGH) and alpha-1 antitrypsin variant (NexP) using anion exchange resin chromatography.
- 제1항에 있어서, 상기 방법은The method of claim 1 wherein the method isa) 전-평형화(pre-equilibration)된 음이온 교환 수지 크로마토그래피(anion exchange chromatography) 컬럼에 지속형 인간 성장호르몬 NexP-hGH 단백질의 혼합액을 포함하는 시료를 주입하는 단계; 및a) injecting a sample comprising a mixture of the sustained human growth hormone NexP-hGH protein into a pre-equilibration anion exchange chromatography column; Andb) pH 6.0 내지 9.0인 용출 완충용액을 이용하여 0M 내지 0.4M의 염 농도 구배에서 지속형 인간 성장호르몬인 NexP-hGH 단백질의 단량체를 용출시키는 단계를 포함하는 것인 방법.b) eluting the monomer of NexP-hGH protein, a sustained human growth hormone, at a salt concentration gradient of 0M to 0.4M using an elution buffer at pH 6.0-9.0.
- 제2항에 있어서, 상기 a) 단계의 지속형 인간 성장 호르몬 NexP-hGH 단백질의 혼합액을 포함하는 시료는 컬럼 주입 전에 여과 단계 없이, pH 6.0 내지 9.0인 완충용액으로 희석한 최종 염 농도가 0 내지 50mM 염화나트륨(NaCl) 또는 0 내지 50mM 염화마그네슘(MgCl2)인 시료인 것인 방법.According to claim 2, wherein the sample containing a mixture of the sustained human growth hormone NexP-hGH protein of step a) is a final salt concentration of 0 to 9.0 diluted with a buffer solution of pH 6.0 to 9.0 without a filtration step before column injection 50 mM sodium chloride (NaCl) or 0-50 mM magnesium chloride (MgCl 2 ).
- 제2항에 있어서, 상기 a) 단계의 지속형 인간 성장호르몬 NexP-hGH 단백질의 혼합액을 포함하는 시료는 주입 전에 음이온 교환 수지 크로마토그래피, 소수성 크로마토그래피 및 안티 알파-1 안티트립신 항체 단편이 부착된 수지로 충진된 친화성 크로마토그래피를 순차적으로 수행하여 부분정제(partially purified)한 것인 방법.According to claim 2, wherein the sample comprising a mixture of the sustained human growth hormone NexP-hGH protein of step a) is attached to the anion exchange resin chromatography, hydrophobic chromatography and anti alpha-1 antitrypsin antibody fragment prior to injection Affinity chromatography filled with a resin is carried out sequentially to partially purified (partially purified).
- 제2항에 있어서, 상기 사용되는 완충 용액은 인산나트륨(sodium phosphate) 완충용액, 인산칼륨(potassium phosphate) 완충용액 또는 트리스(Tris) 완충용액인 것인 방법.The method of claim 2, wherein the buffer solution used is sodium phosphate buffer, potassium phosphate buffer or Tris buffer.
- 제2항에 있어서, 상기 지속형 인간 성장호르몬 NexP-hGH 단백질은 동물세포에서 생산된 단백질인 방법.The method of claim 2, wherein the sustained human growth hormone NexP-hGH protein is a protein produced in an animal cell.
- 제2항에 있어서, 상기 b) 단계의 염 농도구배는 연속적 염 농도구배(continuous salt gradient)인 것인 방법.The method of claim 2, wherein the salt concentration gradient of step b) is a continuous salt gradient.
- 제2항에 있어서, 상기 b) 단계의 염은 염화나트륨(NaCl)인 것인 방법.The method of claim 2, wherein the salt of step b) is sodium chloride (NaCl).
- 제2항에 있어서, 상기 b) 단계의 염 농도 구배는 염화나트륨(NaCl) 100mM 내지 250mM의 염 농도 구배인 것인 방법.The method of claim 2, wherein the salt concentration gradient of step b) is a salt concentration gradient of 100 mM to 250 mM sodium chloride (NaCl).
- 제2항에 있어서, 상기 음이온 교환 수지의 작용기가 Q(Quaternary amine), DEAE(DiEthylAminoEthyl) 및 QAE(Quaternary Amino Ethyl)로 이루어진 군으로부터 선택된 것인 방법.The method of claim 2, wherein the functional group of the anion exchange resin is selected from the group consisting of quaternary amine (Q), diethynaminoethyl (DEAE), and quaternary amino ethynyl (QAE).
- 제2항에 있어서, 상기 방법으로 용출된 지속형 인간 성장호르몬 NexP-hGH 단백질은 단량체가 95% 내지 100%의 순도로 존재하는 것인 방법.The method of claim 2, wherein the sustained human growth hormone NexP-hGH protein eluted by the method is monomeric is present in a purity of 95% to 100%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201380060578.XA CN104812776A (en) | 2012-11-21 | 2013-11-21 | Method for preparing novel long-acting human growth hormone monomer |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR10-2012-0132549 | 2012-11-21 | ||
| KR1020120132549A KR101516054B1 (en) | 2012-11-21 | 2012-11-21 | A novel method for preparing long-acting human growth hormone monomers |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2014081225A1 true WO2014081225A1 (en) | 2014-05-30 |
Family
ID=50776329
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/KR2013/010630 WO2014081225A1 (en) | 2012-11-21 | 2013-11-21 | Method for preparing novel long-acting human growth hormone monomer |
Country Status (3)
| Country | Link |
|---|---|
| KR (1) | KR101516054B1 (en) |
| CN (1) | CN104812776A (en) |
| WO (1) | WO2014081225A1 (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999062936A1 (en) * | 1998-06-01 | 1999-12-09 | Genentech, Inc. | Separation of protein monomers from aggregates by use of ion-exchange chromatography |
| US20080058507A1 (en) * | 2004-02-11 | 2008-03-06 | Hui Liu | Method For The Removal Of Aggregate Proteins From Recombinant Samples Using Ion Exchange Chromatography |
| KR20120013359A (en) * | 2009-04-01 | 2012-02-14 | 바이오제너릭스 에이지 | Method for purifying recombinant fsh |
| KR101183262B1 (en) * | 2009-04-22 | 2012-09-14 | (주)알테오젠 | Fusion protein or fusion peptide with increased half life by keeping long-acting activity, and method for increasing half life using the same |
-
2012
- 2012-11-21 KR KR1020120132549A patent/KR101516054B1/en not_active IP Right Cessation
-
2013
- 2013-11-21 WO PCT/KR2013/010630 patent/WO2014081225A1/en active Application Filing
- 2013-11-21 CN CN201380060578.XA patent/CN104812776A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999062936A1 (en) * | 1998-06-01 | 1999-12-09 | Genentech, Inc. | Separation of protein monomers from aggregates by use of ion-exchange chromatography |
| US20080058507A1 (en) * | 2004-02-11 | 2008-03-06 | Hui Liu | Method For The Removal Of Aggregate Proteins From Recombinant Samples Using Ion Exchange Chromatography |
| KR20120013359A (en) * | 2009-04-01 | 2012-02-14 | 바이오제너릭스 에이지 | Method for purifying recombinant fsh |
| KR101183262B1 (en) * | 2009-04-22 | 2012-09-14 | (주)알테오젠 | Fusion protein or fusion peptide with increased half life by keeping long-acting activity, and method for increasing half life using the same |
Non-Patent Citations (2)
| Title |
|---|
| CHO, E. K. ET AL.: "ATP-independent thermoprotective activity of Nicotian a tabacum heat shock protein 70 in Escherichia coli.", J. BIOCHEM. MOL. BIOL., vol. 40, no. 1, 31 January 2007 (2007-01-31), pages 107 - 112 * |
| ZHANG, C. ET AL.: "Genetic engineering strategies for purification of recombinant proteins from canola by anion exchange chromatography : an example of beta-glucuronidase.", BIOTECHNOL. PROG., vol. 17, no. 1, 2 January 2001 (2001-01-02), pages 161 - 167 * |
Also Published As
| Publication number | Publication date |
|---|---|
| KR20140065780A (en) | 2014-05-30 |
| KR101516054B1 (en) | 2015-05-06 |
| CN104812776A (en) | 2015-07-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11471513B2 (en) | Highly glycosylated human blood-clotting factor VIII fusion protein, and manufacturing method and application of same | |
| US5525472A (en) | Method for production and purification or recombinant Apolipoprotein E from bacteria | |
| JP2019014741A (en) | Purification of erythropoietin | |
| PT1394179E (en) | Process for producing erythropoietin free from animal proteins | |
| NZ295810A (en) | Method of purifying factor viii using hydrophobic interaction chromatography gel with at least one surfactant in the chromatography solution | |
| JPS62223197A (en) | Purification of alpha-1-anti-protease | |
| KR930008093B1 (en) | New polypeptide and its composition | |
| WO2013025079A1 (en) | Method for preparing active form of tnfr-fc fusion protein | |
| EP2138505B1 (en) | Method for producing soluble thrombomodulin of high purity | |
| WO2013183948A1 (en) | Highly glycosylated long-acting human growth hormone protein and production method for same | |
| JP2009502117A (en) | Recombinant method for erythropoiesis stimulating protein production | |
| WO1985003079A1 (en) | HUMAN ERYTHROPOIETIN cDNA CLONES | |
| KR101460266B1 (en) | A novel method for purifying long-acting human growth hormone | |
| CN111499764B (en) | Long-acting fusion protein with erythropoietin activity | |
| WO2015080509A1 (en) | Method for purifying darbepoetin alfa | |
| WO2014081225A1 (en) | Method for preparing novel long-acting human growth hormone monomer | |
| Wojczyk et al. | Purification of a secreted form of recombinant rabies virus glycoprotein: comparison of two affinity tags | |
| CN104292341B (en) | A kind of eight factor fusion protein of blood coagulation and its preparation method and application | |
| US20040106779A1 (en) | Modified factor IX preparation | |
| US6893844B1 (en) | DNA encoding a new human hepatoma derived growth factor and producing method thereof | |
| KR100254574B1 (en) | A method for purifying blood coagulant factor viii by employing anti-von willebrand factor chimeric antibody | |
| CN117126832B (en) | Recombinant human blood coagulation factor IX fusion protein and preparation method thereof | |
| WO2016108569A1 (en) | Method for producing tnfr-fc fusion protein containing target content of impurities | |
| US20110230645A1 (en) | Purification of factor v | |
| WO2023040792A1 (en) | Preparation method for erythropoietin |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 13857586 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 13857586 Country of ref document: EP Kind code of ref document: A1 |