CN114276432A - Purification method of recombinant human insulin C peptide - Google Patents
Purification method of recombinant human insulin C peptide Download PDFInfo
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- 108010075254 C-Peptide Proteins 0.000 title claims abstract description 61
- 101000976075 Homo sapiens Insulin Proteins 0.000 title claims abstract description 43
- PBGKTOXHQIOBKM-FHFVDXKLSA-N insulin (human) Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3NC=NC=3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)[C@@H](C)CC)[C@@H](C)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C1=CN=CN1 PBGKTOXHQIOBKM-FHFVDXKLSA-N 0.000 title claims abstract description 43
- 238000000034 method Methods 0.000 title claims abstract description 41
- 238000000746 purification Methods 0.000 title abstract description 11
- 238000001042 affinity chromatography Methods 0.000 claims abstract description 15
- 229910021645 metal ion Inorganic materials 0.000 claims abstract description 15
- 238000004366 reverse phase liquid chromatography Methods 0.000 claims abstract description 15
- 238000001976 enzyme digestion Methods 0.000 claims abstract description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 70
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims description 63
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical group CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 54
- 239000003480 eluent Substances 0.000 claims description 46
- 239000011780 sodium chloride Substances 0.000 claims description 35
- 239000000243 solution Substances 0.000 claims description 33
- 239000007983 Tris buffer Substances 0.000 claims description 32
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 32
- 239000007788 liquid Substances 0.000 claims description 29
- 238000010828 elution Methods 0.000 claims description 28
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 20
- 239000007853 buffer solution Substances 0.000 claims description 18
- 238000011068 loading method Methods 0.000 claims description 18
- 238000005406 washing Methods 0.000 claims description 17
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 15
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical group N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 15
- 239000005695 Ammonium acetate Substances 0.000 claims description 15
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- 229940043376 ammonium acetate Drugs 0.000 claims description 15
- 235000019257 ammonium acetate Nutrition 0.000 claims description 15
- 229940069078 citric acid / sodium citrate Drugs 0.000 claims description 15
- 235000019270 ammonium chloride Nutrition 0.000 claims description 10
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 8
- 239000003960 organic solvent Substances 0.000 claims description 8
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 6
- 239000000872 buffer Substances 0.000 claims description 6
- 150000003839 salts Chemical class 0.000 claims description 5
- 238000012856 packing Methods 0.000 claims description 4
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 claims description 2
- VEQPNABPJHWNSG-UHFFFAOYSA-N Nickel(2+) Chemical compound [Ni+2] VEQPNABPJHWNSG-UHFFFAOYSA-N 0.000 claims description 2
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 claims description 2
- 229910001429 cobalt ion Inorganic materials 0.000 claims description 2
- XLJKHNWPARRRJB-UHFFFAOYSA-N cobalt(2+) Chemical compound [Co+2] XLJKHNWPARRRJB-UHFFFAOYSA-N 0.000 claims description 2
- 229910001431 copper ion Inorganic materials 0.000 claims description 2
- 229910001453 nickel ion Inorganic materials 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims 1
- VOUAQYXWVJDEQY-QENPJCQMSA-N 33017-11-7 Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)NCC(=O)NCC(=O)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N1[C@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O)CCC1 VOUAQYXWVJDEQY-QENPJCQMSA-N 0.000 abstract description 22
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- 239000012071 phase Substances 0.000 description 73
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 20
- 239000008213 purified water Substances 0.000 description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- 238000004128 high performance liquid chromatography Methods 0.000 description 10
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 10
- 229910052759 nickel Inorganic materials 0.000 description 10
- 238000004440 column chromatography Methods 0.000 description 9
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 229910052725 zinc Inorganic materials 0.000 description 6
- 239000011701 zinc Substances 0.000 description 6
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 5
- 108090001061 Insulin Proteins 0.000 description 5
- 102000004877 Insulin Human genes 0.000 description 5
- 229910017052 cobalt Inorganic materials 0.000 description 5
- 239000010941 cobalt Substances 0.000 description 5
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 5
- 229910052802 copper Inorganic materials 0.000 description 5
- 239000010949 copper Substances 0.000 description 5
- 230000003247 decreasing effect Effects 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 230000036541 health Effects 0.000 description 5
- 229940125396 insulin Drugs 0.000 description 5
- 229920002684 Sepharose Polymers 0.000 description 4
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000010353 genetic engineering Methods 0.000 description 3
- 239000001632 sodium acetate Substances 0.000 description 3
- 235000017281 sodium acetate Nutrition 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 238000000108 ultra-filtration Methods 0.000 description 3
- 208000007342 Diabetic Nephropathies Diseases 0.000 description 2
- 206010054044 Diabetic microangiopathy Diseases 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 241000235058 Komagataella pastoris Species 0.000 description 2
- 108010076181 Proinsulin Proteins 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 201000009101 diabetic angiopathy Diseases 0.000 description 2
- 208000033679 diabetic kidney disease Diseases 0.000 description 2
- LGQLOGILCSXPEA-UHFFFAOYSA-L nickel sulfate Chemical compound [Ni+2].[O-]S([O-])(=O)=O LGQLOGILCSXPEA-UHFFFAOYSA-L 0.000 description 2
- 229910000363 nickel(II) sulfate Inorganic materials 0.000 description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
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- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
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- 229940044175 cobalt sulfate Drugs 0.000 description 1
- KTVIXTQDYHMGHF-UHFFFAOYSA-L cobalt(2+) sulfate Chemical compound [Co+2].[O-]S([O-])(=O)=O KTVIXTQDYHMGHF-UHFFFAOYSA-L 0.000 description 1
- 229910000365 copper sulfate Inorganic materials 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
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- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a purification method of recombinant human insulin C peptide. The purification method comprises the step of carrying out metal ion affinity chromatography and reverse phase chromatography on the enzyme digestion solution containing the recombinant human insulin C peptide to obtain the high-purity recombinant human insulin C peptide. The purification method of the insulin C peptide has simple process, the purity of the obtained insulin C peptide reaches more than 99 percent, the requirement of clinical medication is met, and the insulin C peptide can also be used as a standard substance of the recombinant human insulin C peptide and used for researching related substances of the recombinant human insulin.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a purification method of recombinant human insulin C peptide.
Background
Insulin is a protein hormone involved in blood glucose regulation. Insulin is synthesized in the liver in the form of precursor proinsulin, which is formed by the connection of the A and B chains of insulin via the C peptide. Insulin itself contains only B and A chains. The human proinsulin C peptide is a component of proinsulin, is secreted by islet beta cells, contains 31 amino acids, has a human proinsulin C peptide level of about 1ng/ml in normal human fasting plasma, rapidly rises after eating, reaches a peak after 1 hour, and is about 8 times of the fasting value.
Research has shown that human proinsulin C peptide can be used for treating diabetic microangiopathy such as diabetic nephropathy, which can reduce glomerular extracellular matrix accumulation to cause mesangial expansion, and has therapeutic effect on diabetic microangiopathy such as diabetic nephropathy under the condition of insulin for controlling carbohydrate metabolism. Human studies show that: the proinsulin C peptide can be infused into patients with type I diabetes to reduce glomerular filtration rate in early stage of diabetes, remarkably reduce urinary protein excretion rate, improve peripheral neuropathy and increase utilization of skeletal muscle to diabetes. Human proinsulin C peptide is an endogenous substance, and the use safety is greatly improved. Therefore, human proinsulin C peptide is a safe and effective product.
At present, two methods of chemical synthesis and genetic engineering are available for obtaining proinsulin C peptide. Chemical synthesis methods (e.g. stepwise addition of amino acids to a solid phase) are well established and, although automated, are time consuming and, more importantly, expensive to synthesize and are not economical and reliable due to the limited maximum peptide length that can be synthesized. The chemical synthesis method has been gradually replaced by genetic engineering.
The expression system of genetic engineering mainly comprises two types of pichia pastoris and escherichia coli, Per Jonasson and the like adopt the escherichia coli to express the C peptide, and the method comprises the steps of firstly obtaining multiple copies of C peptide fusion protein by a tandem method, and then carrying out enzyme digestion and purification. The report concatenated 7 copies of C peptide, the expression product was present in the cytoplasm, and the final yield of protein was 400 mg/L. Patent document 01112929.8 discloses that 8 copies of C peptide are secreted and expressed in tandem by Pichia pastoris, and the protein expression level reaches 1 g/L.
The reports of the prior art for purifying the insulin C peptide are less, and a patent CN1854299A discloses a method for preparing the recombinant human proinsulin C peptide, wherein the enzyme digestion solution is subjected to ion exchange chromatography, hydrophobic chromatography and reverse chromatography to obtain a recombinant human proinsulin C peptide product with the purity of more than 95%. Cold snow, recombinant human insulin and C peptide, Beijing university of chemical industry, 2006, volume 33, No. 5, pages 30-33. A process for separating and purifying the recombinant human insulin and C peptide includes such steps as digesting the target protein, and separating by Sephadex G-25 column to obtain the recombinant human insulin and C peptide. The C peptide sample is detected by an enzyme-linked immunosorbent assay, and the mass concentration of the C peptide is 10ng/ml (3.31 nmol/L). Patent CN111363025A discloses a purification method of recombinant human proinsulin C-peptide tandem protein, enzyme digestion solution is subjected to reverse-phase ultrafiltration and normal-phase ultrafiltration in turn to obtain ultrafiltrate; purifying the ultrafiltrate by second column chromatography, and performing normal phase ultrafiltration to obtain recombinant human proinsulin C peptide stock solution with purity of 98.7%.
The method has the advantages of complicated steps, small treatment amount and low product purity, is not suitable for large-scale preparation of the recombinant human insulin C peptide, and cannot meet the clinical development requirement. Therefore, it is necessary to provide a purification method of recombinant human insulin C peptide with higher product purity, which is suitable for large-scale production.
Disclosure of Invention
The invention aims to provide a simple method for purifying recombinant human insulin C peptide, which can obtain high-purity recombinant human insulin C peptide only by two-step purification.
The technical scheme of the invention is as follows:
a method for purifying recombinant human insulin C peptide comprises the step of carrying out metal ion affinity chromatography and reverse phase chromatography on enzyme digestion liquid containing the recombinant human insulin C peptide to obtain the high-purity recombinant human insulin C peptide.
A method for purifying recombinant human insulin C peptide comprises the following steps:
(1) loading the enzyme digestion solution containing the recombinant human insulin C peptide into a metal ion affinity chromatography column, eluting by an eluent, and collecting an effluent liquid containing the recombinant human insulin C peptide;
(2) purifying the effluent obtained in the step (1) by using a reverse phase chromatographic column, eluting by using an eluent, and collecting the effluent containing the recombinant human insulin C peptide to obtain the high-purity recombinant human insulin C peptide.
The metal ions are nickel ions, zinc ions, copper ions or cobalt ions.
Preferably, the metal ion affinity chromatography column is formed by supersaturating and adsorbing inorganic salt solution containing metal ions on the affinity chromatography column.
Preferably, the amount of the metal ion affinity chromatography column is 0.5-3g/L, and the preferable amount of the metal ion affinity chromatography column is 1.5-2 g/L; wherein the mass of the sample is calculated by g, and the volume of the chromatographic column packing is calculated by L.
Preferably, the concentration of the upper column liquid of the metal ion affinity chromatography is 1.0-1.6 g/L.
And eluting the metal ions by steps through a mixed solution containing Tris or PB buffer solution, sodium chloride or ammonium chloride and imidazole after affinity chromatography, and collecting an effluent liquid containing the recombinant human insulin C peptide, wherein the PB buffer solution is a phosphate buffer solution.
Preferably, the elution process of the metal ion affinity chromatography is divided into three steps of elution, wherein the elution process comprises the steps of firstly washing 2-3 column volumes by using an eluent A, then washing 3-8 column volumes by using an eluent B, then eluting by using an eluent C, and collecting an effluent containing the recombinant human insulin C peptide.
The eluent A is 10-50mM Tris or PB buffer solution, 0.15-0.5M sodium chloride or ammonium chloride; in a preferred embodiment, the eluent A is 10-50mM Tris buffer, 0.15-0.5M sodium chloride; further preferably, the eluent A is 25mM Tris buffer, 0.3M sodium chloride.
The eluent B is Tris or PB buffer solution with the pH value of 8.0-11.0 and the concentration of 10-50mM, 0.15-0.5M sodium chloride or ammonium chloride and 0.02-0.05M imidazole; in a preferred embodiment, the eluent B is a Tris buffer solution with the pH value of 8.0-11.0 and the concentration of 10-50mM, 0.15-0.5M sodium chloride and 0.02-0.05M imidazole; further preferably, the eluent B is pH9.0-10.0, 25mM Tris buffer, 0.3M sodium chloride, 0.03M imidazole.
The eluent C is Tris or PB buffer solution with the pH value of 8.0-11.0 and the concentration of 10-50mM, sodium chloride or ammonium chloride with the concentration of 0.15-0.5M and imidazole with the concentration of 0.1-0.3M. In a preferred embodiment, the eluent C is Tris buffer with the pH value of 8.0-11.0 and the concentration of 10-50mM, 0.15-0.5M sodium chloride and 0.1-0.3M imidazole; further preferably, the eluent C is pH9.0-10.0, 25mM Tris buffer, 0.3M sodium chloride, 0.1M imidazole.
The reverse phase chromatography packing is C8 or C18.
Preferably, the amount of the reverse phase chromatography column is 0.8-2g/L, preferably 1.4-1.6 g/L; wherein the sample mass is in g and the filler volume is in L.
Preferably, the concentration of the column liquid on the reverse phase chromatography is 1.2-1.6 g/L.
Preferably, in the step (2), the elution effluent containing the recombinant human insulin C peptide collected in the step (1) is diluted by adding purified water, then ethanol with the final concentration of 10-20% is added, and the pH value is adjusted to 2-6 to be used as the column loading liquid of reverse phase chromatography.
Preferably, the reversed phase chromatography is carried out on the column and then eluted by a mobile phase containing organic/inorganic salts, a buffer solution and an organic solvent; the organic/inorganic salt is selected from ammonium acetate or sodium chloride; the buffer solution is selected from citric acid/sodium citrate or PB buffer solution; the organic solvent is selected from ethanol, acetonitrile, methanol or acetone.
Further preferably, the organic/inorganic salt is selected from ammonium acetate, the buffer is selected from citric acid/sodium citrate, and the organic solvent is selected from ethanol.
Preferably, after adsorption of the reversed-phase chromatography column, the mobile phase A is firstly used for carrying out balance, then 70-60% of the mobile phase A and 30-40% of the mobile phase B are used for washing 1-3 column volumes, and then the mobile phases A and B are used for carrying out gradient elution for 8-12 column volumes. Preferably, the mobile phase B of the gradient elution procedure is increased from 30-40% to 60-70%. In a preferred embodiment, the gradient elution mobile phase B is isocratic from 35% to 60%.
Preferably, the mobile phase A is inorganic salt or organic salt with pH value of 3.0-6.0 and 20-80mM, buffer solution with pH value of 10-50mM and organic solvent with concentration of 5-20%. In a preferred embodiment, mobile phase A is pH3.0-6.0, 20-80mM sodium acetate, 10-50mM citric acid/sodium citrate, 5-20% ethanol; further preferably, the mobile phase A is pH 4.0-5.0, 50mM sodium acetate, 25mM citric acid/sodium citrate, 5-20% ethanol. In a preferred embodiment, mobile phase A is pH4.0,50mM sodium acetate, 25mM citric acid/sodium citrate, 10% ethanol.
Preferably, the mobile phase B is inorganic salt or organic salt with the pH value of 3.0-6.0 and the concentration of 20-80mM, buffer solution with the concentration of 10-50mM and organic solvent with the concentration of 40-80%. In a preferred embodiment, mobile phase B is ammonium acetate, pH3.0-6.0, 20-80mM, citric acid/sodium citrate, 10-50mM, 40-80% ethanol; further preferably, the mobile phase B is pH 4.0-5.0, 50mM ammonium acetate, 25mM citric acid/sodium citrate, 50% ethanol.
The purification method of the insulin C peptide has simple process, the purity of the obtained insulin C peptide reaches more than 99 percent, the requirement of clinical medication is met, and the insulin C peptide can also be used as a standard substance of the recombinant human insulin C peptide and used for researching related substances of the recombinant human insulin.
Detailed Description
The invention will now be further illustrated by reference to specific examples, which are intended to be illustrative only and not to limit the scope of the invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. The raw materials, materials and the like used in the following examples are commercially available unless otherwise specified.
The C-peptide-containing enzyme solutions used in the following examples were obtained by the method disclosed in CN 102994600A.
Example 1
(1) Nickel column chromatography
Column liquid feeding: adding 25mM Tris and 0.3M sodium chloride into the enzyme digestion solution containing the C peptide to adjust the pH to 9.0 +/-0.1, wherein the volume of the solution on the column is 1500ml, and the concentration is 1.38 g/L;
② balancing 1 column volume of affinity chromatographic column (GE Health care chemical Sepharose Fast Flow, 1000ml) by 1M nickel sulfate solution, washing 2 column volumes by purified water to obtain nickel column chromatographic column, balancing 2 column volumes by eluent A (25mM Tris buffer solution, 0.3M sodium chloride) at Flow rate of 60 ml/min;
thirdly, the column feeding liquid is fed into the nickel column at the flow speed of 60 ml/min;
fourthly, after the column loading is finished, the eluent A is continuously used for rebalancing 2 column volumes at the flow rate of 60 ml/min;
fifthly, washing 4 column volumes of the nickel column by eluent B (pH9.0, 25mM Tris buffer solution, 0.3M sodium chloride, 0.03M imidazole) at the flow rate of 60 ml/min;
sixthly, eluting the nickel column by eluent C (pH9.0, 25mM Tris buffer solution, 0.3M sodium chloride and 0.1M imidazole) at the flow rate of 60ml/min, collecting about 600ml of effluent containing the recombinant human insulin C peptide, and performing HPLC detection at the concentration of 3.14g/L, the purity of 82.76 percent and the yield of 91.0 percent.
(2) Reverse phase chromatography
Column liquid feeding: adding 600ml of purified water and 10% ethanol to the effluent collected in the step (1), and adjusting the pH to 3.5 +/-0.1 to obtain a concentration of 1.41 g/L;
② C8 reverse phase column (Kromasil 100, 10 μm, 500ml) was equilibrated with mobile phase A (pH4.0,50mM ammonium acetate, 25mM citric acid/sodium citrate, 10% ethanol) at a flow rate of 50ml/min for 2 column volumes;
thirdly, 500ml of column-loading liquid is loaded on the reversed phase column at the flow rate of 50 ml/min;
fourthly, after the column loading is finished, rebalancing 2 column volumes by using the mobile phase A at 50 ml/min;
fifthly, washing 1 column volume of the reverse phase column by 65% mobile phase A + 35% mobile phase B (pH4.0,50mM ammonium acetate, 25mM citric acid/sodium citrate, 50% ethanol) at the flow rate of 50 ml/min;
sixthly, gradient elution is carried out by using a mobile phase A and a mobile phase B, the isocratic degree of the mobile phase B is increased from 35% to 60%, the isocratic degree of the mobile phase A is decreased from 65% to 40%, the elution flow rate is 50ml/min, about 220ml of effluent containing C peptide is collected, the concentration is 1.64g/L, the HPLC purity is 99.52%, and the yield is 51.2%.
Example 2
(1) Cobalt column chromatography
Column liquid feeding: adding 10mM Tris and 0.5M ammonium chloride into the enzyme digestion solution containing the C peptide to adjust the pH to 10.0 +/-0.1, wherein the volume of the solution on the column is 1500ml, and the concentration is 1.20 g/L;
using 1M cobalt sulfate solution to balance 1 column volume of affinity chromatography column (GE Health care chromatography column Flow, 600ml), then using purified water to wash 2 column volumes to obtain cobalt column chromatography column, using eluent A (10mM Tris buffer solution, 0.5M ammonium chloride) to balance 3 column volumes at Flow rate of 60 ml/min;
thirdly, the column feeding liquid is fed into the cobalt column at the flow speed of 60 ml/min;
fourthly, after the column loading is finished, the eluent A is continuously used for rebalancing 3 column volumes at the flow rate of 60 ml/min;
fifthly, washing the cobalt column by 8 column volumes with eluent B (pH10.0, 10mM Tris buffer solution, 0.5M ammonium chloride, 0.02M imidazole) at the flow rate of 60 ml/min;
sixthly, eluting the cobalt column by eluent C (pH10.0, 10mM Tris buffer solution, 0.5M sodium chloride, 0.2M imidazole) at the flow rate of 60ml/min, collecting about 520ml of effluent containing the recombinant human insulin C peptide, and performing HPLC detection at the concentration of 3.19g/L, the purity of 79.74 percent and the yield of 92.2 percent.
(2) Reverse phase chromatography
Column liquid feeding: adding 520ml of purified water and 20% methanol into the effluent collected in the step (1), and adjusting the pH to 3.5 +/-0.1 to obtain a concentration of 1.28 g/L;
② C18 reverse phase column (Sepax Bio-C18, 10 μm, 800ml) with mobile phase A (pH4.0, 20mM ammonium acetate, 50mMPB buffer, 20% methanol) at a flow rate of 50ml/min balance 3 column volumes;
thirdly, 500ml of column-loading liquid is loaded on the reversed phase column at the flow rate of 50 ml/min;
fourthly, after the column loading is finished, rebalancing 2 column volumes by using the mobile phase A at 50 ml/min;
washing the reverse phase column with 60% mobile phase A + 40% mobile phase B (pH4.0, 20mM ammonium acetate, 50mM PB buffer, 70% methanol) at flow rate of 50ml/min for 1 column volume;
sixthly, gradient elution is carried out by using a mobile phase A and a mobile phase B, the isocratic degree of the mobile phase B is increased from 40 percent to 70 percent, the isocratic degree of the mobile phase A is decreased from 60 percent to 30 percent, the elution flow rate is 50ml/min, about 200ml of effluent containing the C peptide is collected, the concentration is 1.58g/L, the HPLC purity is 99.28 percent, and the yield is 49.3 percent.
Example 3
(1) Zinc column chromatography
Column liquid feeding: adding 50mMPB buffer solution into the enzyme digestion solution containing the C peptide, adjusting the pH to 8.0 +/-0.1 by 0.15M sodium chloride, and adding the column solution with the volume of 1500ml and the concentration of 1.31 g/L;
② 1 column volume of a 1M zinc sulfate solution balance affinity chromatographic column (GE Health care chemical Sepharose Fast Flow, 1200ml), then 2 column volumes of purified water are washed to obtain the zinc column chromatographic column, eluent A (50mM PB buffer solution, 0.15M sodium chloride) is used for balancing 3 column volumes at the Flow rate of 60 ml/min;
thirdly, column feeding liquid to the zinc column at the flow rate of 60 ml/min;
fourthly, after the column loading is finished, the eluent A is continuously used for rebalancing 2 column volumes at the flow rate of 60 ml/min;
fifthly, washing the zinc column by 3 column volumes with eluent B (pH8.0, 50mM PB buffer solution, 0.15M sodium chloride, 0.05M imidazole) at the flow rate of 60 ml/min;
sixthly, eluting the zinc column by eluent C (pH8.0, 50mM PB buffer solution, 0.15M sodium chloride and 0.3M imidazole) at the flow rate of 60ml/min, collecting the effluent containing the recombinant human insulin C peptide, wherein the effluent contains 590ml, the HPLC detection concentration is 3.02g/L, the purity is 78.51 percent, and the yield is 90.6 percent.
(2) Reverse phase chromatography
Column liquid feeding: adding 590ml of purified water and acetonitrile with the final concentration of 5% into the zinc column chromatography effluent, adjusting the pH to 3.5 +/-0.1, and adjusting the concentration to 1.45 g/L;
② C8 reverse phase column (Kromasil 100, 10 μm, 500ml) was equilibrated with mobile phase A (pH3.0, 80mM ammonium acetate, 10mM citric acid/sodium citrate, 5% acetonitrile) at a flow rate of 50ml/min for 2 column volumes;
thirdly, 500ml of column-loading liquid is loaded on the reversed phase column at the flow rate of 50 ml/min;
fourthly, after the column loading is finished, rebalancing 2 column volumes by using the mobile phase A at 50 ml/min;
fifthly, washing 1 column volume of the reverse phase column by 65% mobile phase A + 35% mobile phase B (pH3.0, 80mM ammonium acetate, 10mM citric acid/sodium citrate, 40% acetonitrile) at the flow rate of 50 ml/min;
sixthly, gradient elution is carried out by using a mobile phase A and a mobile phase B, the isocratic degree of the mobile phase B is increased from 35% to 60%, the isocratic degree of the mobile phase A is decreased from 65% to 40%, the elution flow rate is 50ml/min, 190ml of effluent containing the C peptide is collected, the concentration is 1.61g/L, the HPLC purity is 99.06%, and the yield is 42.2%.
Example 4
(1) Nickel column chromatography
Column liquid feeding: adding 25mM Tris and 0.3M sodium chloride into the enzyme digestion solution containing the C peptide to adjust the pH to 10.0 +/-0.1, wherein the volume of the solution on the column is 1000ml, and the concentration is 1.45 g/L;
② balancing 1 column volume of affinity chromatographic column (GE Health care chemical Sepharose Fast Flow, 1000ml) by 1M nickel sulfate solution, washing 2 column volumes by purified water to obtain nickel column chromatographic column, balancing 3 column volumes by eluent A (25mM Tris buffer solution, 0.3M sodium chloride) at Flow rate of 60 ml/min;
thirdly, the column feeding liquid is fed into the nickel column at the flow speed of 60 ml/min;
fourthly, after the column loading is finished, the eluent A is continuously used for rebalancing 3 column volumes at the flow rate of 60 ml/min;
fifthly, washing 6 column volumes of the nickel column by eluent B (pH10.0, 25mM Tris buffer solution, 0.3M sodium chloride, 0.03M imidazole) at the flow rate of 60 ml/min;
sixthly, eluting the nickel column by eluent C (pH10.0, 25mM Tris buffer solution, 0.3M sodium chloride and 0.1M imidazole) at the flow rate of 60ml/min, collecting about 400ml of effluent containing the recombinant human insulin C peptide, and performing HPLC detection at the concentration of 3.3g/L, the purity of 81.36 percent and the yield of 91.0 percent.
(2) Reverse phase chromatography
Column liquid feeding: adding 400ml of purified water and 15% ethanol to the effluent collected in the step (1), and adjusting the pH to 3.5 +/-0.1 to obtain a concentration of 1.40 g/L;
② C8 reverse phase column (Kromasil 100, 10 μm, 500ml) was equilibrated with mobile phase A (pH5.0, 50mM ammonium acetate, 25mM citric acid/sodium citrate, 15% ethanol) at a flow rate of 50ml/min for 3 column volumes;
thirdly, 500ml of column-loading liquid is loaded on the reversed phase column at the flow rate of 50 ml/min;
fourthly, after the column loading is finished, rebalancing 3 column volumes by using the mobile phase A at 50 ml/min;
fifthly, washing 1 column volume of the reverse phase column by 65% mobile phase A + 35% mobile phase B (pH5.0, 50mM ammonium acetate, 25mM citric acid/citrate, 50% ethanol) at the flow rate of 50 ml/min;
sixthly, gradient elution is carried out by using a mobile phase A and a mobile phase B, the isocratic degree of the mobile phase B is increased from 35% to 60%, the isocratic degree of the mobile phase A is decreased from 65% to 40%, the elution flow rate is 50ml/min, about 210ml of effluent containing C peptide is collected, the concentration is 1.66g/L, and the HPLC purity is 99.32%. The yield thereof was found to be 49.8%.
Example 5
(1) Copper column chromatography
Column liquid feeding: adding 25mM Tris and 0.3M sodium chloride into the enzyme digestion solution containing the C peptide to adjust the pH to 9.0 +/-0.1, wherein the volume of the solution on the column is 1500ml, and the concentration is 1.32 g/L;
② 1 column volume of a 1M copper sulfate solution balance affinity chromatography column (GE Health care chemical Sepharose Fast Flow, 1200ml), then 2 column volumes of purified water is washed to obtain a copper column chromatography column, and 2 column volumes are balanced by eluent A (25mM Tris buffer solution, 0.3M sodium chloride) at the Flow rate of 60 ml/min;
thirdly, the column feeding liquid is fed into the copper column at the flow speed of 60 ml/min;
fourthly, after the column loading is finished, the eluent A is continuously used for rebalancing 2 column volumes at the flow rate of 60 ml/min;
fifthly, washing 4 column volumes of the copper column by eluent B (pH9.0, 25mM Tris buffer solution, 0.3M sodium chloride, 0.03M imidazole) at the flow rate of 60 ml/min;
sixthly, eluting the copper column by eluent C (pH9.0, 25mM Tris buffer solution, 0.3M sodium chloride and 0.1M imidazole) at the flow rate of 60ml/min, collecting about 540ml of effluent containing the recombinant human insulin C peptide, and performing HPLC detection at the concentration of 3.31g/L, wherein the purity is 84.1 percent and the yield is 90.3 percent.
(2) Reverse phase chromatography
Column liquid feeding: adding 540ml of purified water and 15% ethanol to the effluent collected in the step (1), and adjusting the pH to 4.0 +/-0.1 to obtain a concentration of 1.41 g/L;
② C8 reverse phase column (Kromasil 100, 10um, 500ml) was equilibrated with mobile phase A (pH4.0,50mM ammonium acetate, 25mM citric acid/sodium citrate, 15% ethanol) at flow rate 50ml/min for 2 column volumes;
thirdly, 500ml of column-loading liquid is loaded on the reversed phase column at the flow rate of 50 ml/min;
fourthly, after the column loading is finished, rebalancing 2 column volumes by using the mobile phase A at 50 ml/min;
fifthly, washing 1 column volume of the reverse phase column by 65% mobile phase A + 35% mobile phase B (pH4.0,50mM ammonium acetate, 25mM citric acid/sodium citrate, 60% ethanol) at the flow rate of 50 ml/min;
sixthly, gradient elution is carried out by using a mobile phase A and a mobile phase B, the isocratic degree of the mobile phase B is increased from 35% to 60%, the isocratic degree of the mobile phase A is decreased from 65% to 40%, the elution flow rate is 50ml/min, about 195ml of effluent containing C peptide is collected, the concentration is 1.72g/L, the HPLC purity is 99.45%, and the yield is 47.6%.
Claims (10)
1. A method for purifying recombinant human insulin C peptide is characterized in that the method comprises the step of carrying out metal ion affinity chromatography and reverse phase chromatography on enzyme digestion liquid containing the recombinant human insulin C peptide to obtain the high-purity recombinant human insulin C peptide.
2. The method of claim 1, comprising the steps of: (1) loading the enzyme digestion solution containing the recombinant human insulin C peptide into a metal ion affinity chromatography column, eluting by an eluent, and collecting an effluent liquid containing the recombinant human insulin C peptide; (2) and (2) passing the effluent obtained in the step (1) through a reversed-phase chromatographic column, eluting a mobile phase, and collecting the effluent containing the recombinant human insulin C peptide to obtain the high-purity recombinant human insulin C peptide.
3. The method according to claim 1 or 2, wherein the metal ions are nickel ions, zinc ions, copper ions or cobalt ions.
4. The method of claim 2, wherein the amount of the metal ion affinity chromatography column is 0.5-3g/L, and the mass of the sample is in g and the volume of the packing is in L.
5. The method as claimed in claim 2, wherein the elution process in step (1) is divided into three steps, wherein the elution is performed by eluting with eluent A, then eluting with eluent B, and then eluting with eluent C, and the effluent containing recombinant human insulin C peptide is collected; wherein the eluent A is 10-50mM Tris or PB buffer solution, 0.15-0.5M sodium chloride or ammonium chloride; eluent B is Tris or PB buffer solution with pH of 8.0-11.0 and 10-50mM, 0.15-0.5M sodium chloride or ammonium chloride and 0.02-0.05M imidazole; the removing agent C is Tris or PB buffer solution with pH of 8.0-11.0 and 10-50mM, 0.15-0.5M sodium chloride or ammonium chloride, and 0.1-0.3M imidazole.
6. The method as claimed in claim 2, wherein the elution process in step (1) is divided into three steps, wherein the elution is performed by eluting with eluent A, then eluting with eluent B, and then eluting with eluent C, and the effluent containing recombinant human insulin C peptide is collected; wherein the eluent A is 10-50mM Tris, 0.15-0.5M sodium chloride; eluent B is pH8.0-11.0, 10-50mM Tris, 0.15-0.5M sodium chloride, 0.02-0.05M imidazole; eluent C is pH8.0-11.0, 10-50mM Tris, 0.15-0.5M sodium chloride, 0.1-0.3M imidazole.
7. The method of claim 1 or 2, wherein the reverse phase chromatography packing is C8 or C18.
8. The method according to claim 2, wherein the elution process in step (2) is carried out in three steps of elution with mobile phase A and elution with mobile phase B, wherein the mobile phase A is pH3.0-6.0, 20-80mM inorganic salt or organic salt, 10-50mM buffer, 5-20% organic solvent; the mobile phase B is inorganic salt or organic salt with pH of 3.0-6.0 and 20-80mM, buffer solution with pH of 10-50mM and organic solvent with pH of 40-80%.
9. The method according to claim 8, characterized in that the organic/inorganic salt is selected from ammonium acetate or sodium chloride, the buffer is selected from citric acid/sodium citrate or PB buffer; the organic solvent is selected from ethanol, acetonitrile, methanol or acetone.
10. The method according to claim 8, wherein the elution process comprises the steps of firstly washing with a mobile phase A, then washing with 70-60% of the mobile phase A and 30-40% of the mobile phase B, and finally performing gradient elution on the mobile phases A and B, and collecting elution liquid containing the recombinant human insulin C peptide, wherein the elution gradient is that the mobile phase B is increased from 30-40% to 60-70%.
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