CN103694339A - Renaturation method of insulin glargine precursor - Google Patents
Renaturation method of insulin glargine precursor Download PDFInfo
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- CN103694339A CN103694339A CN201310754124.4A CN201310754124A CN103694339A CN 103694339 A CN103694339 A CN 103694339A CN 201310754124 A CN201310754124 A CN 201310754124A CN 103694339 A CN103694339 A CN 103694339A
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- COCFEDIXXNGUNL-RFKWWTKHSA-N Insulin glargine 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)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(=O)NCC(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 COCFEDIXXNGUNL-RFKWWTKHSA-N 0.000 title claims abstract description 112
- 108010057186 Insulin Glargine Proteins 0.000 title claims abstract description 110
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- 238000004153 renaturation Methods 0.000 title claims abstract description 61
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- 238000006243 chemical reaction Methods 0.000 claims abstract description 45
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 8
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- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 claims description 7
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 claims description 6
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 6
- TZBAVQKIEKDGFH-UHFFFAOYSA-N n-[2-(diethylamino)ethyl]-1-benzothiophene-2-carboxamide;hydrochloride Chemical compound [Cl-].C1=CC=C2SC(C(=O)NCC[NH+](CC)CC)=CC2=C1 TZBAVQKIEKDGFH-UHFFFAOYSA-N 0.000 claims description 5
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- 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 8
- 102000004877 Insulin Human genes 0.000 description 6
- 108090001061 Insulin Proteins 0.000 description 6
- 239000004475 Arginine Substances 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 5
- 229910021591 Copper(I) chloride Inorganic materials 0.000 description 5
- YAJCHEVQCOHZDC-QMMNLEPNSA-N actrapid 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=3N=CNC=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](NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C(N)=O)C1=CNC=N1 YAJCHEVQCOHZDC-QMMNLEPNSA-N 0.000 description 5
- 238000000137 annealing Methods 0.000 description 5
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 5
- 239000010949 copper Substances 0.000 description 5
- OXBLHERUFWYNTN-UHFFFAOYSA-M copper(I) chloride Chemical compound [Cu]Cl OXBLHERUFWYNTN-UHFFFAOYSA-M 0.000 description 5
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- 229950004152 insulin human Drugs 0.000 description 5
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- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000009322 erkang Substances 0.000 description 2
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- 241000206602 Eukaryota Species 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- LEVWYRKDKASIDU-IMJSIDKUSA-N L-cystine Chemical compound [O-]C(=O)[C@@H]([NH3+])CSSC[C@H]([NH3+])C([O-])=O LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 description 1
- 102000007327 Protamines Human genes 0.000 description 1
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- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 229960003280 cupric chloride Drugs 0.000 description 1
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- 238000001514 detection method Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
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- 238000006703 hydration reaction Methods 0.000 description 1
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- 230000002218 hypoglycaemic effect Effects 0.000 description 1
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-
- 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
- C07K14/62—Insulins
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Endocrinology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Diabetes (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a renaturation method of an insulin glargine precursor, and belongs to the field of biomedical protein folding. The method comprises the following steps: dissolving the insulin glargine precursor in a modifier solution, adding a reducer for reduction, adjusting the pH value to be 9.5-11.5, controlling the temperature of a reaction system to be 35-45 DEG C, and reacting for 30-60 minutes to obtain a modified insulin glargine precursor solution; adding the modified insulin glargine precursor solution into a dilute buffer solution, adding a protein folding additive, adjusting the pH value to be 9.5-11.5, continuously inletting air into the solution, controlling the temperature of the reaction system to be 0-20 DEG C, and reacting for 2-40 hours to obtain an insulin glargine renaturation solution. According to the method, the renaturation reaction time is shortened, the correctly folded protein content is increased, the renaturation efficiency is improved to be 51%-62%, the production cost is reduced, and the large-scale industrialization production and application are facilitated.
Description
Technical field
The invention belongs to biological medicine protein folding field, be specifically related to a kind of refolding method of Lantus precursor, be particularly related to a kind of polyoxyethylene glycol, glycerine and metal ion etc. of utilizing as the atmospheric oxidation dilution refolding method of protein folding additive, the Lantus precursor conversion of false folding is become to have to the precursor of correct space structure, annealing efficiency can surpass 50%, reduced the generation of disulfide linkage mispairing by product, to subsequent purification, work brings great convenience.
Background technology
Lantus (glycine arginine insulin) is a kind of protamine zine insulin kind biological product of being produced and be used for the treatment of I, type II diabetes by recombinant DNA technology.It is to have increased by two arginine at insulin human B chain C-terminal, also the l-asparagine of A chain C-terminal A21 position is replaced to glycine simultaneously, A chain C-terminal is the formation that glycine has prevented deamination product, makes Lantus have good stability; Again because two arginine that increase are positively charged, so the iso-electric point of Lantus is by 5.4(Regular Insulin iso-electric point) left and right is increased to 7.0 left and right, so this insulin analog can be under the condition of meta-acid (pH4.0) be mixed with the injection liquid of stable in properties and clarification.Before Lantus injection, do not need suspendible, after subcutaneous injection, (pH7.4) forms the sustainable slow release Lantus of small precipitation in vivo, and plasma concentration is steady, and peak valley curve is little, and acting duration is long.Blood sugar reducing function of subcutaneous injection every day can maintain 24 hours, and occurs without obvious blood medicine peak value, and secretion that can simulate arm's length basis Regular Insulin, reduces hypoglycemic probability occurs.
The Lantus precursor of producing both at home and abroad at present adopts escherichia coli expression, in its system, lack eukaryote and form the environment of the correct structure of albumen and relevant modified elements, in intestinal bacteria, high level expression often causes protein aggregation and forms inclusion body insoluble, non-activity.Inclusion body is after separation, sex change dissolving and renaturation, be formed with bioactive albumen, in renaturation process, need to provide the folding environment that is beneficial to single chain protein, suitable redox environment is provided simultaneously, make Lantus precursor form correct secondary structure.
The efficiency of renaturation directly affects the height of output.Renaturation is a complicated process, and the protein aggregation reaction in renaturation process is the first cause that causes renaturation yield low.After the concentration of denaturing agent is lower, the protein molecular of strand is folded to form rapidly the intermediate state molecule of a large amount of secondary structures, and the hydrophobic region that intermediate state molecular surface exposes interacts, and congregation occurs.The gathering of albumen and correct be competing reaction between collapsible, therefore reduce the congregation of albumen, improve the correct folding most important to renaturation of albumen.
In prior art relevant for the refolding method of Regular Insulin, for example, at F-J Lu Bailuode C07K14/62, the improved method that obtains the insulin precurosor with correctly bonded cystine linkage is disclosed in Chinese patent CN1132845C, at halfcystine or cysteine hydrochloride with under existing from liquid auxiliary agent, acquire 30%～50% correct renaturation product.The beautiful C07K14/62 of Wang Li, discloses a kind of method of efficient Size Exclusion Chromatograph SEC renaturation Simultaneous purification recombinant insulinum primary in Chinese patent CN103172727A, attempt the method and carry out in renaturation at Lantus precursor, and result shows that the rate of recovery is low.At present, in open source literature, rarely having technique and the parameter report about the high annealing efficiency of Lantus.
Summary of the invention
The object of the invention is to by optimizing renaturation processing parameter, a kind of refolding method of Lantus precursor is provided, with denaturing agent, dissolve inclusion body, add reductive agent to open the disulfide linkage of interchain formation and the nonactive disulfide linkage in chain, the Lantus precursor with primary structure is correctly folded into the activated albumen of tool in diluent, adds the protein folding additives such as polyoxyethylene glycol, glycerine, metal ion in renaturation process.The method can improve efficiency to 51%～62% of renaturation, has reduced the generation of disulfide linkage mispairing by product, and to subsequent purification, work brings great convenience.
The object of the invention is by adopting following technical proposals to realize: a kind of refolding method of Lantus precursor, comprises the steps:
Lantus precursor is dissolved in denaturant solution, obtains solution I; Add reductive agent to reduce, regulating pH value is 9.5～11.5 again, and the temperature of controlling reaction system I is 35 ℃～45 ℃, and reaction 30～60min, obtains the Lantus precursor solution after sex change; Lantus precursor solution after sex change is added in dilution buffer liquid, obtain solution II; In solution II, add protein folding additive, regulating pH value is 9.5～11.5; Then in the solution obtaining, continue to pass into air, the temperature of controlling reaction system II is 0 ℃～20 ℃, reacts 2～40 hours, obtains Lantus renaturation solution.
Described Lantus precursor is with inclusion body form, to be expressed and obtain by recombinant DNA technology by intestinal bacteria, specifically utilize gene recombination technology to form insulin human's the structure of modification that carries out, for 20A-Gly21-C peptide-30B-Arg31-Arg32, the 21st amino acids that is about to insulin human A chain is replaced by glycine, after B chain the 30th amino acids, introduce two arginine simultaneously, then to proceed in intestinal bacteria and to express after C peptide connection A chain and B chain.
The concentration of the Lantus precursor in described solution I is preferably 17～28g/L.
Described denaturing agent is urea or Guanidinium hydrochloride.
Described denaturant solution is preferably 6～8M urea soln or guanidine hydrochloride solution, and pH value is 9.5～11.5; The further preferred urea soln of 6～8M, pH9.5～11.5.
Described reductive agent is selected from one or both in dithiothreitol (DTT) (DTT) and cysteine hydrochloride.
The consumption of described reductive agent calculates for pressing the front body mass ratio 1:2～1:4 of reductive agent and Lantus.
Described dilution buffer liquid is selected from a kind of damping fluid preparing in Tutofusin tris (Tris), glycine, bicarbonate of ammonia and phosphoric acid salt.
Described dilution buffer liquid is preferably the glycine buffer of the Tris-HCl damping fluid of 10～50mM, pH9.5～11.5 or 10～50mM, pH9.5～11.5.
The final concentration of the Lantus precursor in described solution II after sex change is preferably 0.8～2.0g/L, more preferably 1.0～1.4g/L.
Described protein folding additive is at least one in polyoxyethylene glycol, glycerine and metal ion.
Described polyoxyethylene glycol is PEG4000 or PEG6000.
Described metal ion is Ca
2+, Mg
2+, Cu
2+and Fe
3+in a kind of.
Described protein folding additive is preferably that to take the volume of described solution II be denominator, concentration is that the polyoxyethylene glycol of 0.5～2g/L, the metal ion of the glycerine of volume percent 5%～20% and 5～50 μ M form, the Cu of the Macrogol 4000 that more preferably concentration is 0.5～2g/L, the glycerine of volume percent 5%～20% and 5～20 μ M
2+form.
Described Cu
2+by CuSO
4or CuCl
2provide.
Described pass into air be preferably by air by every liter of solution with 2～100cm
3the speed of/H passes in solution.
The reaction conditions of described reaction system II is preferably 4～15 ℃ of reactions 12～25 hours.
The refolding method of described Lantus precursor, more specifically comprises the steps:
(1) denaturation method of Lantus: Lantus precursor is dissolved in the denaturant solution of 6～8M, pH9.5～11.5, obtains solution I; Add reductive agent, regulating pH value is 9.5～11.5, reductive agent and Lantus precursor 1:2～1:4 proportioning in mass ratio wherein, and the temperature of controlling reaction system is 35 ℃～45 ℃, reacts 30～60min, the Lantus precursor solution after acquisition sex change;
(2) refolding method of Lantus: the Lantus precursor solution after sex change is added in dilution buffer liquid, obtain solution II; In solution II, add polyoxyethylene glycol, glycerine and metal ion, wherein, the volume of solution II of take is benchmark, and the concentration of polyoxyethylene glycol is 0.5～2g/L, the concentration of glycerine is that the concentration of volume percent 5%～20%, metal ion is 5～50 μ M, and regulating pH value is 9.5～11.5; Then in gained solution, continue to pass into air, 0 ℃～20 ℃ of the temperature of control reaction system, react 2～40 hours, obtain Lantus renaturation solution;
Lantus precursor described in step (1) is with inclusion body form, to be expressed and obtain by recombinant DNA technology by intestinal bacteria, specifically utilize gene recombination technology to form insulin human's the structure of modification that carries out, for 20A-Gly21-C peptide-30B-Arg31-Arg32, the 21st amino acids that is about to insulin human A chain is replaced by glycine, after B chain the 30th amino acids, introduce two arginine simultaneously, then to proceed in intestinal bacteria and to express after C peptide connection A chain and B chain; Preferably Lantus precursor is dissolved in the denaturant solution of 6～8M by the concentration of 17～28g/L;
Denaturing agent described in step (1) is preferably urea or Guanidinium hydrochloride;
Reductive agent described in step (1) is preferably one or both in DTT and cysteine hydrochloride;
The final concentration of the Lantus precursor in the solution II described in step (2) after sex change is preferably 0.8～2.0g/L, more preferably 1.0～1.4g/L;
Dilution buffer liquid described in step (2) is selected from the damping fluid that a kind of preparation in Tutofusin tris (Tris), glycine, bicarbonate of ammonia and phosphoric acid salt obtains;
Described dilution buffer liquid is preferably the glycine buffer of the Tris-HCl damping fluid of 10～50mM, pH9.5～11.5 or 10～50mM, pH9.5～11.5;
Polyoxyethylene glycol described in step (2) is selected from a kind of in PEG4000 and PEG6000; Be preferably Macrogol 4000;
Metal ion described in step (2) is Ca
2+, Mg
2+, Cu
2+and Fe
3+in a kind of; Be preferably by CuSO
4or CuCl
2the Cu providing
2+; Concentration is preferably 5～20 μ M;
Described in step (2) pass into air be preferably by air by every liter of solution with 2～100cm
3the speed of/H passes in solution;
The temperature of reaction of the reaction system described in step (2) is preferably 4～15 ℃;
The reaction times of the reaction system described in step (2) is preferably 12～25 hours.
The additional proportion of the reductive agent in the present invention is vital at Lantus precursor renaturation process.Lantus precursor contains three pairs of disulfide linkage, at escherichia coli expression, form in inclusion body process, disulfide linkage mispairing in interchain and chain causes albumen non-activity, need to add SH-group reductant to process to reduce these disulfide linkage, the additional proportion of reductive agent need to be considered follow-up renaturation process.When adding the reductive agent of excessive concentrations, not only can reduce and open the disulfide linkage of wrong pairing in inclusion body, can also reduce the disulfide linkage of correct pairing in inclusion body, cause difficulty to follow-up renaturation reaction, and the reductive agent of lower concentration be owing to can not thoroughly reducing the disulfide linkage of wrong pairing in inclusion body, can directly affect equally in renaturation reaction that target protein is correctly folding correctly to be matched with disulfide linkage.The mass ratio of the preferred reductive agent of the present invention and Lantus precursor is preferably 1:3 and adds reductive agent.
In the present invention, improve very large to the efficiency of Lantus precursor renaturation adding of protein folding additives polyethylene glycol, glycerine and metal ion.The dilution oxidizing and refolding system of Lantus precursor is to using airborne oxygen as oxygenant.Protein folding additive can reduce gathering, promotes foldingly, as polyoxyethylene glycol can be protected molten spheroid, increases viscosity, and glycerine has preferential hydration to protein, metal ion can catalytic proteins in the atmospheric oxidation speed of reductive agent residue.Adding of protein folding additive, can accelerate to form the disulfide linkage of correct pairing, thereby effectively increase correct folding target protein content, also greatly shortened the renaturation reaction times of inclusion body simultaneously.The present invention is preferably 0.5～2g/L Macrogol 4000, and metal ion solution is 5～20 μ M CuSO
4or CuCl
2.
The present invention has following advantage and effect with respect to prior art:
(1) the Lantus precursor refolding method the present invention relates to, the by product of disulfide linkage mispairing is few, and the renaturation solution of gained is more conducive to follow-up purifying process.
(2) the present invention adopts protein folding additives polyethylene glycol, glycerine and metal ion to assist renaturation, has shortened the reaction times, has improved correct folding protein content, improves widely annealing efficiency to 51%～62%.
(3) refolding method the present invention relates to, adopts dilution refolding, usings air as oxygenant, has reduced greatly production cost, and applicable industrialized production application.
Accompanying drawing explanation
Sample HPLC figure spectrogram after Fig. 1 embodiment 1 renaturation.
Sample HPLC figure spectrogram after Fig. 2 embodiment 2 renaturation.
Sample HPLC figure spectrogram after Fig. 3 embodiment 3 renaturation.
Sample HPLC figure spectrogram after Fig. 4 embodiment 4 renaturation.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited to this.
The embodiment of the present invention is as follows for detection of the formula of annealing efficiency (%) in renaturation process:
Annealing efficiency (%)=(after renaturation before correct folding Lantus before weight/renaturation before Lantus weight) * 100%
Experiment material:
Lantus precursor: Zhuhai United Laboratories Ltd provides, lot number F18121101; Be specially by US Patent No. 6100376(A
21, B
30, MODIFIED INSULIN DERIVATIVES HAVING AN ALTERED ACTION PROFILE) and middle embodiment 6 preparations.
Urea derives from Guangzhou West Gansu Province Chemical Co., Ltd.; Cysteine hydrochloride derives from Wuhan long-range Hong Yuan limited-liability company; Tutofusin tris derives from Beijing Ke Ao Science and Technology Ltd.; Macrogol 4000 derives from Hu'nan Erkang Pharmaceutical Co., Ltd.; Glycerine derives from Hu'nan Erkang Pharmaceutical Co., Ltd.; Copper sulfate derives from Guangzhou West Gansu Province Chemical Co., Ltd.; Cupric chloride derives from Guangzhou West Gansu Province Chemical Co., Ltd.; Hydrochloric acid derives from Zhuhai City Hua Chengda Chemical Co., Ltd.; Sodium hydroxide derives from Dong Jiang chemical reagent company limited.
Aeration equipment: E+H air flowmeter, pressurized air.
The renaturation of embodiment 1 Lantus precursor
Under room temperature, 10 grams of Lantus precursors are dissolved in the urea soln of 588mL6M, pH9.5, make the protein concentration of Lantus precursor be about 17g/L; Add 5g DTT, regulating pH value is 9.5 again, and the temperature of controlling reaction system is 35 ℃, reaction 30min.
Lantus precursor solution after sex change is added in the Tris-HCl damping fluid of 10mM, pH9.5, with Tris-HCl damping fluid, be settled to 10L, the protein concentration that makes Lantus precursor is 1.0g/L, then adds 5g PEG4000,500mL glycerine, 8mg CuSO
4, regulating pH value is 9.5, with 20cm
3the speed of/H continues to pass into air in renaturation solution, and the temperature of controlling reaction system is 4 ℃, reacts and stops this reaction after 12 hours.Get renaturation solution 20 μ L through HPLC analyzing and testing, Fig. 1 is shown in by sample HPLC collection of illustrative plates after renaturation, the retention time of the Lantus precursor HPLC collection of illustrative plates of correct refolding is 19.98min, according to the actual amount that obtains the Lantus precursor of correct refolding after the areas of peak normalization method calculating renaturation of Lantus precursor, be 6.1g, the yield of Lantus precursor renaturation is about 61% as calculated.
The renaturation of embodiment 2 Lantus precursors
Under room temperature, 10 grams of Lantus precursors are dissolved in the urea soln of 357mL8M, pH11.5, make the protein concentration of Lantus precursor be about 28g/L, add 2.5g DTT, regulating pH value is 11.5, and the temperature of controlling reaction system is 45 ℃, reaction 60min.
Lantus precursor solution after sex change is added in the Tris-HCl damping fluid of 50mM, pH11.5, with Tris-HCl damping fluid, be settled to 7.14L, the protein concentration that makes Lantus precursor is 1.4g/L, then adds 14.28g PEG4000,1428mL glycerine, 22.85mg CuSO
4, regulating pH value is 11.5, with 714cm
3the speed of/H continues to pass into air in renaturation solution, and 15 ℃ of the temperature of control reaction system, react and stop this reaction after 25 hours.Get renaturation solution 20 μ L through HPLC analyzing and testing, Fig. 2 is shown in by sample HPLC collection of illustrative plates after renaturation, the retention time of the Lantus precursor HPLC collection of illustrative plates of correct refolding is 20.71min, according to the actual amount that obtains the Lantus precursor of correct refolding after the areas of peak normalization method calculating renaturation of Lantus precursor, be 5.14g, the yield of Lantus precursor renaturation is about 51.40% as calculated.
The renaturation of embodiment 3 Lantus precursors
Under room temperature, 10 grams of Lantus precursors are dissolved in the urea soln of 588mL6M, pH9.5, make the protein concentration of Lantus precursor be about 17g/L, add 5g cysteine hydrochloride, regulating pH value is 9.5, and the temperature of controlling reaction system is 35 ℃, reaction 30min.
Lantus precursor solution after sex change is added in the glycine dilution buffer liquid of 10mM, pH9.5, with glycine dilution buffer liquid, be settled to 10L, the protein concentration that makes Lantus precursor is 1.0g/L, then adds 5g PEG4000,500mL glycerine, 6.75mg CuCl
2, regulating pH value is 9.5, with 20cm
3the speed of/H continues to pass into air in renaturation solution, and 4 ℃ of the temperature of control reaction system, react and stop this reaction after 12 hours.Get renaturation solution 20 μ L through HPLC analyzing and testing, Fig. 3 is shown in by sample HPLC collection of illustrative plates after renaturation, the retention time of the Lantus precursor HPLC collection of illustrative plates of correct refolding is 19.94min, according to the actual amount that obtains the Lantus precursor of correct refolding after the areas of peak normalization method calculating renaturation of Lantus precursor, be 6.2g, the yield of Lantus precursor renaturation is about 62% as calculated.
The renaturation of embodiment 4 Lantus precursors
Under room temperature, 10 grams of Lantus precursors are dissolved in the urea soln of 357mL8M, pH11.5, make the protein concentration of Lantus precursor be about 28g/L, add 2.5g cysteine hydrochloride, regulating pH value is 11.5, and the temperature of controlling reaction system is 45 ℃, reaction 60min.
Lantus precursor solution after sex change is added in the glycine dilution buffer liquid of 50mM, pH11.5, with glycine dilution buffer liquid, be settled to 7.14L, the protein concentration that makes Lantus precursor is 1.4g/L, then adds the CuCl of 14.28g PEG4000,1428mL glycerine, 19.28mg
2, regulating pH value is 11.5, with 714cm
3the speed of/H continues to pass into air in renaturation solution, and 15 ℃ of the temperature of control reaction system, react and stop this reaction after 25 hours.Get renaturation solution 20 μ L through HPLC analyzing and testing, Fig. 4 is shown in by sample HPLC collection of illustrative plates after renaturation, the retention time of the Lantus precursor HPLC collection of illustrative plates of correct refolding is 20.75min, according to the actual amount that obtains the Lantus precursor of correct refolding after the areas of peak normalization method calculating renaturation of Lantus precursor, be 5.35g, the yield of Lantus precursor renaturation is about 53.5% as calculated.
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
Claims (10)
1. a refolding method for Lantus precursor, is characterized in that comprising the steps: Lantus precursor is dissolved in denaturant solution, obtains solution I; Add reductive agent to reduce, regulating pH value is 9.5～11.5 again, and the temperature of controlling reaction system I is 35 ℃～45 ℃, and reaction 30～60min, obtains the Lantus precursor solution after sex change; Lantus precursor solution after sex change is added in dilution buffer liquid, obtain solution II; In solution II, add protein folding additive, regulating pH value is 9.5～11.5; Then in the solution obtaining, continue to pass into air, the temperature of controlling reaction system II is 0 ℃～20 ℃, reacts 2～40 hours, obtains Lantus renaturation solution.
2. the refolding method of Lantus precursor according to claim 1, is characterized in that:
The concentration of the Lantus precursor in described solution I is 17～28g/L;
The final concentration of the Lantus precursor in described solution II after sex change is 0.8～2.0g/L.
3. the refolding method of Lantus precursor according to claim 2, is characterized in that: the final concentration of the Lantus precursor in described solution II after sex change is 1.0～1.4g/L.
4. the refolding method of Lantus precursor according to claim 1, is characterized in that:
Described denaturing agent is urea or Guanidinium hydrochloride;
Described reductive agent is one or both in dithiothreitol (DTT) and cysteine hydrochloride;
Described dilution buffer liquid is a kind of damping fluid preparing in Tutofusin tris, glycine, bicarbonate of ammonia and phosphoric acid salt;
Described protein folding additive is at least one in polyoxyethylene glycol, glycerine and metal ion.
5. the refolding method of Lantus precursor according to claim 4, is characterized in that:
Described denaturant solution is urea soln or the guanidine hydrochloride solution of 6～8M, pH9.5～11.5;
The consumption of described reductive agent calculates for pressing the front body mass ratio 1:2～1:4 of reductive agent and Lantus;
Described dilution buffer liquid is the glycine buffer of the Tris-HCl damping fluid of 10～50mM, pH9.5～11.5 or 10～50mM, pH9.5～11.5.
6. the refolding method of Lantus precursor according to claim 4, is characterized in that:
Described polyoxyethylene glycol is PEG4000 or PEG6000;
Described metal ion is Ca
2+, Mg
2+, Cu
2+and Fe
3+in a kind of;
Described protein folding additive is served as reasons and be take solution II volume as benchmark, and the metal ion of the glycerine of the polyoxyethylene glycol that concentration is 0.5～2g/L, volume percent 5%～20% and 5～50 μ M forms.
7. the refolding method of Lantus precursor according to claim 6, it is characterized in that: described protein folding additive is served as reasons and be take solution II volume as benchmark the glycerine of the Macrogol 4000 that concentration is 0.5～2g/L, volume percent 5%～20% and the Cu of 5～20 μ M
2+form.
8. the refolding method of Lantus precursor according to claim 1, is characterized in that: described pass into air for by air by every liter of solution with 2～100cm
3the speed of/H passes in solution;
The reaction conditions of described reaction system II is 4～15 ℃ of reactions 12～25 hours.
9. according to the refolding method of the Lantus precursor described in claim 1～8 any one, it is characterized in that comprising the steps:
(1) denaturation method of Lantus: Lantus precursor is dissolved in the denaturant solution of 6～8M, pH9.5～11.5, obtains solution I; Add reductive agent, regulating pH value is 9.5～11.5, reductive agent and Lantus precursor 1:2～1:4 proportioning in mass ratio wherein, and the temperature of controlling reaction system is 35 ℃～45 ℃, reacts 30～60min, the Lantus precursor solution after acquisition sex change;
(2) refolding method of Lantus: the Lantus precursor solution after sex change is added in dilution buffer liquid, obtain solution II; In solution II, add polyoxyethylene glycol, glycerine and metal ion, wherein, the volume of solution II of take is benchmark, and the concentration of polyoxyethylene glycol is 0.5～2g/L, the concentration of glycerine is that the concentration of volume percent 5%～20%, metal ion is 5～50 μ M, and regulating pH value is 9.5～11.5; Then in gained solution, continue to pass into air, 0 ℃～20 ℃ of the temperature of control reaction system, react 2～40 hours, obtain Lantus renaturation solution.
10. the refolding method of Lantus precursor according to claim 9, is characterized in that:
The concentration of the Lantus precursor in the solution I described in step (1) is 17～28g/L;
Denaturing agent described in step (1) is urea or Guanidinium hydrochloride;
Reductive agent described in step (1) is one or both in DTT and cysteine hydrochloride;
The final concentration of the Lantus precursor in the solution II described in step (2) after sex change is 1.0～1.4g/L;
The glycine buffer of the Tris-HCl damping fluid that described dilution buffer liquid is 10～50mM, pH9.5～11.5 or 10～50mM, pH9.5～11.5;
Described polyoxyethylene glycol is PEG4000;
Metal ion described in step (2) is Cu
2+, concentration is 5～20 μ M;
Described in step (2) pass into air for by air by every liter of solution with 2～100cm
3the speed of/H passes in solution;
The temperature of reaction of the reaction system described in step (2) is 4～15 ℃;
The reaction times of the reaction system described in step (2) is 12～25 hours.
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Application publication date: 20140402 Assignee: Zhuhai United Biopharmaceutical Co.,Ltd. Assignor: ZHUHAI UNITED LABORATORIES Co.,Ltd. Contract record no.: X2024980001686 Denomination of invention: A refolding method for proinsulin glargine Granted publication date: 20171013 License type: Common License Record date: 20240131 Application publication date: 20140402 Assignee: Federal Biotechnology (Zhuhai Hengqin) Limited Co. Assignor: ZHUHAI UNITED LABORATORIES Co.,Ltd. Contract record no.: X2024980001558 Denomination of invention: A refolding method for proinsulin glargine Granted publication date: 20171013 License type: Common License Record date: 20240129 |