CN103694338A - Purification method of glucagon hydrochloride - Google Patents
Purification method of glucagon hydrochloride Download PDFInfo
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- CN103694338A CN103694338A CN201310717379.3A CN201310717379A CN103694338A CN 103694338 A CN103694338 A CN 103694338A CN 201310717379 A CN201310717379 A CN 201310717379A CN 103694338 A CN103694338 A CN 103694338A
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- 238000000034 method Methods 0.000 title claims abstract description 25
- 238000000746 purification Methods 0.000 title claims abstract description 23
- 229960004777 glucagon hydrochloride Drugs 0.000 title abstract description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 136
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 53
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 29
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 25
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 23
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000010828 elution Methods 0.000 claims abstract description 14
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 claims abstract description 14
- 150000003839 salts Chemical class 0.000 claims abstract description 14
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical group CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 12
- 235000011114 ammonium hydroxide Nutrition 0.000 claims abstract description 11
- 235000019257 ammonium acetate Nutrition 0.000 claims abstract description 8
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 8
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims abstract description 7
- 239000005695 Ammonium acetate Substances 0.000 claims abstract description 7
- 229940043376 ammonium acetate Drugs 0.000 claims abstract description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 55
- 102000051325 Glucagon Human genes 0.000 claims description 45
- 108060003199 Glucagon Proteins 0.000 claims description 45
- 229960004666 glucagon Drugs 0.000 claims description 45
- MASNOZXLGMXCHN-ZLPAWPGGSA-N glucagon Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 MASNOZXLGMXCHN-ZLPAWPGGSA-N 0.000 claims description 45
- 239000000243 solution Substances 0.000 claims description 45
- 239000007864 aqueous solution Substances 0.000 claims description 14
- 239000000377 silicon dioxide Substances 0.000 claims description 12
- 230000005526 G1 to G0 transition Effects 0.000 claims description 11
- 238000011068 loading method Methods 0.000 claims description 8
- 239000002253 acid Substances 0.000 claims description 7
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 6
- 125000001309 chloro group Chemical group Cl* 0.000 claims description 6
- 238000002203 pretreatment Methods 0.000 claims description 6
- 239000008215 water for injection Substances 0.000 claims description 6
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 5
- 239000010808 liquid waste Substances 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 4
- PWUBONDMIMDOQY-UHFFFAOYSA-N acetonitrile;hydrochloride Chemical compound Cl.CC#N PWUBONDMIMDOQY-UHFFFAOYSA-N 0.000 claims description 3
- 229910021529 ammonia Inorganic materials 0.000 claims description 3
- 238000010025 steaming Methods 0.000 claims description 2
- 238000007865 diluting Methods 0.000 claims 1
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 3
- 238000011033 desalting Methods 0.000 abstract description 2
- 230000001105 regulatory effect Effects 0.000 abstract description 2
- 238000006243 chemical reaction Methods 0.000 abstract 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 abstract 1
- DKAGJZJALZXOOV-UHFFFAOYSA-N hydrate;hydrochloride Chemical compound O.Cl DKAGJZJALZXOOV-UHFFFAOYSA-N 0.000 abstract 1
- 238000005185 salting out Methods 0.000 abstract 1
- 239000000741 silica gel Substances 0.000 abstract 1
- 229910002027 silica gel Inorganic materials 0.000 abstract 1
- 239000012071 phase Substances 0.000 description 23
- 229960000583 acetic acid Drugs 0.000 description 15
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 6
- 238000010790 dilution Methods 0.000 description 4
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- 238000004519 manufacturing process Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- JGSARLDLIJGVTE-UHFFFAOYSA-N 3,3-dimethyl-7-oxo-6-[(2-phenylacetyl)amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid Chemical compound O=C1N2C(C(O)=O)C(C)(C)SC2C1NC(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-UHFFFAOYSA-N 0.000 description 3
- YCKRFDGAMUMZLT-UHFFFAOYSA-N Fluorine atom Chemical compound [F] YCKRFDGAMUMZLT-UHFFFAOYSA-N 0.000 description 3
- 102000004877 Insulin Human genes 0.000 description 3
- 108090001061 Insulin Proteins 0.000 description 3
- 235000019846 buffering salt Nutrition 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 229910052731 fluorine Inorganic materials 0.000 description 3
- 239000011737 fluorine Substances 0.000 description 3
- 238000005374 membrane filtration Methods 0.000 description 3
- 238000010926 purge Methods 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 238000001308 synthesis method Methods 0.000 description 3
- 230000003570 biosynthesizing effect Effects 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 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 2
- 238000011031 large-scale manufacturing process Methods 0.000 description 2
- 238000010532 solid phase synthesis reaction Methods 0.000 description 2
- 206010016803 Fluid overload Diseases 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 150000001343 alkyl silanes Chemical group 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
Classifications
-
- 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
- C07K14/605—Glucagons
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Endocrinology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a purification method of glucagon hydrochloride. The method comprises the following steps: dissolving crude peptides in a water solution consisting of 30-60 vol% acetic acid and 5-20 vol% acetonitrile to obtain a crude peptide solution; purifying the obtained crude peptide solution by chromatographic gradient elution, wherein a solution, which is prepared by regulating the pH value of a water solution consists of 0.1-0.4 vol% sulfuric acid and 0.1-0.4 vol% perchloric acid to 2.5-3.5 with ammonia water, is used as a phase A, acetonitrile is used as a phase B, the elution gradient B% is 20-45%; salting out; carrying out salt conversion and desalting on the salted sample on an octadecylsilane bonded silica gel chromatographic column by chromatography; by using a 0.1-0.8 vol% ammonium acetate water solution as a phase A and acetonitrile as a phase B, carrying out gradient elution at the elution gradient B% of 3-10% for 15-30 minutes; carrying out salt conversion elution by using a hydrochloric acid water solution-acetonitrile system.
Description
Technical field
The present invention relates to a kind of purification process of hydrochloric acid glucagon.Particularly, the present invention relates to that a kind of yield simple to operate, high, purity are high, the purification process of the hydrochloric acid glucagon that is conducive to realize industrialization.
Background technology
Glucagon (glucagon) also claims glucagon or synalbumin or insulin B.It is to follow Regular Insulin by a kind of hormone of the alpha Cell of islet secretion of vertebrates pancreas.Resist mutually with Regular Insulin, play a part to increase blood sugar.The straight-chain polypeptide that glucagon is comprised of 29 amino acid, molecular weight is 3485, it is by a macromolecular precursor cracking.Current domestic two launch that have are respectively the injection biosynthesizing glucagon of Denmark Novo Nordisk Co.,Ltd and the hydrochloride for injection glucagon of Shenzhen writing brush space.It has higher biological activity under the condition of hydrochloride.
The production of glucagon at present mainly adopts biological recombination method, and its yield is not high, and purity is also lower, generally in 90% left and right.About the document of glucagon artificial synthesis aspect seldom, the document about purifying does not almost have.
The object of this invention is to provide that a kind of yield simple to operate, high, purity are high, the purification process of the hydrochloric acid glucagon that is conducive to realize industrialization.
Summary of the invention
For achieving the above object, consider the character of hydrochloric acid glucagon itself, the invention provides a kind of purification process of hydrochloric acid glucagon, comprise the following steps:
-with the aqueous solution of the acetonitrile of the acetic acid of volume ratio 30%-60% and 5%-20%, dissolve thick peptide, obtain thick peptide solution;
-the thick peptide solution of gained is adopted to chromatography gradient elution purifying, take the sulfuric acid of volume ratio 0.1%-0.4% and the aqueous solution of volume ratio 0.1%-0.4% perchloric acid is A phase with the solution after ammoniacal liquor adjust pH 2.5-3.5, acetonitrile is B phase, gradient: B%:20%-45%, preferably 20%-40% wash-out, collects the glucagon sulfate liquor that wash-out obtains;
-gained glucagon sulfate liquor is saltoutd;
-in octadecylsilane chemically bonded silica chromatographic column, adopt reversed-phased high performace liquid chromatographic that the sample after saltouing is turned to salt and desalination, take 0.1～0.8%(w/v) ammonium acetate aqueous solution be A phase, acetonitrile is B phase, gradient: B%:3%～10% gradient or etc. degree rinse after 15～30 minutes, then use aqueous hydrochloric acid--acetonitrile system desalting treatment.
Particularly, the present invention proposes a kind of purification process of hydrochloric acid glucagon, product purity that the method obtains is high and yield good, can reach industrialized requirement.
In one embodiment, the purification process of a kind of glucagon hydrochloride provided by the invention comprises the following steps:
Step 1): the aqueous solution with the acetonitrile of the acetic acid of volume ratio 30%-60% and 5%-20% dissolves thick peptide according to concentration 20g/L-50g/L, water is diluted to 10%-30% by the two bulk volume fraction of the acetic acid in the thick peptide solution of gained and acetonitrile.Step 1) is below also expressed as " pre-treatment ".
Described " thick peptide " refer to and adopt liquid phase synthesizing method or solid-phase synthesis to obtain, and not yet passes through the hydrochloric acid glucagon that the thick peptide of hydrochloric acid glucagon of refinement treatment or purity can not fulfilling medicinals, and in thick peptide, the purity of hydrochloric acid glucagon is more than 20%.
Described " thick peptide solution " refers to thick peptide resulting solution after pre-treatment, and the water using is pure water, and meets water for injection standard, preferably ultrapure water; The acetic acid using is analytically pure Glacial acetic acid, the preferred chromatographically pure of purity grade of " acetonitrile " of the present invention.
The aqueous solution solvent systems of the acetic acid of selection volume ratio 30%-60% and 5%-20% acetonitrile is by simultaneous test and considers that the easy to operate of actual production process draws, the dissolving power of the solvent in this concentration range is not understood step-down, cause the required volume of dissolution of sample dissolution excessive, be unfavorable for the processing (can cause volume overload, purification efficiency is low) of reverse-phase chromatography.
The concentration of the thick peptide of preparation is controlled to 20g/L-50g/L and is conducive to purification efficiency, in the time of can causing follow-up reverse-phase chromatography purifying higher than 50g/L, do not hang chromatographic column, lower than 20g/L, can make purification efficiency low.
Water is diluted to 10%-30% by the two bulk volume fraction of the acetic acid in the thick peptide solution of gained and acetonitrile, when can causing follow-up reverse-phase chromatography purifying higher than the solvent of 30% this scope, do not hang the bulk volume fraction of acetic acid and acetonitrile chromatographic column, when the bulk volume fraction of acetic acid and acetonitrile can make sample separate out lower than 10%, cause follow-up reverse-phase chromatography purifying to carry out.
Step 2): the thick peptide solution of gained is carried out to chromatography gradient elution purifying, the aqueous solution (the volume ratio of the sulfuric acid with 0.1%-0.4% and 0.1%-0.4% perchloric acid, with ammoniacal liquor adjust pH 2.5-3.5) be A phase, acetonitrile is B phase, gradient: B%:20%-45%, preferred 20%-40%, the glucagon sulfate liquor of collection purifying; Step 2) be below also expressed as " purifying ".In a preferred embodiment, described pH is 3.0.
Two kinds of acid of sulfuric acid and 0.1%-0.4% perchloric acid of selection 0.1%-0.4% generate two kinds of buffering salts A after regulating with ammoniacal liquor is the result of optimized choice mutually.In mobile phase A, the concentration of buffering salt dies down moving phase surge capability lower than limited range, cause sample completely the distortion of wash-out and spectrogram do not reach separated requirement.Be greater than this scope, in mobile phase A, the excessive concentration of buffering salt is larger to chromatographic system damage, even can partly produce and saltout, and causing cannot purifying.In addition, the pH value of mobile phase A is also extremely important, in 2.5-3.5 scopes, does not reach separating effect.
In elution system, the ratio of A phase and B phase is sieved preferably and is obtained by great many of experiments, and B% can not rinse lower chromatographic column lower than 20% sample, and B% is greater than 45% sample and can goes out in advance, all can not carry out purifying.Adopt this solvent systems purifying, separating effect can significantly improve compared with prior art, yield 90% left and right of this step, and the purity of gained sample, more than 98%, improves yield and purity greatly, has reduced production cost.
Described step 2) in, use and take the chromatographic column that octadecylsilane chemically bonded silica is stationary phase.In a preferred embodiment, pillar diameter and the length of described chromatographic column are preferably: 5cm * 25cm, 10cm * 25cm or 15cm * 25cm.
Step 3) adopts the method for saltouing to carry out further purifying to glucagon sample, and its purity is reached more than 99%.Described step 3) is below also expressed as " saltouing ".
Described " saltouing " refers to by controlling step 2) the pH value of acetonitrile concentration and solution in the qualified solution of gained, the process that glucagon sample is separated out, described qualified solution refers to the solution that sample purity is greater than 95%.In a preferred embodiment, described saltouing comprises: the method for revolving steaming by normal temperature is by step 2) ratio of acetonitrile is down to below 15% in the qualified solution of gained, after again its pH value being adjusted to 6.0～7.0 with 20% weak ammonia, be placed into 2-8 degree Celsius 2～20 hours, it is separated out.Again turbid solution is carried out to centrifugal treating, supernatant liquor is as liquid waste disposal; After lower floor's solid is dissolved with the aqueous solution of 60%～100% perchloric acid, then proceed to step 4) with after one times of water for injection dilution.
This step improves the purity of glucagon sample, by the purity of glucagon sample from being greater than 95% to being greater than 99.0%.In addition, use salt analysis method to there is very large cost advantage, the cost that the cost of saltouing is processed well below reverse-phase chromatography, particularly particularly evident when large-scale production.
Step 4) adopts reversed-phased high performace liquid chromatographic that the vitriol of glucagon is changed into hydrochloride.Step 4) is below also expressed as " turning salt ".
The stationary phase of described " reversed-phased high performace liquid chromatographic " is octadecylsilane chemically bonded silica.In a preferred embodiment, the described salt method that turns comprises: by the glucagon sample loading of step 3) gained, then take 0.1～0.8%(w/v) ammonium acetate aqueous solution be A phase, acetonitrile is B phase, gradient: B%:3%～10% gradient or etc. degree rinse 15～30 minutes; Then use aqueous hydrochloric acid acetonitrile system wash-out, the concentration of the described aqueous hydrochloric acid that wherein used is 0.1%-0.4%(w/v), be preferably 0.2%(w/v).The acetonitrile concentration that this elution step adopts is 40～80%, collects hydrochloric acid glucagon solution, concentrated, obtains hydrochloric acid glucagon after lyophilize.
In described step 4), use and take the chromatographic column that eight alkyl silane bonded silica gels are stationary phase.In a preferred embodiment, the pillar diameter of described chromatographic column and length are: 5cm * 25cm, 10cm * 25cm or 30cm * 25cm.
Method operation feasible, the purity of purifying hydrochloric acid glucagon provided by the invention high (can reach more than 99.0% and maximum arbitrary impurity is not more than 0.1%), yield high (purification yield can reach more than 80%), reach industrialized requirement (a batch can obtain 200 grams of above smart peptides).
The present invention adopts reverse-phase chromatography and the way of the combination of saltouing is carried out purification process, is applicable to purity at the purifying of more than 20% thick peptide sample, and has very large cost advantage, particularly particularly evident when large-scale production.In addition, the hydrochloric acid glucagon purity that the present invention obtains more than 99.0%, has higher degree compared with biosynthesizing compared with Gao Keda.Purification process of the present invention has significantly improved the quality of product compared with prior art, and the also not rising of purifying cost, and yield is also significantly higher.
The purity of the product that employing the inventive method obtains is more than 99.0%, and purification yield can reach more than 80%.And can accomplish scale production, single batch of output can reach more than 200 grams.Can significantly improve the quality of product and reduce costs.
Accompanying drawing explanation
Fig. 1 is the purifying color atlas that shows hydrochloric acid glucagon purity.
Embodiment
With reference to following examples, the present invention may be better understood.However, it should be understood that, following examples are only for illustrating object, and should not be understood to limit the scope of the invention by any way.
Embodiment
Embodiment 1
1) pre-treatment: the thick peptide obtaining according to the concentration soluble chemistry synthesis method of 20g/L with the acetonitrile solution of the acetic acid of volume ratio 30% and 5%, stir and make sample dissolve the rear inclined to one side fluorine membrane filtration with φ 0.45 μ completely, collect filtrate.By purified water, the two bulk volume fraction of the acetic acid in thick peptide solution and acetonitrile is diluted to 30% standby.Wherein said thick peptide is to adopt conventional solid-phase synthesis to obtain.
2) purifying:
Purification condition: chromatographic column: the chromatographic column that the octadecylsilane chemically bonded silica of take is stationary phase, pillar diameter and length are: 5cm * 25cm.Moving phase: A phase: the aqueous solution of 0.1% sulfuric acid and 0.4% perchloric acid (v/v), with ammoniacal liquor adjust pH 2.5; B phase: acetonitrile, flow velocity: 50-100ml/ minute, gradient: B%:20%～40%(raises 1% in every 3 minutes), 60 minutes, detect wavelength: 280nm.Sample size is 1.0-2.5g.
Purge process: rinse chromatographic column well back balance loading with the acetonitrile of volume ratio 40～60%, applied sample amount is 1.0-2.5g.Linear gradient elution 60 minutes, collect object peak (being maximum peak 30～35 minutes), detect its chromatographic purity, after purity is greater than to more than 95% sample and merges, under the condition of 35 ℃ of water temperatures, on Rotary Evaporators, vacuum rotary steam is concentrated, is concentrated into residue and approximately after half volume, proceeds to the purification step of saltouing.
3) purifying of saltouing
By the solution after concentrated with after ammoniacal liquor adjust pH to 6.0, be placed into 2～8 degrees Celsius lower 5 hours.Again turbid solution is carried out to centrifugal treating, centrifugal condition: 2500 revs/min, 5 minutes, room temperature.Supernatant liquor is as liquid waste disposal; After lower floor's solid is dissolved with the high chloro acid solution of 60 volume %, with after one times of water for injection dilution, proceed to salt step.
4) turn salt: chromatographic column: the chromatographic column that the octadecylsilane chemically bonded silica of take is stationary phase, pillar diameter and length are: 5cm * 25cm.Rinse the acetum of chromatographic column more than 50% acetonitrile by volume ratio well rear loading, applied sample amount 1.0-2.5g, with B, be acetonitrile mutually, A is 0.1%(w/v mutually) the chromatographic condition of ammonium acetate aqueous solution, according to B%, be 3%(volume ratio) condition rinse 25-35 minute, then with B, be acetonitrile mutually, A is 0.1% aqueous hydrochloric acid chromatographic condition mutually, the condition wash-out that is 5% according to B% is after 10 minutes, use B% instead and be 40% condition wash-out, collect object peak, the glucagon solution of collection is no more than under 35 ℃ of conditions to vacuum rotary steam on Rotary Evaporators in water temperature and is concentrated into about 50-200mg/mL, then go to suitable big or small cillin bottle.After lyophilize, can obtain the hydrochloric acid glucagon that purity is greater than 99.0%.
Embodiment 2
1) pre-treatment: the thick peptide obtaining according to the concentration soluble chemistry synthesis method of 30g/L with the acetonitrile solution of the acetic acid of volume ratio 40% and 10%, stir and make sample dissolve the rear inclined to one side fluorine membrane filtration with φ 0.45 μ completely, collect filtrate.Water is diluted to the volume ratio sum of the acetic acid in thick peptide solution and acetonitrile 10% standby.
2) purifying:
Purification condition: chromatographic column: the chromatographic column that the octadecylsilane chemically bonded silica of take is stationary phase, pillar diameter and length are: 10cm * 25cm.Moving phase: A phase: 0.2% sulfuric acid and 0.3% high chloro acid solution (v/v), with ammoniacal liquor adjust pH 3.0; B phase: acetonitrile, flow velocity: 150-300ml/ minute, gradient: B%:20%～40%(raises 1% in every 3 minutes), 60 minutes, detect wavelength: 280nm.Sample size is 10-25g.
Purge process: by chromatographic column by volume ratio more than 50% acetonitrile rinse back balance loading well, applied sample amount is 5-15g.Linear gradient elution 60 minutes, collect object peak (being maximum peak 30-35 minute), detect its chromatographic purity, purity is greater than after more than 95% merging in water temperature, to be no more than under the condition of 35 ℃ vacuum rotary steam concentrated, be concentrated into residue and approximately after half volume, proceed to the purification step of saltouing.
3) purifying of saltouing
By ammoniacal liquor adjust pH to 6.5 for the solution after concentrated, after, be placed into 2～8 degrees Celsius lower 6 hours.Again turbid solution is carried out to centrifugal treating, centrifugal condition: 2500 revs/min, 5 minutes, room temperature.Supernatant liquor is as liquid waste disposal; After lower floor's solid is dissolved with 80% high chloro acid solution, with after one times of water for injection dilution, proceed to salt step.
4) turn salt: chromatographic column: the chromatographic column that the octadecylsilane chemically bonded silica of take is stationary phase, pillar diameter and length are: 10cm * 25cm.By chromatographic column by volume ratio more than 50% acetonitrile acetum rinse rear loading well, applied sample amount 10-25g, with B, be acetonitrile mutually, A is the chromatographic condition of 0.3% ammonium acetate aqueous solution mutually, the condition that is 5% according to B% is rinsed 25-35 minute, then with B, be acetonitrile mutually, A is the chromatographic condition of 0.2% aqueous hydrochloric acid mutually, the condition wash-out that is 5% according to B% is after 10 minutes, use B% instead and be 40% condition wash-out, collect object peak, the glucagon solution of collection is no more than under 35 ℃ of conditions after vacuum rotary steam is concentrated into about 50-200mg/mL and goes to suitable big or small cillin bottle in water temperature.After lyophilize, can obtain the hydrochloric acid glucagon that purity is greater than 99.0%.
Embodiment 3
1) pre-treatment: the thick peptide obtaining according to the concentration soluble chemistry synthesis method of 50g/L with the acetonitrile solution of the acetic acid of volume ratio 60% and 20%, stir and make sample dissolve the rear inclined to one side fluorine membrane filtration with φ 0.45 μ completely, collect filtrate.Water is diluted to the volume ratio sum of the acetic acid in thick peptide solution and acetonitrile 20% standby.
2) purifying:
Purification condition: chromatographic column: the chromatographic column that the octadecylsilane chemically bonded silica of take is stationary phase, pillar diameter and length are: 10cm * 25cm.Moving phase: A phase: 0.4% sulfuric acid and 0.1% high chloro acid solution (v/v), with ammoniacal liquor adjust pH 3.5; B phase: acetonitrile, flow velocity: 300-600ml/ minute, gradient: B%:20%～40%(raises 1% in every 3 minutes), detect wavelength: 280nm.Sample size is 10-25g.
Purge process: by chromatographic column by volume ratio more than 50% acetonitrile rinse back balance loading well, applied sample amount is 10-25g.Linear gradient elution 60 minutes, collect object peak (being maximum peak 30-35 minute), detect its chromatographic purity, purity is greater than after more than 95% merging in water temperature, to be no more than under the condition of 35 ℃ vacuum rotary steam concentrated, be concentrated into residue and approximately after half volume, proceed to the purification step of saltouing.
3) purifying of saltouing
By ammoniacal liquor adjust pH to 7.0 for the solution after concentrated, after, be placed into 2～8 degrees Celsius lower 6 hours.Again turbid solution is carried out to centrifugal treating, centrifugal condition: 2500 revs/min, 5 minutes, room temperature.Supernatant liquor is as liquid waste disposal; After lower floor's solid is dissolved with 100% high chloro acid solution, with after one times of water for injection dilution, proceed to salt step.
4) turn salt: chromatographic column: the chromatographic column that the octadecylsilane chemically bonded silica of take is stationary phase, pillar diameter and length are: 15cm * 25cm.By chromatographic column by volume ratio more than 50% acetonitrile acetum rinse rear loading well, applied sample amount 10-25g, with B, be acetonitrile mutually, A is the chromatographic condition of 0.8% ammonium acetate aqueous solution mutually, the condition that is 10% according to B% is rinsed 25-35 minute, then with B, be acetonitrile mutually, A is the chromatographic condition of 0.4% aqueous hydrochloric acid mutually, the condition wash-out that is 5% according to B% is after 10 minutes, use B% instead and be 50% condition wash-out, collect object peak, the glucagon solution of collection is no more than under 35 ℃ of conditions after vacuum rotary steam is concentrated into about 50-200mg/mL and goes to suitable big or small cillin bottle in water temperature.After lyophilize, can obtain the hydrochloric acid glucagon that purity is greater than 99.0%.
Claims (10)
1. a purification process for hydrochloric acid glucagon, comprises the following steps:
1) pre-treatment: dissolve thick peptide with the acetic acid of volume ratio 30%-60% and 5%-20% acetonitrile solution according to concentration 20g/L-50g/L, water is diluted to 10%-30% by the two bulk volume fraction of the acetic acid in thick peptide solution and acetonitrile;
2) purifying: the thick peptide solution of step 1) gained is carried out to chromatography gradient elution purifying, take the sulfuric acid of volume ratio 0.1%-0.4% and the aqueous solution of volume ratio 0.1%-0.4% perchloric acid is A phase with the solution after ammoniacal liquor adjust pH 2.5-3.5, acetonitrile is B phase, gradient: B%:20%-40% wash-out, the glucagon sulfate liquor of collection purifying;
3) gained glucagon sulfate liquor is saltoutd;
4) turn salt: by reversed-phased high performace liquid chromatographic, the vitriol sample of step 3) gained glucagon is turned to salt, by the glucagon sample loading of step 3) gained, then 0.1%-0.8% the ammonium acetate aqueous solution of take is A phase, acetonitrile is B phase, gradient: B% be 3%-10% gradient or etc. degree rinse 15-30 minutes; With aqueous hydrochloric acid acetonitrile system wash-out, the concentration of described aqueous hydrochloric acid is 0.1%-0.4%, and the acetonitrile concentration that this elution step adopts is more than 40%, collects hydrochloric acid glucagon solution.
2. method claimed in claim 1, wherein said step 2), the pH of A phase is 3.0.
3. the chromatography the method described in claim 1 or 2, wherein said step 2) is used chromatographic column, and its diameter and length are 5cm * 25cm, 10cm * 25cm or 15cm * 25cm.
4. it is stationary phase that the chromatography the method described in any one in claim 1-3, wherein said step 2) is used octadecylsilane chemically bonded silica.
5. the method described in any one in claim 1-4, saltouing of wherein said step 3) comprises:
The method of revolving steaming by normal temperature is by step 2) ratio of acetonitrile is down to below 15% in gained solution,
After the pH value of gained solution is adjusted to 6.0～7.0 with weak ammonia, be placed into 2-8 degree Celsius 2～20 hours, obtain the turbid solution that wherein glucagon sample is separated out
Gained turbid solution is carried out to centrifugal treating, and supernatant liquor, as liquid waste disposal, after lower floor's solid is dissolved with 60%～100% high chloro acid solution, then proceeds to step 4) after diluting one times with water for injection.
6. method claimed in claim 5, wherein said weak ammonia concentration is 20%.
7. the method described in any one in claim 1-6, the chromatography in wherein said step 4) is used chromatographic column, and its diameter and length are 5cm * 25cm, 10cm * 25cm or 30cm * 25cm.
8. the method described in any one in claim 1-7, it is stationary phase that the chromatography in wherein said step 4) is used octadecylsilane chemically bonded silica.
9. the method described in any one in claim 1-8, in wherein said step 4), Spirit of Mindererus is 0.3%.
10. the method described in any one in claim 1-9, the concentration of aqueous hydrochloric acid described in wherein said step 4) is 0.2%.
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| EP3517543A1 (en) | 2018-01-30 | 2019-07-31 | Bachem Holding AG | Manufacture of glucagon peptides |
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| CN113624898B (en) * | 2021-08-23 | 2023-08-25 | 成都诺和晟泰生物科技有限公司 | Purification method of chiral analgesic polypeptide medicine |
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