CN106478804A - A kind of method of purifying salmon calcitonin - Google Patents
A kind of method of purifying salmon calcitonin Download PDFInfo
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- CN106478804A CN106478804A CN201611049524.5A CN201611049524A CN106478804A CN 106478804 A CN106478804 A CN 106478804A CN 201611049524 A CN201611049524 A CN 201611049524A CN 106478804 A CN106478804 A CN 106478804A
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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
- C07K14/585—Calcitonins
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Abstract
The invention discloses a kind of method that utilization reversed-phased high performace liquid chromatographic purifies salmon calcitonin, mainly solves the technical problem currently without a method for being suitable to industrialized production purifying acetic acid redfish calcitonin.Technical solution of the present invention comprises the steps：1）By salmon calcitonin crude product solution with membrane filtration after, with C8 or C18 reverse phase silica gel post, mobile phase A mutually be 0.1% aqueous formic acid of percent by volume, B phase be 0.1% formic acid acetonitrile of percent by volume, carry out gradient elution collect target peak；2）Acetate is changed into using HPLC；3）Freeze-drying after resulting solution vacuum rotary steam is concentrated obtains powder finished product peptide.Method of the present invention process is simple, environmental pollution are low, and high income is easy to industrializing implementation.
Description
Technical field
The invention belongs to reverse-phase HPLC technique field, especially relates to the side that a kind of mass industrialization purifies salmon calcitonin
Method.
Background technology
Salmon calcitonin, English name：Calcitonin salmon, molecular formula：C145H240NllO9S2, molecular weight 3431.85,
Its peptide sequence is such as：H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-
His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser -Gly-Thr-Pro-NH2 .
Acetic acid redfish calcitonin can suppress the activity of osteoclast；Suppression bone salts dissolving, prevents calcium in bone from disengaging；Improve bone
Density, effective pain of alleviation symptom；Reduce the danger of fracture and reduce blood calcium.Therefore clinically it is mainly used in treating old bone
Matter osteoporosis, PMO, fracture move cancer hypercalcinemia.
Industrialized purification process is not found in the document that has delivered, is therefore groped one and is suitable to industrialized production acetic acid salmon
The method of calcitonin is necessary.
Content of the invention
It is an object of the invention to provide a purification process for being suitable to large-scale industrial production salmon calcitonin, main solution
Certainly currently without the technical problem of a method for being suitable to industrialized production purifying acetic acid redfish calcitonin.
For achieving the above object, technical scheme is as follows：
A kind of method of purifying salmon calcitonin, comprises the steps：
1）By salmon calcitonin crude product solution with membrane filtration after, with C8 or C18 reverse phase silica gel post, mobile phase A mutually be volume basis
Than 0.1% aqueous formic acid, B phase is 0.1% formic acid acetonitrile of percent by volume, carries out gradient elution and collects target peak；
2) acetate is changed into using HPLC；
3）Freeze-drying after resulting solution vacuum rotary steam is concentrated obtains powder finished product peptide.
The filter membrane is 0.45 μm of aperture filter membrane.
The gradient elution：B%：Bulk concentration gradient 10-50%, 30-50min, Detection wavelength are 205nm.
Described HPLC turns salt method：The acetum of percent by volume 0.3% and the two phase liquid of trifluoroacetic acid aqueous solution, chromatogram
Post is C8 or C18 reverse phase silica gel post.
The invention has the beneficial effects as follows：A purification technique for being suitable to industrialization salmon calcitonin proposed by the present invention.
Salmon calcitonin produces new impurity in oxidizing process and is very difficult to remove, and the present invention is using acetonitrile as having in HPLC method purge process
Machine solvent flow phase, and turn salt with HPLC, fine peptide of the HPLC purity more than 99.0% can not only be obtained, and effectively can be reduced
The toxicity of organic solvent in production process, reduces cost, so as to reach the purpose of large-scale industrial production.
Description of the drawings
Fig. 1 is the high-efficient liquid phase chromatogram of salmon calcitonin.
Specific embodiment
Embodiment 1
1. sample treatment
In salmon calcitonin oxidation solution, add a little acetonitrile ultrasound to clarify to liquid, be 0.45um membrane filtration standby with aperture
With.
2. purify
Purification condition：Chromatographic column is C8 reverse phase silica gel post, and diameter and length are：5cm × 25cm, mobile phase：A phase：Volume basis
Than 0.1% aqueous formic acid；B phase：0.1% formic acid acetonitrile solution of percent by volume.Flow velocity：60-80mL/min.Detection wavelength：
205nm.Gradient：B%：Bulk concentration gradient 10-50%, 30-50min.Sample size is 2.5-4g.
Purge process：Balanced each other chromatographic column with the B of volumetric concentration 10%, applied sample amount is 0.5-1.5L sample solution.Linear ladder
Degree wash-out collects target peak, the peptide solution for gathering is placed standby.
3. salt is turned
It is neutral with deionized water rinsing C18 reverse phase silica gel post to the efflux of volumetric concentration more than 60%, with percent by volume
0.3% acetate aqueous solution and trifluoroacetic acid aqueous solution solution equilibria chromatographic column, the target peak solution that loading has been gathered.Linear gradient
Wash-out：10-30min, the acetate aqueous solution of bulk concentration gradient 90-95%；30-90min, bulk concentration gradient 30-90%'s
Acetonitrile solution, collects whole target peaks.
4. the solution of collection is carried out being evaporated to 1g/50mL, thickening temperature is less than 37 DEG C, and then freeze-drying is obtained
Salmon calcitonin of the purity more than 99.0%, yield 32.3%.Product chromatogram is shown in Fig. 1.
Embodiment 2
1. sample treatment
In salmon calcitonin oxidation solution, add a little acetonitrile ultrasound to clarify to liquid, be 0.45um membrane filtration standby with aperture
With.
2. purify
Purification condition：Chromatographic column is C18 reverse phase silica gel post, and diameter and length are：10cm × 25cm, mobile phase：A phase：Volume hundred
Divide 0.1% aqueous formic acid of ratio；B phase：0.1% formic acid acetonitrile solution of percent by volume.Flow velocity：180-200mL/min.Detection ripple
Long：205nm.Gradient：B%：Bulk concentration gradient 10-50%, 30-50min.Sample size is 5-10g.
Purge process：Balanced each other chromatographic column with the B of volumetric concentration 10%, applied sample amount is 1.5-2.5L sample solution.Linear ladder
Degree wash-out collects target peak, the peptide solution for gathering is placed standby.
3. salt is turned
It is neutral with deionized water rinsing C18 reverse phase silica gel post to the efflux of volumetric concentration more than 60%, with percent by volume
0.3% acetate aqueous solution and trifluoroacetic acid aqueous solution solution equilibria chromatographic column, the target peak solution that loading has been gathered.Linear gradient
Wash-out：10-30min, the acetate aqueous solution of bulk concentration gradient 90-95%；30-90min, bulk concentration gradient 30-90%'s
Acetonitrile solution, collects whole target peaks.
4. the solution of collection is carried out being evaporated to 1g/50mL, thickening temperature is less than 37 DEG C, and then freeze-drying is obtained
Salmon calcitonin of the purity more than 99.0%, yield 31.6%.Product chromatogram is shown in Fig. 1.
Embodiment 3
1. sample treatment
In salmon calcitonin oxidation solution, add a little acetonitrile ultrasound to clarify to liquid, be 0.45um membrane filtration standby with aperture
With.
2. purify
Purification condition：Chromatographic column is C18 reverse phase silica gel post, and diameter and length are：15cm × 25cm, mobile phase：A phase：Volume hundred
Divide 0.1% aqueous formic acid of ratio；B phase：0.1% formic acid acetonitrile solution of percent by volume.Flow velocity：280-300mL/min.Detection ripple
Long：205nm.Gradient：B%：Bulk concentration gradient 10-50%, 30-50min.Sample size is 12-18g.
Purge process：Balanced each other chromatographic column with the B of volumetric concentration 10%, applied sample amount is 2.0-3.0L sample solution.Linear ladder
Degree wash-out collects target peak, the peptide solution for gathering is placed standby.
3. salt is turned
It is neutral with deionized water rinsing C18 reverse phase silica gel post to the efflux of volumetric concentration more than 60%, with percent by volume
0.3% acetate aqueous solution and trifluoroacetic acid aqueous solution solution equilibria chromatographic column, the target peak solution that loading has been gathered.Linear gradient
Wash-out：10-30min, the acetate aqueous solution of bulk concentration gradient 90-95%；30-90min, bulk concentration gradient 30-90%'s
Acetonitrile solution, collects whole target peaks.
4. the solution of collection is carried out being evaporated to 1g/50mL, thickening temperature is less than 37 DEG C, and then freeze-drying is obtained
Salmon calcitonin of the purity more than 99.0%, yield 30.8%.Product chromatogram is shown in Fig. 1.
Claims (4)
1. a kind of purifying salmon calcitonin method, it is characterised in that adopt reversed-phased high performace liquid chromatographic, comprise the steps：
1）By salmon calcitonin crude product solution with membrane filtration after, with C8 or C18 reverse phase silica gel post, mobile phase A mutually be volume basis
Than 0.1% aqueous formic acid, B phase is 0.1% formic acid acetonitrile of percent by volume, carries out gradient elution and collects target peak；
2) acetate is changed into using HPLC；
3）Freeze-drying after resulting solution vacuum rotary steam is concentrated obtains powder finished product peptide.
2. the method for purifying salmon calcitonin according to claim 1, it is characterised in that：The step 1）Middle filter membrane is aperture
0.45 μm of filter membrane.
3. the method for purifying salmon calcitonin according to claim 1, it is characterised in that：The step 1）Middle gradient elution flowing
The bulk concentration gradient of phase B is 10-50%, and Detection wavelength is 205nm.
4. the method for purifying salmon calcitonin according to claim 1, it is characterised in that：Described HPLC turns salt method, mobile phase
Acetum for percent by volume 0.3% and trifluoroacetic acid aqueous solution solution, chromatographic column are C8 or C18 reverse phase silica gel post.
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| CN201611049524.5A CN106478804A (en) | 2016-11-25 | 2016-11-25 | A kind of method of purifying salmon calcitonin |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107090018A (en) * | 2017-03-09 | 2017-08-25 | 吉尔生化（上海）有限公司 | A kind of purification process of the polypeptide containing 24 amino acid residues |
| CN110746482A (en) * | 2019-11-29 | 2020-02-04 | 安徽科门生物科技有限公司 | Method for purifying transdermal peptide |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103254305A (en) * | 2013-05-31 | 2013-08-21 | 青岛国大生物制药股份有限公司 | Preparation method of acetic acid redfish calcitonin |
| CN104672320A (en) * | 2015-03-18 | 2015-06-03 | 深圳翰宇药业股份有限公司 | Pure solid-phase synthesis method for salmon calcitonin acetate |
-
2016
- 2016-11-25 CN CN201611049524.5A patent/CN106478804A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103254305A (en) * | 2013-05-31 | 2013-08-21 | 青岛国大生物制药股份有限公司 | Preparation method of acetic acid redfish calcitonin |
| CN104672320A (en) * | 2015-03-18 | 2015-06-03 | 深圳翰宇药业股份有限公司 | Pure solid-phase synthesis method for salmon calcitonin acetate |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107090018A (en) * | 2017-03-09 | 2017-08-25 | 吉尔生化（上海）有限公司 | A kind of purification process of the polypeptide containing 24 amino acid residues |
| CN110746482A (en) * | 2019-11-29 | 2020-02-04 | 安徽科门生物科技有限公司 | Method for purifying transdermal peptide |
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