CN107698656A - A kind of purification process of hydrophobic peptides raw material - Google Patents
A kind of purification process of hydrophobic peptides raw material Download PDFInfo
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- CN107698656A CN107698656A CN201710309690.2A CN201710309690A CN107698656A CN 107698656 A CN107698656 A CN 107698656A CN 201710309690 A CN201710309690 A CN 201710309690A CN 107698656 A CN107698656 A CN 107698656A
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
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Abstract
The step of being collected the invention discloses dissolving, forward chromatographic column analysis and the absorption of preparation chromatographic column and elution of a kind of purification process of hydrophobic peptides raw material, including polypeptide raw material.The step of dissolving, includes polypeptide raw material being dissolved in organic solvent obtaining polypeptide material solution；The step of forward chromatographic column analysis, includes polypeptide material solution connecing positive facies analysis chromatographic column progress gradient detection, obtains analyzing collection of illustrative plates；Described the step of preparing chromatographic column absorption and eluting the preparation gradient collected including setting preparation chromatographic column according to the analysis collection of illustrative plates and polypeptide material solution is connect into the preparation chromatographic column progress sample introduction absorption and elutes collection, obtain hydrophobic peptides raw material after purification.The dissolving that solves the problems, such as strong-hydrophobicity raw material during large-scale purification in purge process is difficult, anti-phase preparation when the bad elution of sample and the rate of recovery it is low.
Description
Technical field
The present invention relates to technical field of polypeptide, more particularly to the purification process of strong-hydrophobicity polypeptide raw material.
Background technology
The macromolecular substances for the advantages that polypeptide (Peptide) is active high, rapid-action, degree of safety is high, Small side effects,
Huge work is played in the field such as antitumor, antiviral, antibacterial, vaccine by biological expression or the polypeptide drugs of chemical synthesis
With.With the development of medical science and biochemical technology, polypeptide drug will turn into 21 century and most important monitor, prevents, diagnoses and control
Medicine is treated, in recent years, international drugs general layout has huge change, and the exploitation of small molecule chemical drug is increasingly difficult to, the market share shared by it
Constantly nibbled by institute of Biological Products, wherein polypeptide drugs development is the swiftest and the most violent.At present, nearly 60 polypeptide products of global approved
Listing, the market of global polypeptide drugs is more than 20,000,000,000 dollars.So the synthesis of polypeptide and purification process research are with important
Meaning.
At present, purified polypeptide is more using reversed-phase preparative chromatography, because the raw material of synthesis polypeptide carries blocking group,
Such as the raw material such as Fmoc-cys (pal)-OH, Fmoc-asp-AMC, there is strong-hydrophobicity, water and acetonitrile can not be well crude products
Dissolving has to reverse-phase chromatographic column and greatly injured, greatly reduce reverse-phase chromatographic column, it is necessary to add acid, alkali or DMSO hydrotropies
Service life.Crude product is not easily dissolved, can be relatively difficult on sample adsorption to chromatographic column when purifying especially large-scale purification.
Polypeptide containing protection group retention time in reverse-phase chromatographic column (C18, C8, C4) is oversize, it is not easy to is eluted out, it is possible to
Cause sample to be spread in preparation, cause peak type wider, inferior separating effect, yield is relatively low.It is strong hydrophobic using the purifying of positive phase system
Property raw material, improve the efficiency of purifying and the rate of recovery of target substance, reduce amino acid waste and solvent consumption.
In view of it is above-mentioned the defects of, the design people, be actively subject to research and innovation, to found a kind of hydrophobic peptides raw material
Purification process, make it with more the value in industry.
The content of the invention
In order to solve the above technical problems, solved it is an object of the invention to provide one kind molten in hydrophobic peptides purge process
The difficult purification process with the rate of recovery the problem of low of solution.
The present invention hydrophobic peptides raw material purification process, including polypeptide raw material dissolving, forward chromatographic column analysis and
Positive prepares the step of chromatographic column absorption and elution collection.
Further, the step of dissolving includes polypeptide raw material being dissolved in organic solvent that to obtain polypeptide raw material molten
Liquid.
Further, the step of forward chromatographic column analysis is entered including polypeptide material solution is connect into positive facies analysis chromatographic column
Row gradient detects, and obtains analyzing collection of illustrative plates.
Positive analysis condition is：
Chromatographic column is itrile group post, 5 μm, 4.6*250mm of pillar model Haobo CN,
Wherein, mobile phase：A liquid：N-hexane, B liquid：Isopropanol；
Time：0-20min；
Flow velocity：1.00mL/min；
A：B is：99-90:1-10 to 1-10:99-90；
Wavelength：200-240nm.
Before forward chromatographic column analytical procedure, first HPLC reverse-phase chromatography analytical columns are changed into normal-phase chromatography analytical column, including
Following steps：
(a) reverse-phase chromatographic column rinsed is laid down, connects bilateral, chromatographic system 10min (flow velocity 1- are rinsed with isopropanol
10mL/min), it is empty during this period to dial sampling valve 3 times.
(b) n-hexane or ethyl acetate rinse system 20min (flow velocity 1-10mL/min) are changed, it is empty during this period to dial sampling valve
3 times；
(c) silica gel chromatographic column (can also be itrile group chromatographic column or amino chromatographic column) is loaded onto, flow velocity adjusts 1-10mL/min, uses
N-hexane rinse-system, until system balancing.
Initial Gradient balances chromatographic column, and sample introduction 15-25 μ L carry out gradient detection, preferably 20 μ L.
The eluent gradient of the chromatographic column is A:B is by 99-90:1-10 to 50-70:30-50.
Further, the absorption of preparation chromatographic column and elution collection comprise the following steps：
(1) the preparation gradient for preparing chromatographic column is set according to the analysis collection of illustrative plates.
(2) polypeptide material solution is connect into the preparation chromatographic column progress sample introduction absorption and elutes collection.
(3) all peaks being eluted out are collected and carry out mass spectrum confirmation and product purity detection.
Positive preparation condition is：
Chromatographic column is itrile group post, pillar model Haobo 5 μm of 20*250mm of CN,
Wherein, mobile phase：A liquid：N-hexane, B liquid：Isopropanol；
Time：0-40min；
Flow velocity：20.00mL/min；
A：B is：99-90:1-10 to 50-70:50-30；
Wavelength：200-240nm.
Product purity detection condition is：
Chromatographic column is C18 posts, pillar model Innoval 5 μm of 4.6*250mm of ODS-2,
Wherein, mobile phase：A liquid：Concentration is 0.1-0.2% trifluoroacetic acid aqueous solutions, B liquid：Acetonitrile；
Time：0-40min；
Flow velocity：1.00mL/min；
0-20min A：B is：50-70:50-30 to 1-10：99-90；
20-40min A：B is：1-10：99-90 to 1-10：99-90；
Wavelength：200-240nm.
Before the preparation chromatographic column absorption and elution collection step, the reverse-phase chromatographic column rinsed is first laid down, including with
Lower step：
(I) reverse-phase chromatographic column rinsed is first laid down, connects bilateral, chromatographic system 10min (flow velocitys are rinsed with isopropanol
20-100mL/min), it is empty during this period to dial sampling valve 3 times.
(II) n-hexane or ethyl acetate rinse system 20min (flow velocity 20-100mL/min) are changed, sky is dragged on during this period
Sample valve 3 times.
(III) silica gel chromatographic column (can also be itrile group chromatographic column or amino chromatographic column) is loaded onto, flow velocity adjusts 20-100mL/
Min, with n-hexane rinse-system, until system balancing.
Further, in step (2), the flow velocity of the pump for preparing chromatographic column is 5-50ml/min.
Further, the solution of elution is collected, most of organic solvent, preferably 38 DEG C are removed at 20-50 DEG C.
Removing organic solvent mode includes revolving etc..
Polypeptide material sample is freezed, weighs quality, calculated yield, product purity detects again.
Further, the hydrophobic peptides raw material is the polypeptide raw material with protection group.
Further, the hydrophobic peptides raw material has following amino acid sequence：
Fmoc-cys (pal)-OH, Fmoc-asp-AMC, Fmoc-arg (pbf)-OH or Fmoc-cys (Trt)-OH.
Further, the dissolving crude product concentration of Fmoc-cys (the pal)-OH is 10-100mL, n-hexane and isopropanol
Mixed solution dissolving 100-200mgFmoc-cys (pal)-OH crude products.
Wherein, the volume ratio of n-hexane and isopropanol is 1:2-9.
Further, the preparation chromatographic column is itrile group post, nh 2 column, silicagel column, bonding amide groups silicagel column and bonding
One or more in urea groups silicagel column.
Further, analytical column is one kind in n-hexane, ethyl acetate, isopropanol and ethanol with the mobile phase for preparing post
It is or several.
Further, the organic solvent includes one in n-hexane, isopropanol, ethyl acetate, chloroform and dichloromethane
Kind is several.
Further, the detector of the gradient detection is UV-detector, and Detection wavelength is 200-240nm.
By such scheme, the present invention at least has advantages below：
1st, the present invention proposes a kind of method for purifying strong-hydrophobicity polypeptide raw material with positive phase system, is solved in purge process
Determined large-scale purification when strong-hydrophobicity raw material dissolving is difficult, anti-phase preparation when the bad elution of the sample and rate of recovery is low asks
Topic；
2nd, diffusion of the sample in reversed-phase column is reduced, improves the yield of product, the production cycle is reduced, and reduces energy consumption
And production cost, can a step realize the purifying of product, it is quickly, easy, it is efficiently gentle, shorten operating time and operability；
3rd, purification process provided by the invention, can be widely used in indissoluble peptide purification, this method is very high
Operability, there is very high yield, directly and simply can be amplified to production capacity from research and development scale, be solved in purge process
The problem of crude product indissoluble solution of having determined and anti-phase difficult elution, it is not necessary to acid adding, alkali, DMSO dissolvings, avoid reverse-phase chromatographic column and connect
Thick soda acid and situation about degenerating occurs, reduce the consumption of solvent, while the operating time can be shortened, shorten production week
Phase, so as to improve production efficiency.
Described above is only the general introduction of technical solution of the present invention, in order to better understand the technological means of the present invention,
And can be practiced according to the content of specification, below with presently preferred embodiments of the present invention and coordinate accompanying drawing describe in detail as after.
Brief description of the drawings
Fig. 1 is the positive analysis chart of Fmoc-cys (pal)-OH in embodiments of the invention 1；
The positive that Fig. 2 is Fmoc-cys (pal)-OH in embodiments of the invention 1 prepares figure；
Fig. 3 is the mass spectral analysis figure of Fmoc-cys (pal)-OH sterlings in embodiments of the invention 1；
Fig. 4 is the inverse analysis figure of Fmoc-cys (pal)-OH sterlings in embodiments of the invention 1.
Fig. 5 is the positive analysis chart of Fmoc-cys (pal)-OH in embodiments of the invention 2；
The positive that Fig. 6 is Fmoc-cys (pal)-OH in embodiments of the invention 2 prepares figure；
Fig. 7 is the mass spectral analysis figure of Fmoc-cys (pal)-OH sterlings in embodiments of the invention 2；
Fig. 8 is the inverse analysis figure of Fmoc-cys (pal)-OH sterlings in embodiments of the invention 2.
Embodiment
With reference to the accompanying drawings and examples, the embodiment of the present invention is described in further detail.Implement below
Example is used to illustrate the present invention, but is not limited to the scope of the present invention.
Example 1：
By 1mg Fmoc-cys (pal)-OH 1mL n-hexanes and isopropyl alcohol mixture (v:V=1:9) dissolve, filtering,
Gradient detection and analysis are carried out using 5 μm of 20*250mm of itrile group post Haobo CN.Positive analysis condition is：Mobile phase A liquid is just
Hexane, B liquid are isopropanol；Time：0-20min；Flow velocity：1.00mL/min；A：B is：99:1 to 5:95；Wavelength：220nm.Point
Analysis result is as shown in figure 1, show that the flowing matches Fmoc-cys (pal)-OH analysis, the peak energy in crude product accesses very well
Separation.
The dissolving and processing of Fmoc-cys (pal)-OH crude products：200mg Fmoc-cys (pal)-OH crude products with 20mL just oneself
Alkane isopropyl alcohol mixture (v:V=1:9) ultrasonic dissolution, after dissolving completely, centrifugation, and with after 0.45 μm of organic phase filter membrane filtering
Loading is waited to prepare.
LC3000I high performance liquid chromatograph of the chromatographic column for innovation Tong Heng companies is prepared, filtrate sample introduction is pure with positive phase system
Change, chromatographic column is 5 μm of 20*250mm of Haobo CN.Wherein, in mobile phase：Solvent system is：A liquid is n-hexane, and B liquid is different
Propyl alcohol, 0-40min, A:B is by 99:1 to 60：40 gradient elutions, flow velocity 20mL/min；Wavelength 220nm, collect elution solution.System
It is standby as shown in Fig. 2 showing to match Fmoc-cys (pal)-OH preparation using the flowing, the peak energy in crude product accesses very well
Separation.
The eluting peak mass spectrum of collection confirms and uses reverse-phase chromatographic column purity assay, and mass spectral analysis is as shown in figure 3, show to collect
The eluting peak arrived is target peak (Fmoc-cys (pal)-OH molecular weight is 581.81).Finished product inverse analysis chromatographic condition is innovation
The LC3000I high performance liquid chromatographs of Tong Heng companies, chromatographic column are Innoval ODS-2 5um 4.6*250mm.Wherein, flow
Phase：Concentration is 0.05-0.2% trifluoroacetic acid aqueous solutions (A liquid) and acetonitrile (B liquid)；0 to 20 minutes, A:B is by 70:30 to 10:
90,20 to 40 minutes, A:B is by 10:90 to 1:99；Flow velocity 1mL/min；Wavelength 220nm.Inverse analysis is as shown in figure 4, show to make
Standby obtained finished product purity is 96.92%, and this method can obtain the higher product of purity.
Revolving：Temperature setting is 38 DEG C, rotates out the organic solvent of wherein most.
Lyophilized detection：Polypeptide material sample is freezed, weighs quality, calculated yield, product purity detects again, after freezing
Sample yield is 81%.
Example 2：
1mg Fmoc-cys (pal)-OH 1mL n-hexane isopropyl alcohol mixtures (v:V=1:9) dissolve, filter, use
5 μm of 20*250mm of itrile group post Haobo CN carry out gradient detection and analysis.Positive analysis condition is：Mobile phase A liquid is n-hexane,
B liquid is isopropanol；Time：0-20min；Flow velocity：1.00mL/min；A：B is：95:5 to 5:95；Wavelength：220nm.Analysis result
As shown in figure 5, show that the flowing matches Fmoc-cys (pal)-OH analysis, the peak energy in crude product accesses well point
From.
The dissolving and processing of Fmoc-cys (pal)-OH crude products：400mg Fmoc-cys (pal)-OH crude products with 30mL just oneself
Alkane and isopropyl alcohol mixture (v:V=1:9) ultrasonic dissolution, after dissolving completely, centrifugation, and filtered with 0.45 μm of organic phase filter membrane
Loading is waited to prepare afterwards.
LC3000I high performance liquid chromatograph of the chromatographic column for innovation Tong Heng companies is prepared, filtrate sample introduction is pure with positive phase system
Change, chromatographic column is 5 μm of 20*250mm of Haobo CN.Wherein, in mobile phase：Solvent system is：A liquid is n-hexane, and B liquid is different
Propyl alcohol, 0-40min A:B is by 95:5 to 70：30 gradient elutions, flow velocity 20mL/min；Wavelength 220nm, collect elution solution.System
It is standby as shown in fig. 6, showing that using above-mentioned mobile phase and gradient Fmoc-cys (pal)-OH can be prepared, the peak energy in crude product
Access good separation.
The eluting peak mass spectrum of collection confirms and uses reverse-phase chromatographic column purity assay, and mass spectral analysis is as shown in fig. 7, show to collect
The eluting peak arrived is target peak (Fmoc-cys (pal)-OH molecular weight is 581.81).Finished product inverse analysis chromatographic condition is innovation
The LC3000I high performance liquid chromatographs of Tong Heng companies, chromatographic column are Innoval ODS-2 5um 4.6*250mm.Wherein, flow
Phase：Concentration is 0.05-0.2% trifluoroacetic acid aqueous solutions (A liquid) and acetonitrile (B liquid)；0 to 20 minutes, A:B is by 50:50 to 1:99,
20 to 40 minutes, A:B is by 1:99 to 1:99；Flow velocity 1mL/min；Wavelength 220nm.Inverse analysis is as shown in figure 8, show to be prepared into
The finished product purity arrived is 97.67%, and this method can obtain the higher product of purity.
Revolving：Temperature setting is 38 DEG C, rotates out the organic solvent of wherein most.
Lyophilized detection：Polypeptide material sample is freezed, weighs quality, calculated yield, product purity detects again, after freezing
Sample yield is 75%.
Described above is only the preferred embodiment of the present invention, is not intended to limit the invention, it is noted that for this skill
For the those of ordinary skill in art field, without departing from the technical principles of the invention, can also make it is some improvement and
Modification, these improvement and modification also should be regarded as protection scope of the present invention.
Claims (10)
- A kind of 1. purification process of hydrophobic peptides raw material, it is characterised in that：Dissolving, forward chromatographic column including polypeptide raw material point Analysis and positive prepare chromatographic column absorption and elute the step of collecting.
- 2. purification process according to claim 1, it is characterised in that：The step of dissolving, is included polypeptide dissolution of raw material Polypeptide material solution is obtained in organic solvent.
- 3. purification process according to claim 2, it is characterised in that：The step of forward chromatographic column analysis, includes be more Peptide material solution connects positive facies analysis chromatographic column and carries out gradient detection, obtains analyzing collection of illustrative plates.
- 4. purification process according to claim 3, it is characterised in that the absorption of preparation chromatographic column and elution collection include Following steps：(1) the preparation gradient for preparing chromatographic column is set according to the analysis collection of illustrative plates；(2) polypeptide material solution is connect into the preparation chromatographic column progress sample introduction absorption and elutes collection.
- 5. purification process according to claim 1, it is characterised in that：The hydrophobic peptides raw material is with protection group Polypeptide raw material.
- 6. purification process according to claim 5, it is characterised in that：The hydrophobic peptides raw material has following amino Acid sequence：Fmoc-cys (pal)-OH, Fmoc-asp-AMC, Fmoc-arg (pbf)-OH or Fmoc-cys (Trt)-OH.
- 7. purification process according to claim 1, it is characterised in that：The preparation chromatographic column is itrile group post, nh 2 column, silicon Glue post, bonding amide groups silicagel column and the one or more being bonded in urea groups silicagel column.
- 8. purification process according to claim 1, it is characterised in that：Analytical column and prepare the mobile phase of post for n-hexane, One or more in ethyl acetate, isopropanol and ethanol.
- 9. purification process according to claim 2, it is characterised in that：The organic solvent includes n-hexane, isopropanol, second One or more in acetoacetic ester, chloroform and dichloromethane.
- 10. purification process according to claim 3, it is characterised in that：The detector of the gradient detection is ultraviolet detection Device, and Detection wavelength is 200-240nm.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108864249A (en) * | 2018-06-19 | 2018-11-23 | 南京肽业生物科技有限公司 | A kind of purification process of hydrophobic peptides |
| CN109053859A (en) * | 2018-08-09 | 2018-12-21 | 吉尔生化（上海）有限公司 | A kind of purification process for neutral hydrophilic polypeptide |
| CN111624287A (en) * | 2020-05-28 | 2020-09-04 | 江苏吉泰肽业科技有限公司 | Detection method of insoluble polypeptide |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108864249A (en) * | 2018-06-19 | 2018-11-23 | 南京肽业生物科技有限公司 | A kind of purification process of hydrophobic peptides |
| CN109053859A (en) * | 2018-08-09 | 2018-12-21 | 吉尔生化（上海）有限公司 | A kind of purification process for neutral hydrophilic polypeptide |
| CN111624287A (en) * | 2020-05-28 | 2020-09-04 | 江苏吉泰肽业科技有限公司 | Detection method of insoluble polypeptide |
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Application publication date: 20180216 |