CN103421081A - Method for removing trifluoroacetic acid in polypeptide - Google Patents
Method for removing trifluoroacetic acid in polypeptide Download PDFInfo
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- CN103421081A CN103421081A CN2012101626831A CN201210162683A CN103421081A CN 103421081 A CN103421081 A CN 103421081A CN 2012101626831 A CN2012101626831 A CN 2012101626831A CN 201210162683 A CN201210162683 A CN 201210162683A CN 103421081 A CN103421081 A CN 103421081A
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- polypeptide
- trifluoroacetic
- moving phase
- trifluoroacetic acid
- acetonitrile
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Abstract
The invention provides a method for removing trifluoroacetic acid in polypeptide. The method comprises steps: trifluoroacetic acid-containing polypeptide passes through a reversed phase chromatographic column, and the mobile phases are sodium acetate aqueous solution and acetonitrile; the chromatographic column is washed until the peak of trifluoroacetic acid comes out. The method employs simple devices, has concise production technology, and can remove trifluoroacetic acid in polypeptide effectively.
Description
Technical field
The present invention relates to a kind of high performance liquid chromatography separation method of organic compound, especially relate to a kind of organic acid RPLC removal method in polypeptide compound.
Background technology
Trifluoracetic acid (Trifluoroacetic acid is translated as again trifluoroacetic acid) be Solid-phase Polypeptide synthetic in Fmoc(9-fluorenes carbometoxyl) lytic reagent commonly used in the protection strategy, be also ion pair reagent commonly used in moving phase in the peptide purification process.Trifluoroacetic acidity is stronger, and the salify of generally being combined with polypeptide exists.
Yet, because trifluoracetic acid is harmful, must be after purifying by desalination or change into acetate and remove.
Document (J. Pept. Sci. 2008; 14:354-359) report is removed trifluoroacetic method generally three kinds:
The first, adopt reversed-phased high performace liquid chromatographic, moving phase is 1% aqueous acetic acid, through the wash-out of long period, removes trifluoracetic acid;
The second, adopt ion-exchange-resin process, use reinforcing yin essence ion exchange resin (AG1-X8, quaternary ammonium), after resin is repeatedly cleaned with acetic acid, polypeptide solution is poured on resin, through the Stirring of 1 hour, filter, collect filtrate, can remove trifluoracetic acid;
The 3rd, the proton method of deprotonation/again, be dissolved in polypeptide in the deionized water of 4 ℃, and the sodium hydroxide solution that slowly adds 100 mM is 11 to the pH value of solution, and solution keeps 10 minutes at 4 ℃.In this condition, polypeptide will not dissolve and produce precipitation, by suspension 4 ℃ centrifugal, remove the supernatant liquor that contains sodium trifluoroacetate, particulate matter is dispersed in to all sodium trifluoroacetates of centrifugal removal in 6 times of amount deionized waters, can obtain the polypeptide of neutral form.
Patent CN101372506A discloses the employing ion exchange chromatography and has removed trifluoroacetic method in Eptifibatide.
Patent CN101696236A discloses the employing high performance liquid chromatography and has removed trifluoroacetic method in Atosiban, the aqueous acetic acid that moving phase used is 0.3% and acetonitrile mixed solution.
Patent CN101525382A discloses trifluoroacetic method in the removal tripro-amylin, first with aqueous phosphatic and acetonitrile, as mobile mutual-assistance trifluoroacetate, is converted into phosphoric acid salt, then with anion-exchange chromatography, phosphate transfection is turned to acetate.
In sum, trifluoroacetic method in polypeptide be can industrialization remove and RP-HPLC method and anion-exchange chromatography mainly contained.Two kinds of methods are compared, and the RP-HPLC method has more advantage.At first, the RP-HPLC method does not need to increase the operation that new equipment can complete the removal trifluoracetic acid and change into acetate; Secondly, when anion-exchange chromatography is crossed post, medicine contacts with ion exchange resin, may cause the resin stain liquid, thereby introduces impurity; The 3rd, anion-exchange chromatography has increased processing step, can cause production cost to increase.
In practice, we find that also there is certain problem in the RP-HPLC method while adopting aqueous acetic acid and acetonitrile.If it is lower that aqueous acetic acid concentration is crossed, the aqueous acetic acid such as 0.3%, through wash-out, can't remove trifluoracetic acid fully; If the aqueous acetic acid excessive concentration, particularly surpass after 1%, can cause the hangover of polypeptide chromatographic peak, affect yield.Therefore, need a kind of efficient, simple reversed-phased high performace liquid chromatographic to remove the trifluoracetic acid in polypeptide.
Summary of the invention
In order to solve the problems of the technologies described above, the inventor has carried out lot of experiments, finds that adopting sodium acetate aqueous solution and acetonitrile mixed solution is moving phase, does not need to increase new equipment and can complete the operation of removing trifluoracetic acid and changing into acetate.
The method of this method and bibliographical information is compared, just aqueous acetic acid is changed and become sodium acetate aqueous solution, principle is that sodium-acetate is strong base-weak acid salt, concentration is that the following sodium acetate aqueous solution pH value of 1 mol/L is 7-8, slightly be alkalescence, be under acidic conditions with the vinegar vinegar aqueous solution that to compare acetate ion concentration higher, more easily the trifluoracetic acid negatively charged ion submitted and changed from reverse phase filler.In addition, under alkaline condition, the trifluoracetic acid negatively charged ion is difficult for forming trifluoracetic acid, more easily from chromatographic column, elutes.
The method that we use is, adopts rp-hplc system, and after sample introduction, take sodium acetate aqueous solution and acetonitrile mixed solution is moving phase, after the trifluoracetic acid that is eluted to sample goes out peak, gets final product.
In test, we find, in highly effective liquid phase chromatographic system moving phase, the concentration of sodium acetate soln is that 0.1 mol/L to 1 mol/L is good; Acetonitrile in moving phase: the volume ratio of sodium acetate soln is that 1/99-1/1 is good; Chromatographic column can be selected C4(butyl bonded silica gel according to the difference of polypeptide retention time), C8(heptan alkyl linked silica gel) or the C18(octadecyl silane) reverse-phase chromatographic column.
Trifluoracetic acid is as residual solvent, and in American Pharmacopeia 34 editions, acetic acid gonadorelin (Gonadorelin Acetate) stipulates that its limit must not be 0.25%.In the present invention, trifluoroacetic assay has adopted the acetometry in the synthetic polypeptide of two appendix VII N of Chinese Pharmacopoeia version in 2010, and has carried out corresponding methodology checking, and result shows that this method also is applicable to trifluoroacetic mensuration in synthetic polypeptide.
Concrete grammar is as follows:
It is appropriate that trifluoracetic acid is got in the preparation of reference substance solution, accurately weighed, with the quantitative dilution of the mixing solutions of mobile phase A-Mobile phase B (95:5), makes the solution that approximately contains 0.02 mg in every 1 ml.
It is appropriate that polypeptide is got in the preparation of need testing solution, accurately weighed, and the mixing solutions of mobile phase A-Mobile phase B (95:5) quantitatively dilution is made the solution that approximately contains 5 mg in every 1 ml.
Chromatographic condition and system suitability octadecyl silane are weighting agent (250mm * 4.6mm, 5 μ m); With phosphoric acid solution (add phosphoric acid 0.7ml in 1000ml water, with sodium hydroxide test solution, regulate pH value to 3.0), it is mobile phase A; Methyl alcohol is Mobile phase B; Flow velocity is per minute 1.2ml; The detection wavelength is 210nm.According to the form below carries out gradient elution.Number of theoretical plate calculates and should be not less than 2000 by the trifluoracetic acid peak.The retention time at trifluoracetic acid peak was about 2 ~ 5 minutes.After gradient elution starts, polypeptide is by wash-out, and baseline sharply raises.
| Time/minute | Mobile phase A/% | Mobile phase B/% |
| 0~5 | 95 | 5 |
| 5~10 | 50 | 50 |
| 10~20 | 50 | 50 |
| 20~22 | 95 | 5 |
| 22~30 | 95 | 5 |
The assay method precision measures reference substance solution and each 10 μ l of need testing solution, and the injection liquid chromatography, record color atlas respectively, presses external standard method with trifluoroacetic content in the calculated by peak area polypeptide.
Embodiment
Following examples, only for the present invention is illustrated, are not intended to limit scope of the present invention.
Below for adopting prior art and the inventive method to remove trifluoroacetic comparative example in desmopressin acetate.
Embodiment 1
Learn from else's experience containing the desmopressin acetate solution after trifluoroacetic moving phase purifying, adopt reversed-phased high performace liquid chromatographic, the C18(octadecyl silane) reverse-phase chromatographic column, moving phase is 0.1 mol/L aqueous acetic acid-acetonitrile (95:5), and the wash-out of process long period is until trifluoracetic acid goes out peak.After the sample freeze-drying, according to trifluoroacetic content in two appendix VII working samples of the version in 2010 of the Chinese Pharmacopoeia after above-mentioned checking, result is 1.2%, far above the trifluoroacetic limit of acceptable (0.25%).
Learn from else's experience containing the desmopressin acetate solution after trifluoroacetic moving phase purifying, adopt reversed-phased high performace liquid chromatographic, the C18(octadecyl silane) reverse-phase chromatographic column, moving phase is 0.25mol/L sodium acetate soln-acetonitrile (95:5), the process wash-out is until trifluoracetic acid goes out peak.After the sample freeze-drying, according to trifluoroacetic content in two appendix VII working samples of the version in 2010 of the Chinese Pharmacopoeia after above-mentioned checking, result is not for detecting.
In above-mentioned comparative example 1, employing be that aqueous acetic acid-acetonitrile system is as moving phase; In comparative example 2, employing be sodium acetate soln-acetonitrile; Result shows that the ability that comparative example 2 is removed trifluoroacetic acid obviously is better than comparative example 1.
Embodiment 2
Learn from else's experience containing the Somatostatin solution after trifluoroacetic solution cracking (cutting peptide) oxidation, adopt reversed-phased high performace liquid chromatographic, the C18(octadecyl silane) reverse-phase chromatographic column, moving phase is 0.5mol/L sodium acetate soln-acetonitrile (95:5), the process wash-out is until trifluoracetic acid goes out peak.Sample is purified, after freeze-drying according to trifluoroacetic content in two appendix VII working samples of the version in 2010 of the Chinese Pharmacopoeia after above-mentioned checking, result is not for detecting.
Embodiment 3
Learn from else's experience containing the desmopressin acetate solution after trifluoroacetic moving phase purifying, adopt reversed-phased high performace liquid chromatographic, C4(butane group bonded silica gel) reverse-phase chromatographic column, moving phase is 0.75mol/L sodium acetate soln-acetonitrile (95:5), the process wash-out is until trifluoracetic acid goes out peak.After the sample freeze-drying, according to trifluoroacetic content in two appendix VII working samples of the version in 2010 of the Chinese Pharmacopoeia after above-mentioned checking, result is not for detecting.
Embodiment 4
Learn from else's experience containing the desmopressin acetate solution after trifluoroacetic moving phase purifying, adopt reversed-phased high performace liquid chromatographic, C8(heptan alkyl linked silica gel) reverse-phase chromatographic column, moving phase is 1mol/L sodium acetate soln-acetonitrile (95:5), through wash-out until trifluoracetic acid goes out peak.After the sample freeze-drying, according to trifluoroacetic content in two appendix VII working samples of the version in 2010 of the Chinese Pharmacopoeia after above-mentioned checking, result is not for detecting.
Embodiment 5
Learn from else's experience containing the desmopressin acetate solution after trifluoroacetic moving phase purifying, adopt reversed-phased high performace liquid chromatographic, C4(butyl bonded silica gel) reverse-phase chromatographic column, moving phase is 1mol/L sodium acetate soln-acetonitrile (99:1), the process wash-out is until trifluoracetic acid goes out peak.After the sample freeze-drying, according to trifluoroacetic content in two appendix VII working samples of the version in 2010 of the Chinese Pharmacopoeia after above-mentioned checking, result is not for detecting.
Embodiment 6
Learn from else's experience containing the desmopressin acetate solution after trifluoroacetic moving phase purifying, adopt reversed-phased high performace liquid chromatographic, the C18(octadecyl silane) reverse-phase chromatographic column, moving phase is 1mol/L sodium acetate soln-acetonitrile (50:50), the process wash-out is until trifluoracetic acid goes out peak.After the sample freeze-drying, according to trifluoroacetic content in two appendix VII working samples of the version in 2010 of the Chinese Pharmacopoeia after above-mentioned checking, result is not for detecting.
Embodiment 7
Learn from else's experience containing the desmopressin acetate solution after trifluoroacetic moving phase purifying, adopt reversed-phased high performace liquid chromatographic, the C18(octadecyl silane) reverse-phase chromatographic column, moving phase is 0.5mol/L sodium acetate soln-acetonitrile (95:5), the process wash-out is until trifluoracetic acid goes out peak.After the sample freeze-drying, according to trifluoroacetic content in two appendix VII working samples of the version in 2010 of the Chinese Pharmacopoeia after above-mentioned checking, result is not for detecting.
Above-mentioned test explanation, no matter be the trifluoracetic acid of introducing after polypeptide cracking (cutting peptide), or the trifluoracetic acid of introducing in the peptide purification process, employing the inventive method all can be effectively by its removal, and the inventive method is simple, does not need to increase new installation.
Claims (5)
1. one kind adopts the RP-HPLC method to remove trifluoroacetic method in polypeptide, it is characterized in that moving phase is sodium acetate soln and acetonitrile.
2. employing RP-HPLC method as claimed in claim 1 is removed trifluoroacetic method in polypeptide, it is characterized in that, in described moving phase, the concentration of sodium acetate soln is 0.1mol/L to 1mol/L.
3. employing RP-HPLC method as claimed in claim 1 or 2 is removed trifluoroacetic method in polypeptide, it is characterized in that, in described moving phase, the volume ratio of acetonitrile is 1% ~ 50%.
4. employing RP-HPLC method as claimed in claim 1 or 2 is removed trifluoroacetic method in polypeptide, it is characterized in that, in described moving phase, the volume ratio of sodium acetate soln is 50% ~ 99%.
5. RP-HPLC method as claimed in claim 1 or 2 is removed trifluoroacetic method in polypeptide, it is characterized in that, the filler type of described chromatographic column is C4(butyl bonded silica gel), C8(heptyl bonded silica gel) or the C18(octadecyl silane) reverse-phase chromatographic column.
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| CN2012101626831A CN103421081A (en) | 2012-05-24 | 2012-05-24 | Method for removing trifluoroacetic acid in polypeptide |
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| CN2012101626831A CN103421081A (en) | 2012-05-24 | 2012-05-24 | Method for removing trifluoroacetic acid in polypeptide |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109748954A (en) * | 2017-11-06 | 2019-05-14 | 正大天晴药业集团股份有限公司 | A kind of purification process of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007087253A2 (en) * | 2006-01-23 | 2007-08-02 | Unigene Laboratories, Inc. | Glyoxylate assays and their use for identifying natural amidated compounds |
| CN102382188A (en) * | 2011-11-07 | 2012-03-21 | 深圳翰宇药业股份有限公司 | Method for preparing carperitide acetate |
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- 2012-05-24 CN CN2012101626831A patent/CN103421081A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007087253A2 (en) * | 2006-01-23 | 2007-08-02 | Unigene Laboratories, Inc. | Glyoxylate assays and their use for identifying natural amidated compounds |
| CN102382188A (en) * | 2011-11-07 | 2012-03-21 | 深圳翰宇药业股份有限公司 | Method for preparing carperitide acetate |
Non-Patent Citations (2)
| Title |
|---|
| STEPHANE ROUX ET AL.: "Elimination and exchange of trifluoroacetate counter-ion from cationic peptides:a critical evaluation of different approaches", 《JOURNAL OF PEPTIDE SCIENCE》 * |
| WOJCIECH MROZIK ET AL.: "Determination of counter-ions in synthetic peptides by ion chromatography,capillary isotachophoresis and capillary electrophoresis", 《JOURNAL OF PEPTIDE SCIENCE》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109748954A (en) * | 2017-11-06 | 2019-05-14 | 正大天晴药业集团股份有限公司 | A kind of purification process of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 |
| CN109748954B (en) * | 2017-11-06 | 2022-03-01 | 正大天晴药业集团股份有限公司 | Purification method of degarelix |
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Application publication date: 20131204 |