CN112279895B - Preparation method of chemically synthesized acidic polypeptide - Google Patents
Preparation method of chemically synthesized acidic polypeptide Download PDFInfo
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Abstract
The invention relates to a preparation method for chemically synthesizing acidic polypeptide, which mainly solves the defects of difficult impurity purification and low yield of acidic polypeptide compounds. The purification method is a high performance liquid chromatography, RP-HPLC purification is carried out in two steps or three steps, a solvent used as a mobile phase for elution mainly comprises an ion pair reagent and an organic modifier, and an elution solvent is eluted in a gradient elution mode. The method can separate and purify the crude acid polypeptide without any purification in the whole synthesis and intermediate process, particularly the weak acid polypeptide with poor solubility, obtain the high-purity final product, ensure higher purification yield, have simple and convenient method, are easy to operate, save resources and are particularly suitable for industrial scale preparation.
Description
Technical Field
The invention relates to a preparation method of chemically synthesized acidic polypeptide, in particular to a method for purifying an acidic polypeptide compound synthesized by a chemical method by using reversed-phase preparative chromatography.
Technical Field
The chemically synthesized polypeptide compound has extremely high biological activity, requires a medicinal polypeptide compound, and needs to have extremely high compound purity and extremely low impurity content so as to effectively avoid the discomfort reaction of the patient caused by impurities clinically. During the chemical synthesis of polypeptide, a series of deletion, hydrolysis and racemization impurities are generated, the impurities can cause toxic and side effects of the drug due to the toxicity of the drug in clinical use or the biological activity which is not consistent with that of the expected polypeptide, and the impurities have the characteristics which are very similar to those of a target compound, are difficult to separate and are very serious in industrial preparation.
At the present stage, when an acidic polypeptide compound, particularly a weak acidic polypeptide with a large molecular weight and poor solubility, is separated and purified by adopting a preparative chromatography, most of the existing chromatographic column packing has a limited tolerance range to acid and alkali, the loss of a chromatographic column is very serious when alkaline eluent is used for elution, and the purpose of separating a high-purity target compound by adopting the acidic eluent is difficult to achieve. Acidic compounds are less soluble in acidic solutions than in alkaline solutions. The polypeptide shown in the formula I has very strong hydrophobicity due to the fact that the peptide chain is long, the side chain contains long alkyl modification and hydrophobic amino acid exists, the solubility of the polypeptide in an acidic system is very small, and the purification difficulty of the polypeptide in the acidic buffer system is very large.
In the existing literature, as patent CN103275209A, using 0.1% tfa/aqueous solution-0.1% tfa/acetonitrile solution as eluent, patent CN 201210029818.7, using isopropanol aqueous solution containing 0.1 to 0.2% trifluoroacetic acid as phase a, acetonitrile containing 0.1 to 0.2% trifluoroacetic acid as phase B, first-step purification is carried out, patent CN201410001671.X uses phase a: 0.1% TFA; phase B: acetonitrile was used as the first purification condition and patent CN201810663478.0 used phosphoric acid as the mobile phase. Most of the methods adopt acetic acid, trifluoroacetic acid and phosphoric acid as the components of the mobile phase, but the problems of low recovery rate and the like caused by the adsorption of a sample on a filler in the elution process are not suitable for preparing a mobile phase system; meanwhile, samples are separated out after the prepared and collected fractions are placed for a period of time, and are difficult to redissolve and clear, so that the method is not suitable for industrial production. There are also reports in the literature that purification is carried out using an alkaline mobile phase, but a special column is inevitably required, and the column involves a large number of types of packing and some of the packing is very expensive. Is not suitable for large-scale industrial production.
Disclosure of Invention
In view of the above, the present invention provides a method for purifying an acidic polypeptide compound synthesized by a chemical method by using reverse phase preparative chromatography, in order to solve the problems of poor solubility, difficulty in purifying impurities of a weakly acidic polypeptide compound, and low yield in the prior art.
In order to achieve the purpose, the technical scheme provided by the invention is as follows:
a process for the purification of highly pure acidic polypeptides from a mixture comprising said acidic polypeptides and related impurities, which process is a high performance liquid chromatography, RP-HPLC purification in two or three steps, the solvent used for elution consisting essentially of an ion pair reagent and an organic modifier, said elution solvent eluting in a gradient elution,
wherein the ion-pairing reagent is selected from NH 4 Cl，HCOOH，NH 4 HCO 3 TEAP solution;
wherein the organic modifier is selected from methanol, ethanol, n-propanol, isopropanol and acetonitrile.
In the chemical synthesis of polypeptide, the impurities are mainly byproducts, intermediates, degradation products, oxidation products, salts and various protecting groups in the reaction process. Among them, deletion peptides and racemic peptides are the main impurities to be removed.
In some embodiments of the invention, the acidic polypeptide has the structure:
the acidic polypeptide shown as formula I has a long peptide chain and a complex branched chain structure, and many impurities are generated in the synthesis process, so when the acidic polypeptide is used as a drug active ingredient with high purity requirement, the purity needs to be strictly controlled, and the synthesized crude peptide needs to be purified.
The acidic polypeptide of the invention, in particular to a weak acidic polypeptide with a peptide sequence containing amino acid with stronger hydrophobicity and a longer alkyl side chain modification which causes poor solubility. The elution is difficult in the purification process, so that the loss is caused, the purification difficulty is increased, and the purification yield of the acidic polypeptide is influenced. According to the invention, through RP-HPLC, an elution solution mainly composed of an ion pair reagent and an organic modifier is used alternately for elution, and the elution is carried out in two steps or three steps, so that most isomer impurities and other impurities which are difficult to separate in crude peptide to be purified can be separated and removed through gradient elution; and then, an ammonium bicarbonate solution is used as a mobile phase to carry out gradient elution, so that the difficult-to-remove and tiny impurities are removed, and the problems of difficult elution and low yield are effectively solved. Experimental results prove that the acidic polypeptide obtained by the purification method provided by the invention has high purity and yield, is simple and convenient to operate, and is beneficial to realizing large-scale preparation of the acidic polypeptide.
The combination and collocation scheme of the organic modifier and the concentration ratio of the organic modifier to the ion pair reagent are changed by changing the combination concentration of the organic modifier so as to achieve the maximum separation effect of certain specific impurities in the compound and the target compound in reversed-phase preparation.
Preferably, in the purification method provided by the invention, the elution solvent is NH 4 A mixed solution of a Cl aqueous solution and any one or more organic modifiers of methanol, ethanol, n-propanol, isopropanol and acetonitrile.
Preferably, in the purification method provided by the invention, the elution solvent is a mixed solution of HCOOH aqueous solution and any one or more organic modifiers selected from methanol, ethanol, n-propanol, isopropanol and acetonitrile.
Preferably, in the purification method provided by the invention, the elution solvent is NH 4 HCO 3 The aqueous solution and one or more organic modifiers selected from methanol, ethanol, n-propanol, isopropanol and acetonitrile.
Preferably, in the purification method provided by the invention, the elution solvent is a mixed solution of the TEAP solution and any one or more organic modifiers selected from methanol, ethanol, n-propanol, isopropanol and acetonitrile.
In some embodiments of the invention, RP-HPLC purification is performed in two steps, eluent 1 is NH 4 The mixed solution of Cl aqueous solution and organic modifier, and the eluent 2 is the mixed solution of HCOOH aqueous solution and organic modifier.
In some embodiments of the invention, the RP-HPLC purification is carried out in three steps, eluent 1 is a mixed solution of TEAP solution and organic modifier, eluent 2 is a mixed solution of TEAP solution and organic modifier, eluent 3 is NH 4 HCO 3 A mixed solution of an aqueous solution and an organic modifier.
In some embodiments of the invention, RP-HPLC purification is carried out in three steps, eluent 1 is NH 4 A mixed solution of Cl aqueous solution and an organic modifier, wherein the eluent 2 is a mixed solution of TEAP solution and the organic modifier,eluent 3 is NH 4 HCO 3 A mixed solution of an aqueous solution and an organic modifier.
In some embodiments of the invention, the RP-HPLC purification is carried out in three steps, eluent 1 is a mixed solution of HCOOH aqueous solution and organic modifier, eluent 2 is NH 4 Mixed solution of Cl aqueous solution and organic modifier, and NH 3 as eluent 4 HCO 3 A mixed solution of an aqueous solution and an organic modifier.
In some embodiments of the invention, RP-HPLC purification is carried out in three steps, eluent 1 is NH 4 Mixed solution of Cl aqueous solution and organic modifier, mixed solution of HCOOH aqueous solution and organic modifier as eluent 2, and NH as eluent 3 4 HCO 3 A mixed solution of an aqueous solution and an organic modifier.
The eluent 1 refers to the eluent which is used as a mobile phase during the first step of purification when RP-HPLC purification is carried out step by step; the eluent 2 is used as an eluent of a mobile phase during RP-HPLC purification step by step and during purification of the second step; and the eluent 3 is used as the eluent of the mobile phase during the step-by-step RP-HPLC purification and the third step purification, and so on.
Preferably, the RP-HPLC purification is performed stepwise, with NH 4 When Cl is used as the mobile phase, the pH is 7.0-10.0.
Preferably, the RP-HPLC purification is performed stepwise, with NH 4 When Cl is used as a mobile phase, the volume concentration is 50mmoL/L.
Preferably, the RP-HPLC purification is carried out stepwise with TEAP as the mobile phase and at a pH of 2.5.
Preferably, the RP-HPLC purification is carried out stepwise with TEAP as the mobile phase at a pH of 7.0.
Preferably, the RP-HPLC purification is carried out stepwise, with HCOOH as the mobile phase, at a mass concentration of 0.1 to 10%.
Preferably, the RP-HPLC purification is performed stepwise, with NH 4 HCO 3 When the solid phase is used as a mobile phase, the volume concentration is 1-100 mmoL/L.
In some embodiments of the invention, the organic modifier of eluent 1 or 2 is one or both of acetonitrile, n-propanol, and isopropanol in the stepwise RP-HPLC purification.
In some embodiments of the invention wherein the RP-HPLC purification is performed stepwise, the organic modifier of eluent 3 is n-propanol and/or isopropanol.
After the separation and purification of one or two ion pair reagents, isopropanol or normal propanol is adopted for separation and purification when the last ion pair reagent is used for separation and purification, the isopropanol or normal propanol has lower toxicity, and is used for replacing acetonitrile with higher toxicity in the purification process of the reversed phase polypeptide, so that the clinical risk of the medicine can be avoided.
Preferably, the purification method provided by the present invention is a purification method wherein the acidic polypeptide is purified using an ion pairing reagent having a pH at least 1 pH unit greater than the pKa of the acidic polypeptide compound being isolated.
The principle of reverse phase chromatography is adopted, the characteristic of high resolution of reverse phase silica gel is utilized, an ion pair reagent combination scheme is combined, the ion pair reagent is changed through different pH values, and the pH value of the used ion pair reagent is more than 1 pH value of the pKa value of the compound, so that the structural property of the compound is ensured.
Preferably, in the purification method provided by the invention, when the organic modifier is used as a mobile phase, the content range is 0% to 70%.
Preferably, in the purification method provided by the invention, the gradient range of the mobile phase containing the organic modifier is 20-60% (60 min).
Preferably, the purification method provided by the invention is characterized in that the packing of the chromatographic column of the reversed phase high performance liquid chromatography system (RP-HPLC) is C4, C6, C8 and C18 bonded silica gel or high molecular polymer.
The method can separate and purify the crude acid polypeptide without any purification in the whole synthesis and intermediate process, particularly the weak acid polypeptide with poor solubility, obtain the high-purity final product, ensure higher purification yield, have simple and convenient method, are easy to operate, save resources and are particularly suitable for industrial scale preparation.
Drawings
FIG. 1 is an HPLC chromatogram of the crude peptide of example 1.
FIG. 2 is an HPLC chromatogram of the fine peptide of example 1.
FIG. 3 is an HPLC chromatogram of the fine peptide of example 16.
FIG. 4 is an HPLC chromatogram of the fine peptide of example 17
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The following preparation method of the present invention is further described in detail with reference to specific examples to facilitate the further understanding of the present invention by those skilled in the art, but not to limit the scope of the claims. It is expressly intended that all such similar substitutes and modifications which would be obvious to those skilled in the art are deemed to be included within the invention.
Example 1: preparation of acidic polypeptide shown in formula I
(1) Solubilization and filtration of crude peptide
Weighing 1.03g of crude peptide, grinding, adding 200mL of solvent (acetonitrile: purified water =20, mixed at a volume ratio of 80) for ultrasonic dissolution, adding triethylamine to complete dissolution, adjusting pH to 7.51 with TFA, placing in a 40 ℃ water bath for 1.5h, filtering with a 0.45 μm organic filter membrane, and detecting the crude peptide solution, wherein the HPLC purity of the crude peptide is 71.86% (HPLC detection is shown in figure, table 1).
TABLE 1 HPLC profile data for crude peptides
| Peak number | Retention time | Area of | Area% |
| 1 | 7.477 | 113114 | 1.72 |
| 2 | 7.863 | 8691 | 0.13 |
| 3 | 7.990 | 8802 | 0.13 |
| 4 | 8.220 | 25467 | 0.39 |
| 5 | 8.643 | 5967 | 0.09 |
| 6 | 9.106 | 11030 | 0.17 |
| 7 | 9.607 | 16395 | 0.25 |
| 8 | 9.734 | 8424 | 0.13 |
| 9 | 10.019 | 7246 | 0.11 |
| 10 | 10.215 | 8641 | 0.13 |
| 11 | 10.635 | 111312 | 1.69 |
| 12 | 11.624 | 20236 | 0.31 |
| 13 | 12.091 | 96973 | 1.47 |
| 14 | 12.557 | 49926 | 0.76 |
| 15 | 12.86 | 89864 | 1.36 |
| 16 | 13.071 | 39795 | 0.60 |
| 17 | 13.529 | 23147 | 0.35 |
| 18 | 13.761 | 93692 | 1.42 |
| 19 | 14.499 | 4738261 | 71.86 |
| 20 | 14.854 | 16524 | 0.25 |
| 21 | 15.228 | 38227 | 0.58 |
| 22 | 15.663 | 23598 | 0.36 |
| 23 | 15.885 | 119872 | 1.82 |
| 24 | 16.514 | 92744 | 1.41 |
| 25 | 16.993 | 22970 | 0.35 |
| 26 | 17.510 | 23341 | 0.35 |
| 27 | 18.024 | 77706 | 1.18 |
| 28 | 18.713 | 18458 | 0.28 |
| 29 | 18.893 | 40006 | 0.61 |
| 30 | 19.579 | 28362 | 0.43 |
| 31 | 20.356 | 1576 | 0.02 |
| 32 | 20.808 | 4456 | 0.07 |
| 33 | 21.628 | 425596 | 6.45 |
| 34 | 22.734 | 5783 | 0.09 |
| 35 | 23.525 | 177785 | 2.70 |
(2) Ammonium chloride/acetonitrile as mobile phase for first washing
And loading the filtered crude peptide solution onto a chromatographic column through a sample injection pump, wherein the specification of the chromatographic column is 30 × 250mm, and 10 μm of C8 bonded silica gel filler is filled in the chromatographic column.
Mobile phase a with 50mM ammonium chloride ph8.5, mobile phase a with 50mM ammonium chloride ph8.5: acetonitrile =300 as mobile phase B, the elution flow rate was 25mL/min, and the gradient elution mode was: within 60min, the gradient is changed from 50% A +50% B (V/V) to 40% A +60% B (V/V), the mobile phase elution is started, the spectrum is collected, the change of the absorption value is observed, the main peak is collected and the purity is detected by an analysis liquid phase, the concentration of the acidic polypeptide intermediate shown in formula I is obtained after the collection, sample collection, rotary evaporation and concentration to remove acetonitrile.
(3) Second washing with HCOOH/acetonitrile + n-propanol as mobile phase
And loading the intermediate concentrated solution onto a chromatographic column through a sample injection pump, wherein the specification of the chromatographic column is 30 × 250mm, and 10 μm of C8 bonded silica gel filler is filled in the chromatographic column.
0.1% hcooh as mobile phase a, in n-propanol: acetonitrile =1 (V/V) as mobile phase B, the elution flow rate was 25mL/min, and the gradient elution mode was: within 60min, the gradient is changed from 67% A +33% B (V/V) to 37% A +63% B (V/V), mobile phase elution is started, a spectrum is collected, the change of an absorption value is observed, a main peak is collected and the purity is detected by an analysis liquid phase, fractions with the purity of more than 98% are collected and combined, and after sample evaporation and concentration, freeze drying is carried out, thus obtaining 205.3mg of the pure acid polypeptide product shown by the formula I, the purity is 99.46%, the maximum single impurity content is 0.19% (HPLC detection is shown in figure 2), the spectrum data are shown in table 2, and the total yield is 76.3%.
TABLE 2 Fine peptide HPLC profile data
| Number of peak | Retention time | Area of | Area% |
| 1 | 10.400 | 373 | 0.02 |
| 2 | 10.967 | 1144 | 0.07 |
| 3 | 11.771 | 2589 | 0.15 |
| 4 | 12.292 | 1707706 | 99.46 |
| 5 | 12.913 | 3259 | 0.19 |
| 6 | 13.321 | 1156 | 0.07 |
| 7 | 13.942 | 813 | 0.05 |
Example 2: preparation of acidic polypeptide shown in formula I
(1) Solubilization and filtration of crude peptide
1.05g of the crude peptide was weighed, crushed, added with 200mL of a solvent (acetonitrile: purified water =20, mixed at a volume ratio of 80) and dissolved by sonication, triethylamine was added until completely dissolved, ph7.55 was adjusted with TFA, and then placed in a 40 ℃ water bath for 1.5h, filtered using a 0.45 μm organic filter, and the crude peptide solution was checked for HPLC purity of 71.76%.
(2) Ammonium chloride/acetonitrile as mobile phase for first washing
And loading the filtered crude peptide solution onto a chromatographic column through a sample injection pump, wherein the specification of the chromatographic column is 30 × 250mm, and 10 μm of C8 bonded silica gel filler is filled in the chromatographic column.
Mobile phase a with 50mM ammonium chloride ph7.0, mobile phase a with 50mM ammonium chloride ph7.0: acetonitrile =300 as mobile phase B, the elution flow rate was 25mL/min, and the gradient elution mode was: within 60min, the gradient is changed from 48% A +52% B (V/V) to 38% A +62% B (V/V), the mobile phase elution is started, the spectrum is collected, the change of the absorption value is observed, the main peak is collected and the purity is detected by the analysis liquid phase, the concentration liquid of the acidic polypeptide intermediate shown in formula I is obtained after the collection and the sample rotary evaporation concentration to remove the acetonitrile.
(3) Second washing with HCOOH/acetonitrile + n-propanol as mobile phase
And loading the intermediate concentrated solution onto a chromatographic column through a sample injection pump, wherein the specification of the chromatographic column is 30 × 250mm, and 10 μm of C8 bonded silica gel filler is filled in the chromatographic column.
0.1% hcooh as mobile phase a, in n-propanol: acetonitrile =1 (V/V) as mobile phase B, the elution flow rate was 25mL/min, and the gradient elution mode was: within 60min, the gradient is changed from 67% A +33% B (V/V) to 37% A +63% B (V/V), mobile phase elution is started, the spectrum is collected, the change of the absorption value is observed, the main peak is collected and the purity is detected by an analysis liquid phase, the fractions with the purity of more than 98% are collected and combined, and freeze-dried after sample rotary evaporation concentration, the purity of the sample after freeze-drying is detected to be 99.02%, and the maximum single impurity is 0.34%, and the HPLC detection result chart is similar to that in FIG. 2.
Example 3: preparation of acidic polypeptide shown in formula I
(1) Solubilization and filtration of crude peptide
Weighing 1.02g of crude peptide, grinding, adding 200mL of solvent (acetonitrile: purified water =20, mixed at a volume ratio of 80) for ultrasonic dissolution, adding triethylamine to complete dissolution, adjusting pH to 7.49 with TFA, placing in a 40 ℃ water bath for 1.5h, filtering with a 0.45 μm organic filter membrane, and detecting the crude peptide solution, wherein the HPLC purity of the crude peptide is 71.56%.
(2) Ammonium chloride/acetonitrile as mobile phase for first washing
And loading the filtered crude peptide solution onto a chromatographic column through a sample injection pump, wherein the specification of the chromatographic column is 30 × 250mm, and 10 μm of C8 bonded silica gel filler is filled in the chromatographic column.
Mobile phase a with 50mM ammonium chloride ph10.0, mobile phase a with 50mM ammonium chloride ph10.0: acetonitrile =300 as mobile phase B, the elution flow rate was 25mL/min, and the gradient elution mode was: changing the gradient from 52A + 48B (V/V) to 42% within 60min, starting mobile phase elution, collecting a spectrum, observing the change of an absorption value, collecting a main peak, detecting the purity by using an analysis liquid phase, collecting and carrying out rotary evaporation concentration to remove acetonitrile, and obtaining the acid polypeptide intermediate concentrated solution shown by formula I.
(3) Second washing with HCOOH/acetonitrile + n-propanol as mobile phase
And loading the intermediate concentrated solution onto a chromatographic column through a sample injection pump, wherein the specification of the chromatographic column is 30 × 250mm, and 10 μm of C8 bonded silica gel filler is filled in the chromatographic column.
0.1% hcooh as mobile phase a, in n-propanol: acetonitrile =1 (V/V) as mobile phase B, the elution flow rate was 25mL/min, and the gradient elution mode was: within 60min, the gradient is changed from 67% A +33% B (V/V) to 37% A +63% B (V/V), mobile phase elution is started, the spectrum is collected, the change of the absorption value is observed, the main peak is collected and the purity is detected by an analysis liquid phase, the fractions with the purity of more than 98% are collected and combined, and freeze-dried after sample rotary evaporation concentration, the purity of the sample after freeze-drying is detected to be 98.88%, and the maximum single impurity is 0.30%, and the HPLC detection result chart is similar to that in FIG. 2.
Example 4: preparation of acidic polypeptide shown in formula I
(1) Solubilization and filtration of crude peptide
1.00g of crude peptide was weighed, crushed, added with 200mL of a solvent (acetonitrile: purified water =80 volume ratio mix) and dissolved by sonication, triethylamine was added until completely dissolved, ph7.52 was adjusted with TFA, and then placed in a 40 ℃ water bath for 1.5h, filtered using a 0.45 μm organic filter membrane, and the crude peptide solution was checked for HPLC purity of 71.76%.
(2) Ammonium chloride/acetonitrile as mobile phase for first washing
And loading the filtered crude peptide solution onto a chromatographic column through a sample injection pump, wherein the specification of the chromatographic column is 30 × 250mm, and 10 μm of C8 bonded silica gel filler is filled in the chromatographic column.
Mobile phase a with 50mM ammonium chloride ph8.5, mobile phase a with 50mM ammonium chloride ph8.5: acetonitrile =300 as mobile phase B, the elution flow rate was 25mL/min, and the gradient elution mode was: within 60min, the gradient is changed from 50% A +50% B (V/V) to 40% A +60% B (V/V), the mobile phase elution is started, the spectrum is collected, the change of the absorption value is observed, the main peak is collected and the purity is detected by the analysis liquid phase, the concentration liquid of the acidic polypeptide intermediate shown in formula I is obtained after the collection and the sample rotary evaporation concentration to remove the acetonitrile.
(3) HCOOH/acetonitrile + n-propanol is used as a mobile phase for secondary washing
And loading the intermediate concentrated solution onto a chromatographic column through a sample injection pump, wherein the specification of the chromatographic column is 30 × 250mm, and 10 μm of C8 bonded silica gel filler is filled in the chromatographic column.
5% of HCOOH as mobile phase A, n-propanol: acetonitrile =1 (V/V) as mobile phase B, the elution flow rate was 25mL/min, and the gradient elution mode was: within 60min, the gradient is changed from 70% A +30% B (V/V) to 40% A +60% B (V/V), mobile phase elution is started, the spectrum is collected, the change of the absorption value is observed, the main peak is collected and the purity is detected by an analysis liquid phase, the fractions with the purity of more than 98% are collected and combined, and freeze-dried after sample rotary evaporation concentration, the purity of the sample after freeze-drying is detected to be 98.88%, and the maximum single impurity is 0.30%, and the HPLC detection result chart is similar to that in FIG. 2.
Example 5: preparation of acidic polypeptide shown in formula I
(1) Solubilization and filtration of crude peptide
Weighing 1.00g of crude peptide, grinding, adding 200mL of solvent (acetonitrile: purified water =20, mixed at a volume ratio of 80) for ultrasonic dissolution, adding triethylamine to complete dissolution, adjusting pH to 7.85 with TFA, placing in a water bath at 40 ℃ for 1.0h, filtering with a 0.45 μm organic filter membrane, and detecting the crude peptide solution with HPLC purity of 71.46%.
(2) Ammonium chloride/ethanol as mobile phase for the first washing
And loading the filtered crude peptide solution onto a chromatographic column through a sample injection pump, wherein the specification of the chromatographic column is 30 × 250mm, and 10 μm of C8 bonded silica gel filler is filled in the chromatographic column.
Mobile phase a with 50mM ammonium chloride ph8.5, mobile phase a with 50mM ammonium chloride ph8.5: ethanol =300 as mobile phase B, elution flow rate 25mL/min, gradient elution mode: within 60min, the gradient is changed from 42% A +58% B (V/V) to 32% A +68% B (V/V), the mobile phase elution is started, the spectrum is collected, the change of the absorption value is observed, the main peak is collected and the purity is detected by the analysis liquid phase, the concentration liquid of the acidic polypeptide intermediate shown in formula I is obtained after the collection and the sample rotary evaporation concentration to remove the acetonitrile.
(3) Second washing with HCOOH/acetonitrile + n-propanol as mobile phase
And loading the intermediate concentrated solution onto a chromatographic column through a sample injection pump, wherein the specification of the chromatographic column is 30 × 250mm, and 10 μm of C8 bonded silica gel filler is filled in the chromatographic column.
0.1% hcooh as mobile phase a, in n-propanol: acetonitrile =1 (V/V) as mobile phase B, the elution flow rate was 25mL/min, and the gradient elution mode was: within 60min, the gradient is changed from 72% A +28% to 42% A +58% B (V/V), mobile phase elution is started, the spectrum is collected, the change of the absorption value is observed, the main peak is collected and the purity is detected by an analytical liquid phase, and freeze-drying is carried out after rotary evaporation concentration, the purity of the sample after freeze-drying is detected to be 98.59%, and the maximum single impurity is 0.41%, and the HPLC detection result graph is similar to that in FIG. 2.
Example 6: preparation of acidic polypeptide shown in formula I
(1) Solubilization and filtration of crude peptide
1.00g of crude peptide was weighed, crushed, added with 200mL of a solvent (acetonitrile: purified water =80 volume ratio mixture) and dissolved by sonication, triethylamine was added until completely dissolved, ph7.25 was adjusted with TFA, and then placed in a 40 ℃ water bath for 2.0h, filtered using a 0.45 μm organic filter membrane, and the crude peptide solution was checked for HPLC purity of 71.86%.
(2) Ammonium chloride/methanol as mobile phase for the first washing
And loading the filtered crude peptide solution onto a chromatographic column through a sample injection pump, wherein the specification of the chromatographic column is 30 × 250mm, and 10 μm of C8 bonded silica gel filler is filled in the chromatographic column.
Mobile phase a with 50mM ammonium chloride ph8.5, mobile phase a with 50mM ammonium chloride ph8.5: methanol =300 as mobile phase B, the elution flow rate was 25mL/min, and the gradient elution mode was: changing the gradient from 35A + 65B (V/V) to 25% within 60min, starting mobile phase elution, collecting a spectrum, observing the change of an absorption value, collecting a main peak, detecting the purity by using an analysis liquid phase, collecting and carrying out rotary evaporation concentration to remove acetonitrile, and obtaining the acid polypeptide intermediate concentrated solution shown in the formula I.
(3) Second washing with HCOOH/acetonitrile + n-propanol as mobile phase
And loading the intermediate concentrated solution onto a chromatographic column through a sample injection pump, wherein the specification of the chromatographic column is 30 x 250mm, and 10 mu m of C8 bonded silica gel filler is filled in the chromatographic column.
0.1% hcooh as mobile phase a, in n-propanol: acetonitrile =1 (V/V) as mobile phase B, the elution flow rate was 25mL/min, and the gradient elution mode was: within 60min, the gradient is changed from 67% A +33% B (V/V) to 37% A +63% B (V/V), mobile phase elution is started, the spectrum is collected, the change of the absorption value is observed, the main peak is collected and the purity is detected by an analysis liquid phase, the fractions with the purity of more than 98% are collected and combined, and freeze-dried after sample rotary evaporation concentration, the purity of the sample after freeze-drying is detected to be 98.88%, and the maximum single impurity is 0.30%, and the HPLC detection result chart is similar to that in FIG. 2.
Example 7: preparation of acidic polypeptide shown in formula I
(1) Solubilization and filtration of crude peptide
Weighing 1.02g of crude peptide, grinding, adding 200mL of solvent (acetonitrile: purified water =20, mixed at a volume ratio of 80) for ultrasonic dissolution, adding triethylamine to complete dissolution, adjusting pH to 7.5 +/-0.5 with TFA, placing in a water bath at 40 ℃ for 1.5h, filtering with a 0.45 mu m organic filter membrane, and detecting the crude peptide solution with the HPLC purity of 71.56%.
(2) Ammonium chloride/acetonitrile as mobile phase for first washing
The filtered crude peptide solution was loaded onto a column with a specification of 30 x 250mm containing 10 μm of C18-bonded silica gel packing via a sample pump.
Mobile phase a with 50mM ammonium chloride ph8.5, mobile phase a with 50mM ammonium chloride ph8.5: acetonitrile =300 as mobile phase B, the elution flow rate was 25mL/min, and the gradient elution mode was: changing the gradient from 50% A +50% B (V/V) to 40% A +60% B (V/V) within 60min, starting mobile phase elution, collecting a map, observing the change of an absorption value, collecting a main peak, detecting the purity by using an analysis liquid phase, collecting and carrying out rotary evaporation concentration to remove acetonitrile, and obtaining the acid polypeptide intermediate concentrated solution shown in the formula I.
(3)NH 4 HCO 3 Acetonitrile as mobile phase for second washing
And loading the intermediate concentrated solution onto a chromatographic column through a sample injection pump, wherein the specification of the chromatographic column is 30 × 250mm, and 10 μm of C8 bonded silica gel filler is filled in the chromatographic column.
10mM ammonium bicarbonate as mobile phase A, acetonitrile: purified water =800 (V/V) as mobile phase B, elution flow rate 25mL/min, gradient elution mode: within 60min, the gradient is changed from 60% A +40% B (V/V) to 50% A +50% B (V/V), the mobile phase elution is started, the spectrum is collected, the change of the absorption value is observed, the main peak is collected and the purity is detected by an analysis liquid phase, the fractions with the purity of more than 98% are collected and combined, and freeze-dried after rotary evaporation and concentration to obtain 198.6mg of the pure acidic polypeptide shown in formula I, the purity of the sample after freeze-drying is detected to be 98.49%, the maximum single impurity content is 0.63%, and the HPLC detection result graph is similar to that in figure 2.
Example 8: preparation of acidic polypeptide shown in formula I
(1) Solubilization and filtration of crude peptide
Weighing 1.03g of crude peptide, grinding, adding 200mL of solvent (acetonitrile: purified water =20, mixed at a volume ratio of 80) for ultrasonic dissolution, adding triethylamine to complete dissolution, adjusting pH to 7.95 with TFA, placing in a 40 ℃ water bath for 1.5h, filtering with a 0.45 μm organic filter membrane, and detecting the crude peptide solution, wherein the HPLC purity of the crude peptide is 71.86%.
(2) 1% TEAP pH7.0/acetonitrile as mobile phase
And loading the filtered crude peptide solution onto a chromatographic column through a sample injection pump, wherein the specification of the chromatographic column is 30 × 250mm, and 10 μm of C8 bonded silica gel filler is filled in the chromatographic column.
1% TEAP pH7.0 as mobile phase A, 1% TEAP pH7.0: acetonitrile =300 as mobile phase B, the elution flow rate was 25mL/min, and the gradient elution mode was: changing the gradient from 45 to 55 percent A + B (V/V) to 40 percent A +60 percent B (V/V) within 60min, starting mobile phase elution, collecting a map, observing the change of an absorption value, collecting a main peak, detecting the purity by using an analysis liquid phase, collecting and carrying out rotary evaporation concentration to remove acetonitrile, and obtaining the acid polypeptide intermediate concentrated solution 1 shown in the formula I.
(3) 1% TEAP pH2.5/n-propanol as the mobile phase for a second wash
And (3) loading the intermediate concentrated solution 1 onto a chromatographic column through a sample injection pump, wherein the specification of the chromatographic column is 30 x 250mm, and a C8 bonded silica gel filler with the particle size of 10 microns is filled in the chromatographic column.
1% teap ph2.5 as mobile phase a, 1% teap ph2.5: n-propanol =300 (V/V) as mobile phase B, the elution flow rate was 25mL/min, and the gradient elution mode was: changing the gradient from 62 to 38% A + B (V/V) to 52% by weight in 60min, starting mobile phase elution, collecting a spectrum, observing the change of an absorption value, collecting a main peak, detecting the purity by using an analysis liquid phase, collecting and carrying out rotary evaporation concentration to remove acetonitrile, and obtaining the acidic polypeptide intermediate concentrated solution 2 shown in formula I.
(4)NH 4 HCO 3 Acetonitrile as mobile phase for the third washing
And loading the intermediate concentrated solution 2 onto a chromatographic column through a sample injection pump, wherein the specification of the chromatographic column is 30 x 250mm, and a C8 bonded silica gel filler with the particle size of 10 mu m is filled in the chromatographic column.
10mM ammonium bicarbonate as mobile phase A, acetonitrile: purified water =800 (V/V) as mobile phase B, elution flow rate 25mL/min, gradient elution mode: within 60min, the gradient is changed from 60% A +40% B (V/V) to 50% A +50% B (V/V), the mobile phase elution is started, the spectrum is collected, the change of the absorption value is observed, the main peak is collected and the purity is detected by an analytical liquid phase, the fractions with the purity of more than 98% are collected and combined, and freeze-dried after rotary evaporation and concentration to obtain 178.5mg of pure acidic polypeptide shown in formula I, the purity of the sample after freeze-drying is detected to be 98.56%, and the maximum single impurity content is 0.49% (the HPLC detection result graph is similar to FIG. 2).
Example 9: preparation of acidic polypeptide shown in formula I
(1) Solubilization and filtration of crude peptide
1.04g of the crude peptide was weighed, crushed, added with 200mL of a solvent (acetonitrile: purified water =80 volume ratio mix) and dissolved by sonication, triethylamine was added until complete dissolution was achieved, ph7.38 was adjusted with TFA, and then placed in a 40 ℃ water bath for 1.0h and filtered using a 0.45 μm organic filter. The crude peptide solution was checked for HPLC purity of 70.86%.
(2) 1% TEAP pH2.5/n-propanol as the mobile phase for the first washing
The filtered crude peptide solution was loaded onto a column with a specification of 30 x 250mm containing 10 μm of C8-bonded silica gel packing via a sample pump.
1% of teap ph2.5 as mobile phase a, 1% of teap ph2.5: n-propanol =300 (V/V) as mobile phase B, the elution flow rate is 25mL/min, the gradient elution regime is: changing the gradient from 62 to 38% A + B (V/V) to 52% by weight in 60min, starting mobile phase elution, collecting a spectrum, observing the change of an absorption value, collecting a main peak, detecting the purity by using an analysis liquid phase, collecting and carrying out rotary evaporation concentration to remove acetonitrile, and obtaining the acidic polypeptide intermediate concentrated solution 1 shown in formula I.
(3) 1% TEAP pH7.0/acetonitrile as mobile phase for a second washing
And loading the intermediate concentrated solution 1 onto a chromatographic column through a sample injection pump, wherein the specification of the chromatographic column is 30 x 250mm, and 10 mu m of C8 bonded silica gel filler is filled in the chromatographic column.
1% TEAP pH7.0 as mobile phase A, 1% TEAP pH7.0: acetonitrile =300 as mobile phase B, the elution flow rate was 25mL/min, and the gradient elution mode was: within 60min, the gradient is changed from 45% A +55% B (V/V) to 40% A +60% B (V/V), the mobile phase elution is started, the spectrum is collected, the change of the absorption value is observed, the main peak is collected and the purity is detected by the analysis liquid phase, the concentration liquid 2 of the preparation intermediate of the acidic polypeptide shown in formula I is obtained after the acetonitrile is removed by collection and rotary evaporation concentration.
(4)NH 4 HCO 3 Acetonitrile as mobile phase for the third washing
And loading the intermediate concentrated solution 2 onto a chromatographic column through a sample injection pump, wherein the specification of the chromatographic column is 30 x 250mm, and 10 mu m of C8 bonded silica gel filler is filled in the chromatographic column.
10mM ammonium bicarbonate as mobile phase A, acetonitrile: purified water =800 (V/V) as mobile phase B, elution flow rate 25mL/min, gradient elution mode: within 60min, the gradient is changed from 60% A +40% B (V/V) to 50% A +50% B (V/V), mobile phase elution is started, the spectrum is collected, the change of the absorption value is observed, the main peak is collected and the purity is detected by an analytical liquid phase, the fractions with the purity of more than 98% are collected and combined, and freeze-dried after sample rotary evaporation concentration, the purity of the sample after freeze-drying is detected to be 98.56%, and the maximum single impurity is 0.49% (the HPLC detection result chart is similar to that in FIG. 2).
Example 10: preparation of acidic polypeptide shown in formula I
(1) Solubilization and filtration of crude peptide
1.03g of the crude peptide was weighed, crushed, added with 200mL of a solvent (acetonitrile: purified water =20, mixed at a volume ratio of 80) and dissolved by sonication, triethylamine was added until completely dissolved, ph7.68 was adjusted with TFA, and then placed in a 40 ℃ water bath for 1.5h, filtered using a 0.45 μm organic filter membrane, and the crude peptide solution was checked for HPLC purity of 71.66%.
(2) Ammonium chloride/acetonitrile as mobile phase for the first washing
The filtered crude peptide solution was loaded onto a column with a specification of 30 x 250mm containing 10 μm of C8-bonded silica gel packing via a sample pump.
Mobile phase a was performed with 50mM ammonium chloride ph8.5, mobile phase a was performed with 50mM ammonium chloride ph8.5: acetonitrile =300 as mobile phase B, the elution flow rate was 25mL/min, and the gradient elution mode was: within 60min, the gradient is changed from 50% A +50% B (V/V) to 40% A +60% B (V/V), the mobile phase elution is started, the spectrum is collected, the change of the absorption value is observed, the main peak is collected and the purity is detected by the analysis liquid phase, the concentration liquid 1 of the acidic polypeptide intermediate shown in formula I is obtained after collection and rotary evaporation concentration to remove acetonitrile.
(3) 1% TEAP pH7.0/acetonitrile as mobile phase for a second washing
And (3) loading the intermediate concentrated solution 1 onto a chromatographic column through a sample injection pump, wherein the specification of the chromatographic column is 30 x 250mm, and a C8 bonded silica gel filler with the particle size of 10 microns is filled in the chromatographic column.
1% TEAP pH7.0 as mobile phase A, 1% TEAP pH7.0: acetonitrile =300 as a mobile phase B, the elution flow rate is 25mL/min, and the gradient elution mode is: within 60min, the gradient is changed from 45% A +55% B (V/V) to 40% A +60% B (V/V), mobile phase elution is started, a graph is collected, the change of an absorption value is observed, a main peak is collected and the purity is detected by an analysis liquid phase, and after collection and sample rotary evaporation concentration are carried out to remove acetonitrile, the acid polypeptide intermediate concentrated solution 2 shown in the formula I is obtained.
(4)NH 4 HCO 3 Acetonitrile as mobile phase for the third washing
And loading the intermediate concentrated solution 2 onto a chromatographic column through a sample injection pump, wherein the specification of the chromatographic column is 30 x 250mm, and a C8 bonded silica gel filler with the particle size of 10 mu m is filled in the chromatographic column.
10mM ammonium bicarbonate as mobile phase A, acetonitrile: purified water =800 (V/V) as mobile phase B, elution flow rate 25mL/min, gradient elution mode: within 60min, the gradient is changed from 60% A +40% B (V/V) to 50% A +50% B (V/V), mobile phase elution is started, the spectrum is collected, the change of the absorption value is observed, the main peak is collected and the purity is detected by an analytical liquid phase, the fractions with the purity of more than 98% are collected and combined, and freeze-dried after sample rotary evaporation concentration, the purity of the sample after freeze-drying is detected to be 98.85%, and the maximum single impurity is 0.34% (the HPLC detection result chart is similar to that in FIG. 2).
Example 11: preparation of acidic polypeptide shown in formula I
(1) Solubilization and filtration of crude peptide
Weighing 1.01g of crude peptide, grinding, adding 200mL of solvent (acetonitrile: mixed with purified water =20:80 by volume ratio) for ultrasonic dissolution, adding triethylamine to complete dissolution, adjusting pH to 7.5 +/-1.0 with TFA, placing in a water bath at 40 +/-10 ℃ for 1.5 +/-0.5 h, filtering by using a 0.45 mu m organic filter membrane, and detecting the crude peptide solution with the HPLC purity of 72.46%.
(2) Ammonium chloride/acetonitrile as mobile phase for the first washing
And loading the filtered crude peptide solution onto a chromatographic column through a sample injection pump, wherein the specification of the chromatographic column is 30 × 250mm, and 10 μm of C8 bonded silica gel filler is filled in the chromatographic column.
Mobile phase a with 50mM ammonium chloride ph8.5, mobile phase a with 50mM ammonium chloride ph8.5: acetonitrile =300 as mobile phase B, the elution flow rate was 25mL/min, and the gradient elution mode was: within 60min, the gradient is changed from 50% A +50% B (V/V) to 40% A +60% B (V/V), the mobile phase elution is started, the spectrum is collected, the change of the absorption value is observed, the main peak is collected and the purity is detected by the analysis liquid phase, the concentration liquid 1 of the acidic polypeptide intermediate shown in formula I is obtained after collection and rotary evaporation concentration to remove acetonitrile.
(3) 1% TEAP pH2.5/n-propanol as mobile phase for the second washing
And loading the intermediate concentrated solution 1 onto a chromatographic column through a sample injection pump, wherein the specification of the chromatographic column is 30 x 250mm, and 10 mu m of C8 bonded silica gel filler is filled in the chromatographic column.
1% teap ph2.5 as mobile phase a, 1% teap ph2.5: n-propanol =300 (V/V) as mobile phase B, the elution flow rate was 25mL/min, and the gradient elution mode was: changing the gradient from 62 to 38% A + B (V/V) to 52% by weight in 60min, starting mobile phase elution, collecting a spectrum, observing the change of an absorption value, collecting a main peak, detecting the purity by using an analysis liquid phase, collecting and carrying out rotary evaporation concentration to remove acetonitrile, and obtaining the acidic polypeptide intermediate concentrated solution 2 shown in formula I.
(4)NH 4 HCO 3 Acetonitrile as mobile phase for the third washing
And loading the intermediate concentrated solution 2 onto a chromatographic column through a sample injection pump, wherein the specification of the chromatographic column is 30 x 250mm, and 10 mu m of C8 bonded silica gel filler is filled in the chromatographic column.
10mM ammonium bicarbonate as mobile phase A, acetonitrile: purified water =800 (V/V) as mobile phase B, elution flow rate 25mL/min, gradient elution mode: within 60min, the gradient is changed from 60% A +40% B (V/V) to 50% A +50% B (V/V), mobile phase elution is started, the spectrum is collected, the change of the absorption value is observed, the main peak is collected and the purity is detected by an analysis liquid phase, the fractions with the purity of more than 98% are collected and combined, and the sample is subjected to freeze drying after being subjected to rotary evaporation concentration and is subjected to freeze drying after being subjected to rotary evaporation concentration, the sample purity after being subjected to freeze drying is 98.97%, and the maximum single impurity is 0.34% (the HPLC detection result chart is similar to that in FIG. 2).
Example 12: preparation of acidic polypeptide shown in formula I
(1) Solubilization and filtration of crude peptide
1.03g of the crude peptide was weighed, crushed, added with 200mL of a solvent (acetonitrile: purified water =80 volume ratio mix) and dissolved by sonication, triethylamine was added until complete dissolution was achieved, ph7.58 was adjusted with TFA, and then placed in a 45 ℃ water bath for 1.5h and filtered using a 0.45 μm organic filter. The crude peptide solution was assayed for 71.16% HPLC purity.
(2) HCOOH/acetonitrile is used as a mobile phase for carrying out first flushing
And loading the filtered crude peptide solution onto a chromatographic column through a sample injection pump, wherein the specification of the chromatographic column is 30 × 250mm, and 10 μm of C8 bonded silica gel filler is filled in the chromatographic column.
0.1% hcooh as mobile phase a, in n-propanol: acetonitrile =1 (V/V) as mobile phase B, the elution flow rate was 25mL/min, and the gradient elution mode was: within 60min, the gradient is changed from 67% A +33% B (V/V) to 37% A +63% B (V/V), mobile phase elution is started, a graph is collected, the change of an absorption value is observed, a main peak is collected and the purity is detected by an analysis liquid phase, and after collection and sample rotary evaporation concentration are carried out to remove acetonitrile, the acid polypeptide intermediate concentrated solution 1 shown in formula I is obtained.
(3) Ammonium chloride/acetonitrile as mobile phase for second washing
And loading the intermediate concentrated solution 1 onto a chromatographic column through a sample injection pump, wherein the specification of the chromatographic column is 30 x 250mm, and 10 mu m of C8 bonded silica gel filler is filled in the chromatographic column.
Mobile phase a with 50mM ammonium chloride ph8.5, mobile phase a with 50mM ammonium chloride ph8.5: acetonitrile =300 as mobile phase B, the elution flow rate was 25mL/min, and the gradient elution mode was: within 60min, the gradient is changed from 50% A +50% B (V/V) to 40% A +60% B (V/V), mobile phase elution is started, a graph is collected, the change of an absorption value is observed, a main peak is collected and the purity is detected by an analysis liquid phase, and after collection and sample rotary evaporation concentration are carried out to remove acetonitrile, the acid polypeptide intermediate concentrated solution 2 shown in the formula I is obtained.
(4)NH 4 HCO 3 Acetonitrile as mobile phase for the third washing
And loading the intermediate concentrated solution 2 onto a chromatographic column through a sample injection pump, wherein the specification of the chromatographic column is 30 x 250mm, and 10 mu m of C8 bonded silica gel filler is filled in the chromatographic column.
10mM ammonium bicarbonate as mobile phase A, acetonitrile: purified water =800 (V/V) as mobile phase B, elution flow rate 25mL/min, gradient elution mode: within 60min, the gradient is changed from 60% A +40% B (V/V) to 50% A +50% B (V/V), mobile phase elution is started, a graph is collected, the change of an absorption value is observed, a main peak is collected and purity is detected by an analysis liquid phase, fractions with purity being more than 98% are collected and combined, and freeze drying is carried out after sample rotary evaporation and concentration, thus obtaining 198.7mg of the pure acid polypeptide shown by formula I, the purity of the sample after detection and freeze drying is 99.14%, and the maximum single impurity is 0.31% (an HPLC detection result graph is similar to that in FIG. 2).
Example 13: preparation of acidic polypeptide shown in formula I
(1) Solubilization and filtration of crude peptide
1.02g of crude peptide was weighed, crushed, added with 200mL of a solvent (acetonitrile: purified water =80 volume ratio mix) and dissolved by sonication, triethylamine was added until completely dissolved, ph7.68 was adjusted with TFA, and then placed in a 45 ℃ water bath for 1.5h and filtered using a 0.45 μm organic filter. The crude peptide solution was assayed for HPLC purity 71.06%.
(2) Ammonium chloride/acetonitrile as mobile phase for first washing
And loading the filtered crude peptide solution onto a chromatographic column through a sample injection pump, wherein the specification of the chromatographic column is 30 × 250mm, and 10 μm of C8 bonded silica gel filler is filled in the chromatographic column.
Mobile phase a was performed with 50mM ammonium chloride ph8.5, mobile phase a was performed with 50mM ammonium chloride ph8.5: acetonitrile =300 as a mobile phase B, the elution flow rate is 25mL/min, and the gradient elution mode is: within 60min, the gradient is changed from 50% A +50% B (V/V) to 40% A +60% B (V/V), the mobile phase elution is started, the spectrum is collected, the change of the absorption value is observed, the main peak is collected and the purity is detected by the analysis liquid phase, the concentration liquid 1 of the acidic polypeptide intermediate shown in formula I is obtained after collection and rotary evaporation concentration to remove acetonitrile.
(3) Second washing with HCOOH/acetonitrile as mobile phase
And (3) loading the intermediate concentrated solution 1 onto a chromatographic column through a sample injection pump, wherein the specification of the chromatographic column is 30 x 250mm, and a C8 bonded silica gel filler with the particle size of 10 microns is filled in the chromatographic column.
Hcooh as mobile phase a, 0.1%: acetonitrile =1 (V/V) as mobile phase B, the elution flow rate was 25mL/min, and the gradient elution mode was: within 60min, the gradient is changed from 67% A +33% B (V/V) to 37% A +63% B (V/V), mobile phase elution is started, a graph is collected, the change of an absorption value is observed, a main peak is collected and the purity is detected by an analysis liquid phase, and after collection and sample rotary evaporation concentration are carried out to remove acetonitrile, the acid polypeptide intermediate concentrated solution 2 shown in formula I is obtained.
(4)NH 4 HCO 3 Acetonitrile as mobile phase for the third washing
And loading the intermediate concentrated solution 2 onto a chromatographic column through a sample injection pump, wherein the specification of the chromatographic column is 30 x 250mm, and a C8 bonded silica gel filler with the particle size of 10 mu m is filled in the chromatographic column.
10mM ammonium bicarbonate as mobile phase A, acetonitrile: purified water =800 (V/V) as mobile phase B, elution flow rate 25mL/min, gradient elution mode: within 60min, the gradient is changed from 60% A +40% B (V/V) to 50% A +50% B (V/V), the mobile phase elution is started, the spectrum is collected, the change of the absorption value is observed, the main peak is collected and the purity is detected by an analytical liquid phase, the fractions with the purity of more than 98% are collected and combined, and freeze-dried after rotary evaporation and concentration to obtain the pure acidic polypeptide product 203.6mg shown in formula I, the purity of the sample after freeze-drying is 99.62%, and the maximum single impurity content is 0.13% (the HPLC detection result chart is similar to that in FIG. 2).
Example 14: preparation of acidic polypeptide shown in formula I
(1) Solubilization and filtration of crude peptide
1.01g of crude peptide was weighed, crushed, added with 200mL of a solvent (acetonitrile: purified water =80 volume ratio mix) and dissolved by sonication, triethylamine was added until completely dissolved, ph7.55 was adjusted with TFA, and then placed in a 45 ℃ water bath for 1.5h and filtered using a 0.45 μm organic filter. The crude peptide solution was checked for HPLC purity of 72.06%.
(2) Ammonium chloride/acetonitrile as mobile phase for first washing
The filtered crude peptide solution was loaded onto a column with a specification of 30 x 250mm containing 10 μm of C8-bonded silica gel packing via a sample pump.
Mobile phase a with 50mM ammonium chloride ph8.5, mobile phase a with 50mM ammonium chloride ph8.5: acetonitrile =300 as mobile phase B, the elution flow rate was 25mL/min, and the gradient elution mode was: within 60min, the gradient is changed from 50% A +50% B (V/V) to 40% A +60% B (V/V), the mobile phase elution is started, the spectrum is collected, the change of the absorption value is observed, the main peak is collected and the purity is detected by the analysis liquid phase, the concentration liquid 1 of the acidic polypeptide intermediate shown in formula I is obtained after collection and rotary evaporation concentration to remove acetonitrile.
(3) Second washing with HCOOH/acetonitrile as mobile phase
And loading the intermediate concentrated solution 1 onto a chromatographic column through a sample injection pump, wherein the specification of the chromatographic column is 30 x 250mm, and 10 mu m of C8 bonded silica gel filler is filled in the chromatographic column.
0.1% hcooh as mobile phase a, in n-propanol: acetonitrile =1 (V/V) as mobile phase B, the elution flow rate was 25mL/min, and the gradient elution mode was: within 60min, the gradient is changed from 67% A +33% B (V/V) to 37% A +63% B (V/V), mobile phase elution is started, a graph is collected, the change of an absorption value is observed, a main peak is collected and the purity is detected by an analysis liquid phase, and after collection and sample rotary evaporation concentration are carried out to remove acetonitrile, the acid polypeptide intermediate concentrated solution 2 shown in formula I is obtained.
(4)NH 4 HCO 3 Taking n-propanol as mobile phase to carry out third washing
And loading the intermediate concentrated solution 2 onto a chromatographic column through a sample injection pump, wherein the specification of the chromatographic column is 30 x 250mm, and a C8 bonded silica gel filler with the particle size of 10 mu m is filled in the chromatographic column.
10mM ammonium bicarbonate as mobile phase A, purified water: n-propanol =1 (V/V) as mobile phase B, the elution flow rate was 25mL/min, and the gradient elution mode was: within 60min, the gradient is changed from 61% A +39% B (V/V) to 41% A +59% B (V/V), mobile phase elution is started, a graph is collected, the change of an absorption value is observed, a main peak is collected and purity is detected by an analysis liquid phase, fractions with purity being more than 98% are collected and combined, and freeze drying is carried out after sample rotary evaporation concentration, so that 209.9mg of the pure acid polypeptide shown by formula I is obtained, the purity of the sample after freeze drying is detected to be 99.55%, and the maximum single impurity content is 0.12% (an HPLC detection result graph is similar to that in FIG. 2).
Example 15: preparation of acidic polypeptide shown in formula I
(1) Solubilization and filtration of crude peptide
1.05g of the crude peptide was weighed, crushed, added with 200mL of a solvent (acetonitrile: purified water =80 volume ratio mix) and dissolved by sonication, triethylamine was added until complete dissolution, ph7.63 was adjusted with TFA, and then placed in a 45 ℃ water bath for 1.5h and filtered using a 0.45 μm organic filter. The crude peptide solution was checked for HPLC purity of 70.86%.
(2) Ammonium chloride/acetonitrile as mobile phase for the first washing
The filtered crude peptide solution was loaded onto a column with a specification of 30 x 250mm containing 10 μm of C8-bonded silica gel packing via a sample pump.
Mobile phase a with 50mM ammonium chloride ph8.5, mobile phase a with 50mM ammonium chloride ph8.5: acetonitrile =300 as mobile phase B, the elution flow rate was 25mL/min, and the gradient elution mode was: within 60min, the gradient is changed from 50% A +50% B (V/V) to 40% A +60% B (V/V), the mobile phase elution is started, the spectrum is collected, the change of the absorption value is observed, the main peak is collected and the purity is detected by the analysis liquid phase, the concentration liquid 1 of the acidic polypeptide intermediate shown in formula I is obtained after collection and rotary evaporation concentration to remove acetonitrile.
(3) Using HCOOH/acetonitrile + and n-propanol as mobile phase to make secondary washing
And (3) loading the intermediate concentrated solution 1 onto a chromatographic column through a sample injection pump, wherein the specification of the chromatographic column is 30 x 250mm, and a C8 bonded silica gel filler with the particle size of 10 microns is filled in the chromatographic column.
0.1% hcooh as mobile phase a, in n-propanol: acetonitrile =1 (V/V) as mobile phase B, the elution flow rate was 25mL/min, and the gradient elution mode was: within 60min, the gradient is changed from 67% A +33% B (V/V) to 37% A +63% B (V/V), mobile phase elution is started, a graph is collected, the change of an absorption value is observed, a main peak is collected and the purity is detected by an analysis liquid phase, and after collection and sample rotary evaporation concentration are carried out to remove acetonitrile, the acid polypeptide intermediate concentrated solution 2 shown in formula I is obtained.
(4)NH 4 HCO 3 Taking isopropanol as mobile phase to carry out third washing
And loading the intermediate concentrated solution 2 onto a chromatographic column through a sample injection pump, wherein the specification of the chromatographic column is 30 x 250mm, and a C8 bonded silica gel filler with the particle size of 10 mu m is filled in the chromatographic column.
10mM ammonium bicarbonate as mobile phase A, purified water: isopropanol =1 (V/V) as mobile phase B, elution flow rate 25mL/min, gradient elution mode: within 60min, the gradient is changed from 55A + 45B (V/V) to 35% A + 65B (V/V), mobile phase elution is started, a graph is collected, the change of an absorption value is observed, a main peak is collected and purity is detected by an analysis liquid phase, fractions with purity being more than 98% are collected and combined, and freeze drying is carried out after sample rotary evaporation and concentration, so that 208.8mg of a pure acid polypeptide product shown by formula I is obtained, the purity of the sample after freeze drying is detected to be 99.62%, and the maximum single impurity content is 0.14% (an HPLC detection result graph is similar to that in FIG. 2).
Example 16: preparation of acidic polypeptide shown in formula I
(1) Solubilization and filtration of crude peptide
1.02g of the crude peptide was weighed, crushed, added with 200mL of a solvent (acetonitrile: purified water =80 volume ratio mix) and dissolved by sonication, triethylamine was added until complete dissolution, ph7.76 was adjusted with TFA, and then placed in a 45 ℃ water bath for 1.5h and filtered using a 0.45 μm organic filter. The crude peptide solution was checked for HPLC purity 72.06%.
(2) 0.1% of TFA/acetonitrile as mobile phase
And loading the filtered crude peptide solution onto a chromatographic column through a sample injection pump, wherein the specification of the chromatographic column is 30 × 250mm, and 10 μm of C8 bonded silica gel filler is filled in the chromatographic column.
Using 0.1% TFA as mobile phase A, acetonitrile as mobile phase B, an elution flow rate of 25mL/min, a gradient elution profile: within 60min, the gradient is changed from 63% A +37% B (V/V) to 43% A +57% B (V/V), mobile phase elution is started, a graph is collected, the change of an absorption value is observed, a main peak is collected and the purity is detected by an analysis liquid phase, after acetonitrile is removed by collection and rotary evaporation concentration, the pure acid polypeptide shown by the formula I is obtained, the detection purity is 96.39, and the maximum single impurity is 2.65% (shown in figure 3 by HPLC detection, and graph data are shown in Table 3).
TABLE 3 Fine peptide HPLC profile data
Example 17: preparation of acidic polypeptide shown in formula I
(1) Solubilization and filtration of crude peptide
1.05g of the crude peptide was weighed, crushed, added with 200mL of a solvent (acetonitrile: purified water =80 volume ratio mix) and dissolved by sonication, triethylamine was added until complete dissolution, ph7.59 was adjusted with TFA, and then placed in a 45 ℃ water bath for 1.5h and filtered using a 0.45 μm organic filter. The crude peptide solution was checked for HPLC purity 72.86%.
(2) Washing with 0.1% phosphoric acid/acetonitrile as mobile phase
And loading the filtered crude peptide solution onto a chromatographic column through a sample injection pump, wherein the specification of the chromatographic column is 30 × 250mm, and 10 μm of C8 bonded silica gel filler is filled in the chromatographic column.
Taking 0.1% phosphoric acid as a mobile phase A, acetonitrile as a mobile phase B, and performing gradient elution at an elution flow rate of 25mL/min in a manner that: within 60min, the gradient is changed from 65% A +35% B (V/V) to 45% A +55% B (V/V), the mobile phase elution is started, the spectrum is collected, the change of the absorption value is observed, the main peak is collected and the purity is detected by analyzing the liquid phase, after the acetonitrile is removed by collection and rotary evaporation concentration, the pure acidic polypeptide shown in formula I is obtained, the detection purity is 93.05%, the maximum single impurity is 2.60% (shown in figure 4 by HPLC detection, the spectrum data are shown in table 4).
TABLE 4 Fine peptide HPLC profile data
| Number of peak | Retention time | Area of | Area% |
| 1 | 7.400 | 1108 | 0.08 |
| 2 | 7.819 | 1237 | 0.09 |
| 3 | 8.875 | 1395 | 0.10 |
| 4 | 9.108 | 1267 | 0.09 |
| 5 | 9.404 | 3010 | 0.22 |
| 6 | 9.715 | 3143 | 0.23 |
| 7 | 10.306 | 1794 | 0.13 |
| 8 | 11.689 | 6891 | 0.49 |
| 9 | 12.440 | 6216 | 0.45 |
| 10 | 12.792 | 10026 | 0.72 |
| 11 | 13.198 | 11802 | 0.85 |
| 12 | 14.074 | 1296929 | 93.05 |
| 13 | 14.871 | 36274 | 2.60 |
| 14 | 16.268 | 8933 | 0.64 |
| 15 | 18.726 | 2253 | 0.16 |
| 16 | 21.184 | 1602 | 0.11 |
Claims (6)
1. A method for the purification of a highly pure acidic polypeptide from a mixture comprising the acidic polypeptide and related impurities, characterized in that the method is an RP-HPLC chromatography method, the solvent used for elution as mobile phase consists essentially of an ion-pair reagent and an organic modifier, the elution solvent being eluted in the form of a gradient elution,
the purification is carried out in two steps, wherein ammonium chloride is taken as a mobile phase A in one step, and an ammonium chloride-organic modifier is taken as a mobile phase B; one step with HCOOH or NH 4 HCO 3 The organic modifier is used as a mobile phase A and an organic modifier is used as a mobile phase B;
wherein the organic modifier is selected from one or more of methanol, ethanol, n-propanol, isopropanol and acetonitrile;
wherein the pH value of the ammonium chloride is 7.0-10.0, and the volume concentration is 50mmoL/L;
wherein, the mass concentration of HCOOH is 0.1-10%;
wherein NH 4 HCO 3 The volume concentration of (A) is 1-100 mmoL/L;
wherein the acidic polypeptide is a compound with the following structure:
the formula I is shown in the specification.
2. A process for the purification preparation of a highly pure acidic polypeptide from a mixture comprising the acidic polypeptide and related impurities, characterized in that the process is an RP-HPLC chromatography, the solvent used for elution as mobile phase consists essentially of an ion-pair reagent and an organic modifier, the elution solvent being eluted in a gradient elution,
the purification is carried out in three steps, wherein in one step, ammonium chloride is used as a mobile phase A, and ammonium chloride-organic modifier is used as a mobile phase B; one step processHCOOH or TEAP is used as a mobile phase A, and an organic modifier is used as a mobile phase B; one step with NH 4 HCO 3 The organic modifier is used as a mobile phase A and an organic modifier is used as a mobile phase B;
wherein the organic modifier is selected from one or more of methanol, ethanol, n-propanol, isopropanol and acetonitrile;
wherein the pH value of the ammonium chloride is 7.0-10.0, and the volume concentration is 50mmoL/L;
wherein, the mass concentration of HCOOH is 0.1-10%;
wherein NH 4 HCO 3 The volume concentration of (A) is 1-100 mmoL/L;
wherein the pH of TEAP is 2.5 + -1.0 or 7.0 + -1.0;
wherein the acidic polypeptide is a compound of the following structure:
formula I.
3. The method of any one of claims 1-2, wherein NH is used 4 HCO 3 When the phase is the mobile phase A, the organic modifier-purified water is also used as the mobile phase B.
4. The method according to any one of claims 1-2, wherein the pH of the ion pairing reagent used is at least 1 pH unit greater than the pKa of the isolated acidic polypeptide compound.
5. The method of any of claims 1-2, wherein the mobile phase gradient with the organic modifier is in the range of 20% to 60%.
6. The method of any one of claims 1-2, wherein the packing material of the column of the reverse phase high performance liquid chromatography system is C4, C6, C8 or C18 bonded silica gel.
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