CN114405065B

CN114405065B - Method for preparing chiral polypeptide medicine by dynamic thermodynamic equilibrium purification

Info

Publication number: CN114405065B
Application number: CN202210061819.3A
Authority: CN
Inventors: 高剑; 钟国庆; 年贺凤
Original assignee: Hangzhou Hetai Jianyu Biotechnology Co ltd
Current assignee: Hangzhou Hetai Jianyu Biotechnology Co ltd
Priority date: 2022-01-19
Filing date: 2022-01-19
Publication date: 2023-08-01
Anticipated expiration: 2042-01-19
Also published as: CN114405065A

Abstract

The invention provides a method for preparing chiral polypeptide type medicines by dynamic thermodynamic equilibrium purification, which comprises the following steps: s1) dissolving a chiral polypeptide crude drug product by using a solvent, mixing with an aqueous solution of an alkaline inorganic buffer salt, and standing to obtain a chiral polypeptide crude drug product solution; s2) purifying the chiral polypeptide medicine crude product solution by a reverse phase chromatography method to obtain a refined solution of the chiral polypeptide medicine. Compared with the prior art, the method has the advantages that the dynamic thermodynamic equilibrium method is utilized to pretreat the crude chiral polypeptide medicine solution, so that the impurities and the medicine components reach a special thermodynamic stable state, a limited separation mode on the conventional chromatograph is formed, and other process impurities can be effectively controlled; meanwhile, the dynamic thermodynamic equilibrium method is utilized to realize the effective separation mode, and the separation effect which cannot be achieved by the conventional chromatographic materials and methods is breakthrough realized.

Description

Method for preparing chiral polypeptide medicine by dynamic thermodynamic equilibrium purification

Technical Field

The invention belongs to the technical field of medicine purification, and particularly relates to a method for preparing chiral polypeptide medicines by dynamic thermodynamic equilibrium purification.

Background

Polypeptide drugs, which are widely used in biological systems, are signal molecules, transport molecules, and digestive molecules. As signal molecules, polypeptides control biological functions of organisms such as cell division, mating, chemotaxis, pain, growth and immunity, and polypeptide synthesis has become one of the most powerful tools for biochemical, pharmacological, immunological and biophysical studies; as a transport molecule, the polypeptide can promote the passage of ions through the cell membrane; as digestive molecules, polypeptides play an important role in nutrient absorption by cells and intact organisms. In addition, the polypeptides may also act as protective agents, such as antibiotics, which have excellent antibacterial and antiviral properties. Polypeptide drugs are now an important commercial entity in the pharmaceutical market, and have been used in different therapeutic fields, such as diabetes, allergy, anti-infection, obesity, diagnosis, tumor, arthritis, cardiovascular diseases, etc.

Because polypeptide drugs are mainly derived from endogenous polypeptides or other natural polypeptides, in general, the structure is clear, the action mechanism is clear, and a plurality of properties are often between that of small molecular chemical drugs and that of macromolecular protein/antibody drugs, for example, compared with small molecular chemical drugs, the half-life is generally short, unstable, the in vivo is easy to degrade rapidly, the preparation is unstable and needs to be stored at low temperature, the cost is low, the molecular drug is high (especially long-chain polypeptide), and the like; compared with macromolecular protein, the polypeptide has better stability, less dosage, higher unit activity and lower cost, and the chemical synthesis technology of the polypeptide is mature, easy to separate from impurities or byproducts and high in purity.

Large scale, economical purification of polypeptides is an increasingly important issue for the biotechnology industry. Often, a combination of different chromatographic techniques is used to isolate a polypeptide of interest from other products produced by a biosynthetic or chemical synthesis. These techniques separate mixtures of polypeptides according to their charge, degree of hydrophobicity, size, or specific interactions between the polypeptide of interest and the immobilized capture agent. For each of these techniques, several different chromatography resins are available, allowing for precise adjustment of the purification scheme depending on the specific polypeptide involved. The essence of each of these separation methods is that the polypeptides can either be moved down the long column at different rates, to achieve physical separation that increases as they move further down the column, or selectively adhere to the separation medium, and then elute by different solvent differences. In some cases, the polypeptide of interest is separated from the impurity, i.e., the polypeptide of interest is present in a "flow-through" when the impurity specifically adheres to the column and the polypeptide of interest does not adhere to the column. Large scale, low cost purification of polypeptides to a purity sufficient for use as human pharmaceuticals remains a significant challenge.

The separation and purification method is generally determined by the nature of the substance to be separated. Common methods for protein and polypeptide extraction and separation include: salting-out method, ultrafiltration method, gel filtration method, isoelectric precipitation method, ion exchange chromatography, affinity chromatography, adsorption chromatography, countercurrent precipitation, enzymolysis method, etc. These methods are often combined to separate and purify a particular substance. The usual method for separating and purifying polypeptides by industrial application is as follows:

high Performance Liquid Chromatography (HPLC)

The advent of HPLC provides an advantageous means for the separation of peptides, since proteins, polypeptides can be produced on a preparative scale with biologically active polypeptides compared to other compounds under suitable chromatographic conditions. Therefore, researchers have made a lot of work on the method of separation and preparation of polypeptides. How to maintain the activity of the polypeptide, how to select the stationary phase material, the type of eluent and how to analyze and measure are all the contents of the current research.

Reversed phase high performance liquid chromatography (RP-HPLC)

Separation of polypeptides by RP-HPLC first resulted in determination of the retention of polypeptides of different structures on the column. The composition of different amino acids has a certain influence on the retention coefficient, wherein the retention time on the column can be reduced by the polar amino acid residues in the peptide consisting of 2-20 amino acids; in peptides consisting of 10 to 60 amino acids, more nonpolar amino acids can also reduce the retention time on the column, whereas in small peptides containing 5 to 25 amino acids, an increase in nonpolar amino acids can extend the retention time on the column.

In addition, various documents at home and abroad report the influence of conditions such as peptide chain length, amino acid composition, temperature and the like on the retention condition, and proper conditions for separating and simulating the obtained polypeptide are utilized for computer processing analysis.

Hydrophobic interaction chromatography (Hydrophobic interaction chromatogrphy, HIC)

HIC is characterized in that polypeptide contains hydrophobic gene, which can generate hydrophobic effect with stationary phase to achieve separation and analysis, and has less polypeptide denaturation than RP-GPLC.

Size exclusion chromatography (Sizs-Exclusion chromatogrphy, SEC)

The SEC uses the difference of the size and shape of polypeptide molecules to separate and purify polypeptide substances, is particularly convenient for some larger aggregated molecules, and has completely different separation behaviors of compounds with different structures and configurations on a SEC column, so that variants with different configurations or slight differences in amino acid sequences can be separated.

Ion exchange chromatography (ion-Exchange chromatography, IEXC)

IEXC can separate and purify polypeptides having biological activity under neutral conditions by utilizing the difference in chargeability of the polypeptides. It can be divided into two major classes of cationic column and anionic column, and some new resins such as macroporous resin, homopore resin, ion exchange cellulose, sephadex, sepharose resin, etc. In the separation analysis research of polypeptide substances, the properties, the eluent and the elution conditions of the polypeptides are more studied, different polypeptide separation conditions are different, and particularly the ion strength, the salt concentration and the like of the eluent have great influence on purification.

High performance displacement chromatography (High-Performance Displacement Chromatography, HPDC)

HPDC is to exchange the sample on the chromatographic column by using a small molecular high-efficiency displacer, thereby achieving the purpose of separation. It has the characteristic of separating components with low content. Studies have shown that the lower the relative molecular mass of the displacer, the more readily it binds to the stationary phase, and therefore, when isolating polypeptides of small relative molecular mass, smaller displacers are required to be displacement purified.

Perfusion chromatography (Perfusion Chromatography, PC)

PC is a chromatographic separation method based on a molecular sieve principle and a mobile phase flowing at a high speed, and the pore size of a stationary phase and the speed of the mobile phase directly influence the separation effect. The test proves that the material has the characteristics of low investment and high output in the production and preparation processes. The PC stationary phase available in the market at present is more in variety and suitable for polypeptide separation of different molecular weights.

Affinity chromatography (Affinity Chromatography, AC)

AC is a chromatographic method for separating substances using specific affinity between ligands attached to a stationary phase matrix and ligands that can act with their specificity. Since the concept of affinity chromatography was proposed in 1968, many combinations have been found in the search for specific affinity substances, such as antigen-antibodies, enzyme-catalytic substrates, lectin-polysaccharides, oligonucleotides and their complementary strands, etc. The separation of polypeptides is currently mainly carried out by applying monoclonal antibodies or biomimetic ligands thereof to the polypeptides, wherein the ligands are synthesized from natural or artificial materials according to the structures of the ligands.

Immobilized metal affinity chromatography (Immobilized Metal Affinity Chromatography, IMAC)

IMAC has been developed in recent yearsIs an affinity method of (a). Having some metal ions, e.g. Cu, sequestered in the stationary phase matrix ²⁺ 、Ni ²⁺ 、Fe ³⁺ And the like, the column can chelate the polypeptide containing Lys, met, asp, arg, tyr, glu and His in the side chain through coordination bonds, particularly the structure containing His-X-X-X-His in the peptide sequence is most easily combined to the metal ion affinity column, and the purification effect is better.

System application of polypeptide separation engineering

The above-mentioned techniques for separating polypeptides are combined with each other in the practical application process, and different separation means are adopted according to the different properties of the separated polypeptides. In particular, in the post-genome era, intensive research on proteome is conducted, means for separating polypeptides are continuously improved, various properties of polypeptides are comprehensively utilized, and the novel separation technologies such as high performance liquid chromatography and the like and the combination of the traditional technologies are utilized simultaneously by adopting the conventional polypeptide extraction method, so that the separation and purification of different types of polypeptides are realized.

The chiral of the polypeptide medicine is more, the conventional separation method is to use reversed phase chromatography to realize separation by utilizing the retention difference of the medicine and impurities, but when the chromatographic method faces the enantiomer, diastereomer, cis-trans isomer and epimer impurities, the structural difference and the medicine are very small, the physicochemical property is almost not different, the effective separation can not be realized under the conventional reversed phase chromatography material and the conventional chromatography process, the chiral separation method becomes the key for restricting the quality of the chiral polypeptide medicine, so the isomer impurities become the difficulty of the purification preparation of the chiral polypeptide medicine, and generally, the chromatographic separation or other special purification separation process is required to be carried out by using extremely expensive chiral preparation materials, the material and the production cost are 10-20 times or more than those of the conventional purification preparation materials and the cost, and the market price of the chiral polypeptide medicine is indirectly influenced.

And because of the specificity of the biosynthesis process or the chemical synthesis process, various process impurities and degradation impurities of polypeptide medicaments known in the industry are very difficult to purify and remove and effectively control, while various chiral columns, chromatographic conditions and other combinations can be used in analysis and detection, the analysis and detection can possibly effectively detect related impurities, and the analysis and detection are very strict in chromatographic conditions, very small in loading capacity, very fine in chromatographic filler particle size, very high in material cost and very high in equipment and instrument corresponding requirements, so that the analysis method can hardly realize amplified production in industrial-grade purification preparation.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a method for preparing chiral polypeptide drugs by dynamic thermodynamic equilibrium purification, which uses the dynamic thermodynamic equilibrium relationship of chemical substances under the condition of a certain temperature and pressure in the form of solution to make the chiral polypeptide drugs and the isomer impurities thereof have obvious chromatographic retention difference, so that the conventional industrial chromatographic separation (the enantiomer, diastereomer, epimer and other isomer impurities) is difficult to effectively and simply separate, the other processes and degradation impurities are not more than 0.1%, the total impurities are not more than 1.0%, and the yield reaches more than 50% when the purity is more than 99.0%.

The invention provides a method for preparing chiral polypeptide type medicines by dynamic thermodynamic equilibrium purification, which comprises the following steps:

s1) dissolving a chiral polypeptide crude drug product by using a solvent, mixing with an aqueous solution of an alkaline inorganic buffer salt, and standing to obtain a chiral polypeptide crude drug product solution;

s2) purifying the chiral polypeptide drug crude product solution by a reverse phase chromatography to obtain a chiral polypeptide drug refined solution;

the chiral polypeptide type drug comprises a group shown in a formula (1):

wherein R is ₁ One or more selected from amino, hydroxyl, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C6-C10 aryl and substituted or unsubstituted C6-C10 heterocyclic groups;

R ₂ selected from amino or H;

or R is ₁ And R is R ₂ Connected into a ring;

n is 0 or 1.

Preferably, the chiral polypeptide type drug is represented by formula (A) or formula (B):

the substituent groups in the substituted C1-C10 alkyl, the substituted C6-C10 aryl and the substituted C6-C10 heterocyclic group are selected from one or more of amino, hydroxyl, C6-C10 aryl and C6-C10 heterocyclic group;

the A-A-A is a peptide chain formed by 1 to 25 chiral amino acids.

Preferably, the chiral polypeptide type medicine is shown in the formulas (I) to (VI):

The A-A-A is 3-25 chiral amino acids to form a peptide chain.

Preferably, the solvent is selected from one or more of methanol, ethanol, isopropanol, water, acetonitrile, an aqueous inorganic acid solution and an aqueous inorganic salt solution; the concentration of the chiral polypeptide crude drug after dissolution is 100-200 g/L;

the alkaline inorganic buffer salt is selected from one or more of sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate and ammonium bicarbonate;

the concentration of the aqueous solution of the alkaline inorganic buffer salt is 10-250 mmol/L;

the pH value of the aqueous solution of the alkaline inorganic buffer salt is 8-10;

the ratio of the alkaline inorganic buffer salt to the chiral polypeptide crude drug is (0.8-1.5) mmol:1g.

Preferably, the temperature of the placing is 20-30 ℃; the time for placing is 30-60 min.

Preferably, the stationary phase purified by the reverse phase chromatography is octadecylsilane chemically bonded silica gel; the mobile phase purified by the reversed phase chromatography adopts an aqueous solution of an alkaline inorganic buffer salt as a mobile phase A and adopts an alcohol solvent and/or acetonitrile as a mobile phase B; the reversed phase chromatography purification adopts gradient elution; the gradient elution process is that the mobile phase B is 0-5 min 5% by volume percent, the mobile phase B is 5-60% by volume percent after 5.1-50 min, and then the mobile phase B is maintained at 60% by volume percent for at least 10min; the detection wavelength of the reversed phase chromatography purification is 200-280 nm.

Preferably, the mobile phase A is 50-250 mmol/L bicarbonate water solution or 10-20 mmol/L carbonate water solution; the pH value of the mobile phase A is 8-9;

the linear velocity and flow rate of the mobile phase during purification by reverse phase chromatography are 3.5-6.5 cm/min.

Preferably, the method further comprises:

and (3) carrying out salt conversion on the refined solution of the chiral polypeptide type medicine through a chromatographic method, and concentrating under reduced pressure, and freeze-drying to obtain the chiral polypeptide type medicine.

Preferably, the salt transfer is performed by chromatography, specifically by reverse phase chromatography exchange using a buffer system consisting of acetate and acetic acid;

the mobile phase of reverse phase chromatographic exchange salt transfer comprises a mobile phase A1, a mobile phase A2 and a mobile phase B; the mobile phase A1 is 10-30 mM acetate water solution, and the mobile phase A2 is 0.002-0.01 wt% acetate water solution; the mobile phase B is an alcohol solvent and/or acetonitrile;

reversed phase chromatographic exchange salt transfer takes octadecylsilane chemically bonded silica gel as a stationary phase;

reversed phase chromatographic exchange salt transferring adopts gradient elution; the gradient elution procedure is that, in terms of volume percent, 95% of mobile phase A1 and 5% of mobile phase B are sequentially eluted for 30-60 min,95% of mobile phase A2 and 5% of mobile phase B are eluted for 15-25 min, then mobile phase B and mobile phase A2 are adopted to elute within 15-40 min, and the mobile phase B is raised from 5% to 60%, and then the mobile phase B is maintained to be 60% of mobile phase B for at least 20min.

Preferably, the temperature of the reduced pressure concentration is not more than 35 ℃, and the vacuum degree is more than-0.08 MPa;

the freeze drying process comprises the following steps: pre-freezing to-40 to-30 ℃ and keeping for 2-4 hours, first-stage drying at-5 to 0 ℃ for 24-36 hours, sublimation drying at 20 to 25 ℃ for 24-36 hours, and vacuum degree stabilizing at 0.01 to 1.00MPa.

The invention provides a method for preparing chiral polypeptide type medicines by dynamic thermodynamic equilibrium purification, which comprises the following steps: s1) dissolving a chiral polypeptide crude drug product by using a solvent, mixing with an aqueous solution of an alkaline inorganic buffer salt, and standing to obtain a chiral polypeptide crude drug product solution; s2) purifying the chiral polypeptide medicine crude product solution by a reverse phase chromatography method to obtain a refined solution of the chiral polypeptide medicine. Compared with the prior art, the method has the advantages that the dynamic thermodynamic equilibrium method is utilized to pretreat the crude chiral polypeptide medicine solution, so that the impurities and the medicine components reach a special thermodynamic stable state, a limited separation mode on the conventional chromatograph is formed, other process impurities can be effectively controlled, and the effective separation can be realized without other purification processes; meanwhile, the mode of realizing effective separation by utilizing a dynamic thermodynamic equilibrium method breaks through the separation effect which cannot be achieved by the conventional chromatographic materials and methods, and the cost is far lower than that of a complex purification method which needs chiral resolution or special chromatographic materials and processes to realize the separation of isomer impurities.

Drawings

FIG. 1 is a diagram showing the purity detection of crude chiral polypeptide-type drug YH-21101201;

FIG. 2 is a chart showing the purity detection of the chiral polypeptide type drug obtained in example 1 of the present invention;

FIG. 3 is a diagram showing the separation and purification preparation of chiral polypeptide type drugs obtained in example 1 of the present invention;

FIG. 4 is a chart showing the purity detection of the chiral polypeptide type drug obtained in example 2 of the present invention;

FIG. 5 is a chart showing the purity detection of crude chiral polypeptide-type drug YH-21101801;

FIG. 6 is a chart showing the purity detection of the chiral polypeptide type drug obtained in example 3 of the present invention;

FIG. 7 is a chart showing the purity detection of the chiral polypeptide type drug finally obtained in comparative example 1 of the present invention;

FIG. 8 is a diagram showing the separation and purification preparation of the chiral polypeptide type drug finally obtained in comparative example 1 of the present invention.

Detailed Description

The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The invention provides a method for preparing chiral polypeptide type medicines by dynamic thermodynamic equilibrium purification, which comprises the following steps: s1) dissolving a chiral polypeptide crude drug product by using a solvent, mixing with an aqueous solution of an alkaline inorganic buffer salt, and standing to obtain a chiral polypeptide crude drug product solution; s2) purifying the chiral polypeptide drug crude product solution by a reverse phase chromatography to obtain a chiral polypeptide drug refined solution; the chiral polypeptide type drug comprises a group shown in a formula (1):

wherein R is ₁ Is one or more of amino, hydroxyl, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted C6-C10 heterocyclic, preferably amino, hydroxyl, substituted or unsubstituted C1-C5 alkyl, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted C6-C10 heterocyclic, more preferably amino, hydroxyl, substituted or unsubstituted C1-C3 alkyl, substituted or unsubstituted phenyl, substituted or unsubstituted indolyl; the heteroatom in the heterocyclyl is preferably one or more of O, S and N; the substituents in the substituted C1-C10 alkyl, substituted C6-C10 aryl and substituted C6-C10 heterocyclic group are preferably amino, hydroxyl and C1-to-amino One or more of C3 alkoxy, C6-C10 aryl, C6-C10 heterocyclic group, more preferably one or more of amino, hydroxy, methylalkoxy, ethylalkoxy and phenyl.

R ₂ Amino or H;

or R is ₁ And R is R ₂ Connected into a ring;

n is 0 or 1.

The chiral polypeptide type medicine provided by the invention comprises two adjacent or separated carbonyl groups of one methylene, wherein one carbonyl group is on an amide bond, and the other carbonyl group which is not amide can form a hemiacetal ketone structure in a protonic solution.

In the present invention, preferably, the chiral polypeptide-type drug is represented by formula (a) or formula (B):

the A-A-A is a peptide chain formed by 1 to 25 chiral amino acids, preferably a peptide chain formed by 3 to 25 chiral amino acids, more preferably a peptide chain formed by 3 to 20 chiral amino acids, still more preferably a peptide chain formed by 3 to 15 chiral amino acids, still more preferably a peptide chain formed by 3 to 10 chiral amino acids, and most preferably a peptide chain formed by 3 to 6 chiral amino acids.

Further preferably, the chiral polypeptide type drug is shown as the formula (I) to the formula (VI):

the A-A-A is 3-25 chiral amino acids to form a peptide chain; the A-A-A is the same as that described above and will not be described in detail herein.

In the embodiment provided by the invention, the chiral polypeptide type drug is specifically shown as YH-21101201 or YH-21101801:

Dissolving the chiral polypeptide crude drug with a solvent; the source of the crude chiral polypeptide-type drug is not particularly limited, and it can be synthesized by biosynthesis, chemical synthesis or a combination of both; the solvent is preferably one or more of methanol, ethanol, isopropanol, water, acetonitrile, an aqueous inorganic acid solution and an aqueous inorganic salt solution; the inorganic acid aqueous solution is preferably acetic acid aqueous solution; the inorganic salt aqueous solution is preferably an acetate salt aqueous solution; the ratio of the chiral polypeptide crude drug to the solvent is preferably 1g: (5-8) mL; the concentration of the chiral polypeptide type crude drug after dissolution is preferably 100-200 g/L, more preferably 100-180 g/L, still more preferably 120-150 g/L.

Then mixing with aqueous solution of alkaline inorganic buffer salt; the alkaline inorganic buffer salt is preferably one or more of sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate and ammonium bicarbonate; the concentration of the aqueous solution of the alkaline inorganic buffer salt is preferably 10-250 mmol/L; in the present invention, the aqueous solution of the basic inorganic buffer salt is preferably 50 to 250mmol/L of an aqueous bicarbonate solution or 10 to 20mmol/L of an aqueous carbonate solution, more preferably 100 to 250mmol/L of an aqueous bicarbonate solution or 10 to 15mmol/L of an aqueous carbonate solution, still more preferably 150 to 200mmol/L of an aqueous bicarbonate solution or 10 to 15mmol/L of an aqueous carbonate solution; the pH value of the aqueous solution of the alkaline inorganic buffer salt is preferably 8 to 10, more preferably 8 to 9; in the present invention, it is preferable to adjust the pH of the alkaline inorganic buffer salt aqueous solution with carbonic acid or sodium hydroxide; the ratio of the alkaline inorganic buffer salt to the chiral polypeptide crude drug is (0.8-1.5) mmol:1g; the concentration of the crude chiral polypeptide drug in the solution obtained after mixing is preferably 10 to 40mg/mL, more preferably 10 to 30mg/mL, still more preferably 10 to 20mg/mL, and most preferably 12 to 15mg/mL.

Preferably filtering after mixing, and standing the filtrate for a period of time to obtain chiral polypeptide crude drug solution; the filtration is preferably performed using an organic-based filter membrane, more preferably an organic-based filter membrane of 0.45 μm; the temperature of the standing is preferably 20-30 ℃, more preferably 22-28 ℃, still more preferably 24-36 ℃, and most preferably 25-26 ℃; the time for the placement is preferably 30 to 60 minutes, more preferably 30 to 50 minutes, and still more preferably 30 to 40 minutes; the placing is preferably performed under slow stirring. The placing temperature is inversely proportional to the placing time, slow stirring can accelerate the mass transfer and heat transfer process, but a severe heating or cooling mode cannot be used for temperature control; after the solution is placed, the pH value of the solution is reduced by 0.2 to 0.5. The chiral polypeptide medicine provided by the invention has a specific structure, can form an enol tautomeric compound under the conditions of a certain temperature and buffer salt existence, and has a certain thermodynamic dynamic equilibrium relationship in the tautomerism, and can be placed for a period of time to enable a solution to reach a thermodynamic equilibrium state at the temperature.

Purifying the chiral polypeptide crude drug solution by reverse phase chromatography to obtain a refined solution of chiral polypeptide drug; the stationary phase purified by the reversed phase chromatography is octadecylsilane chemically bonded silica, more preferably YMC Triart prep C-S or Nano Unisil C18; the particle size of the stationary phase is preferably 7-10 mu m; the pore size of the stationary phase is preferably The mobile phase purified by the reversed phase chromatography adopts an aqueous solution of an alkaline inorganic buffer salt as a mobile phase A and adopts an alcohol solvent and/or acetonitrile as a mobile phase B; the concentration of the mobile phase A is preferably 10-250 mmol/L; in the present invention, it is further preferred that the mobile phase A is 50 to 250mmol/L of an aqueous bicarbonate solution or 10 to 20mmol/L of an aqueous carbonate solution, more preferably 100 to 250mmol/L of an aqueous bicarbonate solution or 10 to 20mmol/L of an aqueous carbonate solution, still more preferably 150 to 250mmol/L of an aqueous bicarbonate solution or 10 to 15mmol/L of an aqueous carbonate solution, most preferablySelected as 200mmol/L bicarbonate aqueous solution or 10mmol/L carbonate aqueous solution; the bicarbonate aqueous solution is preferably an aqueous solution of one or more of ammonium bicarbonate, potassium bicarbonate and sodium carbonate; the aqueous carbonate solution is preferably an aqueous solution of potassium carbonate and/or sodium carbonate; the pH value of the mobile phase A is preferably 8-9, more preferably 8-8.5; the pH value can be regulated by carbonic acid or sodium hydroxide; the alcohol solvent is not particularly limited as long as it is well known to those skilled in the art, and is preferably one or more of methanol, ethanol and isopropanol, more preferably methanol; the linear velocity and flow rate of the mobile phase are preferably 3.5-6.5 cm/min, more preferably 4-6 cm/min; in the present invention, it is preferable to equilibrate the column and then load the column for elution; the equilibrium chromatographic column is preferably carried out with 5% mobile phase B and 95% mobile phase a; the time for balancing the chromatographic column is preferably 10-20 min; in the present invention, the reversed phase chromatography purification preferably employs gradient elution; the gradient elution process is that the mobile phase B is 0-5 min 5% by volume percent, the mobile phase B is 5-60% by volume percent after 5.1-50 min, and then the mobile phase B is maintained at 60% by volume percent for at least 10min; the detection wavelength of the reverse phase chromatography purification is preferably 200 to 280nm, more preferably 210 to 260nm, still more preferably 220 to 254nm.

The crude filtrate of chiral polypeptide medicine tends to form a certain thermodynamic equilibrium state in chromatographic column under a certain temperature condition because of the buffer action of carbonate, namely, the chiral polypeptide medicine of target product has a certain nucleophilic reaction between active carbonyl in the structure and water/alcohol in mobile phase (lower alcohol, ketone solvent or lower alcohol, ketone aqueous solution or other lower alcohol, ketone solution combination and other proton solvents) under a certain pH value condition because of the buffer action of carbonate, at this moment, there is a relative thermodynamic equilibrium state of the substance in the solution, namely, 2 unstable intermediate states of carbonate form, ionic form, molecular form and enol interconversion of the chiral polypeptide compound, and stable state formed by the active carbonyl and the residual inorganic salt in other crude products, the chiral polypeptide compounds are different forms, and the mother body is the chiral compound, but under the conditions of a certain temperature, a certain carbonate concentration and a certain sample concentration, the specific thermodynamic equilibrium relationship of various ions, molecular forms and salt forms is formed, the chromatographic behavior of the same compound form is concentrated, but the chromatographic retention behavior caused by the chemical structure difference of different compound forms (ionic, molecular, different salt forms and the like) is obviously different, and the isomer impurities (enantiomer, diastereomer and the like, epimer and cis-trans isomer) are obviously different due to the fact that the nucleophilic reaction with water/alcohol can also occur, and the compound concentration of the isomer impurities is lower than that of the target polypeptide compound, the reaction intensity and degree of the isomer impurities are different, under the same temperature and pressure conditions, the method is characterized in that the method comprises the steps of carrying out a chromatographic separation on a target chiral polypeptide drug, wherein the target chiral polypeptide drug is prepared from a solution of an isomer impurity, a target chiral polypeptide drug and a main component of the target chiral polypeptide drug, wherein the solution of the isomer impurity is prepared from a solution of the isomer impurity, the solution of the isomer impurity and the target chiral polypeptide drug, and the solution of the isomer impurity are prepared from a solution of the isomer impurity. The dynamic change process is utilized to realize the retention difference of chiral isomer impurities on the conventional chromatograph, the chiral isomer impurities which are effectively separated on the conventional chromatographic method and chromatographic conditions are breakthrough realized, and the method is simple, convenient and low-cost, and is extremely easy to realize the separation and purification preparation of chiral polypeptide medicines on an industrial scale.

In the present invention, the chirality is preferably selected fromThe salt conversion is carried out on the refined solution of the polypeptide drug by chromatography, more preferably, the salt conversion is carried out by reverse phase chromatography by using a buffer system consisting of inorganic salt and acid, and even more preferably, the salt conversion is carried out by reverse phase chromatography by using a buffer system consisting of acetate and acetic acid; the acetate is preferably one or more of ammonium acetate, sodium acetate and potassium acetate; in the present invention, the reversed phase chromatography preferably uses octadecylsilane chemically bonded silica as a stationary phase; more preferably YMC Triart prep C-S or Nano Unisil C18; the particle size of the stationary phase is preferably 7-10 mu m; the pore size of the stationary phase is preferablyThe mobile phase of the reversed phase chromatographic exchange salt comprises a mobile phase A1, a mobile phase A2 and a mobile phase B; the mobile phase A1 is preferably 10-30 mM acetate water solution, more preferably 15-25 mM acetate water solution, and still more preferably 20mM acetate water solution; the pH value of the mobile phase A1 is preferably 3-4, more preferably 3.5-4; the pH value is preferably adjusted by acetic acid; the mobile phase A2 is preferably 0.002 to 0.01wt% aqueous acetic acid solution, more preferably 0.005 to 0.01wt% aqueous acetic acid solution; the mobile phase B is preferably an alcohol solvent and/or acetonitrile; the alcohol solvent is preferably one or more of methanol, ethanol and isopropanol; in the present invention, the reversed phase chromatography exchange salt is preferably eluted with a gradient; the gradient elution process comprises the steps of sequentially eluting 95% of mobile phase A1 and 5% of mobile phase B for 30-60 min, eluting 95% of mobile phase A2 and 5% of mobile phase B for 15-25 min, then eluting with mobile phase B and mobile phase A2 within 15-40 min, and maintaining the mobile phase B at 60% for at least 20min after the mobile phase B is raised from 5% to 60%; more preferably, the elution is carried out for 35-60 min by sequentially eluting 95% of mobile phase A1 and 5% of mobile phase B, the elution is carried out for 18-22 min by 95% of mobile phase A2 and 5% of mobile phase B, then the elution is carried out by adopting mobile phase B and mobile phase A2 within 15-40 min, the mobile phase B is increased from 5% to 60%, and then the mobile phase B is maintained to be 60% of mobile phase B for at least 20min; in the embodiment provided by the invention, the gradient elution program is specifically that 0-60 min 95% mobile phase A1 and 5% mobile phase B, 60.1-80 min mobile phase A2 and 5% mobile phase B, and 80.1-120 min are adopted Mobile phase B eluted with mobile phase A2 and mobile phase B increased from 5% to 60%, after which it remained at mobile phase B60% for at least 20min; or specifically, 0-35 min 95% of mobile phase A1 and 5% of mobile phase B, 35.1-55 min of mobile phase A2 and 5% of mobile phase B, 55.1-70 min eluting by adopting the mobile phase B and the mobile phase A2, and the mobile phase B is raised from 5% to 60%, and then is maintained to be 60% of the mobile phase B for at least 20min; or specifically, 0 to 60min 95% of mobile phase A1 and 5% of mobile phase B,60.1 to 80min of mobile phase A2 and 5% of mobile phase B,80.1 to 120min eluting by adopting the mobile phase B and the mobile phase A2, and the mobile phase B is raised from 5% to 60%, and then is maintained to be 60% of the mobile phase B for at least 20min. The linear velocity and flow rate of the mobile phase at the time of reverse phase chromatography salt transfer are preferably 3.5 to 6.5cm/min, more preferably 3 to 5cm/min.

After salt conversion, collecting the purified solution required by the compounding of related substances, concentrating under reduced pressure, and freeze-drying to obtain chiral polypeptide medicine; the temperature of the reduced pressure concentration is preferably not more than 35 ℃, and the vacuum degree is more than-0.08 MPa; the freeze-drying process is preferably specifically as follows: pre-freezing to-40 to-30 ℃ and keeping for 2 to 4 hours, first-stage drying at-5 to 0 ℃ for 24 to 36 hours, sublimation drying at 20 to 25 ℃ for 24 to 36 hours, and stabilizing the vacuum degree at 0.01 to 1.00MPa; more preferably specifically: pre-freezing to-40 to-30 ℃ and keeping for 2 to 4 hours, first-stage drying at-5 to-2 ℃ for 30 to 36 hours, sublimation drying at 20 to 25 ℃ for 24 to 30 hours, and stabilizing the vacuum degree at 0.01 to 1.00MPa; the more preferred specific ones are: pre-freezing to-40 to-30 ℃ and keeping 2 to 4 hours, first-stage drying at-5 to-2 ℃ for 36 hours, sublimation drying at 25 ℃ for 24 hours, and stabilizing the vacuum degree at 0.01 to 1.00MPa.

According to the invention, the crude chiral polypeptide type medicine solution is pretreated by utilizing a dynamic thermodynamic equilibrium method, so that impurities and medicine components of the crude chiral polypeptide type medicine solution reach a special thermodynamic stable state, a limited separation mode on conventional chromatography is formed, other process impurities can be effectively controlled, and effective separation can be realized without other purification processes; meanwhile, the mode of realizing effective separation by utilizing a dynamic thermodynamic equilibrium method breaks through the separation effect which cannot be achieved by the conventional chromatographic materials and methods, and the cost is far lower than that of a complex purification method which needs chiral resolution or special chromatographic materials and processes to realize the separation of isomer impurities.

To further illustrate the present invention, a method for preparing chiral polypeptide-type drugs using dynamic thermodynamic equilibrium purification is provided in the present invention as follows.

The reagents used in the examples below are all commercially available.

Example 1

A method for preparing a chiral polypeptide drug using dynamic thermodynamic equilibrium purification comprising:

(1) Sample treatment: the crude chiral polypeptide medicine crude product (YH-21101201) with the purity of 64.71 percent is obtained by adopting a solid phase synthesis method, 32.25g of the crude chiral polypeptide medicine crude product is dissolved by methanol according to the concentration of 150g/L, 10mmmol/L potassium carbonate solution is used for diluting to about 15mg/ml, an organic filter membrane with the concentration of 0.45um is used for filtering, the crude filtrate is obtained, the detection pH value is 8.4, the filtrate is placed at 26 ℃, and the detection pH value is 7.9 after slow stirring for 30 min.

(2) Purifying: about 2000ml of the crude solution was packed with a DAC150 liquid phase purification system, packing YMC Triart prep C-S, 7 μm,12nm, packing height 250mm, and room temperature to 26 ℃. The mobile phase A phase is: 10mmol/L potassium carbonate aqueous solution, and sodium hydroxide solution to adjust the pH value to 8.1; the mobile phase B phase is: methanol; volumetric flow rate: 1100ml/min; the column was equilibrated with 5% phase B, 95% phase a for about 10min. Gradient elution procedure: 5% -5% of B phase in 5min, 5.1min to 60min gradient of B phase from 5% to 60%, then 60.01-70.01 min maintaining B phase 60% gradient for at least 10min, detecting wavelength: 220nm.

The purity detection spectrum of chiral polypeptide crude YH-21101201 is shown in figure 1. The chiral medicine of the polypeptide and the isomer impurity thereof can be seen to be very close to each other in chromatographic behavior in analytical detection, and can not be effectively separated in analytical detection, and during industrial separation and purification, the performance and equipment performance of a chromatographic packing are far inferior to those of an analytical detection by at least 2 orders of magnitude due to the fact that the treatment capacity is enlarged by more than the analytical detection, and the separation degree of a theoretical preparation chromatograph can be poorer, so that a large broadening of both a main peak and an isomer peak thereof can be observed remarkably in the preparation chromatograph, wherein the main peak and the isomer peak thereof can be reduced in a cross part due to the fact that the total quantity of the isomer impurity is dispersed in a fixed manner in the total quantity, the main peak is highly overlapped in the preparation chromatograph, and the crude isomer impurity content is about 30.12%, a mixture of various forms of the main component and the isomer impurity is formed in the pretreatment of a sample, the chromatographic behavior of the crude isomer impurity is changed into a mixed compound form which is relatively close to be relatively similar to the single substance form, the chromatographic behavior is shown as a large peak broadening of the chromatograph, but the total quantity is consistent with the substance conservation, the main peak is adopted in the separation, the separation is carried out by adopting the sectional collection, the large-stage separation, the main peak broadening is adopted, the large broadening is adopted, the main peak broadening is fixed, and the main peak broadening is adopted, and the main peak is fixed in the large-stage, and the main peak broadening is fixed, and the main peak is highly coincident after main component and the main component is highly coincident with the main component and the main component can be highly.

(3) Salt conversion

Reversed phase chromatography conditions: high performance liquid chromatography with octadecylsilane chemically bonded silica as stationary phase is used to prepare column with particle diameter of 10 μm and pore diameter of 100A. The column diameter and length were: 150x250mm. The mobile phase A1 phase is: 10mM ammonium acetate, pH was adjusted to 3.5 with acetic acid; the mobile phase A2 phase is: 0.005% aqueous acetic acid; the mobile phase B phase is: methanol; volumetric flow rate: 700ml/min; the A1 phase maintains a gradient of 95% in 60min, the A2 phase is switched, the A2 phase maintains a gradient of 95% from 60.1min to 80min, the B phase increases from 5% to 60% in 80.1min to 120min, and then a gradient of 60% of the B phase is maintained for 30min, wherein the B phase is methanol.

Concentrating the collected purified solution meeting the requirements of related substances by reduced pressure rotary evaporation at the temperature below 35 ℃ until the concentration is about 20 mg/ml-40 mg/ml, and then freeze-drying to obtain chiral polypeptide type medicinal solution with any single impurity not more than 0.5%, total impurities not more than 1.0% and purity higher than 99%.

The purity detection spectrum of the chiral polypeptide type drug finally obtained in example 1 is shown in fig. 2. The method comprises the following steps of: the maximum single impurity of the finished product of this example was 0.43% chiral diastereomeric impurity, total impurity was 0.46%, purity was 99.54%, and purification yield was 58.2%.

The chiral polypeptide type drug finally obtained in example 1 was analyzed by preparative chromatography to obtain its separation and purification preparation profile as shown in fig. 3.

Example 2

(1) Sample treatment: 28.56g of crude chiral polypeptide medicine (YH-21101201) with the purity of 64.71% is obtained by adopting a solid-phase synthesis method, the crude chiral polypeptide medicine is dissolved by methanol according to the concentration of 130g/L, the crude chiral polypeptide medicine is diluted to about 15mg/ml by 200mmol/L ammonium bicarbonate solution, then the crude chiral polypeptide medicine is filtered by an organic filter membrane with the concentration of 0.45 mu m, the crude chiral polypeptide medicine filtrate is obtained, the pH value of the crude chiral polypeptide medicine filtrate is detected to be 8.0, the filtrate is placed between 25 ℃ and is slowly stirred for 30min, and then the pH value of the crude chiral polypeptide medicine filtrate is detected to be 7.8.

(2) Purifying: about 2000ml of the crude solution was purified using DAC150 liquid phase purification system with packing of Nano Unisil C18, 10 μm,the filling height was 250mm. The mobile phase A phase is: 200mmol/L ammonium bicarbonate aqueous solution, and adjusting the pH to 8.1 with sodium hydroxide; the mobile phase B phase is: methanol; volumetric flow rate: 1000ml/min; gradient elution procedure: 5% -5% of phase B in 5min, 5.1min to 50min gradient from 5% to 60%, maintaining 60% gradient of phase B for at least 10min, and detecting wavelength: 220nm; and collecting main peaks in sections, combining the section purified liquid with any single impurity not more than 0.5%, combining and concentrating the unqualified sections, and repeating the purification steps until the purified liquid is qualified.

(3) Salt conversion

Chromatographic column: octadecylsilane chemically bonded silica (Nano university C18, 10 μm,) Is a stationary phase, the column is->

Mobile phase: phase A1, 20mM aqueous ammonium acetate solution, phase A2, 0.01% aqueous acetic acid solution, phase B methanol;

detection wavelength: 220nm;

the volume flow rate is 800ml/min;

the salt transfer elution gradient is shown in table 1:

TABLE 1 salt-transition elution gradient procedure

Time (minutes)	Mobile phase A1 (%)	Mobile phase A2 (%)	Mobile phase B (%)
				0	95	0	5
35	95	0	5
				35.1	0	95	5
55	0	95	5
				55.1	0	95	5
70	0	40	60
				120	0	40	60

Concentrating the collected purified solution meeting the requirements of related substances by rotary evaporation under reduced pressure at the temperature below 30 ℃ until the concentration is about 20 mg/ml-40 mg/ml, and then freeze-drying to obtain chiral polypeptide type drug solution with any single impurity not more than 0.5%, total impurities not more than 1.0% and purity higher than 99.0%.

The purity detection spectrum of the chiral polypeptide type drug finally obtained in example 2 is shown in fig. 4. The method comprises the following steps of: the largest single impurity in this example was 0.49% chiral diastereomeric impurity, total impurity 0.57%, purity 99.43% and purification yield 61.6%.

Example 3

(1) Sample treatment: the chiral polypeptide crude drug (YH-21101801) with the crude purity of 64.55% is obtained by adopting a solid phase synthesis method, 29.88g of the crude drug is dissolved by methanol according to the concentration of 140g/L, 10mmol/L sodium carbonate solution is diluted to about 12mg/ml, and then an organic filter membrane with the concentration of 0.45 mu m is used for filtering to obtain crude filtrate, the pH value is detected to be 8.5, the filtrate is placed between 26 ℃ and is slowly stirred for 30min, and then the pH value is detected to be 8.3.

(2) Purifying: about 2000ml of the crude solution was purified using DAC150 liquid phase purification system with packing of Nano Unisil C18, 10 μm,the filling height was 250mm. The mobile phase A phase is: 10mmol/L sodium carbonate aqueous solution; the mobile phase B phase is:methanol; volumetric flow rate: 1100ml/min; gradient: 5% -5% of phase B in 5min, 5.1min to 60min gradient from 5% to 60%, and maintaining 60% gradient of phase B for at least 10min, and detecting wavelength: 220nm; and collecting main peaks in sections, combining the section purified liquid with any single impurity not more than 0.5%, combining and concentrating the unqualified sections, and repeating the purification steps until the purified liquid is qualified.

(3) Salt conversion

Reversed phase chromatography conditions: high performance liquid chromatography preparation column with octadecylsilane chemically bonded silica as stationary phase (packing Nano Unisil C18, 10 μm,150mm diameter, 250mm packing height), the A1 phase in the mobile phase is: 20mM ammonium acetate, pH was adjusted to 3.5 with acetic acid; the mobile phase A2 phase is: 0.002% aqueous acetic acid; the mobile phase B phase is: methanol; volumetric flow rate: 750ml/min; phase A1 maintains a 95% gradient over 60min, phase A2 is switched, phase A2 maintains a 95% gradient from 60.1min to 80min, phase B increases from 5% to 60% gradient over 80.1min to 120min and maintains a 60% gradient of phase B for at least 30min, the phase B being methanol.

Concentrating the collected purified solution meeting the requirements of related substances under reduced pressure and rotary evaporation at the temperature of below 30 ℃ until the concentration is about 20 mg/ml-40 mg/ml, and then freeze-drying to obtain a chiral polypeptide type drug finished product.

The purity detection spectrum of the chiral polypeptide crude YH-21101801 drug is shown in figure 5; the purity detection spectrum of the chiral polypeptide type drug finally obtained in example 3 is shown in fig. 6. The method comprises the following steps of: the largest single impurity in this example was chiral diastereomeric impurity 0.31%, total impurity 0.31%, purity 99.69% and purification yield 69.78%.

Comparative example 1

The polypeptide sample was purified using only conventional reverse phase chromatography, and the specific procedure and experimental results were as follows:

(1) Sample treatment: the chiral polypeptide crude drug (YH-21101801) with crude purity reaching 64.55% is obtained by adopting a chemical (solid phase) synthesis method, 25.34g of the crude drug is dissolved by stirring with methanol according to the concentration of 120g/L, after the crude drug is completely dissolved, the crude drug is diluted to about 6mg/ml by using 0.1% trifluoroacetic acid aqueous solution, and then the crude drug is filtered by using an organic filter membrane with 0.45um, insoluble matters are filtered, and crude filtrate is obtained.

(2) Purifying: about 2000ml of the crude solution was purified by DAC150 liquid phase purification system with a packing of FUJI C18, 10 μm, The filling height was 250mm. The mobile phase A phase is: 0.1% aqueous trifluoroacetic acid; the mobile phase B phase is: methanol; volumetric flow rate: 1100ml/min; gradient: 5% -5% of B phase in 5min, 5.1 min-60 min gradient of B phase from 5% to 60%, and maintaining 60% gradient of B phase for at least 10min, detecting wavelength: 220nm; the main peak is collected in segments.

(3) Salt conversion

Chromatographic column: octadecylsilane chemically bonded silica is used as a stationary phase (FUJI C18, 10 μm,) The column is

Mobile phase: phase A1, 30mM aqueous ammonium acetate, phase A2, 0.02% aqueous acetic acid, phase B methanol;

detection wavelength: 220nm;

volumetric flow rate: 750ml/min;

the salt transfer elution gradient is shown in table 2.

TABLE 2 salt-transition elution gradient procedure

Time (minutes)	Mobile phase A1 (%)	Mobile phase A2 (%)	Mobile phase B (%)
				0	95	0	5
60	95	0	5
				60.1	0	95	5
80	0	95	5
				80.1	0	95	5
120	0	40	60
				150	0	40	60

Concentrating the collected main peak under reduced pressure and rotary evaporation at the temperature below 30 ℃ until the concentration is about 20 mg/ml-40 mg/ml, and then freeze-drying to obtain the chiral polypeptide type drug finished product.

Since the experiment was not performed with a specific sample pretreatment and according to specific purification process conditions, the purification effect was significantly different from that of the comparative example, and the final product could not effectively separate the isomer impurities.

The purity detection spectrum of the chiral polypeptide type drug finally obtained in comparative example 1 is shown in figure 7; the method comprises the following steps of: the final product of this example had a maximum single impurity of 8.18% isomer impurity (diastereomer), total impurity of 14.02%, purity of 85.98% and recovery of 65.52% of the total major component.

The chiral polypeptide type drug finally obtained in comparative example 1 was analyzed by preparative chromatography to obtain its separation and purification preparation profile as shown in fig. 8.

Comparative example 2

A number of tests are carried out on the sample treatment process in the step (1), screening and optimizing conditions are carried out to confirm that the chiral polypeptide type drug can be effectively separated from the isomer impurities thereof, and the treatment condition screening is carried out by taking a crude chiral polypeptide type drug (YH-21101801) as an example, and the results are shown in Table 3.

TABLE 3 sample processing condition screening results

Only in the case of basic buffer salts, and when the molar concentration ratio of the basic buffer salts to the target compound is at a proper concentration, the compound is beneficial to promote the formation of various forms of the compound, neither the dissociation of the compound nor the conversion of the compound to the same type is inhibited, and only when the concentration, the salt concentration and the temperature and the time of the compound are right, the compound forms at least 5 thermodynamic equilibrium states (namely, molecular state, ionic state, carbonate form and 2 unstable states of enol interconversion formed by carbonyl), so that one substance forms 5 different types of forms, and the compound changes from a theoretical sharp single peak form to a wide and fleshy peak form on the chromatograph. This dwarf peak shape provides a possibility for the separation of chiral polypeptide type drugs from the isomer impurities (enantiomers, diastereomers, etc., epimers, cis-trans isomers) whose physicochemical properties are very close.

The method utilizes a dynamic thermodynamic equilibrium relation to purify and transform chiral polypeptide drugs under a sample pretreatment mode and a reversed phase chromatographic system, solves the problem that the quality of industrial products is influenced by isomer impurities (enantiomer, diastereomer, epimer and the like) of the chiral polypeptide drugs, can effectively control other process impurities, utilizes the fact that the chiral polypeptide drugs capable of forming enol tautomeric groups in the structure have a certain thermodynamic dynamic equilibrium relation under the condition that certain temperature and buffer salt exist, actively and skillfully constructs different compound forms formed by the drugs and the impurities to realize different retention behaviors on the chromatograph, finally realizes the chromatographic separation through simple mobile phase configuration and temperature control, realizes the differentiated thermodynamic equilibrium relation between the compound and the isomer impurities which are most difficult to separate by the relative relation between the inorganic salt concentration and the compound content, and realizes the effective separation and the effective control of the chiral polypeptide drugs and the isomer impurities by utilizing the relation.

Claims

1. A method for preparing chiral polypeptide type medicine by dynamic thermodynamic equilibrium purification, which is characterized by comprising the following steps:

the solvent is selected from one or more of methanol, ethanol, isopropanol, water, acetonitrile, inorganic acid aqueous solution and inorganic salt aqueous solution; the concentration of the chiral polypeptide crude drug after dissolution is 100-200 g/L;

the ratio of the alkaline inorganic buffer salt to the chiral polypeptide crude drug is (0.8-1.5) mmol:1g;

the temperature of the placing is 20-30 ℃; the time for placing is 30-60 min;

the stationary phase purified by the reversed phase chromatography is octadecylsilane chemically bonded silica gel; the mobile phase purified by the reversed phase chromatography adopts an aqueous solution of an alkaline inorganic buffer salt as a mobile phase A and adopts an alcohol solvent and/or acetonitrile as a mobile phase B; the reversed phase chromatography purification adopts gradient elution; the gradient elution process is that the mobile phase B is 0-5 min 5% by volume percent, the mobile phase B is 5-60% by volume percent after 5.1-50 min, and then the mobile phase B is maintained at 60% by volume percent for at least 10min; the detection wavelength of the reversed phase chromatography purification is 200-280 nm;

The chiral polypeptide type drug is shown as a formula (A) or a formula (B):

R ₂ selected from amino or H;

or R is ₁ And R is R ₂ Connected into a ring;

n is 0 or 1;

the A-A-A is a peptide chain formed by 1 to 25 chiral amino acids.

2. The method of claim 1, wherein the chiral polypeptide-type drug is represented by formula (I) to formula (VI):

the A-A-A is 3-25 chiral amino acids to form a peptide chain.

3. The process according to claim 1, wherein the mobile phase a is 50 to 250mmol/L of aqueous bicarbonate or 10 to 20mmol/L of aqueous carbonate; the pH value of the mobile phase A is 8-9;

4. The method as recited in claim 1, further comprising:

5. The method according to claim 4, wherein the salt transfer is performed by chromatography, in particular by reverse phase chromatography using a buffer system consisting of acetate and acetic acid;

6. The method according to claim 4, wherein the reduced pressure concentration is carried out at a temperature of not more than 35 ℃ and a vacuum degree of-0.08 MPa or more;