CN114405065A - Method for preparing chiral polypeptide type medicine by dynamic thermodynamic equilibrium purification - Google Patents
Method for preparing chiral polypeptide type medicine by dynamic thermodynamic equilibrium purification Download PDFInfo
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Abstract
The invention provides a method for preparing a chiral polypeptide type medicament by dynamic thermodynamic equilibrium purification, which comprises the following steps: s1) dissolving the chiral polypeptide type crude drug product with a solvent, mixing with an aqueous solution of alkaline inorganic buffer salt, and standing to obtain a chiral polypeptide type crude drug product solution; s2) purifying the crude chiral polypeptide drug solution by reverse phase chromatography to obtain a pure chiral polypeptide drug solution. Compared with the prior art, the method utilizes a dynamic thermodynamic equilibrium method to pretreat the chiral polypeptide type crude drug solution, so that impurities and drug components of the solution reach a special thermodynamic stable state, thereby forming a mode of limited separation on conventional chromatography and effectively controlling other process impurities; meanwhile, the mode of realizing effective separation by using the dynamic thermodynamic equilibrium method realizes the separation effect which cannot be achieved by the conventional chromatographic material and the conventional method in a breakthrough manner.
Description
Technical Field
The invention belongs to the technical field of medicine purification, and particularly relates to a method for preparing a chiral polypeptide type medicine by dynamic thermodynamic equilibrium purification.
Background
Polypeptide drugs, which are widely present in biological systems, are signal molecules, transport molecules, and also digestive molecules. As signaling molecules, polypeptides control biological functions of organisms such as cell division, mating, chemotaxis, pain sensation, 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 facilitate the passage of ions through cell membrane channels; as a digestive molecule, polypeptides play an important role in nutrient uptake by cells and intact organisms. In addition, the polypeptides may also be used 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 areas, such as diabetes, allergy, anti-infection, obesity, diagnostics, tumors, arthritis and cardiovascular diseases.
Because polypeptide drugs are mainly derived from endogenous polypeptides or other natural polypeptides, generally, the structure is clear, the action mechanism is clear, and many properties are often between those of small molecule chemical drugs and macromolecular protein/antibody drugs, for example, compared with small molecule chemical drugs, the polypeptide drugs have the advantages of short half-life, instability, easy rapid degradation in vivo, unstable preparation, low-temperature storage, low cost, high molecular chemical drug (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. Attempts are often made to isolate the polypeptide of interest from other products produced by biological or chemical synthesis methods using a combination of different chromatographic techniques. These techniques separate mixtures of polypeptides based on their charge, degree of hydrophobicity, size, or specific interaction between the polypeptide of interest and the immobilized capture agent. For each of these techniques, several different chromatography resins are available, allowing precise adjustment of the purification scheme according to the specific polypeptide involved. The essence of each of these separation methods is that the polypeptides can either be made to move down a long column at different rates, achieve an enhanced physical separation as they move further down the column, or selectively adhere to the separation medium, and then be eluted differentially by different solvents. In some cases, the polypeptide of interest is separated from the impurities when the impurities specifically adhere to the column and the polypeptide of interest does not adhere to the column, i.e., the polypeptide of interest is present in a "flow-through". Large-scale, low-cost purification of polypeptides to a purity sufficient for use as a human drug remains a significant challenge.
The method of separation and purification is generally determined by the nature of the material being separated. The method commonly used for extracting and separating the protein and the polypeptide comprises the following steps: salting out, ultrafiltration, gel filtration, isoelectric precipitation, ion exchange chromatography, affinity chromatography, adsorption chromatography, reverse flow dissolution, enzymolysis, etc. These methods are often combined to separate and purify a particular substance. The separation and purification method of the polypeptide in the industrial application is as follows:
high Performance Liquid Chromatography (HPLC)
The advent of HPLC has provided an advantageous means for the isolation of peptidic materials because proteins, polypeptides and other compounds can be produced on a preparative scale under appropriate chromatographic conditions to produce biologically active polypeptides. Therefore, researchers have done a lot of work on the methods for the isolation and preparation of polypeptides. How to maintain polypeptide activity, how to select stationary phase materials, eluent types, and how to analyze the assay are all the subject of current research.
Reversed phase high Performance liquid chromatography (RP-HPLC)
The RP-HPLC separation of polypeptides first allows the determination of the retention of polypeptides of different structures on the column. Different amino acid compositions have certain influence on retention coefficients, wherein polar amino acid residues in peptides consisting of 2-20 amino acids can reduce retention time on a column; in peptides consisting of 10-60 amino acids, the retention time on the column can also be reduced by a larger amount of non-polar amino acids, while in small peptides containing 5-25 amino acids, the retention time on the column can be extended by an increase in non-polar amino acids.
In addition, the influence of conditions such as peptide chain length, amino acid composition, temperature and the like on retention conditions is reported by a plurality of documents at home and abroad, and proper conditions for separating and extracting the polypeptide are obtained by computer processing analysis simulation.
Hydrophobic Interaction Chromatography (HIC)
HIC achieves the purpose of separation and analysis by utilizing the fact that polypeptide contains hydrophobic genes and can generate hydrophobic effect with a stationary phase, and compared with RP-GPLC, HIC has the characteristic of less polypeptide denaturation.
Size Exclusion chromatography (Sizs-Exclusion chromatography, SEC)
SEC is to separate and purify polypeptide substance by using the difference of size and shape of polypeptide molecule, especially for some molecules in larger aggregation state, and the separation of compounds with different structures and configurations on SEC column is completely different, so that variants with different configurations or slight difference in amino acid sequence can be separated.
Ion Exchange chromatography (Iron-Exchange chromatography, IEXC)
The IEXC can separate and purify the polypeptide with biological activity by utilizing the charge difference of the polypeptide under neutral conditions. It can be divided into two types of cation column and anion column, and some new resins, such as macroporous resin, homogeneous porous resin, ion exchange cellulose, sephadex, sepharose resin, etc. In the research of the separation and analysis of polypeptide substances, the research on the properties of polypeptides, eluents and elution conditions is more, different polypeptide separation conditions are different, and particularly, the ionic strength, salt concentration and the like of the eluents have larger influence on the purification.
High Performance Displacement Chromatography (HPDC)
HPDC is a method for separating by exchanging samples on a chromatographic column by using a small-molecule high-efficiency displacer. It has the characteristic of separating components with a small content of components. Studies have shown that the lower the relative molecular mass of the displacer, the easier it is to bind to the stationary phase, and therefore smaller displacers are required to purify the polypeptides by displacement when separating them with a smaller relative molecular mass.
Perfusion Chromatography (Perfusion Chromatography, PC)
PC is a chromatographic separation method of a mobile phase based on the principle of a molecular sieve and high-speed flow, and the separation effect is directly influenced by the size of the aperture of a stationary phase and the speed of the mobile phase. The test proves that the compound has the characteristics of low input and high output in the production and preparation processes. The available PC stationary phase in the market has more types, and is suitable for separating and using polypeptides with different molecular weights.
Affinity Chromatography (Affinity Chromatography, AC)
AC is a chromatographic method for separating substances by utilizing the specific affinity between ligands attached to a matrix of a stationary phase and ligands that specifically interact with the ligands. Since the concept of affinity chromatography was proposed in 1968, many combinations were found in search of specific affinity interaction substances, such as antigen-antibody, enzyme-catalytic substrate, lectin-polysaccharide, oligonucleotide and its complementary strand, and so on. The separation of polypeptides is currently mainly carried out by using monoclonal antibodies or biomimetic ligands with affinity, wherein the ligands are natural and artificially synthesized according to the structures.
Immobilized Metal Affinity Chromatography (IMAC)
IMAC is an affinity method that has been developed in recent years. The stationary phase matrix has some metal ions, such as Cu, chelated thereon2+、Ni2+、Fe3+The column can chelate the polypeptide with Lys, Met, Asp, Arg, Tyr, Glu and His in the side chain through coordination bond, especially the structure containing His-X-X-X-His in the peptide sequence is easy to be combined on the metal ion affinity column, and the purification effect is better.
Systematic application of polypeptide separation engineering
The above-mentioned techniques for separating polypeptides are combined with each other in practical application, and different separation means are adopted according to different properties of the separated polypeptides. In particular, in the post-genome era, intensive research on proteome has led to continuous improvement of means for separating polypeptides, comprehensive utilization of various properties of polypeptides, and realization of separation and purification of different types of polypeptides by using the aforementioned conventional polypeptide extraction method and simultaneously using a combination of emerging separation techniques such as high performance liquid chromatography and conventional techniques.
The polypeptide drugs have more chirality, the conventional separation method is to use reversed phase chromatography to realize separation by utilizing the retention difference of drugs and impurities, however, when facing the impurities of enantiomer, diastereoisomer, cis-trans isomer and epimer, the chromatographic method has little difference between the structure and the medicament and almost no difference between the physicochemical properties, the effective separation can hardly be realized under the conventional reversed phase chromatography material and the conventional chromatography process, which becomes the key for restricting the quality of the chiral polypeptide type medicine, so the isomer impurities become the difficulty of the purification and preparation of the chiral polypeptide type medicine, the extremely expensive chiral preparation material is generally required to be used for chromatographic resolution or other special purification and separation processes, the material and production cost of the chiral polypeptide type medicine is 10-20 times or even higher than the conventional purification preparation material and cost, and the market price of the chiral polypeptide type medicine is influenced indirectly.
Due to the particularity of the biological synthesis process or the chemical synthesis process, various process impurities and degradation impurities of the polypeptide drugs known in the industry are extremely difficult to purify, remove and effectively control, and although various combinations of chiral columns, chromatographic conditions and the like can be used in analysis and detection to possibly effectively detect related impurities, the analysis and detection is harsh in chromatographic conditions, extremely small in loading capacity, extremely fine in chromatographic packing particle size, extremely high in material cost and extremely high in corresponding requirements of equipment and instruments, so that the analysis method is almost impossible to realize amplification production in industrial purification and 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 drug by dynamic thermodynamic equilibrium purification, which utilizes the dynamic thermodynamic equilibrium relationship of chemical substances under a certain temperature and pressure condition in a solution form to make obvious chromatographic retention difference exist between chiral polypeptide drug and isomer impurities thereof, thereby realizing that the conventional industrial chromatographic separation is difficult to effectively and simply separate (enantiomer, diastereomer, epimer and other isomer impurities) by no more than 0.5%, other process and degraded impurities by no more than 0.1%, all total impurities by no more than 1.0%, and the yield by more than 50% when the purity is more than 99.0%.
The invention provides a method for preparing a chiral polypeptide type medicament by dynamic thermodynamic equilibrium purification, which comprises the following steps:
s1) dissolving the chiral polypeptide type crude drug product with a solvent, mixing with an aqueous solution of alkaline inorganic buffer salt, and standing to obtain a chiral polypeptide type crude drug product solution;
s2) purifying the chiral polypeptide type medicine crude product solution by reverse phase chromatography to obtain a pure chiral polypeptide type medicine solution;
the chiral polypeptide-type drug comprises a group represented by formula (1):
wherein R is1One 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 group;
R2selected from amino or H;
or R1And R2Connecting to form a ring;
n is 0 or 1.
Preferably, the chiral polypeptide drug is represented by formula (A) or formula (B):
the substituent in the substituted C1-C10 alkyl, substituted C6-C10 aryl and substituted C6-C10 heterocyclic group is 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 1-25 chiral amino acids.
Preferably, the chiral polypeptide drug is represented by formula (I) to formula (VI):
the A-A-A is a peptide chain formed by 3-25 chiral amino acids.
Preferably, the solvent is selected from one or more of methanol, ethanol, isopropanol, water, acetonitrile, aqueous inorganic acid solution and aqueous inorganic salt solution; the concentration of the dissolved crude chiral polypeptide drug 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 proportion of the alkaline inorganic buffer salt to the chiral polypeptide type crude drug is (0.8-1.5) mmol: 1g of the total weight of the composition.
Preferably, the temperature of the placement is 20-30 ℃; the standing time is 30-60 min.
Preferably, the stationary phase purified by the reverse phase chromatography is octadecylsilane chemically bonded silica; the mobile phase purified by the reverse phase chromatography takes an aqueous solution of alkaline inorganic buffer salt as a mobile phase A and takes an alcohol solvent and/or acetonitrile as a mobile phase B; the reversed phase chromatography purification adopts gradient elution; the procedure of gradient elution is that the volume percentage is calculated, the procedure of gradient elution is 0-5 min, the content of the mobile phase B is 5%, 5.1-50 min is that the content of the mobile phase B is increased from 5% to 60%, and then the content of the mobile phase B60% is maintained for at least 10 min; the detection wavelength of the purification by the reversed phase chromatography is 200-280 nm.
Preferably, the mobile phase A is 50-250 mmol/L bicarbonate aqueous solution or 10-20 mmol/L carbonate aqueous solution; the pH value of the mobile phase A is 8-9;
the linear velocity flow rate of the mobile phase during purification by the reversed phase chromatography is 3.5-6.5 cm/min.
Preferably, the method further comprises the following steps:
and (3) carrying out salt conversion on the pure solution of the chiral polypeptide drug by chromatography, carrying out reduced pressure concentration, and carrying out freeze drying to obtain the chiral polypeptide drug.
Preferably, the salt conversion is carried out by chromatography, in particular, the salt conversion is carried out by reverse phase chromatography by using a buffer system consisting of acetate and acetic acid;
the mobile phase of the reverse phase chromatographic exchange salt conversion comprises a mobile phase A1, a mobile phase A2 and a mobile phase B; the mobile phase A1 is a 10-30 mM acetate aqueous solution, and the mobile phase A2 is a 0.002-0.01 wt% acetic acid aqueous solution; the mobile phase B is an alcohol solvent and/or acetonitrile;
the reversed-phase chromatographic exchange salt transfer takes octadecylsilane chemically bonded silica as a stationary phase;
performing reverse phase chromatography, exchanging and salt conversion by gradient elution; the procedure of the gradient elution comprises the following steps of eluting 95% of mobile phase A1 and 5% of mobile phase B for 30-60 min and eluting 95% of mobile phase A2 and 5% of mobile phase B for 15-25 min in sequence in percentage by volume, then eluting with the mobile phase B and the mobile phase A2 within 15-40 min, increasing the content of the mobile phase B from 5% to 60%, and then maintaining the content of the mobile phase B60% for at least 20 min.
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 specifically comprises the following steps: pre-freezing to-40 to-30 ℃ for 2 to 4 hours, drying for 24 to 36 hours at the first-order drying temperature of-5 to 0 ℃, carrying out sublimation drying for 24 to 36 hours at the temperature of 20 to 25 ℃, and stabilizing the vacuum degree at 0.01 to 1.00 MPa.
The invention provides a method for preparing a chiral polypeptide type medicament by dynamic thermodynamic equilibrium purification, which comprises the following steps: s1) dissolving the chiral polypeptide type crude drug product with a solvent, mixing with an aqueous solution of alkaline inorganic buffer salt, and standing to obtain a chiral polypeptide type crude drug product solution; s2) purifying the crude chiral polypeptide drug solution by reverse phase chromatography to obtain a pure chiral polypeptide drug solution. Compared with the prior art, the method utilizes a dynamic thermodynamic equilibrium method to pretreat the chiral polypeptide type crude drug solution, so that impurities and drug components of the solution reach a special thermodynamic stable state, thereby forming a mode of limited separation on conventional chromatography, effectively controlling other process impurities, and realizing effective separation without other purification processes; meanwhile, the mode of realizing effective separation by using the dynamic thermodynamic equilibrium method realizes the separation effect which cannot be achieved by the conventional chromatographic materials and methods in a breakthrough manner, and the cost is far lower than that of a complex purification method which needs chiral separation or special chromatographic materials and processes to realize the separation of isomer impurities.
Drawings
FIG. 1 is a purity determination map of chiral polypeptide type crude drug YH-21101201;
FIG. 2 is a diagram of the purity detection of the chiral polypeptide drug finally obtained in example 1 of the present invention;
FIG. 3 is a diagram of the separation, purification and preparation of chiral polypeptide drug obtained in example 1 of the present invention;
FIG. 4 is a diagram of the purity detection of the chiral polypeptide drug finally obtained in example 2 of the present invention;
FIG. 5 is a purity determination map of chiral polypeptide type crude drug YH-21101801;
FIG. 6 is a diagram of the purity test of the chiral polypeptide drug finally obtained in example 3 of the present invention;
FIG. 7 is a spectrum of purity measurement of a chiral polypeptide-type drug finally obtained in comparative example 1 of the present invention;
FIG. 8 is a diagram showing the separation and purification of chiral polypeptide drug in comparative example 1.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a method for preparing a chiral polypeptide type medicament by dynamic thermodynamic equilibrium purification, which comprises the following steps: s1) dissolving the chiral polypeptide type crude drug product with a solvent, mixing with an aqueous solution of alkaline inorganic buffer salt, and standing to obtain a chiral polypeptide type crude drug product solution; s2) purifying the chiral polypeptide type medicine crude product solution by reverse phase chromatography to obtain a pure chiral polypeptide type medicine solution; the chiral polypeptide-type drug comprises a group represented by formula (1):
wherein R is1Is one or more of amino, hydroxyl, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C6-C10 aryl and substituted or unsubstituted C6-C10 heterocyclic radical, preferably amino, hydroxyl, substituted or unsubstituted C1-C5 alkyl, substituted or unsubstituted C6-C10 aryl and substituted or unsubstituted C6-C10 heterocyclic radical, more preferably amino, hydroxyl, substituted or unsubstituted C1-C3 alkyl, substituted or unsubstituted phenyl and substituted or unsubstituted indolyl; the heteroatom in the heterocyclic group is preferably one or more of O, S and N; the substituent of the substituted C1-C10 alkyl, substituted C6-C10 aryl and substituted C6-C10 heterocyclic group is preferably one or more of amino, hydroxyl, C1-C3 alkoxy, C6-C10 aryl and C6-C10 heterocyclic group, and more preferably one or more of amino, hydroxyl, methanoxy, ethanoxy and phenyl.
R2Is amino or H;
or R1And R2Connecting to form a ring;
n is 0 or 1.
The chiral polypeptide type medicine provided by the invention comprises two adjacent or separated carbonyl groups with a methylene, wherein one carbonyl group is in an amido bond, and the other non-amide carbonyl group can form a hemiacetal ketone structure in a protic solution.
In the present invention, preferably, the chiral polypeptide drug is represented by formula (a) or formula (B):
the A-A-A is a peptide chain formed by 1-25 chiral amino acids, preferably a peptide bond formed by 3-25 chiral amino acids, more preferably a peptide chain formed by 3-20 chiral amino acids, further preferably a peptide chain formed by 3-15 chiral amino acids, further preferably a peptide chain formed by 3-10 chiral amino acids, and most preferably a peptide chain formed by 3-6 chiral amino acids.
Further preferably, the chiral polypeptide drug is represented by formula (I) to formula (VI):
the A-A-A is a peptide chain formed by 3-25 chiral amino acids; the A-A-A is the same as the above and is not described in detail herein.
In the embodiment provided by the invention, the chiral polypeptide drug is specifically shown as YH-21101201 or YH-21101801:
dissolving a chiral polypeptide type medicine crude product by using a solvent; the source of the crude chiral polypeptide drug is not particularly limited, and the crude chiral polypeptide drug can be synthesized by a biological synthesis method, a chemical synthesis method or a combination method of the two; the solvent is preferably one or more of methanol, ethanol, isopropanol, water, acetonitrile, inorganic acid aqueous solution and inorganic salt aqueous solution; the inorganic acid aqueous solution is preferably acetic acid aqueous solution; the aqueous solution of inorganic salt is preferably an aqueous acetate solution; the ratio of the crude chiral polypeptide drug to the solvent is preferably 1 g: (5-8) mL; the concentration of the dissolved crude chiral polypeptide drug is preferably 100-200 g/L, more preferably 100-180 g/L, and further preferably 120-150 g/L.
Then mixing with the 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 bicarbonate aqueous solution or 10 to 20mmol/L of carbonate aqueous solution, more preferably 100 to 250mmol/L of bicarbonate aqueous solution or 10 to 15mmol/L of carbonate aqueous solution, and still more preferably 150 to 200mmol/L of bicarbonate aqueous solution or 10 to 15mmol/L of carbonate aqueous solution; the pH value of the aqueous solution of the alkaline inorganic buffer salt is preferably 8-10, and more preferably 8-9; in the present invention, it is preferable to adjust the pH of the alkaline inorganic buffer aqueous solution with carbonic acid or sodium hydroxide; the proportion of the alkaline inorganic buffer salt to the chiral polypeptide type crude drug is (0.8-1.5) mmol: 1g of a compound; the concentration of the crude chiral polypeptide drug in the mixed solution is preferably 10-40 mg/mL, more preferably 10-30 mg/mL, even more preferably 10-20 mg/mL, and most preferably 12-15 mg/mL.
Mixing, filtering preferably, and standing the filtrate for a period of time to obtain a crude chiral polypeptide drug solution; the filtration is preferably performed with an organic filter membrane, more preferably with an organic filter membrane of 0.45 μm; the temperature of the standing is preferably 20-30 ℃, more preferably 22-28 ℃, further preferably 24-36 ℃ and most preferably 25-26 ℃; the standing time is preferably 30-60 min, more preferably 30-50 min, and further preferably 30-40 min; the standing is preferably carried out under slow stirring. The standing temperature is inversely proportional to the standing time, the mass transfer and heat transfer processes can be accelerated by slow stirring, but the temperature control cannot be carried out by using a violent heating or cooling mode; after standing, the pH value of the solution is reduced by 0.2-0.5. The chiral polypeptide type medicine provided by the invention has a specific structure, can form a compound in an enol tautomeric form under the conditions of a certain temperature and the existence of buffer salts, has a certain thermodynamic equilibrium relationship in the tautomeric form, and can be placed for a period of time to enable a solution to reach a thermodynamic equilibrium state at the temperature.
Passing the crude chiral polypeptide drug solution throughPurifying by reversed phase chromatography to obtain pure solution of chiral polypeptide type medicine; the stationary phase purified by the reverse phase chromatography is octadecylsilane chemically bonded silica, and is more preferably YMC Triart prep C18-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 reverse phase chromatography takes an aqueous solution of alkaline inorganic buffer salt as a mobile phase A and takes 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 bicarbonate aqueous solution or 10 to 20mmol/L carbonate aqueous solution, more preferably 100 to 250mmol/L bicarbonate aqueous solution or 10 to 20mmol/L carbonate aqueous solution, still more preferably 150 to 250mmol/L bicarbonate aqueous solution or 10 to 15mmol/L carbonate aqueous solution, and most preferably 200mmol/L bicarbonate aqueous solution or 10mmol/L carbonate aqueous solution; the aqueous bicarbonate solution is preferably an aqueous solution of one or more of ammonium bicarbonate, potassium bicarbonate and sodium carbonate; the aqueous solution of the carbonate is preferably an aqueous solution of potassium carbonate and/or sodium carbonate; the pH value of the mobile phase A is preferably 8-9, and more preferably 8-8.5; the pH value can be adjusted by adopting carbonic acid or sodium hydroxide; the alcohol solvent is an alcohol solvent well known to those skilled in the art, and is not particularly limited, and in the present invention, one or more of methanol, ethanol and isopropanol are preferable, and methanol is more preferable; the linear velocity flow rate of the mobile phase is preferably 3.5-6.5 cm/min, and more preferably 4-6 cm/min; in the present invention, it is preferred to equilibrate the column prior to loading for elution; the equilibrium chromatography 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 reverse phase chromatography purification preferably employs gradient elution; the procedure of gradient elution is that the volume percentage is calculated, the procedure of gradient elution is 0-5 min, the content of the mobile phase B is 5%, 5.1-50 min is that the content of the mobile phase B is increased from 5% to 60%, and then the content of the mobile phase B60% is maintained for at least 10 min; the detection wavelength of the purification by the reversed phase chromatography is preferably 200-280 nm, and is more preferablyThe particle size is selected to be 210-260 nm, and preferably 220-254 nm.
The crude filtrate of chiral polypeptide drug tends to form a certain thermodynamic equilibrium state under certain temperature condition due to the buffering action of carbonate in chromatographic column, i.e. the target product chiral polypeptide drug can generate a certain nucleophilic reaction with water/alcohol (lower alcohol, ketone solvent or lower alcohol, ketone aqueous solution or other lower alcohol, ketone solution combination and other protonic solvents) in mobile phase under certain pH value condition due to the buffering action of carbonate, at this time, a relative thermodynamic equilibrium state of the substance exists in the solution, i.e. carbonate form, ion form, molecular form, 2 unstable intermediate states of enol interconversion of the chiral polypeptide compound, and stable form with residual inorganic salt in other crude products, which are different forms of the chiral polypeptide compound, the parent is the chiral compound, but under the conditions of certain temperature, certain carbonate concentration and sample concentration, the chiral compound forms a specific thermodynamic equilibrium relationship of various ions, molecular forms and salt forms, the chromatographic behavior of the same compound form is more concentrated, but the chromatographic retention behavior caused by the chemical structure difference of different compound forms (ionic forms, molecular forms, different salt forms and the like) has obvious difference, while the isomer impurities (enantiomer, diastereoisomer and the like, epimer, cis-trans-isomer and the like) can generate nucleophilic reaction with water/alcohol because the compound concentration of the isomer impurities is lower than that of the target polypeptide compound, the strength and the degree of the reaction are different to form difference, under the same temperature and pressure conditions, the isomer impurities in the solution state also form a thermodynamic equilibrium relationship of various compound forms, the difference between the chromatographic retention behaviors of the isomer impurities and the chromatographic retention difference between the isomer impurities and the target chiral polypeptide medicament are caused, and the isomer impurities are differentiated into the impurities with different structures under the thermodynamic equilibrium state, so that the chromatographic retention difference is enhanced with main components in the chromatographic retention, so that the target chiral polypeptide medicament and the isomer impurities can be skillfully distinguished in the chromatographic separation, the separation is comprehensively formed on the basis of the chemical property difference of compounds, the sample concentration difference, the mobile phase concentration difference and the like under a certain temperature condition, the various variable conditions have mutual influence but independent single factors, the thermodynamic equilibrium relation is not invariable, and fluctuates along with the change of the compound structure, the change of the concentration, the fluctuation of the temperature, the concentration change of the mobile phase and the like, this particular thermodynamic equilibrium relationship exhibits an interactive, dynamic process of change. The retention difference of chiral isomer impurities on the conventional chromatogram is realized by utilizing the dynamic change process, the chiral isomer impurities effectively separated on the conventional chromatogram method and the chromatogram condition are realized in a breakthrough manner, and the method is simple, convenient and cheap, and is very easy to realize the separation, purification and preparation of the chiral polypeptide type medicine on an industrial scale.
In the present invention, the chiral polypeptide type drug is preferably converted into a salt by chromatography in a pure solution, more preferably by reverse phase chromatography using a buffer system composed of an inorganic salt and an acid, and still more preferably by reverse phase chromatography using a buffer system composed of an acetate salt 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 C18-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 of the reverse phase chromatographic exchange salt conversion comprises a mobile phase A1, a mobile phase A2 and a mobile phase B; the mobile phase A1 is preferably 10-30 mM acetate aqueous solution, more preferably 15-25 mM acetate aqueous solution, and still more preferably 20mM acetate aqueous solution; the pH value of the mobile phase A1 is preferably 3-4, and more preferably 3.5-4; the pH value is preferably adjusted by using acetic acid; the mobile phase A2 is preferably 0.002-0.01 wt% acetic acid aqueous solution, more preferably 0.005-0.01 wt% acetic acid aqueous solution; the mobile phase B is preferablyAlcohol solvent and/or acetonitrile; the alcohol solvent is preferably one or more of methanol, ethanol and isopropanol; in the present invention, the reverse phase chromatographic salt exchange is preferably performed by gradient elution; according to the volume percentage, the gradient elution procedure comprises the steps of eluting 95% of mobile phase A1 and 5% of mobile phase B for 30-60 min and eluting 95% of mobile phase A2 and 5% of mobile phase B for 15-25 min in sequence, then eluting by adopting the mobile phase B and the mobile phase A2 within 15-40 min, increasing the content of the mobile phase B from 5% to 60%, and then maintaining the content of the mobile phase B60% for at least 20 min; more preferably, the elution is sequentially performed by eluting 95% of mobile phase A1 and 5% of mobile phase B for 35-60 min, eluting 95% of mobile phase A2 and 5% of mobile phase B for 18-22 min, then eluting by adopting the mobile phase B and the mobile phase A2 within 15-40 min, increasing the content of the mobile phase B from 5% to 60%, and then maintaining the content of the mobile phase B60% for at least 20 min; in the embodiment provided by the invention, the gradient elution procedure specifically comprises 0-60 min 95% of mobile phase A1 and 5% of mobile phase B, 60.1-80 min of mobile phase A2 and 5% of mobile phase B, and 80.1-120 min of elution with mobile phase B and mobile phase A2, wherein the mobile phase B rises from 5% to 60%, and then is maintained at the mobile phase B60% for at least 20 min; 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, and 55.1-70 min of elution by adopting the mobile phase B and the mobile phase A2, wherein the mobile phase B is increased from 5% to 60%, and then the mobile phase B60% is maintained for at least 20 min; or specifically 0-60 min 95% of mobile phase A1 and 5% of mobile phase B, 60.1-80 min of mobile phase A2 and 5% of mobile phase B, and 80.1-120 min of elution by adopting the mobile phase B and the mobile phase A2, wherein the mobile phase B is increased from 5% to 60%, and then the mobile phase B60% is maintained for at least 20 min. The linear velocity flow rate of the mobile phase during the reverse phase chromatography salt exchange conversion is preferably 3.5-6.5 cm/min, and more preferably 3-5 cm/min.
After the salt is converted, collecting a purified solution with related substance compound requirements, and performing reduced pressure concentration and freeze drying to obtain the chiral polypeptide type drug; 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 as follows: pre-freezing to-40 to-30 ℃ for 2 to 4 hours, drying for 24 to 36 hours at the first-order drying temperature of-5 to 0 ℃, carrying out sublimation drying for 24 to 36 hours at the temperature of 20 to 25 ℃, and stabilizing the vacuum degree at 0.01 to 1.00 MPa; more preferably specifically: pre-freezing to-40 ℃ to-30 ℃ for 2-4 h, drying at-5 ℃ to-2 ℃ for 30-36 h in a first-order manner, carrying out sublimation drying at 20-25 ℃ for 24-30 h, and stabilizing the vacuum degree at 0.01-1.00 MPa; more preferably specifically: pre-freezing to-40 to-30 ℃ for 2 to 4 hours, drying for 36 hours at-5 to-2 ℃ in a first-order manner, carrying out sublimation drying for 24 hours at 25 ℃, and stabilizing the vacuum degree at 0.01 to 1.00 MPa.
The invention utilizes a dynamic thermodynamic equilibrium method to pretreat the chiral polypeptide type crude drug solution, so that impurities and drug components of the solution reach a special thermodynamic stable state, thereby forming a mode of limited separation on conventional chromatography, effectively controlling other process impurities, and realizing effective separation without other purification processes; meanwhile, the mode of realizing effective separation by using the dynamic thermodynamic equilibrium method realizes the separation effect which cannot be achieved by the conventional chromatographic materials and methods in a breakthrough manner, and the cost is far lower than that of a complex purification method which needs chiral separation or special chromatographic materials and processes to realize the separation of isomer impurities.
In order to further illustrate the present invention, a method for preparing chiral polypeptide-type drugs by dynamic thermodynamic equilibrium purification according to the present invention is described in detail below with reference to the following examples.
The reagents used in the following examples are all commercially available.
Example 1
A method for preparing a chiral polypeptide type drug by dynamic thermodynamic equilibrium purification, comprising:
(1) sample treatment: 32.25g of chiral polypeptide type drug crude product (YH-21101201) with crude product purity (area normalization) of 64.71% is obtained by a solid phase synthesis method, the crude product is dissolved by methanol according to the concentration of 150g/L, diluted to about 15mg/ml by 10mmmol/L potassium carbonate solution, filtered by an organic system filter membrane of 0.45um to obtain crude filtrate, the pH value is detected to be 8.4, the filtrate is placed at 26 ℃, and the pH value is detected to be 7.9 after the filtrate is slowly stirred for 30 min.
(2) And (3) purification: about 2000ml of the crude solution is purified by a DAC150 liquid phase purification system, the packing material is YMC Triart prep C18-S, the diameter is 7 mu m, the diameter is 12nm, the packing height is 250mm, and the temperature is controlled to be 26 ℃. The mobile phase A is: regulating the pH value to 8.1 by using 10mmol/L potassium carbonate aqueous solution and sodium hydroxide solution; the mobile phase B is as follows: methanol; volume flow rate: 1100 ml/min; the column was equilibrated with 5% phase B, 95% phase a for about 10 min. Gradient elution procedure: 5-5% of phase B within 5min, increasing the gradient of phase B from 5% to 60% within 5.1min to 60min, then maintaining the gradient of phase B60% for at least 10min within 60.01-70.01 min, and detecting the wavelength: 220 nm.
The purity detection map of the chiral polypeptide type drug crude product YH-21101201 is shown in FIG. 1. The chiral polypeptide medicine and its isomer impurity are very similar in chromatographic behavior and can not be effectively separated in analytical detection, and in the course of industrial separation and purification, because the treatment quantity is enlarged by at least 2 orders of magnitude compared with analytical detection, the performance of chromatographic packing and equipment performance are far less precise than analytical grade, and the separation degree of theoretical preparation chromatogram is poorer, so that it is obvious in preparation chromatogram that its main peak and its isomer peak are both greatly widened, in which because the crude isomer impurity content is about 30.12%, in the pretreatment of sample the mixture of main component and isomer impurity with several forms is formed, its chromatographic behavior is changed from single substance form to mixed and relatively close compound form, and on chromatogram it shows a large peak broadening, but its total quantity is according to material conservation, therefore, sectional collection is adopted during separation, multi-section segmentation is adopted for a main peak with larger broadening, isomer impurities are fixed in the total amount, the total amount is dispersed after various forms are formed, the part which is highly overlapped and crossed with the main component is reduced, limited separation and purification of the main component can be realized, the yield of qualified samples of the main component is also improved, sectional purification liquid with any single isomer impurity of not more than 0.5 percent is combined after analysis and detection, and the purification step (including the pretreatment step of the sample) is repeated after unqualified sections are combined and concentrated until the purification liquid is qualified.
(3) Salt conversion
Reverse phase chromatography conditions: the high performance liquid chromatography column using octadecylsilane chemically bonded silica as stationary phase has particle size of 10 μm and pore diameter of 100A. The column diameter and length were: 150x250 mm. The mobile phase A1 is: 10mM ammonium acetate, pH adjusted to 3.5 by acetic acid; the mobile phase A2 is: 0.005% acetic acid aqueous solution; the B phase in the mobile phase is: methanol; volume flow rate: 700 ml/min; maintaining 95% gradient of A1 phase within 60min, switching A2 phase, maintaining 95% gradient of A2 phase from 60.1min to 80min, increasing B phase from 5% to 60% gradient within 80.1min to 120min, and maintaining at least 60% gradient of B phase for 30min, wherein the B phase is methanol.
Concentrating the collected purified liquid with the relevant substances meeting the requirements to the concentration of about 20 mg/ml-40 mg/ml under reduced pressure and rotary evaporation at the temperature of below 35 ℃, and then freeze-drying to obtain the chiral polypeptide type medicine solution with any single impurity of not more than 0.5 percent, total impurities of not more than 1.0 percent and the purity of more than 99 percent.
The purity detection spectrum of the chiral polypeptide type drug finally obtained in example 1 is shown in fig. 2. Through calculation: the maximum single impurity of the finished product in this example is 0.43% of chiral diastereoisomer impurity, 0.46% of total impurities, 99.54% of purity and 58.2% of purification yield.
The chiral polypeptide drug finally obtained in example 1 was analyzed by preparative chromatography to obtain its separation and purification preparative spectrum, as shown in fig. 3.
Example 2
(1) Sample treatment: 28.56g of chiral polypeptide type drug crude product (YH-21101201) with the crude product purity of 64.71% is obtained by adopting a solid phase synthesis method, the chiral polypeptide type drug crude product is dissolved by methanol according to the concentration of 130g/L, the solution is diluted to about 15mg/ml by 200mmol/L ammonium bicarbonate solution, then the solution is filtered by a 0.45 mu m organic filter membrane to obtain crude filtrate, the pH value is detected to be 8.0, the filtrate is placed at 25 ℃, the pH value is detected to be 7.8 after the filtrate is slowly stirred for 30 min.
(2) And (3) purification: about 2000ml of the crude solution was purified by a liquid phase purification system of DAC150 using Nano Unisil C18, 10 μm,
the filling height is 250 mm. The mobile phase A is: 200mmol/L ammonium bicarbonate water solution, and adjusting the pH to 8.1 by sodium hydroxide; the mobile phase B is as follows: methanol; volume flow rate: 1000 ml/min; gradient elution procedure: 5-5% of phase B within 5min, and 5% of phase B within 5.1-50 minRising to 60% gradient, then maintaining B phase 60% gradient for at least 10min, detecting wavelength: 220 nm; and (4) collecting main peaks in a segmented mode, combining any segmented purified liquid with single impurity not more than 0.5%, combining unqualified segments, concentrating, and repeating the purification steps until the purified liquid is qualified.
(3) Salt conversion
A chromatographic column: octadecylsilane bonded silica gel (Nano Unisil C18, 10 μm,
) Is a stationary phase and the column is
Mobile phase: a1 phase 20mM ammonium acetate acid aqueous solution, A2 phase 0.01% acetic acid aqueous solution, B phase methanol;
detection wavelength: 220 nm;
the volume flow rate is 800 ml/min;
the salt transfer elution gradient is shown in table 1:
TABLE 1 salt transfer 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 liquid meeting the requirement of related substances to the concentration of about 20 mg/ml-40 mg/ml under reduced pressure and rotary evaporation at the temperature of below 30 ℃, and then freeze-drying to obtain the chiral polypeptide type medicine solution with any single impurity of not more than 0.5 percent, total impurities of not more than 1.0 percent and purity of more than 99.0 percent.
The purity detection spectrum of the chiral polypeptide type drug finally obtained in example 2 is shown in fig. 4. Through calculation: the largest single impurity in this example was 0.49% chiral diastereomeric impurity, 0.57% total impurity, 99.43% purity, 61.6% purification yield.
Example 3
(1) Sample treatment: 29.88g of chiral polypeptide type drug crude product (YH-21101801) with crude product purity of 64.55% is obtained by a solid phase synthesis method, the chiral polypeptide type drug crude product is dissolved by methanol according to the concentration of 140g/L, diluted to about 12mg/ml by 10mmol/L sodium carbonate solution, filtered by an organic system filter membrane with the diameter of 0.45 mu m to obtain crude filtrate, the pH value is detected to be 8.5, the filtrate is placed at 26 ℃, and the pH value is detected to be 8.3 after the filtrate is slowly stirred for 30 min.
(2) And (3) purification: about 2000ml of the crude solution was purified by a liquid phase purification system of DAC150 using Nano Unisil C18, 10 μm,
the filling height is 250 mm. The mobile phase A is: 10mmol/L sodium carbonate aqueous solution; the mobile phase B is as follows: methanol; volume flow rate: 1100 ml/min; gradient: 5-5% of phase B within 5min, increasing the gradient of phase B from 5% to 60% within 5.1min to 60min, maintaining the gradient of phase B60% for at least 10min, and detecting the wavelength: 220 nm; and (4) collecting main peaks in a segmented mode, combining any segmented purified liquid with single impurity not more than 0.5%, combining unqualified segments, concentrating, and repeating the purification steps until the purified liquid is qualified.
(3) Salt conversion
Reverse phase chromatography conditions: high performance liquid chromatography column with octadecylsilane chemically bonded silica as stationary phase (Nano Unisil C18 as filler, 10 μm,
150mm in diameter and 250mm in filling height), the A1 phase in the mobile phase is: 20mM ammonium acetate, pH adjusted to 3.5 by acetic acid; the mobile phase A2 is: 0.002% acetic acid aqueous solution; the B phase in the mobile phase is: methanol; volume flow rate: 750 ml/min; maintaining 95% gradient of A1 phase within 60min, switching A2 phase, maintaining 95% gradient of A2 phase from 60.1min to 80min, increasing B phase from 5% to 60% gradient within 80.1min to 120min, and maintaining at least 60% gradient of B phase for 30min, wherein the B phase is methanol.
Concentrating the collected purified liquid with the relevant substances meeting the requirements 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 the chiral polypeptide type medicine finished product.
The purity detection map of the chiral polypeptide type drug crude product YH-21101801 is shown in FIG. 5; the purity detection spectrum of the chiral polypeptide type drug finally obtained in example 3 is shown in fig. 6. Through calculation: the largest single impurity of this example was 0.31% chiral diastereomer impurity, 0.31% total impurity, 99.69% purity, 69.78% purification yield.
Comparative example 1
Only the conventional reversed phase chromatography is adopted to purify the polypeptide sample, and the specific process and the experimental result are as follows:
(1) sample treatment: 25.34g of chiral polypeptide type drug crude product (YH-21101801) with the crude product purity of 64.55% is obtained by adopting a chemical (solid phase) synthesis method, methanol is used for stirring and dissolving according to the concentration of 120g/L, after the chiral polypeptide type drug crude product is completely dissolved, 0.1% trifluoroacetic acid aqueous solution is used for diluting to about 6mg/ml, then 0.45um organic system filter membrane is used for filtering insoluble substances, and crude product filtrate is obtained.
(2) And (3) purification: about 2000ml of the crude solution was purified by using a liquid phase purification system of DAC150 packed with FUJI C18, 10 μm,
the filling height is 250 mm. The mobile phase A is: 0.1% aqueous trifluoroacetic acid; the mobile phase B is as follows: methanol; volume flow rate: 1100 ml/min; gradient: 5-5% of phase B within 5min, increasing the gradient of phase B from 5% to 60% within 5.1min to 60min, maintaining the gradient of phase B60% for at least 10min, and detecting the wavelength: 220 nm; and collecting main peaks in sections.
(3) Salt conversion
A chromatographic column: octadecylsilane chemically bonded silica was used as a stationary phase (FUJI C18, 10 μm,
) The pillar is
Mobile phase: a1 phase 30mM ammonium acetate acid aqueous solution, A2 phase 0.02% acetic acid aqueous solution, and B phase methanol;
detection wavelength: 220 nm;
volume flow rate: 750 ml/min;
the salt transfer elution gradient is shown in table 2.
TABLE 2 salt transfer 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 at the temperature below 30 ℃ under reduced pressure by rotary evaporation to a concentration of about 20 mg/ml-40 mg/ml, and then freeze-drying to obtain the chiral polypeptide type medicine finished product.
Since the experiment is not carried out with specific sample pretreatment and according to specific purification process conditions, the purification effect is obviously different from that of the comparative example, and the final finished product can not effectively separate isomer impurities.
The purity detection spectrum of the chiral polypeptide type drug finally obtained in the comparative example 1 is shown in fig. 7; through calculation: the final product of this example had a maximum single impurity of 8.18% as the isomeric impurity (diastereomer), a total impurity of 14.02%, a purity of 85.98%, and a recovery of 65.52% for all major components.
The chiral polypeptide drug finally obtained in comparative example 1 was analyzed by preparative chromatography to obtain its separation and purification preparative spectrum, as shown in fig. 8.
Comparative example 2
A large number of tests are carried out on the sample processing process in the step (1), optimized conditions are screened to determine that the chiral polypeptide drug can be effectively separated from isomer impurities thereof, the processing conditions are screened by taking a chiral polypeptide drug crude product (YH-21101801) as an example, and the results are shown in Table 3.
TABLE 3 screening results of sample treatment conditions
Only in the case of basic buffer salts, and at a suitable concentration ratio to the target compound, is it advantageous to promote the formation of multiple forms of the compound, neither inhibit the dissociation of the compound, nor promote its conversion to the same type, only when and exactly the concentration of the compound, the salt concentration, and the temperature, time are appropriate, is the compound in at least 5 thermodynamic equilibrium states (i.e. the molecular state, the ionic state, the carbonate form, and the 2 unstable states of enol tautomerism by carbonyl groups), so that one species forms 5 different types of forms, which chromatographically changes from a theoretically sharp single peak to a more broadened dwarf peak. The dwarf peak provides a possibility for separating chiral polypeptide drugs from isomer impurities (enantiomers, diastereomers and the like, epimers and cis-trans isomers) with extremely close physical and chemical properties.
The method utilizes the dynamic thermodynamic equilibrium relationship to purify and convert the chiral polypeptide drug in a sample pretreatment mode and a reversed-phase chromatographic system, solves the problem of quality influence of isomer impurities (enantiomers, diastereomers and the like, epimers and cis-trans isomers) of the chiral polypeptide drug on industrial products, and can effectively control other process impurities Forms a differential dynamic thermodynamic equilibrium relationship and utilizes the relationship to realize the effective separation and the effective control of chiral polypeptide drugs and isomer impurities.
Claims (10)
1. A method for preparing a chiral polypeptide type drug by dynamic thermodynamic equilibrium purification is characterized by comprising the following steps:
s1) dissolving the chiral polypeptide type crude drug product with a solvent, mixing with an aqueous solution of alkaline inorganic buffer salt, and standing to obtain a chiral polypeptide type crude drug product solution;
s2) purifying the chiral polypeptide type medicine crude product solution by reverse phase chromatography to obtain a pure chiral polypeptide type medicine solution;
the chiral polypeptide-type drug comprises a group represented by formula (1):
wherein R is1One 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 group;
R2selected from amino or H;
or R1And R2Connecting to form a ring;
n is 0 or 1.
2. The method of claim 1, wherein the chiral polypeptide drug is represented by formula (a) or formula (B):
the substituent in the substituted C1-C10 alkyl, substituted C6-C10 aryl and substituted C6-C10 heterocyclic group is 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 1-25 chiral amino acids.
3. The method according to claim 1, wherein the chiral polypeptide drug is represented by formula (I) to formula (VI):
the A-A-A is a peptide chain formed by 3-25 chiral amino acids.
4. The method according to claim 1, wherein the solvent is selected from one or more of methanol, ethanol, isopropanol, water, acetonitrile, aqueous solutions of inorganic acids and inorganic salts; the concentration of the dissolved crude chiral polypeptide drug 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 proportion of the alkaline inorganic buffer salt to the chiral polypeptide type crude drug is (0.8-1.5) mmol: 1g of the total weight of the composition.
5. The method of claim 1, wherein the temperature of said placing is 20 ℃ to 30 ℃; the standing time is 30-60 min.
6. The method of claim 1, wherein the stationary phase purified by reverse phase chromatography is octadecylsilane chemically bonded silica; the mobile phase purified by the reverse phase chromatography takes an aqueous solution of alkaline inorganic buffer salt as a mobile phase A and takes an alcohol solvent and/or acetonitrile as a mobile phase B; the reversed phase chromatography purification adopts gradient elution; the procedure of gradient elution is that the volume percentage is calculated, the procedure of gradient elution is 0-5 min, the content of the mobile phase B is 5%, 5.1-50 min is that the content of the mobile phase B is increased from 5% to 60%, and then the content of the mobile phase B60% is maintained for at least 10 min; the detection wavelength of the purification by the reversed phase chromatography is 200-280 nm.
7. The method according to claim 6, wherein 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 flow rate of the mobile phase during purification by the reversed phase chromatography is 3.5-6.5 cm/min.
8. The method of claim 1, further comprising:
and (3) carrying out salt conversion on the pure solution of the chiral polypeptide drug by chromatography, carrying out reduced pressure concentration, and carrying out freeze drying to obtain the chiral polypeptide drug.
9. The method according to claim 8, wherein the salt transfer by chromatography is carried out by reverse phase chromatography using a buffer system of acetate and acetic acid;
the mobile phase of the reverse phase chromatographic exchange salt conversion comprises a mobile phase A1, a mobile phase A2 and a mobile phase B; the mobile phase A1 is a 10-30 mM acetate aqueous solution, and the mobile phase A2 is a 0.002-0.01 wt% acetic acid aqueous solution; the mobile phase B is an alcohol solvent and/or acetonitrile;
the reversed-phase chromatographic exchange salt transfer takes octadecylsilane chemically bonded silica as a stationary phase;
performing reverse phase chromatography, exchanging and salt conversion by gradient elution; the procedure of the gradient elution comprises the following steps of eluting 95% of mobile phase A1 and 5% of mobile phase B for 30-60 min and eluting 95% of mobile phase A2 and 5% of mobile phase B for 15-25 min in sequence in percentage by volume, then eluting with the mobile phase B and the mobile phase A2 within 15-40 min, increasing the content of the mobile phase B from 5% to 60%, and then maintaining the content of the mobile phase B60% for at least 20 min.
10. The method according to claim 8, wherein 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 specifically comprises the following steps: pre-freezing to-40 to-30 ℃ for 2 to 4 hours, drying for 24 to 36 hours at the first-order drying temperature of-5 to 0 ℃, carrying out sublimation drying for 24 to 36 hours at the temperature of 20 to 25 ℃, and stabilizing the vacuum degree at 0.01 to 1.00 MPa.
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