CN113929743A - Method for preparing daptomycin impurity RS-1 and impurity RS-3 - Google Patents
Method for preparing daptomycin impurity RS-1 and impurity RS-3 Download PDFInfo
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- 239000012535 impurity Substances 0.000 title claims abstract description 151
- 108010013198 Daptomycin Proteins 0.000 title claims abstract description 141
- DOAKLVKFURWEDJ-QCMAZARJSA-N daptomycin Chemical compound C([C@H]1C(=O)O[C@H](C)[C@@H](C(NCC(=O)N[C@@H](CCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@H](CO)C(=O)N[C@H](C(=O)N1)[C@H](C)CC(O)=O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](CC(N)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)CCCCCCCCC)C(=O)C1=CC=CC=C1N DOAKLVKFURWEDJ-QCMAZARJSA-N 0.000 title claims abstract description 141
- 229960005484 daptomycin Drugs 0.000 title claims abstract description 141
- 238000000034 method Methods 0.000 title claims abstract description 32
- 238000002360 preparation method Methods 0.000 claims abstract description 31
- 239000003513 alkali Substances 0.000 claims abstract description 17
- 238000004237 preparative chromatography Methods 0.000 claims abstract description 15
- 230000002378 acidificating effect Effects 0.000 claims abstract description 11
- 239000000243 solution Substances 0.000 claims description 127
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- 238000006243 chemical reaction Methods 0.000 claims description 39
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 30
- 239000005695 Ammonium acetate Substances 0.000 claims description 30
- 229940043376 ammonium acetate Drugs 0.000 claims description 30
- 235000019257 ammonium acetate Nutrition 0.000 claims description 30
- 238000010438 heat treatment Methods 0.000 claims description 26
- 239000000047 product Substances 0.000 claims description 26
- 239000000413 hydrolysate Substances 0.000 claims description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 23
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- 238000004108 freeze drying Methods 0.000 claims description 19
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 15
- 239000007788 liquid Substances 0.000 claims description 15
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 14
- 239000002253 acid Substances 0.000 claims description 13
- 238000000746 purification Methods 0.000 claims description 11
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 10
- 238000007710 freezing Methods 0.000 claims description 10
- 230000008014 freezing Effects 0.000 claims description 10
- 238000000926 separation method Methods 0.000 claims description 10
- 238000011068 loading method Methods 0.000 claims description 9
- 239000008213 purified water Substances 0.000 claims description 9
- 239000007787 solid Substances 0.000 claims description 9
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 8
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 claims description 8
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 7
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 238000002347 injection Methods 0.000 claims description 4
- 239000007924 injection Substances 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 239000012670 alkaline solution Substances 0.000 claims description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 3
- 238000005903 acid hydrolysis reaction Methods 0.000 claims description 2
- 238000011097 chromatography purification Methods 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- 230000007062 hydrolysis Effects 0.000 claims description 2
- 238000006460 hydrolysis reaction Methods 0.000 claims description 2
- 230000007935 neutral effect Effects 0.000 claims description 2
- 238000006386 neutralization reaction Methods 0.000 claims description 2
- 238000013375 chromatographic separation Methods 0.000 claims 1
- 238000011160 research Methods 0.000 abstract description 13
- 239000003814 drug Substances 0.000 abstract description 10
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 230000003301 hydrolyzing effect Effects 0.000 abstract 2
- 230000003472 neutralizing effect Effects 0.000 abstract 1
- 238000001514 detection method Methods 0.000 description 34
- 239000012071 phase Substances 0.000 description 23
- 238000004128 high performance liquid chromatography Methods 0.000 description 11
- 239000007791 liquid phase Substances 0.000 description 11
- 238000000855 fermentation Methods 0.000 description 8
- 230000004151 fermentation Effects 0.000 description 8
- 239000011259 mixed solution Substances 0.000 description 8
- 108010009736 Protein Hydrolysates Proteins 0.000 description 7
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- 229940079593 drug Drugs 0.000 description 6
- 239000012043 crude product Substances 0.000 description 4
- 230000006872 improvement Effects 0.000 description 4
- 230000014759 maintenance of location Effects 0.000 description 4
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- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000003908 quality control method Methods 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- RJQXTJLFIWVMTO-TYNCELHUSA-N Methicillin Chemical compound COC1=CC=CC(OC)=C1C(=O)N[C@@H]1C(=O)N2[C@@H](C(O)=O)C(C)(C)S[C@@H]21 RJQXTJLFIWVMTO-TYNCELHUSA-N 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- -1 daptomycin lactone Chemical class 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 229960003085 meticillin Drugs 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- GYSCBCSGKXNZRH-UHFFFAOYSA-N 1-benzothiophene-2-carboxamide Chemical compound C1=CC=C2SC(C(=O)N)=CC2=C1 GYSCBCSGKXNZRH-UHFFFAOYSA-N 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- GHVNFZFCNZKVNT-UHFFFAOYSA-N Decanoic acid Natural products CCCCCCCCCC(O)=O GHVNFZFCNZKVNT-UHFFFAOYSA-N 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 108010028921 Lipopeptides Proteins 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 241000958215 Streptomyces filamentosus Species 0.000 description 1
- 108010059993 Vancomycin Proteins 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 238000005904 alkaline hydrolysis reaction Methods 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
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- 230000003115 biocidal effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
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- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
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- 239000012362 glacial acetic acid Substances 0.000 description 1
- 230000008676 import Effects 0.000 description 1
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- 238000009776 industrial production Methods 0.000 description 1
- 229960003907 linezolid Drugs 0.000 description 1
- TYZROVQLWOKYKF-ZDUSSCGKSA-N linezolid Chemical compound O=C1O[C@@H](CNC(=O)C)CN1C(C=C1F)=CC=C1N1CCOCC1 TYZROVQLWOKYKF-ZDUSSCGKSA-N 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
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- 238000001728 nano-filtration Methods 0.000 description 1
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- 238000012827 research and development Methods 0.000 description 1
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- 238000003860 storage Methods 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 229960003165 vancomycin Drugs 0.000 description 1
- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 description 1
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
Abstract
The invention belongs to the field of medicine preparation, and relates to a method for preparing daptomycin impurities RS-1 and RS-3, in particular to a method for preparing daptomycin impurities RS-1 and RS-3, which comprises the steps of hydrolyzing a daptomycin sample under an alkaline condition, hydrolyzing under an acidic condition, neutralizing with alkali, and finally purifying by preparative chromatography to obtain high-purity daptomycin impurities RS-1 and RS-3. The method for preparing daptomycin impurities RS-1 and RS-3 is simple and convenient to operate, low in cost and capable of being prepared in an enlarged mode according to needs and meeting the needs of research and production.
Description
Technical Field
The invention belongs to the field of medicine preparation, and particularly relates to a preparation method of daptomycin impurity RS-1 and impurity RS-3.
Background
Daptomycin (Daptomycin) is the first novel cyclic lipopeptide antibiotic containing ten carbon side chains extracted from streptomyces (S.roseosporus) fermentation liquor, can destroy the function of bacterial cell membranes in many aspects and quickly kill gram-positive bacteria, and particularly has good bactericidal effect on high-pathogenicity drug-resistant bacteria such as Methicillin (Methicillin), vancomycin and linezolid in vitro and small toxic and side effects, thereby providing a new treatment means for clinical critically infected patients.
The research on daptomycin impurities is the premise of ensuring the product quality and safety, so that daptomycin impurities can be separatedIt is essential to purify impurities in daptomycin and to perform systematic studies. Daptomycin impurity RS-1 (C)44H66N10O15) And impurity RS-3 (C)35H51N7O11) Is an important impurity in the quality control of daptomycin, and the structural formula of the impurity is as follows:
the production of daptomycin is mainly realized by a microbial fermentation method, and the method has the remarkable advantages of high yield and low pollution, and is the preferred technology for the current industrial production. The complexity and specificity of the fermentation product and the strict requirements of the pharmaceutical industry on the purity and yield of the fermentation product make the separation and purification of the fermentation product play a key role in the whole biological processing process (including breeding of high-yield strains, microbial fermentation, enzymatic reaction and the like). The fermentation liquor is an extremely complex multi-phase and multi-component system, contains various unknown components, and the research on impurities is the premise of ensuring the safety of products, so that the research on the sources, detection methods, limits, separation and purification and the like of the medicine impurities has important significance, and particularly influences the research on dosage form selection, prescription composition, process determination, analysis methods, product storage and the like in the research and development of medicines. The impurities are the key points of quality research, quality control and safety research of innovative medicaments, and directly reflect the research level of innovation. The daptomycin product often contains impurities such as dehydrated daptomycin, isomeric daptomycin, daptomycin lactone hydrolysate and the like, and the quality and safety of the product are seriously affected, so that the impurities in the daptomycin can be prepared and the system research is very necessary.
European patent 1586580A2 and CN01805212.6 disclose 14 daptomycin impurities and attribution thereof, and do not particularly relate to a daptomycin impurity separation method.
In China journal of antibiotics (vol. 38, No. 10, 2013, 760-764), 5 main related impurities generated by daptomycin under different acid-base (pH2, pH4, pH6, pH8, pH10) conditions are reported, and the impurities are prepared and the structure of the impurities is researched by analytical and preparative HPLC, wherein the liquid phase preparation needs to consume a large amount of mobile phase, the yield is low, the production period is long, the operation is complex and the preparation cost is high. CN106866791A, CN 105699554A and Zhou Xuyan "identification and separation control of impurity components of daptomycin product" disclose separation, purification and preparation methods of high-purity daptomycin lactone hydrolysate.
CN104387444A discloses a preparation method of a high-purity sample of daptomycin impurity RS-2, which uses resin chromatography, ultrafiltration/nanofiltration, silica gel chromatography and high performance liquid chromatography, and has complex method and great operation difficulty.
CN106866790A discloses a preparation method of daptomycin RS-5/6, RS-7 and RS-7a/7b impurities, a chiral chromatographic column Ib-SitC8/6045-1 is required during liquid phase purification, and the preparation cost is high.
Chinese journal of antibiotics (vol. 44, No. 5, No. 44, 2019, 574-579) reports a new impurity with a molecular weight of 1617, which is presumed to be derived from the supplemented decanoic acid in the fermentation process according to the chemical structure of the impurity, and the preparation of the impurity is still prepared by using a liquid phase, and also consumes a large amount of mobile phase, and has low yield and high preparation cost.
The preparation and detection of each impurity in daptomycin are crucial to the improvement of product quality and the guarantee of medication safety, and the preparation of the impurity is beneficial to the improvement of product purity, the establishment of quality standard and the improvement of medication safety. Related impurities RS-1 and RS-3 of daptomycin are the important impurities in daptomycin quality control, and high-purity impurity samples play an important role in research on the RS-1 and RS-3 impurities. According to the description of the national food and drug administration import drug registration standard (standard number JX20070250) about daptomycin for injection, when daptomycin is detected by a high pressure liquid phase, an impurity with the retention time of 17.69 minutes (relative to the retention time of daptomycin of 0.48) is named as RS-1, and an impurity with the retention time of 21.68 minutes (relative to the retention time of daptomycin of 0.59) is named as RS-3. The invention researches the preparation methods of daptomycin impurities RS-1 and RS-3 so as to obtain high-purity impurities, improve the product quality and accelerate the high-level industrialization of daptomycin.
Disclosure of Invention
The invention aims to provide a preparation method which is simple and convenient to operate, low in cost and capable of quickly preparing high-purity daptomycin impurities RS-1 and RS-3 from a daptomycin finished product.
The technical scheme of the invention is as follows: the daptomycin sample is hydrolyzed under alkaline conditions, then hydrolyzed under acidic conditions, and then neutralized by alkali to obtain feed liquid containing impurities RS-1 and RS-3, and then purified by preparative chromatography to obtain purified daptomycin impurities RS-1 and RS-3.
A method for preparing daptomycin impurity RS-1 and impurity RS-3 specifically comprises the following steps:
A. alkali hydrolysis: dissolving a daptomycin sample by using purified water, adding alkaline solution to adjust the solution to be alkaline, and heating for reaction;
B. acid hydrolysis: b, adding acid liquid into the hydrolysate obtained in the step A to adjust the hydrolysate to be acidic, and heating for reaction;
C. alkali neutralization: b, adding alkaline liquor into the hydrolysate obtained in the step B to adjust the hydrolysate to be neutral, so as to obtain a solution containing daptomycin impurities RS-1 and RS-3;
D. and (3) purification: c, loading the solution obtained in the step C onto a preparative chromatographic column, separating and purifying, and respectively collecting a preparation solution containing a daptomycin impurity RS-1 and a preparation solution containing RS-3;
E. and (3) freeze drying: and D, respectively freezing and drying the preparation liquid of the daptomycin impurities RS-1 and RS-3 obtained in the step D to obtain daptomycin impurity RS-1 and RS-3 solids.
Preferably, the daptomycin sample in step A is a crude daptomycin product, a finished daptomycin product or a daptomycin powder injection.
Preferably, the alkali solution in step a is selected from one of sodium hydroxide, potassium hydroxide, sodium carbonate or potassium carbonate solution.
Further preferably, the alkali solution in the step A is sodium carbonate or potassium carbonate solution.
Preferably, the concentration of the alkali liquor in the step A is 0.1-1 mol/L.
Preferably, the pH value of the alkali liquor added in the step A is adjusted to 9.0-11.0.
Further preferably, the pH value of the solution added with alkali in the step A is adjusted to 9.5-10.5.
Preferably, the heating temperature in the step A is 55-70 ℃.
Further preferably, the heating reaction temperature in the step A is 60-65 ℃.
Preferably, the heating reaction time in the step A is 3-9 h.
Further preferably, the heating reaction time in the step A is 4-6 h.
Preferably, the acid solution in step B is selected from one of hydrochloric acid, sulfuric acid or phosphoric acid.
Preferably, the concentration of the acid solution in the step B is 0.1-1 mol/L.
Preferably, the pH value of the acid-adding liquid in the step B is adjusted to 1.6-4.0.
Further preferably, the pH value of the acid solution added in the step B is adjusted to 1.6-3.0.
Preferably, the heating temperature in the step B is 70-85 ℃.
Further preferably, the heating reaction temperature in the step B is 75-80 ℃.
Preferably, the heating reaction time in the step B is 3-9 h.
Further preferably, the heating reaction time in the step B is 6-8 h.
Preferably, the alkali solution in step C is selected from one of sodium hydroxide, potassium hydroxide, sodium carbonate or potassium carbonate solution.
Further preferably, the alkali liquor in step C is sodium hydroxide or potassium hydroxide.
Preferably, the concentration of the alkali liquor in the step C is 0.01-0.1 mol/L.
Preferably, in the step D, the mobile phase is a mixture of acetonitrile and ammonium acetate solution.
More preferably, in the separation and purification by preparative chromatography described in step D, the mobile phase is a mixture of acetonitrile and 0.05 to 0.2mol/L ammonium acetate solution.
More preferably, in the step D, the mobile phase is a mixture of acetonitrile and 0.05-0.2 mol/L ammonium acetate solution with pH of 3.5-4.5.
Preferably, in the separation and purification of the preparative chromatography described in the step D, the volume ratio of acetonitrile to ammonium acetate solution in the mobile phase is 25: 75-50: 50.
Further preferably, in the separation and purification by preparative chromatography described in step D, the volume ratio of acetonitrile to ammonium acetate solution in the mobile phase is 25: 75-40: 60.
Preferably, the chromatographic conditions for the preparative chromatographic purification described in step D are:
a chromatographic column: LP-C18, 250X 21.2 mm;
wavelength: 214 nm;
mobile phase: a mixed solution of acetonitrile and 0.05-0.2 mol/L ammonium acetate solution with the pH value of 3.5-4.5;
flow rate: 19 mL/min;
column temperature: the room temperature is 20-30 ℃.
Preferably, the freeze-drying conditions in step E are: freezing for 2-8 h at-60 to-45 ℃ and under the pressure of less than 20 Pa; then freeze-drying for 50-80 h at-40 to-10 ℃ and under the pressure of 20-60 Pa; and then freeze-drying for 2-8 h at the temperature of 10-30 ℃ and under the pressure of less than 10 Pa.
Compared with the prior art, the invention has the following outstanding advantages:
1. through the research, the chemical structure and the generation source of the impurity are determined, the related impurity and the generation background thereof are deeply known, the production process is controlled, and the product quality is improved;
2. the method for preparing the daptomycin impurity RS-1 and the impurity RS-3, which is provided by the invention, has a simple process, can improve the content of the impurity RS-1 and the impurity RS-3 from about 0.1% to more than 14%, obtains an impurity sample with higher concentration before preparation, and saves time and preparation cost;
3. the method has low requirement on the source of the starting material of the daptomycin sample, is easy to obtain, and can be used for preparing impurities in an enlarged way according to the requirement, thereby meeting the requirements of research and production;
4. according to the method for preparing daptomycin impurity RS-1 and impurity RS-3, the chromatographic purity of the obtained impurity is higher than 95%, and ESI-MS structure identification is carried out on the impurity RS-1 and the impurity RS-3.
Drawings
FIG. 1 is an HPLC detection profile of a finished daptomycin preparation.
FIG. 2 is an HPLC detection profile of purified daptomycin impurity RS-1.
FIG. 3 is an HPLC detection profile of purified daptomycin impurity RS-3.
FIG. 4 is an ESI-MS mass spectrum of purified daptomycin impurity RS-1.
FIG. 5 is an ESI-MS mass spectrum of purified daptomycin impurity RS-3.
Detailed Description
The present invention is further described by the following embodiments, which do not limit the scope of the present invention in any way, and those skilled in the art can make various modifications or improvements based on the basic idea of the present invention, but within the scope of the present invention, as long as they do not depart from the basic idea of the present invention.
Example 1
Taking a daptomycin finished product (5g) with the chromatographic purity of 95 percent, dissolving the daptomycin finished product into 50mg/mL solution by using purified water, then adding 1mol/L sodium carbonate solution into the daptomycin-containing aqueous solution to adjust the pH of the reaction solution to 10.0, placing the reaction solution in a thermostatic water bath kettle at 60 ℃, and heating and reacting for 5 hours to obtain alkaline hydrolysate; then adding 0.1mol/L hydrochloric acid solution into the alkaline hydrolysate to adjust the pH of the reaction solution to 2.0, placing the reaction solution in a water bath kettle with constant temperature of 80 ℃, and heating and reacting for 7 hours; then adding 0.02mol/L sodium hydroxide solution into the acidic hydrolysate, and adjusting the pH of the reaction system to 7.0 to obtain solutions containing daptomycin impurities RS-1 and RS-3 (the purities of the impurities RS-1 and RS-3 in the HPLC detection solution are 16.7 percent and 21.6 percent respectively); separating and purifying the solution containing daptomycin impurities RS-1 and RS-3 by preparative chromatography (chromatographic column is LP-C18, 250 multiplied by 21.2mm), wherein the mobile phase is a mixed solution of acetonitrile and 0.1mol/L ammonium acetate solution (the volume ratio of the acetonitrile to the ammonium acetate solution is 30: 70, and the pH value of the ammonium acetate solution is 4.0), the sample loading flow rate of the mobile phase is 19mL/min, the column temperature is 25 ℃, and the detection is carried out by high performance liquid phase tracking (detection wavelength is 214nm), the detection conditions are the same as the method disclosed by European patent EP1586580A2, and the prepared solutions containing the daptomycin impurities RS-1 and RS-3 are respectively collected; freezing the prepared preparation liquid containing daptomycin impurities RS-1 and RS-3 for 5 hours at-60 to-45 ℃ and under the pressure of less than 20Pa, then freeze-drying for 65 hours at-40 to-10 ℃ and under the pressure of 20 to 60Pa, and freeze-drying for 5 hours at 10 to 30 ℃ and under the pressure of less than 10Pa to obtain daptomycin impurity RS-1 and RS-3 solids, wherein the purity of the daptomycin impurity RS-1 is 97.8 percent, and the purity of the impurity RS-3 is 98.6 percent.
Example 2
Taking daptomycin powder injection (2g), dissolving the daptomycin powder injection into 50mg/mL solution by using purified water, then adding 0.5mol/L potassium carbonate solution into daptomycin-containing aqueous solution to adjust the pH of reaction liquid to 10.5, placing the solution in a constant-temperature water bath kettle at 65 ℃, and heating and reacting for 6 hours to obtain alkaline hydrolysis liquid; then adding 0.2mol/L sulfuric acid solution into the alkaline hydrolysate to adjust the pH of the reaction solution to 1.6, placing the reaction solution in a water bath kettle with the constant temperature of 75 ℃, and heating and reacting for 8 hours; then adding 0.01mol/L potassium hydroxide solution into the acidic hydrolysate, and adjusting the pH of the reaction system to 7.0 to obtain solutions mainly containing daptomycin impurities RS-1 and RS-3 (the purities of the impurities RS-1 and RS-3 in the HPLC detection solution are 16.2 percent and 21.1 percent respectively); separating and purifying the solution containing daptomycin impurities RS-1 and RS-3 by preparative chromatography (chromatographic column is LP-C18, 250 multiplied by 21.2mm), wherein the mobile phase is a mixed solution of acetonitrile and 0.1mol/L ammonium acetate solution (the volume ratio of acetonitrile to ammonium acetate solution is 25: 75, the pH value of the ammonium acetate solution is 4.0), the sample loading flow rate of the mobile phase is 19mL/min, the column temperature is 25 ℃, and the detection is carried out by high performance liquid phase tracking (detection wavelength is 214nm), the detection conditions are the same as the method disclosed by European patent EP1586580A2, and the prepared solutions containing the daptomycin impurities RS-1 and RS-3 are respectively collected; freezing the prepared preparation liquid containing daptomycin impurities RS-1 and RS-3 for 3 hours at-60 to-45 ℃ and under the pressure of less than 20Pa, then freeze-drying for 60 hours at-40 to-10 ℃ and under the pressure of 20 to 60Pa, and freeze-drying for 3 hours at 10 to 30 ℃ and under the pressure of less than 10Pa to obtain daptomycin impurity RS-1 and RS-3 solids, wherein the purity of the daptomycin impurity RS-1 is 97.2 percent, and the purity of the impurity RS-3 is 98.1 percent.
Example 3
Taking a daptomycin finished product (5g) with the chromatographic purity of 90 percent, dissolving the daptomycin finished product into 50mg/mL solution by using purified water, then adding 0.8mol/L potassium carbonate solution into the daptomycin-containing aqueous solution to adjust the pH of the reaction solution to 9.5, placing the reaction solution in a thermostatic water bath kettle at 63 ℃, and heating and reacting for 4 hours to obtain alkaline hydrolysate; then adding 0.3mol/L sulfuric acid solution into the alkaline hydrolysate to adjust the pH of the reaction solution to 3.0, placing the reaction solution in a water bath kettle with constant temperature of 77 ℃, and heating and reacting for 6 hours; then adding 0.03mol/L potassium hydroxide solution into the acidic hydrolysate, and adjusting the pH of the reaction system to 7.0 to obtain solutions mainly containing daptomycin impurities RS-1 and RS-3 (the purities of the impurities RS-1 and RS-3 in the HPLC detection solution are 16.3 percent and 21.0 percent respectively); separating and purifying the solution containing daptomycin impurities RS-1 and RS-3 by preparative chromatography (chromatographic column is LP-C18, 250 multiplied by 21.2mm), wherein the mobile phase is a mixed solution of acetonitrile and 0.15mol/L ammonium acetate solution (the volume ratio of the acetonitrile to the ammonium acetate solution is 40: 60, and the pH value of the ammonium acetate solution is 4.0), the sample loading flow rate of the mobile phase is 19mL/min, the column temperature is 25 ℃, and the detection is carried out by high performance liquid phase tracking (detection wavelength is 214nm), the detection conditions are the same as the method disclosed by European patent EP1586580A2, and the prepared solutions containing the daptomycin impurities RS-1 and RS-3 are respectively collected; freezing the prepared preparation liquid containing daptomycin impurities RS-1 and RS-3 for 6h under the conditions of-60 to-45 ℃ and the pressure of lower than 20Pa, then freeze-drying for 70h under the conditions of-40 to-10 ℃ and the pressure of 20 to 60Pa, and freeze-drying for 6h under the conditions of 10 to 30 ℃ and the pressure of lower than 10Pa to obtain daptomycin impurity RS-1 and RS-3 solids, wherein the purity of the daptomycin impurity RS-1 is 97.1 percent, and the purity of the impurity RS-3 is 98.4 percent.
Example 4
Taking a daptomycin crude product (10g) with the chromatographic purity of 88 percent, dissolving the daptomycin crude product into 50mg/mL solution by using purified water, then adding 0.1mol/L sodium hydroxide solution into the daptomycin-containing aqueous solution to adjust the pH of a reaction solution to 9.0, placing the reaction solution in a thermostatic water bath kettle at 55 ℃, and heating and reacting for 9 hours to obtain alkaline hydrolysate; then adding 0.5mol/L hydrochloric acid solution into the alkaline hydrolysate to adjust the pH of the reaction solution to 3.5, placing the reaction solution in a constant-temperature water bath kettle at 70 ℃, and heating and reacting for 9 hours; then adding 0.05mol/L sodium carbonate solution into the acidic hydrolysate, and adjusting the pH value of the reaction system to 7.0 to obtain a solution mainly containing daptomycin impurities RS-1 and RS-3 (the purities of the impurities RS-1 and RS-3 in the acid solution are respectively 15.4 percent and 19.6 percent through HPLC detection); separating and purifying the solution containing daptomycin impurities RS-1 and RS-3 by preparative chromatography (chromatographic column is LP-C18, 250 multiplied by 21.2mm), wherein the mobile phase is a mixed solution of acetonitrile and 0.05mol/L ammonium acetate solution (the volume ratio of the acetonitrile to the ammonium acetate solution is 45: 55, and the pH value of the ammonium acetate solution is 3.5), the sample loading flow rate of the mobile phase is 19mL/min, the column temperature is 30 ℃, and the detection is carried out by high performance liquid phase tracking (the detection wavelength is 214nm), the detection conditions are the same as the method disclosed by European patent EP1586580A2, and the prepared solutions containing the daptomycin impurities RS-1 and RS-3 are respectively collected; freezing the prepared preparation liquid containing daptomycin impurities RS-1 and RS-3 for 2 hours at-60 to-45 ℃ and under the pressure of less than 20Pa, then freeze-drying for 50 hours at-40 to-10 ℃ and under the pressure of 20 to 60Pa, and freeze-drying for 8 hours at 10 to 30 ℃ and under the pressure of less than 10Pa to obtain daptomycin impurity RS-1 and RS-3 solids, wherein the purity of the daptomycin impurity RS-1 is 96.8 percent, and the purity of the impurity RS-3 is 98.0 percent.
Example 5
Taking a daptomycin crude product (10g) with the chromatographic purity of 85 percent, dissolving the daptomycin crude product into 50mg/mL solution by using purified water, then adding 0.2mol/L potassium hydroxide solution into the daptomycin-containing aqueous solution to adjust the pH of a reaction solution to 11.0, placing the reaction solution in a constant-temperature water bath kettle at 70 ℃, and heating and reacting for 3 hours to obtain alkaline hydrolysate; then adding 1mol/L phosphoric acid solution into the alkaline hydrolysate to adjust the pH of the reaction solution to 4.0, placing the reaction solution in a water bath kettle with constant temperature of 85 ℃, and heating and reacting for 3 hours; then adding 0.1mol/L potassium carbonate solution into the acidic hydrolysate, and adjusting the pH value of the reaction system to 7.0 to obtain a solution mainly containing daptomycin impurities RS-1 and RS-3 (the purities of the impurities RS-1 and RS-3 in the acid solution are 14.2 percent and 19.7 percent respectively by HPLC detection); separating and purifying the solution containing daptomycin impurities RS-1 and RS-3 by preparative chromatography (chromatographic column is LP-C18, 250 multiplied by 21.2mm), wherein the mobile phase is a mixed solution of acetonitrile and 0.2mol/L ammonium acetate solution (the volume ratio of acetonitrile to ammonium acetate solution is 50: 50, the pH value of the ammonium acetate solution is 4.5), the sample loading flow rate of the mobile phase is 19mL/min, the column temperature is 20 ℃, and the detection is carried out by high performance liquid phase tracking detection (detection wavelength is 214nm), the detection conditions are the same as the method disclosed by European patent EP1586580A2, and the prepared solutions containing the daptomycin impurities RS-1 and RS-3 are respectively collected; freezing the prepared preparation liquid containing daptomycin impurities RS-1 and RS-3 for 8 hours at-60 to-45 ℃ and under the pressure of less than 20Pa, then freeze-drying for 80 hours at-40 to-10 ℃ and under the pressure of 20 to 60Pa, and freeze-drying for 2 hours at 10 to 30 ℃ and under the pressure of less than 10Pa to obtain daptomycin impurity RS-1 and RS-3 solids, wherein the purity of the daptomycin impurity RS-1 is 96.6 percent, and the purity of the impurity RS-3 is 97.4 percent.
Example 6
Taking a daptomycin finished product (5g) with the chromatographic purity of 90 percent, dissolving the daptomycin finished product into 50mg/mL solution by using purified water, then adding 0.2mol/L ammonia water solution into the daptomycin-containing aqueous solution to adjust the pH of the reaction solution to 8.0, placing the reaction solution in a thermostatic water bath kettle at 40 ℃, and heating and reacting for 10 hours to obtain alkaline hydrolysate; then adding 1mol/L hydrochloric acid solution into the alkaline hydrolysate to adjust the pH of the reaction solution to 1.0, placing the reaction solution in a water bath kettle with the constant temperature of 90 ℃, and heating and reacting for 2 hours; then adding 0.1mol/L potassium carbonate solution into the acidic hydrolysate, and adjusting the pH value of the reaction system to 7.0 to obtain a solution mainly containing daptomycin impurities RS-1 and RS-3 (the purities of the impurities RS-1 and RS-3 in the acid solution are respectively 12.6 percent and 17.1 percent through HPLC detection); separating and purifying the solution containing daptomycin impurities RS-1 and RS-3 by preparative chromatography (chromatographic column is LP-C18, 250 multiplied by 21.2mm), wherein the mobile phase is a mixed solution of acetonitrile and 0.1mol/L ammonium acetate solution (the volume ratio of the acetonitrile to the ammonium acetate solution is 60: 40, and the pH value of the ammonium acetate solution is 4.0), the sample loading flow rate of the mobile phase is 19mL/min, the column temperature is 25 ℃, and the detection is carried out by high performance liquid phase tracking (detection wavelength is 214nm), the detection conditions are the same as the method disclosed by European patent EP1586580A2, and the prepared solutions containing the daptomycin impurities RS-1 and RS-3 are respectively collected; freezing the prepared preparation liquid containing daptomycin impurities RS-1 and RS-3 for 1h at-60 to-45 ℃ and under the pressure of less than 20Pa, then freeze-drying for 40h at-40 to-10 ℃ and under the pressure of 20 to 60Pa, and freeze-drying for 9h at 10 to 30 ℃ and under the pressure of less than 10Pa to obtain daptomycin impurity RS-1 and RS-3 solids, wherein the purity of the daptomycin impurity RS-1 is 86.3 percent, and the purity of the impurity RS-3 is 87.9 percent.
Example 7
Taking a daptomycin finished product (10g) with the chromatographic purity of 90 percent, dissolving the daptomycin finished product into 50mg/mL solution by using purified water, then adding 0.1mol/L sodium hydroxide solution into the daptomycin-containing aqueous solution to adjust the pH of the reaction solution to 12.0, placing the reaction solution in a water bath kettle with the constant temperature of 80 ℃, and heating and reacting for 2 hours to obtain alkaline hydrolysate; then adding 1mol/L glacial acetic acid solution into the alkaline hydrolysate to adjust the pH of the reaction solution to 5.0, placing the reaction solution in a constant-temperature water bath kettle at 60 ℃, and heating and reacting for 10 hours; then adding 0.05mol/L sodium carbonate solution into the acid hydrolysate, and adjusting the pH of the reaction system to 7.0 to obtain a solution mainly containing daptomycin impurities RS-1 and RS-3 (the purities of the impurities RS-1 and RS-3 in the acid hydrolysate are respectively 11.8 percent and 17.9 percent by HPLC detection); separating and purifying the solution containing daptomycin impurities RS-1 and RS-3 by preparative chromatography (chromatographic column is LP-C18, 250 multiplied by 21.2mm), wherein the mobile phase is a mixed solution of acetonitrile and 0.1mol/L ammonium acetate solution (the volume ratio of the acetonitrile to the ammonium acetate solution is 15: 85, the pH value of the ammonium acetate solution is 4.0), the sample loading flow rate of the mobile phase is 19mL/min, the column temperature is 25 ℃, and the detection is carried out by high performance liquid phase tracking (detection wavelength is 214nm), the detection conditions are the same as the method disclosed by European patent EP1586580A2, and the prepared solutions containing the daptomycin impurities RS-1 and RS-3 are respectively collected; freezing the prepared preparation liquid containing daptomycin impurities RS-1 and RS-3 for 9 hours at-60 to-45 ℃ and under the pressure of less than 20Pa, then freeze-drying for 90 hours at-40 to-10 ℃ and under the pressure of 20 to 60Pa, and freeze-drying for 1 hour at 10 to 30 ℃ and under the pressure of less than 10Pa to obtain daptomycin impurity RS-1 and RS-3 solids, wherein the purity of the daptomycin impurity RS-1 is 85.8 percent, and the purity of the impurity RS-3 is 88.0 percent.
Claims (10)
1. A method for preparing daptomycin impurity RS-1 and impurity RS-3 is characterized by comprising the following steps: the daptomycin sample is hydrolyzed under alkaline conditions, then hydrolyzed under acidic conditions, and then neutralized by alkali to obtain feed liquid containing impurities RS-1 and RS-3, and then purified by preparative chromatography to obtain purified daptomycin impurities RS-1 and RS-3.
2. The method for preparing daptomycin impurity RS-1 and impurity RS-3 according to claim 1, comprising the steps of:
A. alkali hydrolysis: dissolving a daptomycin sample by using purified water, adding alkaline solution to adjust the solution to be alkaline, and heating for reaction;
B. acid hydrolysis: b, adding acid liquid into the hydrolysate obtained in the step A to adjust the hydrolysate to be acidic, and heating for reaction;
C. alkali neutralization: b, adding alkaline liquor into the hydrolysate obtained in the step B to adjust the hydrolysate to be neutral, so as to obtain a solution containing daptomycin impurities RS-1 and RS-3;
D. and (3) purification: c, loading the solution obtained in the step C onto a preparative chromatographic column, separating and purifying, and respectively collecting a preparation solution containing a daptomycin impurity RS-1 and a preparation solution containing RS-3;
E. and (3) freeze drying: and D, respectively freezing and drying the preparation liquid of the daptomycin impurities RS-1 and RS-3 obtained in the step D to obtain daptomycin impurity RS-1 and RS-3 solids.
3. The method for preparing impurity RS-1 and impurity RS-3 of daptomycin according to claim 2, wherein the sample of daptomycin in step A is a crude daptomycin product, a finished daptomycin product or a daptomycin powder injection.
4. The method for preparing daptomycin impurity RS-1 and impurity RS-3 as claimed in claim 2, wherein the alkali solution in step A is one selected from sodium hydroxide, potassium hydroxide, sodium carbonate or potassium carbonate solution, and the concentration of the alkali solution is 0.1-1 mol/L.
5. The method for preparing the daptomycin impurity RS-1 and the impurity RS-3 as claimed in claim 2, wherein the pH is adjusted to 9.0-11.0 by adding the alkaline solution in the step A.
6. The method for preparing the daptomycin impurity RS-1 and the impurity RS-3 as claimed in claim 2, wherein the acid solution in step B is selected from one of hydrochloric acid, sulfuric acid or phosphoric acid, and the concentration of the acid solution is 0.1-1 mol/L.
7. The method for preparing daptomycin impurity RS-1 and impurity RS-3 as claimed in claim 2, wherein the pH of the acid solution in step B is adjusted to 1.6-4.0.
8. The method for preparing impurity RS-1 and impurity RS-3 of daptomycin according to claim 2, wherein the mobile phase is a mixture of acetonitrile and ammonium acetate solution during the preparative chromatography separation and purification in step D.
9. The method for preparing daptomycin impurity RS-1 and impurity RS-3 as claimed in claim 2, wherein the mobile phase is a mixture of acetonitrile and 0.05-0.2 mol/L ammonium acetate solution during the separation and purification of the preparative chromatography in step D.
10. The method for preparing daptomycin impurity RS-1 and impurity RS-3 as claimed in claim 2, wherein the volume ratio of acetonitrile to ammonium acetate solution in the mobile phase during the preparative chromatographic separation and purification in step D is 25: 75-50: 50.
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