CN103122023B - A kind of purification process of triptorelin - Google Patents
A kind of purification process of triptorelin Download PDFInfo
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- CN103122023B CN103122023B CN201310074154.0A CN201310074154A CN103122023B CN 103122023 B CN103122023 B CN 103122023B CN 201310074154 A CN201310074154 A CN 201310074154A CN 103122023 B CN103122023 B CN 103122023B
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Abstract
The invention provides a kind of purification process of triptorelin, first with containing the aqueous dissolution sample of organic solvent, take octadecylsilane chemically bonded silica as stationary phase, and with the buffering salt of phosphoric acid or sulfuric acid for A phase, acetonitrile is B phase, carries out gradient elution; Then, adopt Reversed phase HPLC method, take octadecylsilane chemically bonded silica as stationary phase, triptorelin phosphoric acid salt is changed into acetate, freeze-drying obtains purity and be greater than 99.7% single assorted triptorelin acetate being less than 0.05%, total recovery is up to more than 30%.The invention provides a kind of yield with low cost, high, technological process is simple, sample impurity is few and stablizes controlled, to be conducive to realizing industrialization triptorelin purifying process.
Description
Technical field
The present invention relates to a kind of purification process of polypeptide drugs, particularly the purification process of triptorelin, belongs to medicinal chemistry art.
Background technology
Triptorelin, English name: Triptorelin, commodity are called look ammonia Rayleigh, Wy-42462, Decapeptyl etc., it is a kind of gonadotropin releasing hormone (GnRH) the decapeptide congener of 10 amino acid compositions, its similar goserelin, its peptide sequence is: Pyr-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH
2, clinical in its embonate (storage preparation).
Triptorelin is as a kind of Amino-Cerv, and its indication is mainly: 1. the palliative treatment of advanced prostate cancer; 2. sexual prematurity; 3. endometriosis, female infertility and hysteromyoma.When continuing medication with therapeutic dose, this product can suppress the secretion of short sexual hormoue forcefully, and the natural GnRH of its specific activity is strong.After starting administration, the LH during blood follows, FSH, the level of testosterone and estradiol there will be transience peak.After continuous long term administration, generally obviously reduce at the secretory volume continuous decrease that can be observed LH and FSH for 2 ~ 4 weeks of begin treatment and the steroid synthesis of testis and ovary.Triptorelin treats the optimal medicine of Central precocious puberty at present, can suppress the speed that the maturation of secondal sexual character and health linearly grow quickly and effectively, and after drug withdrawal, the natural process of Development in Puberty biology is unaffected.Clinically be mainly used in sexual prematurity, endometriosis, female acyesis and hysteromyoma etc.
Prior art CN101357936A discloses a kind of separation purification method of triptorelin, concrete steps are as follows: be dissolved in by triptorelin crude product in 5% acetic acid, and filter, filtrate is through C18 column purification, moving phase: 0.1MNH4Ac: acetonitrile (7.5: 2.5, volume ratio); Flow velocity is 500ml/min; Determined wavelength is: 280nm; Follow the tracks of the effluent liquid required for collecting with liquid chromatograph, sample peak desalts after merging, freeze-drying, about obtains 20g white chunks thing finished product (MW:1311.5,15.25mmol), total recovery 25.4% (60mmol in resin).This separation purification method is difficult to the Light absorbing impurty controlling raw material, is difficult to reach medicinal required purity.
For effectively controlling the Light absorbing impurty of triptorelin raw material, improving purity, needing the separation purification method that research is new further.
Summary of the invention
The present invention is directed to the physicochemical characteristics of triptorelin, provide a kind of yield with low cost, high, technological process is simple, sample impurity is few and stablize controlled, to be conducive to realizing industrialization triptorelin purifying process.
For achieving the above object, the present invention takes following technical scheme: a kind of purification process of triptorelin, is characterized in that:
1) with the aqueous solution of the organic solvent of volume ratio content 5%-15%, triptorelin sample is dissolved according to concentration 100g/L, take octadecylsilane chemically bonded silica as the chromatographic column of stationary phase, take buffering salt as A phase, acetonitrile is B phase, carries out gradient elution (this step is aftermentioned referred to as " purifying ");
2) adopt Reversed phase HPLC method, take octadecylsilane chemically bonded silica as stationary phase, triptorelin phosphoric acid salt is changed into acetate, freeze-drying obtains triptorelin (this step is aftermentioned referred to as " turning salt ").
Step 1), preferably with the aqueous solution of the organic solvent of volume ratio content 8%-10% in described step 1), triptorelin sample is dissolved according to concentration 100g/L, wherein, described sample dissolution organic solvent used is the organic reagent that polarity is stronger, comprise methyl alcohol, acetonitrile, dimethyl sulfoxide (DMSO) (DMSO), Virahol etc., wherein preferred acetonitrile.
Applicant finds, due to the feature of this body structure of triptorelin composition, therefore the solubilising reagent of thick peptide sample requires very harsh in crude product easily containing various synthesis not exclusively or racemization impurity.Find after applicant's repetition test, when organic solvent content is in 5% ~ 15% scope, sample dissolution is better, and wherein organic solvent content is when 8% ~ 10% scope, and effect is more obvious; And find when groping different organic reagent, the organic reagent of the stronger polarity such as DMSO, methyl alcohol, acetonitrile, Virahol is better to the solute effect of sample, wherein again with acetonitrile best results.
Wherein, described 1), the buffering salt of A phase is phosphate-buffered salt or sulfuric acid buffering salt.
Wherein, described phosphate-buffered salt is 0.1%-0.8%(v/v) phosphate buffered saline buffer, the solvent of damping fluid is water; Described sulfuric acid buffer salt system is 0.2%-0.6%(v/v) sulfate buffer.
Wherein, described phosphate-buffered salt comprises the buffer salt system such as phosphoric acid triethylamine, Sodium phosphate dibasic, dipotassium hydrogen phosphate, SODIUM PHOSPHATE, MONOBASIC, potassium primary phosphate; Sulfuric acid buffer salt system is ammonium sulfate buffering salt.
Carrying out in purge process to triptorelin, applicant analyzes different flow visualizing, contrasts, and looks for best flow visualizing.Find in process, what most of flow visualizing can only be simple removes partial impurities, once in purge process in order to ensure purity, just loaded down with trivial details recovery purifying often repeatedly will be carried out repeatedly can reach the purity meeted the requirements; Repeatedly groping through applicant, 0.1% final-0.8%(v/v) phosphate buffer and 0.2%-0.6%(v/v) vitriol buffer system, to the impurity successful in the thick peptide of removing, only need purifying, once reclaim, save multistep repetitive operation.Purifying can be obtained purity and be greater than 95%, carry out turning salt after single assorted sample being less than 0.3% removes most of acetonitrile.Purity be less than 95% be greater than 80% once partially recycled, get its moderate purity and be greater than 95%, single assorted be less than 0.3% part and other salable product one to run up salt, reclaim purity undesirable discarded.
Wherein, described phosphate-buffered salt and sulfuric acid buffering salt sodium hydroxide or potassium hydroxide or ammoniacal liquor adjust pH1.5-3.2.Applicant find, triptorelin is comparatively harsh for the requirement of pH value, pH value higher than 5 time, sample is easily separated out, pH value lower than 1 time, sample stability be deteriorated.Through repetition test, when finding pH2.0-3.0, sample stability is best.
In step 1), the gradient of described wash-out reversed-phase HPLC is B phase 20%-40%(v/v), linear gradient elution time 60min.
Step 2), wherein, the described salt that turns comprises two steps: turn salt with acetate buffer salts solution; Be A phase with aqueous acetic acid afterwards, acetonitrile is that B phase carries out gradient elution.
Wherein, acetate buffer salts solution is the aqueous solution of acetate buffer salt and the mixing solutions of acetonitrile, and in the aqueous solution of described acetate buffer salt, acetate buffer salt is ammonium acetate; The aqueous solution of acetate buffer salt and the volume ratio of acetonitrile are 95:5.
Wherein, the concentration of described acetate buffer salt is 0.02-0.1%(v/v), the pH value range of acetate buffer salt is 1.5-4.5.Applicant find, acetate buffer salt concn lower than 0.02% time, higher than 0.1% time, sample is all unstable.When acetate buffer salt pH value range is 1.5-4.5, sample is all very stable.
Wherein, in aqueous acetic acid, the volume by volume concentration of acetic acid is 0.02%-0.65%, and the gradient of described reversed-phase HPLC is B%:5% ~ 25%, and the linear gradient elution time is 30min.Applicant find, because the first step sample purity is high, therefore turning 30min in salt process, just can purifying obtain high purity 99.8%, maximum list mix be less than 0.02% triptorelin acetate, obviously shorten the time turning salt purifying, saved production cost.
The chromatographic column of to be octadecylsilane chemically bonded silica the be stationary phase of described reversed-phase column.Also be referred to as volume ratio in (v/v) in specification sheets, the ratio of two kinds of solvents of same volume unit, as 0.02%(v/v) aqueous acetic acid refers to that volume is the mixing solutions of 0.02L acetic acid and 1L water.
Present method is by two step purifying, and the distance removed in triptorelin crude product is mixed and made purity be greater than 99.7%, be singly assortedly less than 0.03% and change into stable acetate respectively, and process stabilizing is controlled, purity is high, productive rate is high, has practical value and application prospect widely.
Embodiment
Embodiment 1
Embodiment 1 the first step purifying
Dissolve thick peptide with the acetonitrile solution of volume ratio 5% according to the concentration of 100g/L, stir and make sample dissolve rear membrane filtration completely, collect filtrate.
Purification condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 5cm × 25cm.Moving phase: A phase: 0.1% phosphate buffered (v/v), adjusts pH2.0 with sodium hydroxide; B phase: acetonitrile, flow velocity: 60-80ml/min, gradient: B%:20% ~ 40%, linear gradient elution 60min; Determined wavelength: 230nm.Sample size is 1.5g.Collect object peak, purifying can be obtained purity and be greater than 95%, carry out turning salt after single assorted sample being less than 0.3% removes most of acetonitrile.Purity be less than 95% be greater than 80% once partially recycled, get its moderate purity and be greater than 95%, single assorted be less than 0.3% part and other salable product one to run up salt, reclaim purity undesirable discarded.Satisfactory triptorelin solution vacuum rotary steam under the condition of water temperature 30 DEG C is for subsequent use after being concentrated into about 15mg/mL.
Embodiment 2 the first step purifying
Dissolve thick peptide with the DMSO aqueous solution of volume ratio 15% according to the concentration of 100g/L, stir and make sample dissolve rear membrane filtration completely, collect filtrate.
Purification condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 5cm × 25cm.Moving phase: A phase: 0.8% phosphate buffered (v/v), adjusts pH3.0 with ammoniacal liquor; B phase: acetonitrile, flow velocity: 60-80ml/min, gradient: B%:20%-40%, linear gradient elution 60min; Determined wavelength: 230nm.Sample size is 1.5g.Collect object peak, purifying can be obtained purity and be greater than 95%, carry out turning salt after single assorted sample being less than 0.3% removes most of acetonitrile.Purity be less than 95% be greater than 80% once partially recycled, get its moderate purity and be greater than 95%, single assorted be less than 0.3% part and other salable product one to run up salt, reclaim purity undesirable discarded.Triptorelin solution vacuum rotary steam under the condition of water temperature 30 DEG C that satisfactory song is collected is for subsequent use after being concentrated into about 15mg/mL.
Embodiment 3 the first step purifying
Dissolve thick peptide with the isopropanol water solution of volume ratio 10% according to the concentration of 100g/L, stir and make sample dissolve rear membrane filtration completely, collect filtrate.
Purification condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 5cm × 25cm.Moving phase: A phase: 0.2% vitriol buffering (v/v), adjusts pH2.0 with phosphoric acid; B phase: acetonitrile, flow velocity: 60-80ml/min, gradient: B%:20%-40%, linear gradient elution 60min; Determined wavelength: 230nm.Sample size is 1.5g.Collect object peak, purifying can be obtained purity and be greater than 95%, carry out turning salt after single assorted sample being less than 0.3% removes most of acetonitrile.Purity be less than 95% be greater than 80% once partially recycled, get its moderate purity and be greater than 95%, single assorted be less than 0.3% part and other salable product one to run up salt, reclaim purity undesirable discarded.Triptorelin solution vacuum rotary steam under the condition of water temperature 30 DEG C that satisfactory song is collected is for subsequent use after being concentrated into about 15mg/mL.
Embodiment 4 the first step purifying
Dissolve thick peptide with the acetonitrile solution of volume ratio 15% according to the concentration of 100g/L, stir and make sample dissolve rear membrane filtration completely, collect filtrate.
Purification condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 10cm × 25cm.Moving phase: A phase: 0.6% vitriol buffering (v/v), adjusts pH3.0 with phosphoric acid; B phase: acetonitrile, flow velocity: 200-250ml/min, gradient: B%:20%-40%, linear gradient elution 60min; Determined wavelength: 230nm.Sample size is 15-20g.Collect object peak, purifying can be obtained purity and be greater than 95%, carry out turning salt after single assorted sample being less than 0.3% removes most of acetonitrile.Purity be less than 95% be greater than 80% once partially recycled, get its moderate purity and be greater than 95%, single assorted be less than 0.3% part and other salable product one to run up salt, reclaim purity undesirable discarded.Triptorelin solution vacuum rotary steam under the condition of water temperature 30 DEG C that satisfactory song is collected is for subsequent use after being concentrated into about 15mg/mL.
Embodiment 5 the first step purifying
Dissolve thick peptide with volume ratio 8% methanol aqueous solution according to the concentration of 100g/L, stir and make sample dissolve rear membrane filtration completely, collect filtrate.
Purification condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 10cm × 25cm.Moving phase: A phase: 0.5% phosphate buffered (v/v), adjusts pH2.5 with sodium hydroxide; B phase: acetonitrile, flow velocity: 220-250ml/min, gradient: B%:20%-40%, linear gradient elution 60min; Determined wavelength: 230nm.Sample size is 15-20g.Collect object peak, purifying can be obtained purity and be greater than 95%, carry out turning salt after single assorted sample being less than 0.3% removes most of acetonitrile.Purity be less than 95% be greater than 80% once partially recycled, get its moderate purity and be greater than 95%, single assorted be less than 0.3% part and other salable product one to run up salt, reclaim purity undesirable discarded.Triptorelin solution vacuum rotary steam under the condition of water temperature 30 DEG C that satisfactory song is collected is for subsequent use after being concentrated into about 15mg/mL.
Embodiment 6 the first step purifying
Dissolve thick peptide with the isopropanol water solution of volume ratio 5% according to the concentration of 100g/L, stir and make sample dissolve rear membrane filtration completely, collect filtrate.
Purification condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 10cm × 25cm.Moving phase: A phase: 0.5% vitriol buffering (v/v), adjusts pH2.3 with phosphoric acid; B phase: acetonitrile, flow velocity: 220-250ml/min, gradient: B%:20%-40%, linear gradient elution 60min; Determined wavelength: 230nm.Sample size is 15-20g.Receive purifying can obtain purity and be greater than 95%, carry out turning salt after single assorted sample being less than 0.3% removes most of acetonitrile.Purity be less than 95% be greater than 80% once partially recycled, get its moderate purity and be greater than 95%, single assorted be less than 0.3% part and other salable product one to run up salt, reclaim purity undesirable discarded.Satisfactory bent Ji Mufeng, by for subsequent use after being concentrated into about 15mg/mL for the triptorelin solution of collection vacuum rotary steam under the condition of water temperature 30 DEG C.
Embodiment 7 the first step purifying
Dissolve thick peptide with the DMSO aqueous solution of volume ratio 8% according to the concentration of 100g/L, stir and make sample dissolve rear membrane filtration completely, collect filtrate.
Purification condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 15cm × 30cm.Moving phase: A phase: 0.3% phosphate buffered (v/v), adjusts pH2.5 with potassium hydroxide; B phase: acetonitrile, flow velocity: 450-550ml/min, gradient: B%:20%-40%, linear gradient elution 30min; Determined wavelength: 230nm.Sample size is 20-25g.Collect object peak, purifying can be obtained purity and be greater than 95%, carry out turning salt after single assorted sample being less than 0.3% removes most of acetonitrile.Purity be less than 95% be greater than 80% once partially recycled, get its moderate purity and be greater than 95%, single assorted be less than 0.3% part and other salable product one to run up salt, reclaim purity undesirable discarded.Triptorelin solution vacuum rotary steam under the condition of water temperature 30 DEG C that satisfactory song is collected is for subsequent use after being concentrated into about 15mg/mL.
Embodiment 8 the first step purifying
Dissolve thick peptide with the methanol aqueous solution of volume ratio 6% according to the concentration of 100g/L, stir and make sample dissolve rear membrane filtration completely, collect filtrate.
Purification condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 15cm × 30cm.Moving phase: A phase: 0.8% phosphate buffered (v/v), adjusts pH3.0 with ammoniacal liquor; B phase: acetonitrile, flow velocity: 450-550ml/min, gradient: B%:20%-40%, linear gradient elution 60min; Determined wavelength: 230nm.Sample size is 20-25g.Collect object peak, purifying can be obtained purity and be greater than 95%, carry out turning salt after single assorted sample being less than 0.3% removes most of acetonitrile.Purity be less than 95% be greater than 80% once partially recycled, get its moderate purity and be greater than 95%, single assorted be less than 0.3% part and other salable product one to run up salt, reclaim purity undesirable discarded.Triptorelin solution vacuum rotary steam under the condition of water temperature 30 DEG C that satisfactory song is collected is for subsequent use after being concentrated into about 15mg/mL.
Embodiment 9 the first step purifying
Dissolve thick peptide with the acetonitrile solution of volume ratio 5% according to the concentration of 100g/L, stir and make sample dissolve rear membrane filtration completely, collect filtrate.
Purification condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 30cm × 30cm.Moving phase: A phase: 0.5% phosphate buffered (v/v), adjusts pH2.0 with potassium hydroxide; B phase: acetonitrile, flow velocity: 1900-2200ml/min, gradient: B%:20%-40%, linear gradient elution 60min; Determined wavelength: 230nm.Sample size is 60-85g.Collect object peak, purifying can be obtained purity and be greater than 95%, carry out turning salt after single assorted sample being less than 0.3% removes most of acetonitrile.Purity be less than 95% be greater than 80% once partially recycled, get its moderate purity and be greater than 95%, single assorted be less than 0.3% part and other salable product one to run up salt, reclaim purity undesirable discarded.Triptorelin solution vacuum rotary steam under the condition of water temperature 30 DEG C that satisfactory song is collected is for subsequent use after being concentrated into about 15mg/mL.
Embodiment 10 the first step purifying
Dissolve thick peptide with the DMSO aqueous solution of volume ratio 15% according to the concentration of 100g/L, stir and make sample dissolve rear membrane filtration completely, collect filtrate.
Purification condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 30cm × 30cm.Moving phase: A phase: 0.2% vitriol buffering (v/v), adjusts pH2.5 with phosphoric acid; B phase: acetonitrile, flow velocity: 1900-2200ml/min, gradient: B%:20-40%, linear gradient elution 60min; Determined wavelength: 230nm.Sample size is 60-85g.Collect object peak, purifying can be obtained purity and be greater than 95%, carry out turning salt after single assorted sample being less than 0.3% removes most of acetonitrile.Purity be less than 95% be greater than 80% once partially recycled, get its moderate purity and be greater than 95%, single assorted be less than 0.3% part and other salable product one to run up salt, reclaim purity undesirable discarded.Triptorelin solution vacuum rotary steam under the condition of water temperature 30 DEG C that satisfactory song is collected is for subsequent use after being concentrated into about 15mg/mL.
Embodiment 11 turns of salt
Chromatographic condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 5cm × 25cm.Moving phase: A phase: 0.02% aqueous acetic acid (v/v); B phase: acetonitrile; C phase: 0.02% Ammoniom-Acetate, pH2.0, flow velocity: 80ml/min, determined wavelength: 230nm.Sample size is 1.5g.
Step: after turning salt 20min with 5%B+95%C, with 5%B+95%A ~ 25%B+75%A, linear gradient elution 30min; Collect object peak, triptorelin solution enforcement 1 collected goes to 50ml cillin bottle after vacuum rotary steam is concentrated into about 50-200mg/mL under water temperature 30 DEG C of conditions.Can obtain the triptorelin that purity is greater than 99.75% after lyophilize, other impurity is all less than 0.02%.
Embodiment 12 turns of salt
Chromatographic condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 5cm × 25cm.Moving phase: A phase: 0.05% aqueous acetic acid (v/v); B phase: acetonitrile, C phase: 0.05% Ammoniom-Acetate, pH2.5, flow velocity: 60ml/min, determined wavelength: 230nm.Sample size is 1.5g.
Step: after turning salt 20min with 5%B+95%C, with 5%B+95%A ~ 25%B+75%A, linear gradient elution 30min; Collect object peak, triptorelin solution enforcement 2 collected goes to 50ml cillin bottle after vacuum rotary steam is concentrated into about 50-200mg/mL under water temperature 30 DEG C of conditions.Can obtain the triptorelin that purity is greater than 99.78% after lyophilize, other impurity is all less than 0.02%.
Embodiment 13 turns of salt
Chromatographic condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 5cm × 25cm.Moving phase: A phase: 0.1% aqueous acetic acid (v/v); B phase: acetonitrile, C phase: 0.08% Ammoniom-Acetate, pH3.0, flow velocity: 80ml/min; Determined wavelength: 230nm.Sample size is 1.5g.
Step: after turning salt 20min with 5%B+95%C, with 5%B+95%A ~ 25%B+75%A, linear gradient elution 30min; Collect object peak, triptorelin solution enforcement 3 collected goes to 50ml cillin bottle after vacuum rotary steam is concentrated into about 50-200mg/mL under water temperature 30 DEG C of conditions.Can obtain the triptorelin that purity is greater than 99.76% after lyophilize, other impurity is all less than 0.02%.
Embodiment 14 turns of salt
Chromatographic condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 10cm × 25cm.Moving phase: A phase: 0.06% aqueous acetic acid (v/v); B phase: acetonitrile, flow velocity: 220ml/min, determined wavelength: 230nm.Sample size is 10-15g.
Step: after turning salt 20min with 5%B+95%C, with 5%B+95%A ~ 25%B+75%A, linear gradient elution 30min; Collect object peak, triptorelin solution enforcement 4 collected goes to 50ml cillin bottle after vacuum rotary steam is concentrated into about 50-200mg/mL under water temperature 30 DEG C of conditions.Can obtain the triptorelin that purity is greater than 99.76% after lyophilize, other impurity is all less than 0.02%.
Embodiment 15 turns of salt
Chromatographic condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 10cm × 25cm.Moving phase: A phase: 0.03% aqueous acetic acid (v/v); B phase: acetonitrile, C phase: 0.10% Ammoniom-Acetate, pH4.5, flow velocity: 250ml/min, determined wavelength: 230nm.Sample size is 15-20g.
Step: after turning salt 20min with 5%B+95%C, with 5%B+95%A ~ 25%B+75%A, linear gradient elution 30min; Collect object peak, triptorelin solution enforcement 5 collected goes to 50ml cillin bottle after vacuum rotary steam is concentrated into about 50-200mg/mL under water temperature 30 DEG C of conditions.Can obtain the triptorelin that purity is greater than 99.72% after lyophilize, other impurity is all less than 0.02%.
Embodiment 16 turns of salt
Chromatographic condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 15cm × 30cm.Moving phase: A phase: 0.09% aqueous acetic acid (v/v); B phase: acetonitrile, C phase: 0.07% Ammoniom-Acetate, pH3.2, flow velocity: 400ml/min, determined wavelength: 230nm.Sample size is 15-20g.
Step: after turning salt 20min with 5%B+95%C, with 5%B+95%A ~ 25%B+75%A, linear gradient elution 30min; Collect object peak, triptorelin solution enforcement 6 collected goes to 50ml cillin bottle after vacuum rotary steam is concentrated into about 50-200mg/mL under water temperature 30 DEG C of conditions.Can obtain the triptorelin that purity is greater than 99.75% after lyophilize, other impurity is all less than 0.02%.
Embodiment 17 turns of salt
Chromatographic condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 15cm × 30cm.Moving phase: A phase: 0.65% aqueous acetic acid (v/v); B phase: acetonitrile, C phase: 0.06% Ammoniom-Acetate, pH2.0, flow velocity: 450ml/min, determined wavelength: 230nm.Sample size is 15-20g.
Step: after turning salt 20min with 5%B+95%C, with 5%B+95%A ~ 25%B+75%A, linear gradient elution 30min; Collect object peak, triptorelin solution enforcement 7 collected goes to 50ml cillin bottle after vacuum rotary steam is concentrated into about 50-200mg/mL under water temperature 30 DEG C of conditions.Can obtain the triptorelin that purity is greater than 99.78% after lyophilize, other impurity is all less than 0.02%.
Embodiment 18 turns of salt
Chromatographic condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 30cm × 30cm.Moving phase: A phase: 0.32% aqueous acetic acid (v/v); B phase: acetonitrile, C phase: 0.05% Ammoniom-Acetate, pH2.5, flow velocity: 1900ml/min, determined wavelength: 230nm.Sample size is 60-85g.
Step: after turning salt 20min with 5%B+95%C, with 5%B+95%A ~ 25%B+75%A, linear gradient elution 30min; Collect object peak, triptorelin solution enforcement 8 collected goes to 50ml cillin bottle after vacuum rotary steam is concentrated into about 50-200mg/mL under water temperature 30 DEG C of conditions.Can obtain the triptorelin that purity is greater than 99.75% after lyophilize, other impurity is all less than 0.02%.
Embodiment 19 turns of salt
Chromatographic condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 30cm × 30cm.Moving phase: A phase: 0.10% aqueous acetic acid (v/v); B phase: acetonitrile, C phase: 0.02% Ammoniom-Acetate, pH2.0, flow velocity: 2200ml/min, determined wavelength: 230nm.Sample size is 60-85g.
Step: after turning salt 20min with 5%B+95%C, with 5%B+95%A ~ 25%B+75%A, linear gradient elution 30min; Collect object peak, triptorelin solution enforcement 9 collected goes to 50ml cillin bottle after vacuum rotary steam is concentrated into about 50-200mg/mL under water temperature 30 DEG C of conditions.Can obtain the triptorelin that purity is greater than 99.74% after lyophilize, other impurity is all less than 0.02%.
Embodiment 20 turns of salt
Chromatographic condition: chromatographic column: the chromatographic column taking octadecylsilane chemically bonded silica as stationary phase, pillar diameter and length are: 30cm × 30cm.Moving phase: A phase: 0.08% aqueous acetic acid (v/v); B phase: acetonitrile, C phase: 0.68% Ammoniom-Acetate, pH2.3, flow velocity: 2000ml/min, determined wavelength: 230nm.Sample size is 60-85g.
Step: after turning salt 20min with 5%B+95%C, with 5%B+95%A ~ 25%B+75%A, linear gradient elution 30min; Collect object peak, triptorelin solution enforcement 10 collected goes to 50ml cillin bottle after vacuum rotary steam is concentrated into about 50-200mg/mL under water temperature 30 DEG C of conditions.Can obtain the triptorelin that purity is greater than 99.75% after lyophilize, other impurity is all less than 0.02%.
Claims (1)
1. the purification process of a triptorelin:
Step 1): with the aqueous solution of the organic solvent of volume ratio content 8%-10%, dissolve triptorelin sample according to concentration 100g/L, described organic solvent is acetonitrile; Take octadecylsilane chemically bonded silica as the chromatographic column of stationary phase, be A phase by the sulfuric acid buffer salt solution adjusting PH with base 2.0-3.0 of the phosphate-buffered salt of volume ratio 0.1%-0.8% or volume ratio 0.2%-0.6%, acetonitrile is that B phase carries out gradient elution purifying, gradient is B%:20%-40%, and described phosphate-buffered salt is phosphoric acid triethylamine, Sodium phosphate dibasic, dipotassium hydrogen phosphate, SODIUM PHOSPHATE, MONOBASIC or potassium primary phosphate buffer salt system; Sulfuric acid buffering salt is ammonium sulfate buffering salt; Described alkali is sodium hydroxide, potassium hydroxide or ammoniacal liquor;
Step 2): turn salt with acetate buffer salts solution, described acetate buffer salts solution is the aqueous solution of acetate buffer salt and the mixing solutions of acetonitrile, in the aqueous solution of described acetate buffer salt, acetate buffer salt is ammonium acetate, and the volume by volume concentration of acetate buffer salt is 0.02-0.1%; The aqueous solution of acetate buffer salt and the volume ratio of acetonitrile are 95:5, and the pH value range of the aqueous solution of described acetate buffer salt is 1.5-4.5;
Be that the chromatographic column of stationary phase carries out gradient elution purifying with octadecylsilane chemically bonded silica, be A phase with aqueous acetic acid afterwards, acetonitrile is that B phase carries out gradient elution, in aqueous acetic acid, the volume by volume concentration of acetic acid is 0.02%-0.65%, gradient is B%:5% ~ 25%, linear gradient elution 30min.
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| CN104761620A (en) * | 2015-01-06 | 2015-07-08 | 苏州天马医药集团天吉生物制药有限公司 | Triptorelin purification preparation method |
| RU2585105C1 (en) * | 2015-03-26 | 2016-05-27 | Индивидуальный предприниматель Михайлов Олег Ростиславович | Method of purifying triptorelin |
| CN106932498B (en) * | 2015-12-29 | 2019-12-03 | 深圳翰宇药业股份有限公司 | A kind of detection method of ganirelix acetate |
| CN111057141B (en) * | 2018-10-17 | 2023-11-14 | 深圳市健元医药科技有限公司 | Tripeptide refining process |
| CN109438561B (en) * | 2018-12-26 | 2020-10-30 | 苏州天马医药集团天吉生物制药有限公司 | Purification method of triptorelin |
| CN114014913B (en) * | 2022-01-10 | 2022-03-29 | 浙江湃肽生物有限公司南京分公司 | Purification method of triptorelin acetate |
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| CN101357936A (en) * | 2007-07-31 | 2009-02-04 | 崔颀 | Method for synthesizing triptorelin from solid phase polypeptide |
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| CN101357936A (en) * | 2007-07-31 | 2009-02-04 | 崔颀 | Method for synthesizing triptorelin from solid phase polypeptide |
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