CN106279368A - A kind of acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 prepares post-processing approach - Google Patents
A kind of acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 prepares post-processing approach Download PDFInfo
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- CN106279368A CN106279368A CN201510254773.7A CN201510254773A CN106279368A CN 106279368 A CN106279368 A CN 106279368A CN 201510254773 A CN201510254773 A CN 201510254773A CN 106279368 A CN106279368 A CN 106279368A
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Landscapes
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to a kind of acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 preparation and post-processing approach thereof, solve the intermediate self aggregation that prior art exists, purity is low, and technique is unstable, the problem being not suitable for large-scale production.The present invention provides the method for preparation injection acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 while providing production acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 lyophilizing crude drug method.Wherein acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude drug is dissolved in water viscosity at 20mg/mL and is less than 3.0mPas, and injection acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 is dissolved in water for injection range of viscosities at 2.0 ~ 15.0mPas at 20mg/mL.
Description
Technical field
The present invention relates to a kind of acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 prepares post-processing approach.
Background technology
U.S. FDA approval Ferring
Pharmaceuticals company degarelix powder pin (Firmagon) lists, and is used for treating advanced prostate cancer.Chemical name: N-acetyl group-3-(2-naphthyl)-D-alanyl-4-chloro-D-phenylalanyl-3-(3-pyridine radicals)-D-alanyl-L seryl-4-[[[(4S)-hexahydro-2; 6-dioxo-4-pyrimidine radicals] carbonyl] amino]-L-phenylalanyl-4-[(amino carbonyl) amino]-D phenylalanyl-L-leucyl-N6-(1-Methylethyl)-L lysyl-L-prolyl-D-aminopropanamide, structure is as follows:
Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(L-Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2。
Wherein Ac is acetyl group; 2Nal is 2-naphthylalanine; 4Cpa is 4-chlorophenylalanine, and 3Pal is 3-pyriylalanine, and Ser is serine; 4Aph is 4-amino phenylalanine; Hor is hydroorotic acid, and Cbm is carbamyl amine, and Leu is leucine; Lys(iPr) being N6-isopropyl lysine, Pro is proline and Ala is alanine.
This strain one gonadotropin releasing hormone (GnRH) acceptor inhibitor class medicine, reversible inhibition hypophysis GnRH receptor reduces gonadotropin releasing hormone and then suppresses the release of testosterone.This product delays growth and the deterioration of carcinoma of prostate by suppressing testosterone vital to carcinoma of prostate continued propagation.The initial stage reducing testosterone concentration with hormone therapy carcinoma of prostate can cause testosterone concentration to increase sharply, this hormone receptor of this initial impulse can temporary promotion tumor growth rather than suppress it, Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 then will not.III phase clinical studies show, this product reduces the effect of testosterone concentration and at least can compare favourably with leuprorelin depot controlled release injection (Lupron Depot), and it is notable fast statistically to reduce testosterone concentration.At the 3rd day for the treatment of, this product group 96% reached gonadal testosterone concentration, and leuprorelin group effect is 0%.14th, this product group 99% reached gonadal testosterone concentration, and leuprorelin group is 18%.In clinical studies, prostate specific antigen (PSA) concentration can be as the 2nd Outcome measure terminal of monitoring.Degarelix is used after 2 weeks, to reduce PSA
64%, after January 85%, after March 95%, in whole 1 year for the treatment of, suppress PSA all the time.
This product dosage strengths: degarelix 80 mg, 120
Mg/ bottle.This dosage strengths is higher in polypeptide kind, easily stores so being badly in need of a kind of intermediate, and product is more stable, it is easy to amplify, and is suitable for the preparation method of large-scale production.
Chinese patent CN102428097 describes the preparation method of a kind of acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude drug and CN102204889 describes the preparation method of a kind of Degarelix acetate lyophilized powder injection, all it is not directed to the integrated gelatin phenomenon of autohemagglutination that Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 easily produces in crude drug and follow-up formulation manufacturing processes, the most the raw material of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 and the viscosity of preparation is not carried out corresponding Detection & Controling.
Chinese patent CN104334182 discloses the manufacture method of a kind of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, concretely comprises the following steps: be purified by liquid phase or Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 that Solid phase peptide synthesis is obtained is to obtain the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 solution with at least 95% purity；Evaporation solvent is to concentrate described Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 solution to obtain the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 assembled；The Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 acetic acid depolymerization that will assemble；The Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 of lyophilizing depolymerization is to provide described Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 drug substance.
In existing Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 preparation process, due to the physicochemical characteristics of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, self aggregation the most in aqueous, and ultimately form gel solution.30% acetic acid depolymerized sample needs more low temperature (<-20 DEG C) just can freeze full pattern product, and lyophilizing cryogenic temperature requires that low and effect is not fine.Therefore in mass commercial production, Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 intermediate deposit and all there is considerable influence in the stability of product quality.The present inventor studies discovery, although some method can be with the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 of depolymerization self aggregation, but lyophilizing difficulty can be increased while adding a large amount of acetic acid and affect effect, and long-time Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 aqueous solution concentration process also easily causes the bad stability of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, so that finished product quality level and yield reduce.To this end, do not affect quality and the yield problem of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 product while how the present inventor's primary study solves Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 self aggregation problem.
The present inventor all cannot effectively prevent Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 aqueous solution self aggregation phenomenon after purification from producing by existing preparation method, i.e. allows to depolymerization also high to freeze-drier requirement and be difficult to ensure that effect.To this end, the preparation method of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 is studied by the present inventor, thus having obtained technical scheme so that acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 is not the most produced from gathering, product is stable, it is easy to lyophilizing, and final products purity is high, is suitable for large-scale production.
Summary of the invention
It is an object of the invention to provide a kind of high efficiency, high-purity and low impurity Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 prepare post-processing approach.The technical issues that need to address of the present invention are: select the preparation method of a kind of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, solve self aggregation phenomenon during (1) preparation method, (2) product purity is the highest, and (3) lyophilizing cryogenic temperature requires height, and (4) technique is unstable.
The technical scheme is that
Use before Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 intermediate revolves steaming, add a small amount of acetic acid every time, and multiple after steaming freezing melting is revolved in employing in batches, then after sample mixing, merge the technique of lyophilizing, can effectively solve Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 self aggregation problem in large-scale production.
Post-processing approach is prepared, it is characterised in that step is as follows for what this present invention provided a kind of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2:
Step 1, is purified by the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 being obtained solid-phase synthesis, thus obtains the purity Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 solution more than 99.0%；
Step 2, takes the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 solution that steaming to be revolved after turning salt processes, and adds 5～the acetic acid of 20%, after stir；
Step 3, carries out concentrated by rotary evaporation under 30～35 DEG C of water bath condition by step 2 gained solution by portions and removes organic modifiers so that the concentration of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 solution reaches 30～50g/l；
Step 4, freezen protective at step 3 gained concentrated solution is placed in-20 DEG C, merge after being placed in the concentrated by rotary evaporation liquid of all freezen protective under 20～30 DEG C of water-baths carrying out multiple melting after all batch step 2 solution concentrated by rotary evaporations are complete；
Step 5, obtains sterling acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 by step 4 gained merging solution through lyophilization.
Preferably operating procedure is as follows:
Step 1, is purified by the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 that solid-phase synthesis is obtained, thus obtains the purity Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 solution more than 99.0%；
Step 2, takes the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 solution that steaming to be revolved after turning salt processes, and adds the acetic acid of 5%, after stir；
Step 3, carries out concentrated by rotary evaporation under 30～35 DEG C of water bath condition by step 2 gained solution by portions and removes organic modifiers so that the concentration of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 solution reaches 50g/l；
Step 4, freezen protective at step 3 gained concentrated solution is placed in-20 DEG C, merge after being placed in the concentrated by rotary evaporation liquid of all freezen protective under 20 DEG C of water-baths carrying out multiple melting after all batch step 2 solution concentrated by rotary evaporations are complete；
Step 5, obtains acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude drug by step 4 gained merging solution through lyophilization.
Beneficial effects of the present invention is further illustrated below by way of experimental data:
Using acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 prepared by Chinese patent CN104334182 method, fine peptide purity has reached 97.50%, and yield is 29%, and the viscosity of acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 is 2.3mPas；
Using acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 prepared by the method for the present invention, fine peptide purity has reached 99.76%, and yield is 53.2%, and the viscosity of acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 is 2.5mPas.
The method of the present invention obtains through screening, and screening process is as follows:
Optimization experiment one:
The ratio adding acetic acid selects: be added without；5%；10%；20%
4 kinds of experiment conditions are proposed for this:
Experiment condition 1: after turning salt, solution is without acetic acid；
Experiment condition 2: after turning salt, solution adds 5% acetic acid；
Experiment condition 3: after turning salt, solution adds 10% acetic acid；
Experiment condition 4: after turning salt, solution adds 20% acetic acid.
The method using experiment condition 1 prepares Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, and during rotation is steamed, acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 solution starts self aggregation, and ultimately forms gel, it is impossible to lyophilizing；
The method using experiment condition 2 prepares Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, and after concentrated by rotary evaporation is complete, lyophilizing obtains fine peptide purity and reached 99.70%, and yield is 52%, and the viscosity of acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 is 2.4mPas；
The method using experiment condition 3 prepares Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, and after concentrated by rotary evaporation is complete, lyophilizing obtains fine peptide purity and reached 99.70%, and yield is 50%, and the viscosity of acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 is 2.4mPas；
The method using experiment condition 4 prepares Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, and after concentrated by rotary evaporation is complete, in lyophilizing, acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 solution is difficult to crystallization and freezes reality, it is more difficult to lyophilizing, there is high requirement for freeze-drier.
It is to turn after salt the 5% ~ 10% of acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 liquor capacity according to the amount adding acetic acid before above experimental phenomena, preferably concentrated by rotary evaporation.
Optimization experiment two:
Turning saline solution rotation and steam temperature, before time and acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 lyophilizing, concentration selects:
Temperature: 30 DEG C；35℃
Time: 2h；4h
Concentration: 30g/l；50g/l
4 kinds of experiment conditions are proposed for this:
Experiment condition 5: after turning salt, solution adds 5% acetic acid, steams 2 hours at 30 DEG C of backspins, and the concentration making acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 solution is 30g/L；
Experiment condition 6: after turning salt, solution adds 5% acetic acid, steams 4 hours at 30 DEG C of backspins, and the concentration making acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 solution is 50g/L；
Experiment condition 7: after turning salt, solution adds 5% acetic acid, steams 2 hours at 35 DEG C of backspins, and the concentration making acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 solution is 30g/L；
Experiment condition 8: after turning salt, solution adds 5% acetic acid, steams 4 hours at 35 DEG C of backspins, and the concentration making acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 solution is 50g/L.
The method using experiment condition 5 prepares Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, and after concentrated by rotary evaporation is complete, lyophilizing obtains fine peptide purity and reached 99.70%, and yield is 52%, and the viscosity of acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 is 2.4mPas；
The method using experiment condition 6 prepares Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, and after concentrated by rotary evaporation is complete, lyophilizing obtains fine peptide purity and reached 99.68%, and yield is 51%, and the viscosity of acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 is 2.7mPas；
The method using experiment condition 7 prepares Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, and after concentrated by rotary evaporation is complete, lyophilizing obtains fine peptide purity and reached 99.71%, and yield is 52%, and the viscosity of acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 is 2.1mPas；
The method using experiment condition 8 prepares Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, and after concentrated by rotary evaporation is complete, lyophilizing obtains fine peptide purity and reached 99.65%, and yield is 51%, and the viscosity of acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 is 2.4mPas.
According to above experimental phenomena, in conjunction with actual process demand, it is preferably 30～35 DEG C in temperature, revolves steaming 2 ~ 4h so that the concentration of acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 is 50g/L.
Optimization experiment three:
Melt temperature again, selection of time:
Temperature: 20 DEG C；30℃
Time: 2h；4h
2 kinds of experiment conditions are proposed for this:
Experiment condition 9: be placed in the water-bath of 20 DEG C by solution after the concentration of pre-freeze in batches, after melting 4h again, merges solution, carries out lyophilizing；
Experiment condition 10: be placed in the water-bath of 30 DEG C by solution after the concentration of pre-freeze in batches, after melting 2h again, merges solution, carries out lyophilizing；
The method using experiment condition 9 prepares Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, and lyophilizing obtains fine peptide purity and reached 99.67%, and yield is 51%, and the viscosity of acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 is 2.3mPas；
The method using experiment condition 10 prepares Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2, and lyophilizing obtains fine peptide purity and reached 99.66%, and yield is 51%, and the viscosity of acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 is 2.3mPas.
According to above experimental phenomena, melting temperature the most again is 20 ~ 30 DEG C, and the time of melting is 2 ~ 4h again.
The invention has the beneficial effects as follows: the acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 providing a kind of high-purity (99.76%), high yield (more than 50%) and finished product viscosity controlled prepares post-processing approach.
Accompanying drawing explanation
After the preparation of Fig. 1 patent CN104334182 method, acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 HPLC detects collection of illustrative plates；
Acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 HPLC detection collection of illustrative plates (1mmol) after the preparation of Fig. 2 the inventive method；
Acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 HPLC detection collection of illustrative plates (300mmol) after the preparation of Fig. 3 the inventive method.
Detailed description of the invention
Embodiment 1:
(1) take 2.0g(1.0mmol) Rink amide MBHA
(substitution value=0.50mmol/g) resin joins in reaction column, adds the swelling 30min of 15mlDCM, deprotection: add the V of 15mlPiperidines:VDMF =20:80 mixed solution, pumps after stirring reaction 5min, again adds above-mentioned mixed solution, pumps after stirring reaction 10min, so carries out 2 deprotections, then wash 6 times with DMF, each 15mL.
(2) coupling Fmoc-D-Ala-OH: 0.94g (3.0mmol) Fmoc-D-Ala-OH and 0.51g (3.0mmol) 6-Cl-HOBt solvent DMF is dissolved; ice bath chilling temperature is 0～10 DEG C; add 465 μ l (3.0mmol) DIC; join in reaction column after activating 5 minutes and react with the carrier having taken off protection; react 2 hours at 25～35 DEG C; extracting liquid, DMF washs 3 times, each 15mL.
(3) coupling is repeated: repeated removal Fmoc protection group and connect the operation of reactive polypeptide step; according to Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 aminoacid sequence, coupling successively: Fmoc-Pro-OH, Fmoc-Ilys (Boc)-OH, Fmoc-Leu-OH, Fmoc-D-4Aph (Dde)-OH, Fmoc-4Aph (Teoc)-OH, Fmoc-Ser (tBu)-OH, Fmoc-D-3Pal-OH, Fmoc-D-Phe (4Cl)-OH, Fmoc-D-2Nal-OH.
(4) acetic anhydride end-blocking: first deprotection base Fmoc, adds the V of 15mlPiperidines:VDMF =20:80 mixed solution, pumps after stirring reaction 5min, again adds above-mentioned mixed solution, pumps after stirring reaction 10min, so carries out 2 deprotections, then wash 6 times with DMF, each 15mL.Taking 1ml acetic anhydride, 1ml pyridine and 1mlDMF, ice bath cools down, and is slowly added in carrier, reacts 2 hours, extract liquid at 25～35 DEG C, washs 6 times with DMF, each 15mL.
(5) Dde protection group is gone: add the V of 15mlHydrazine hydrate:VDMF =2:98 mixed solution, pumps after stirring reaction 5min, again adds above-mentioned mixed solution, pumps after stirring reaction 10min, so carries out 2 deprotections, then wash 6 times with DMF, each 15mL.
(6) coupling Cbm: add 270 μ l(2mmol) DMF solution of trimethyl silicane based isocyanate, ice bath cools down, is slowly added in carrier, low-temp reaction 2 hours, reacts 24 hours, extract liquid at 25～35 DEG C, washs 3 times with DMF, each 15mL.
(7) Teoc protection group is gone: add 1.04g(4mmol) the DMF mixed solution of tetrabutyl ammonium fluoride, reacts 2 hours at 25～35 DEG C, extracts liquid, wash 3 times with DMF, each 15mL.
(8) coupling L-Hor: by 0.73g (3.0mmol) L-Hor-OH and 0.51g
(3.0mmol) 6-Cl-HOBt solvent DMF is dissolved; ice bath chilling temperature is 0～10 DEG C; add 465 μ l (3.0mmol) DIC; join in reaction column after activating 5 minutes and react with the carrier having taken off protection; react 2 hours at 25-35 DEG C; extracting liquid, DMF washs 3 times, each 15mL.
(9) cracking: by above-mentioned peptide resin 15ml
VTIS:VPhSMe:VTFA=5:5:90 lysate cracks, pyrolysis time: 2 hours, filters, and rotation is steamed, and rotation is steamed in the ether that mother solution adds 90ml, centrifuge washing 5 times after sedimentation, each 45mL.Prepare Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 thick peptide 1.72g.
(10) purification for the first time: by 5% acetonitrile balance system 5min after chromatographic column being rinsed well with 50% acetonitrile, carrying out HPLC purification with the applied sample amount of 1-3g/ time, phase: the A that flows is 0.1%TFA aqueous solution；Mobile phase B is acetonitrile, and Gradient program is: original state Mobile phase B is 10%, keeps 5 minutes, then in 60 minutes, Mobile phase B ratio is increased to 50%, and flow velocity is: 20 mL/min.Detecting at 226nm wavelength, according to chromatographic peak Fractional Collections fraction, and detected by the fraction analytical liquid chromatograph of collection, purity >=99.0% and single miscellaneous≤0.2% fraction are the qualified fraction of the first step.Remaining is slop cut, and slop cut moderate purity carries out more than 80% part reclaiming purification again, and purity is given up less than 80%.
(11) purification for the second time: by 5% acetonitrile balance system 5min after chromatographic column is rinsed well with 50% acetonitrile, HPLC purification is carried out with the applied sample amount of 1-3g/ time, flowing phase: A is 0.3% aqueous acetic acid, Mobile phase B is acetonitrile, Gradient program is: original state Mobile phase B is 10%, keeping 5 minutes, then in 60 minutes, Mobile phase B ratio is increased to 50%, flow velocity is: 20 mL/min.Detecting at 226nm wavelength, according to chromatographic peak Fractional Collections fraction, and detected by the fraction analytical liquid chromatograph of collection, purity >=99.5% and single miscellaneous≤0.1% fraction are the qualified fraction of second step.Remaining is slop cut, and slop cut moderate purity carries out more than 80% part reclaiming purification again, and purity is given up less than 80%.
(12) condition of salt is turned: flow phase: A phase: 50
The aqueous solution of mmol/L ammonium acetate: acetonitrile=95:5；B phase: 0.1 % vinegar aqueous acid: acetonitrile=95:5；C phase: 0.1 % vinegar aqueous acid: acetonitrile=50:50, Gradient program is: with mobile phase A Gradient elution 20 minutes, be converted into Mobile phase B Gradient elution 10 minutes, is converted into flowing phase C Gradient elution 25 minutes.Carry out turning salt with the applied sample amount of 1-3g/ time, require to carry out eluting according to above-mentioned gradient, detect at 226nm wavelength, the sample collection having ultraviolet absorption value is merged.
(13) concentration and lyophilizing: merge, after turning salt, the acetic acid that fraction adds 5%, distinguish concentrated by rotary evaporation 1h in two batches, it is about 50mg/ml to Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 concentration, every batch of rotation all puts into refrigerator freezen protective at-20 DEG C after having steamed, before treating lyophilizing, it is placed in two batches of concentrated solutions merging after 2h is melted in 20 DEG C of water-baths again, after lyophilizing, obtains acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 fine peptide: 0.90g, purity: 99.76%, yield: 53.2%.The viscosity of acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 is 2.5mPas.
Embodiment 2:
(1) take 600.0g(300.0mmol) Rink amide MBHA
(substitution value=0.50mmol/g) resin joins in reaction column, adds the swelling 30min of 4000mlDCM, deprotection: add the V of 4000mlPiperidines:VDMF =20:80 mixed solution, pumps after stirring reaction 5min, again adds above-mentioned mixed solution, pumps after stirring reaction 10min, so carries out 2 deprotections, then wash 6 times with DMF, each 4000mL.
(2) coupling Fmoc-D-Ala-OH: 283.6g (900.0mmol) Fmoc-D-Ala-OH and 153.6g (900.0mmol) 6-Cl-HOBt solvent DMF being dissolved, ice bath chilling temperature is 0～10 DEG C, adds 144ml
(900.0mmol) DIC, joins in reaction column after activating 5 minutes and reacts with the carrier having taken off protection, reacts 2 hours at 25-35 DEG C, extracts liquid, and DMF washs 3 times, each 4000mL.
(3) coupling is repeated: repeated removal Fmoc protection group and connect the operation of reactive polypeptide step; according to Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 aminoacid sequence, coupling successively: Fmoc-Pro-OH, Fmoc-Ilys (Boc)-OH, Fmoc-Leu-OH, Fmoc-D-4Aph (Dde)-OH, Fmoc-4Aph (Teoc)-OH, Fmoc-Ser (tBu)-OH, Fmoc-D-3Pal-OH, Fmoc-D-Phe (4Cl)-OH, Fmoc-D-2Nal-OH.
(4) acetic anhydride end-blocking: first deprotection base Fmoc, adds the V of 4000mlPiperidines:VDMF =20:80 mixed solution, pumps after stirring reaction 5min, again adds above-mentioned mixed solution, pumps after stirring reaction 10min, so carries out 2 deprotections, then wash 6 times with DMF, each 4000mL.Taking 300ml acetic anhydride, 300ml pyridine and 300mlDMF, ice bath cools down, and is slowly added in carrier, reacts 2 hours, extract liquid at 25-35 DEG C, washs 6 times with DMF, each 4000mL.
(5) Dde protection group is gone: add the V of 4000mlHydrazine hydrate:VDMF =2:98 mixed solution, pumps after stirring reaction 5min, again adds above-mentioned mixed solution, pumps after stirring reaction 10min, so carries out 2 deprotections, then wash 6 times with DMF, each 4000mL.
(6) DMF solution of trimethyl silicane based isocyanate, ice bath cools down, is slowly added in carrier, low-temp reaction 2 hours, reacts 24 hours, extract liquid at 25～35 DEG C, washs 3 times with DMF, each 4000mL coupling Cbm: addition 84ml(600mmol).
(7) Teoc protection group is gone: add 312g(1200mmol) the DMF mixed solution of tetrabutyl ammonium fluoride, reacts 2 hours at 25～35 DEG C, extracts liquid, wash 3 times with DMF, each 4000mL.
(8) coupling L-Hor: by 220.2g (900.0mmol) L-Hor-OH and 153.6g
(900.0mmol) 6-Cl-HOBt solvent DMF is dissolved, and ice bath chilling temperature is 0～10 DEG C, adds 144ml
(900.0mmol) DIC, joins in reaction column after activating 5 minutes and reacts with the carrier having taken off protection, reacts 2 hours at 25～35 DEG C, extracts liquid, and DMF washs 3 times, each 4000mL.
(9) cracking: by above-mentioned peptide resin 4500ml
VTIS:VPhSMe:VTFA=5:5:90 lysate cracks, pyrolysis time: 2 hours, filters, and rotation is steamed, and rotation is steamed in the ether that mother solution adds 9000ml, centrifuge washing 5 times after sedimentation, each 13500mL.Prepare Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 thick peptide 534g.
(10) purification for the first time: by 5% acetonitrile balance system 5min after chromatographic column being rinsed well with 50% acetonitrile, carrying out HPLC purification with the applied sample amount of 20-35g/ time, phase: the A that flows is 0.1%TFA aqueous solution；Mobile phase B is acetonitrile, and Gradient program is: original state Mobile phase B is 10%, keeps 5 minutes, then in 60 minutes, Mobile phase B ratio is increased to 50%;Flow velocity is: 80 mL/min.Detecting at 226nm wavelength, according to chromatographic peak Fractional Collections fraction, and detected by the fraction analytical liquid chromatograph of collection, purity >=99.0% and single miscellaneous≤0.2% fraction are the qualified fraction of the first step.Remaining is slop cut, and slop cut moderate purity carries out more than 80% part reclaiming purification again, and purity is given up less than 80%.
(11) purification for the second time: by 5% acetonitrile balance system 5min after chromatographic column being rinsed well with 50% acetonitrile, carrying out HPLC purification with the applied sample amount of 20-35g/ time, phase: the A that flows is 0.1% aqueous acetic acid；Mobile phase B is acetonitrile, and Gradient program is: original state Mobile phase B is 10%, keeps 5 minutes, then in 60 minutes, Mobile phase B ratio is increased to 50%, and flow velocity is: 80 mL/min.Detecting at 226nm wavelength, according to chromatographic peak Fractional Collections fraction, and detected by the fraction analytical liquid chromatograph of collection, purity >=99.5% and single miscellaneous≤0.1% fraction are the qualified fraction of second step.Remaining is slop cut, and slop cut moderate purity carries out more than 80% part reclaiming purification again, and purity is given up less than 80%.
(12) condition of salt is turned: flow phase: A phase: 50
The aqueous solution of mmol/L ammonium acetate: acetonitrile=95:5；B phase: 0.1 % vinegar aqueous acid: acetonitrile=95:5；C phase: 0.1 % vinegar aqueous acid: acetonitrile=50:50, Gradient program is: with mobile phase A Gradient elution 25 minutes, be converted into Mobile phase B Gradient elution 15 minutes, is converted into flowing phase C Gradient elution 40 minutes.Carry out turning salt with the applied sample amount of 20-35g/ time, require to carry out eluting according to above-mentioned gradient, detect at 226nm wavelength, the sample collection having ultraviolet absorption value is merged.
(13) concentration and lyophilizing: merge, after turning salt, the acetic acid that fraction adds 5%, divide ten batches of concentrated by rotary evaporations 4h respectively, it is about 50g/l to Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 concentration, every batch of rotation all puts into refrigerator freezen protective at-20 DEG C after having steamed, before treating lyophilizing, it is placed in ten batches of concentrated solutions merging after 4h is melted in 20 DEG C of water-baths again, after lyophilizing, obtains acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 fine peptide: 286.1g, purity: 99.89%, yield: 56.4%.The viscosity of acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 is 2.1mPas.
Embodiment 3:
(1) in rustless steel solution preparation tank, add the water for injection of 3.528L,
(2) add the mannitol of 220.5g, be kept stirring for making material dissolution,
(3) add the acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude drug of 88.2g, be kept stirring for making material dissolution,
(4) 0.882L water for injection is added to full dose.Respectively through 0.45 μm and 0.22 μm filtering with microporous membrane, subpackage.Lyophilization, jumps a queue.
Embodiment 4:
(1) in rustless steel solution preparation tank, add the water for injection of 5.120L,
(2) add the mannitol of 160.0g, be kept stirring for making material dissolution,
(3) add the acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude drug of 128.0g, be kept stirring for making material dissolution,
(4) water for injection of 1.280L is added to full dose.Respectively through 0.45 μm and 0.22 μm filtering with microporous membrane, subpackage.Lyophilization, jumps a queue.
Embodiment 5:
Using BROOKFIELD DV-II type rotational viscometer, the viscosity for acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 and injection Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 measures, and method is as follows:
(1) configuration of test sample: take acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 dissolved in purified water respectively and be configured to the uniform solution of 20mg/ml or injection Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 water for injection is configured to the uniform solution of 20mg/ml.
(2) container filling test sample is immersed 20 DEG C of water-baths.
(3) rotor is immersed in sample at the grooved score in rotor shaft.
(4) setting speed value is 50rpm, opens stirring detection test sample viscosity, after instrument readings is stable, records data.
Wherein in embodiment 2, acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 k value is: 2.1mPas, and in embodiment 3, injection Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 k value is 9.8mPas, and in embodiment 4, injection Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 k value is 10.4mPas.
Claims (1)
1. an acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 prepare post-processing approach; can effectively prevent the self aggregation of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 during large-scale production and make acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 viscosity be dissolved in water less than 3.0mPas(20mg/mL); injection acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 is dissolved in water for injection range of viscosities at 2.0 ~ 15.0mPas at 20mg/mL; it is characterized in that, said method comprising the steps of:
Step 1, is purified by the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 crude product that solid-phase synthesis is obtained, thus obtains the purity acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 solution more than 99.0%；
Step 2, adds 5 ~ 20% acetic acid of acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 overall solution volume in the Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 solution of step one gained, be preferably added to acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 overall solution volume 5 ~ 10% acetic acid；
Step 3, carries out concentrated by rotary evaporation 2 ~ 4h under 30～35 DEG C of water bath condition by step 2 gained solution by portions and removes organic modifiers so that the concentration of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 solution reaches 30 ~ 50g/l, preferably 50g/l；
Step 4, freezen protective at step 3 gained concentrated solution is placed in-20 DEG C, merge after being placed in the concentrated by rotary evaporation liquid of all freezen protective under 20 ~ 30 DEG C of water-baths melting 2 ~ 4h again after all batch step 2 solution concentrated by rotary evaporations are complete；
Step 5, within a specified time obtains sterling acetic acid Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 through lyophilization by step 4 gained merging solution.
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| CN112423776A (en) * | 2018-07-05 | 2021-02-26 | 安迪威有限公司 | Freeze-drying method and teverelix-TFA freeze-dried substance obtained by same |
| CN112423776B (en) * | 2018-07-05 | 2024-05-31 | 安迪威有限公司 | Freeze-drying method and tivalia-TFA freeze-dried product obtained by method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112423776A (en) * | 2018-07-05 | 2021-02-26 | 安迪威有限公司 | Freeze-drying method and teverelix-TFA freeze-dried substance obtained by same |
| CN112423776B (en) * | 2018-07-05 | 2024-05-31 | 安迪威有限公司 | Freeze-drying method and tivalia-TFA freeze-dried product obtained by method |
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