CN104447942A - Method for purifying polypeptide containing amino acid susceptible to oxidation - Google Patents
Method for purifying polypeptide containing amino acid susceptible to oxidation Download PDFInfo
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Abstract
The present invention relates to the field of peptide purification, and in particular to a method of purifying polypeptides containing amino acids susceptible to oxidation. The purification method comprises the steps of: acquiring crude peptide, conducting pretreatment to obtain a first crude peptide solution, and conducting first adjustment to adjust the pH value to 6-8, so as to obtain a second crude peptide solution; mixing the second crude peptide solution with a reducing agent, conducting second adjustment to adjust the pH value to 4.0-7.0, and carrying out filtration and purification. The reducing agent is selected from the group including organic acid consisting of thiol, acid or inorganic salt containing sulfide ion or sulfhydryl ion, inorganic salt containing reductive metal ions, inorganic salt containing sulfite radical or bisulfite radical, and sulfur dioxide; and the amino acid susceptible to oxidation is amino acid susceptible to oxidation reaction. The purification method provided by the invention can effectively remove the color in the polypeptides containing amino acids susceptible to oxidation, and improve the drug quality and yield of fine peptide, has the advantages of simple operation and low cost, and is suitable for industrialized production.
Description
Technical field
The present invention relates to field of polypeptide purification, particularly a kind of purification process containing easy oxidized amino acid whose polypeptide.
Background technology
Mammary cancer is the malignant tumour occurring in breast epithelial tissue.Whole world breast cancer incidence starts always in rising trend from late 1970s.In mammary cancer, 99% occurs in women, and the male sex only accounts for 1%.Pathogenesis of breast carcinoma data presentation in 2009 according to National Cancer Center and prevention and control of diseases office of the Ministry of Health are announced for 2012: national tumour registers the 1st that regional breast cancer incidence occupies female malignant, the female mammary gland cancer morbidity whole nation adds up to and is about 42.55/10 ten thousand, city is about 51.91/10 ten thousand, and rural area is about 23.12/10 ten thousand.Current mammary cancer has become the able-bodied kinds of tumor of threat women.
The medicine of common treatment mammary cancer comprises medroxyprogesterone acetate tablets, methyltestosterone tablets, xeloda, premarin, goserelin etc.Wherein, goserelin is a kind of decapeptide compound of synthesis, belongs to the analogue of luteinizing hormone-releasing hormone, and life-time service can suppress the secretion of the prolan B of hypophysis, thus cause the decline of women's serum estradiol, reach the effect for the treatment of mammary cancer.
Have tryptophan residue in goserelin peptide sequence, tryptophane is very easily oxidized and produce obviously color, is generally redness or red-purple.When adopting solid-phase synthesis, the liquid phase synthesizing method synthesis thick peptide of goserelin, because building-up process is comparatively complicated, can use multiple condensing agent and catalyzer, the tryptophan residue therefore in peptide sequence is easily oxidized and make product with red or red-purple, affects the quality of goserelin medicine.Also oxidation in various degree can be there is and make product with color in other the easy oxidized amino acid whose polypeptide drugs such as tryptophane that contain when synthesizing, therefore contain in easy oxidized amino acid whose polypeptide drug process in synthesis, need to remove color to ensure the quality of medicine.General employing adopts HPLC method of purification and active carbon adsorption oxidation impurities to be removed.Utilize HPLC method of purification remove color time, due to oxidation do not make whole peptide sequence change, its character and oxidized front difference very little, therefore need repeatedly HPLC purifying by colour removal, to cause the yield of product lower; When utilizing active carbon adsorption to remove color, because the adsorption of gac does not possess selectivity, while absorption color, also can adsorbed target product, just be difficult to disintegrate down once the upper target product of absorption, cause the yield of target product on the low side, this situation is particularly evident for the polypeptide of molecular weight below 2000.Therefore, provide a kind of color can effectively removed in polypeptide products, can ensure that again the more much higher peptide purification method of the yield of product has important practical significance.
Summary of the invention
In view of this, the invention provides a kind of purification process containing easy oxidized amino acid whose polypeptide.The method by pH value to 6.0 ~ 8.0 of the thick peptide solution of adjustment, then adds reductive agent, thus can remove the color in thick peptide solution, and improves the yield of smart peptide.
In order to realize foregoing invention object, the invention provides following technical scheme:
The invention provides a kind of purification process containing easy oxidized amino acid whose polypeptide, comprise the steps:
Obtain thick peptide, through pre-treatment, obtain the first thick peptide solution, through the first adjust ph to 6.0 ~ 8.0, obtain the second thick peptide solution;
Get the second thick peptide solution to mix with reductive agent, through the second adjust ph to 4.0 ~ 7.0.
Reductive agent is selected from the organic acid containing sulfydryl, containing sulfonium ion or the hydrionic acid of sulphur or inorganic salt, and the inorganic salt containing reducing metal ion, the inorganic salt containing inferior sulfate radical or bisulfite, or sulfurous gas;
Easy oxidized amino acid is the amino acid that oxidizing reaction easily occurs.
Polypeptide is as medicine, there is the plurality of advantages such as physiologically active is strong, immunogenicity is low, curative effect is high, but the intrinsic feature of polypeptides matter self is as lower in Oral availability, enzyme liberating is high and the transformation period is extremely short etc., makes its Application and Development as medicine be subject to many limitations.And cause polypeptide drugs instability major reason to be exactly very easily oxidized containing easy oxidized amino acid whose polypeptide.Polypeptide can be caused with color after oxidized in building-up process containing easy oxidized amino acid whose polypeptide, affect the quality of polypeptide products, therefore need to remove oxidized polypeptide.The invention provides a kind of method utilizing the reducing property of reductive agent to remove oxidized polypeptide, and provide the kind of reductive agent, additionally provide pH value range suitable when oxidized polypeptide and reductive agent react, purification process provided by the invention can avoid polypeptide by excessive reduction, the structure of polypeptide can not be destroyed, maintain the original activity of polypeptide.In order to avoid polypeptide is by excessive reduction, the structure of polypeptide can not be destroyed, keep the original active function of polypeptide, as preferably, reductive agent is selected from the organic acid containing sulfydryl, containing sulfonium ion or the hydrionic acid of sulphur or inorganic salt, and the inorganic salt containing reducing metal ion, inorganic salt containing inferior sulfate radical or bisulfite, or sulfurous gas.
Different types of amino acid stability has very big difference, and some amino acid contains easily oxidized structure, under natural storage condition, easily oxidizing reaction occurs, and these easily oxidized amino acid comprise tryptophane, tyrosine, halfcystine, methionine(Met) etc.
In embodiments more provided by the invention; containing the polypeptide that easy oxidized amino acid whose polypeptide is containing tryptophane; but be not limited to the polypeptide containing tryptophane containing easy oxidized amino acid whose polypeptide; as long as what those skilled in the art approved contains easy oxidized amino acid whose polypeptide all within protection scope of the present invention, the present invention does not limit at this.
In embodiments more provided by the invention, be goserelin containing easy oxidized amino acid whose polypeptide.
In embodiments more provided by the invention; organic acid containing sulfydryl is halfcystine or reduced glutathion; but those skilled in the art think that the feasible organic acid containing sulfydryl is all within protection scope of the present invention; kinds of organic acids containing sulfydryl is not limited thereto, and the present invention does not limit at this.
In embodiments more provided by the invention; be hydrosulphuric acid containing sulfonium ion or the hydrionic acid of sulphur; but those skilled in the art think feasible containing sulfonium ion or the hydrionic acid of sulphur all within protection scope of the present invention; be not limited thereto containing sulfonium ion or the hydrionic sour kind of sulphur, the present invention does not limit at this.
In embodiments more provided by the invention; inorganic salt containing sulfonium ion are sodium sulphite; but those skilled in the art think that the feasible inorganic salt containing sulfonium ion are all within protection scope of the present invention, and the Inorganic Salts containing sulfonium ion is not limited thereto, and the present invention does not limit at this.
In embodiments more provided by the invention; be Sodium sulfhydrate containing the hydrionic inorganic salt of sulphur; but those skilled in the art think feasible containing the hydrionic inorganic salt of sulphur all within protection scope of the present invention; be not limited thereto containing the hydrionic Inorganic Salts of sulphur, the present invention does not limit at this.
In embodiments more provided by the invention; inorganic salt containing reducing metal ion are the inorganic salt containing ferrous ion or the inorganic salt containing cuprous ion; but those skilled in the art think that the feasible inorganic salt containing reducing metal ion are all within protection scope of the present invention; Inorganic Salts containing reducing metal ion is not limited thereto, and the present invention does not limit at this.
In embodiments more provided by the invention; inorganic salt containing ferrous ion are ferrous sulfate or iron protochloride; but those skilled in the art think that the feasible inorganic salt containing ferrous ion are all within protection scope of the present invention; Inorganic Salts containing ferrous ion is not limited thereto, and the present invention does not limit at this.
In embodiments more provided by the invention; inorganic salt containing cuprous ion are cuprous sulfate or cuprous chloride; but those skilled in the art think that the feasible inorganic salt containing cuprous ion are all within protection scope of the present invention; Inorganic Salts containing cuprous ion is not limited thereto, and the present invention does not limit at this.
In embodiments more provided by the invention; inorganic salt containing inferior sulfate radical are S-WAT or potassium sulfite; but those skilled in the art think that the feasible inorganic salt containing inferior sulfate radical are all within protection scope of the present invention; Inorganic Salts containing inferior sulfate radical is not limited thereto, and the present invention does not limit at this.
In embodiments more provided by the invention; inorganic salt containing bisulfite are sodium bisulfite or Potassium hydrogen sulfite; but those skilled in the art think that the feasible inorganic salt containing bisulfite are all within protection scope of the present invention; Inorganic Salts containing bisulfite is not limited thereto, and the present invention does not limit at this.
In order to ensure that oxidized polypeptide is fully removed, make the color of polypeptide disappear or shoal, improve the quality of polypeptide products, in embodiments more provided by the invention, the mass ratio of reductive agent and thick peptide is (5 ~ 30): 100.
As preferably, the mass ratio of reductive agent and thick peptide is 1:10.
In embodiments more provided by the invention, reductive agent need be mixed with reductant solution and use, and as preferably, the mass body volume concentrations of reductant solution is 10g/L.
In order to ensure that reductive agent mixes with the second thick peptide solution, oxidized polypeptide is fully reduced, in embodiments more provided by the invention, the time of mixing is 2 ~ 5min.
In embodiments more provided by the invention, the pH value of the first thick peptide solution is less than 6.0, in order to ensure that reductive agent can be good at playing reductive action, needs pH value to 6.0 ~ 8.0 of the thick peptide solution of adjustment first, as preferably, the reagent that the first adjust ph adopts is alkaline solution.
As preferably, the alkali in the alkaline solution that the first adjust ph adopts is alkali soluble in water.
In embodiments more provided by the invention, the alkali in the alkaline solution that the first adjust ph adopts is ammoniacal liquor, sodium hydroxide or potassium hydroxide.But those skilled in the art think that feasible alkali soluble in water is all within protection scope of the present invention, and the kind of alkali soluble in water is not limited thereto, and the present invention does not limit at this.
In embodiments more provided by the invention, the mass percentage concentration of alkaline solution is 5% ~ 40%.
In embodiments more provided by the invention, the pre-treatment in purification process is by thick peptide dissolving, dilution.
In embodiments more provided by the invention, in pre-treatment by thick peptide dissolving, dilution concrete operations be: acetic acid, acetonitrile and water are mixed, obtains mixed solvent; Thick peptide is dissolved in mixed solvent, then adds water and dilute, obtain the first thick peptide solution.
In embodiments more provided by the invention, by the mixed solvent of acquisition after the mixing of acetic acid, acetonitrile and water, the volume ratio of acetic acid, acetonitrile and water is (6 ~ 12): (1 ~ 4): (4 ~ 13).
After thick peptide being dissolved in mixed solvent in pre-treatment, the mass body volume concentrations of thick peptide is preferably 20 ~ 50g/L.
In embodiments more provided by the invention, after thick peptide being dissolved in mixed solvent, the mass body volume concentrations of thick peptide is 20 ~ 25g/L.
In the purification process of polypeptide, thick peptide obtains the first thick peptide solution through dissolving, after dilution, and as preferably, the mass body volume concentrations of the first thick peptide solution is 2 ~ 45g/L.
In embodiments more provided by the invention, the mass body volume concentrations of the first thick peptide solution is 5 ~ 14.3g/L.
In embodiments more provided by the invention, the purifying after filter operation specifically adopts HPLC system purifying, and mobile phase A is the 0.1%TFA aqueous solution, and Mobile phase B is acetonitrile, according to B% be 18% ~ 38%, the gradient elution Fractional Collections sample of 40min.
In other embodiments provided by the invention, the purifying after filter operation specifically adopts HPLC system purifying, and mobile phase A is the 0.1%TFA aqueous solution, and Mobile phase B is acetonitrile, according to B% be 25% ~ 45%, the gradient elution Fractional Collections sample of 40min.
The invention provides a kind of purification process containing easy oxidized amino acid whose polypeptide, this purification process comprises: obtain thick peptide, through pre-treatment, obtains the first thick peptide solution, through the first adjust ph to 6.0 ~ 8.0, obtains the second thick peptide solution; Get the second thick peptide solution to mix with reductive agent, through the second adjust ph to 4.0 ~ 7.0, after filtration, purifying, to obtain final product; Reductive agent is selected from the organic acid containing sulfydryl, containing sulfonium ion or the hydrionic acid of sulphur or inorganic salt, and the inorganic salt containing reducing metal ion, the inorganic salt containing inferior sulfate radical or bisulfite, or sulfurous gas; Easy oxidized amino acid is the amino acid that oxidizing reaction easily occurs.Utilize purification process provided by the invention can effectively remove containing the color in easy oxidized amino acid whose polypeptide, improve the quality of medicine; The smart peptide yield utilizing method provided by the invention to obtain can reach 90.9%, and the smart peptide yield that the method utilizing charcoal absorption to remove color obtains only has 43.1%, the smart peptide yield that the method utilizing HPLC purifying to remove color obtains only has 49.8%, shows that purification process provided by the invention substantially increases the yield of smart peptide.As can be seen here, purification process provided by the invention can effectively be removed containing the color in easy oxidized amino acid whose polypeptide, and improve the quality of medicine, and improve the yield of smart peptide, simple to operate, cost is low, is suitable for suitability for industrialized production.
Accompanying drawing explanation
Fig. 1 shows the mass spectrum of the goserelin that embodiment 1 provides.
Embodiment
The invention discloses a kind of purification process containing easy oxidized amino acid whose polypeptide, those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope methods and applications as herein described are changed or suitably change with combination, realize and apply the technology of the present invention.
In purification process containing easy oxidized amino acid whose polypeptide provided by the invention, agents useful for same or raw material all can be buied by market.
Below in conjunction with embodiment, set forth the present invention further:
The preparation of the thick peptide of embodiment 1 goserelin
Be the Sieber Resin of 0.7mmol/g by 10g substitution degree, join in solid state reaction post, after adding DCM swellable resins 30min, remove Fmoc protection for twice with 20% piperidines/DMF solution, the time is respectively 10min and 15min.DMF washs six times, by 12g N, N-bis-succinimdyl carbonate 20mL DMF dissolve, add 0.51g DMAP ice-water bath pre-activate 5min, add in solid phase reactor, room temperature reaction 2.5h.Reaction end detects with ninhydrin method and is as the criterion.Reaction terminates rear DMF and washs 6 times, adds 25mL DMF as solvent, slowly adds 14.1mL hydrazine hydrate, room temperature reaction 3h, and after reaction terminates, DMF washs 6 times.
1.15g Fmoc-Pro-OH, 4.86g HOAt is dissolved in DMF in ice bath situation, adds in above-mentioned resin after molecular balance 10min, add 5.6mL DIC, room temperature reaction 2h.After DMF washs 3 times, DCM washes 3 times, and shrink 3 times with methyl alcohol, the time is respectively 3min, 5min, 8min, obtains 11.9g Fmoc-Pro-Azagly-Sieber Resin, and detection substitution degree is 0.495mmol/g.
Take the Fmoc-Pro-Azagly-Sieber Resin(10mmol that 20.2g substitution degree is 0.495mmol/g) add in reactor, with the swelling 0.5h of DCM, then use 20% piperidines/DMF twice to remove Fmoc protection, the time is respectively 10min and 15min.Fmoc-Arg (NO is connected after washing
2)-OH.By 12.9g Fmoc-Arg (NO
2)-OH, 4.9g HOBt, 6.1mL DIC is dissolved in DCM and (can adds a small amount of DMF hydrotropy), after ice-water bath activation 5min, adds in solid phase reactor, room temperature reaction 2h.Reaction end detects with ninhydrin method and is as the criterion.According to above-mentioned connection Fmoc-Arg (NO
2) method coupling Fmoc-Leu-OH, Fmoc-D-Ser (tBu)-OH, Fmoc-Tyr (Bzl)-OH, Fmoc-Ser (Trt)-OH, Fmoc-Trp-OH, Fmoc-His (Trt)-OH, the Pyr-OH successively of-OH, obtain the peptide resin 37.2g of sequence as shown in SEQ ID NO:1, its sequence is Pyr-His (Trt)-Trp-Ser (Trt)-Tyr (Bzl)-D-Ser (tBu)-Leu-Arg (NO
2)-Pro-AzaGly-Sieber Resin.
Preparation lytic reagent 380mL, wherein trifluoroacetic acid 38mL, water 19mL, DCM323mL, precooling 30min under the condition of-5 DEG C; Join in 500mL round-bottomed flask by above-mentioned peptide resin 37.2g, joined by lytic reagent in above-mentioned round-bottomed flask, ice-water bath while stirring, passes into nitrogen.Withdraw ice bath after reaction 30min, under room temperature, continue reaction 2h.Filter resin, collect filtrate.With a small amount of DCM washing resin, merging filtrate.Concentrating under reduced pressure is to 60mL, concentrated solution is slowly added in 600mL ice ether and precipitate, centrifugal, ice washed with diethylether 5 times, drying under reduced pressure obtains the thick peptide 16.2g of sequence as shown in SEQ ID NO:1, and its sequence is Pyr-His-Trp-Ser-Tyr (Bzl)-D-Ser (tBu)-Leu-Arg (NO
2)-Pro-Azagly-NH
2.
By molten for above-mentioned thick peptide 16.2g in 160mL4.4% formic acid methanol solution, 16.2g10% palladium carbon, room temperature reaction 0.5h, detection raw material disappears, filter palladium carbon, filtrate inspection is concentrated into 30mL, join 500mL ice ether sedimentation, centrifugal, dry, obtain the thick peptide 11.0g of the goserelin of sequence as shown in SEQ ID NO:1, color takes on a red color, thick peptide yield 86.7%, HPLC purity 93.7%, in the thick peptide of goserelin, the content of goserelin essence peptide is 5.28g, be Pyr-His-Trp-Ser-Tyr-D-Ser (tBu)-Leu-Arg-Pro-Azagly-NH by mass spectrum determination structure
2, its mass-spectrometric data is 1269.7, and mass spectrum is shown in Fig. 1.
The preparation of embodiment 2 goserelin essence peptide
The proportions acetic acid of 6/1/13, the mixed solvent 500ml of acetonitrile and water by volume, the goserelin thick peptide 10g that Example 1 obtains is dissolved in the above-mentioned mixed solvent of 200mL, add 500mL water to dilute, obtain the thick peptide solution of goserelin first that mass body volume concentrations is 14.3g/L.Dripping mass percentage concentration is the ammonia soln of 30%, regulates the pH value to 7.0 of the thick peptide solution of goserelin first, obtains the thick peptide solution of goserelin second.
In the thick peptide solution of above-mentioned goserelin second, add the sodium sulfite solution of 200mL10g/L, stir 4min, color is transferred to faint yellow by redness.Drip acetic acid adjust ph to 6.0, filter, by HPLC system purifying on the thick peptide solution of goserelin second of above-mentioned process, mobile phase A is the 0.1%TFA aqueous solution, and Mobile phase B is acetonitrile, according to B% be 18% ~ 38%, the gradient elution Fractional Collections sample of 40min, detect purity, namely obtain the goserelin essence peptide solution that purity is 99%, in goserelin essence peptide solution, the content of goserelin essence peptide is 4.36g, and yield is 90.8%.
The preparation of embodiment 3 goserelin essence peptide
The proportions acetic acid of 4/1/5, the mixed solvent 500ml of acetonitrile and water by volume, the goserelin thick peptide 10g that Example 1 obtains is dissolved in the above-mentioned mixed solvent of 400mL, add 600mL water to dilute, obtain the thick peptide solution of goserelin first that mass body volume concentrations is 10g/L.Dripping mass percentage concentration is the sodium hydroxide solution of 10%, regulates the pH value to 6.0 of the thick peptide solution of goserelin first, obtains the thick peptide solution of goserelin second.
In the thick peptide solution of above-mentioned goserelin second, add the cysteine solution of 50mL10g/L, stir 5min, color is transferred to faint yellow by redness.Drip acetic acid adjust ph to 5.5, filter, by HPLC system purifying on the thick peptide solution of goserelin second of above-mentioned process, mobile phase A is the 0.1%TFA aqueous solution, and Mobile phase B is acetonitrile, according to B% be 25% ~ 45%, the gradient elution Fractional Collections sample of 40min, detect purity, namely obtain the goserelin essence peptide solution that purity is 99.1%, in goserelin essence peptide solution, the content of goserelin essence peptide is 4.22g, and yield is 87.9%.
The preparation of embodiment 4 goserelin essence peptide
The proportions acetic acid of 3/1/1, the mixed solvent 500ml of acetonitrile and water by volume, the goserelin thick peptide 10g that Example 1 obtains is dissolved in the above-mentioned mixed solvent of 500mL, add 1500mL water to dilute, obtain the thick peptide solution of goserelin first that mass body volume concentrations is 5g/L.Dripping mass percentage concentration is the potassium hydroxide solution of 40%, regulates the pH value to 8.0 of the thick peptide solution of goserelin first, obtains the thick peptide solution of goserelin second.
In the thick peptide solution of above-mentioned goserelin second, add the sodium sulfide solution of 300mL10g/L, stir 2min, color disappears.Drip acetic acid adjust ph to 5.7, filter, by HPLC system purifying on the thick peptide solution of goserelin second of above-mentioned process, mobile phase A is the 0.1%TFA aqueous solution, and Mobile phase B is acetonitrile, according to B% be 18% ~ 38%, the gradient elution Fractional Collections sample of 40min, detect purity, namely obtain the goserelin essence peptide solution that purity is 99.3%, in goserelin essence peptide solution, the content of goserelin essence peptide is 4.31g, and yield is 90.1%.
The preparation of embodiment 5 goserelin essence peptide
The proportions acetic acid of 3/1/1, the mixed solvent 500ml of acetonitrile and water by volume, the goserelin thick peptide 10g that Example 1 obtains is dissolved in the above-mentioned mixed solvent of 500mL, add 1000mL water to dilute, obtain the thick peptide solution of goserelin first that mass body volume concentrations is 6.7g/L.Dripping mass percentage concentration is the ammonia soln of 20%, regulates the pH value to 6.5 of the thick peptide solution of goserelin first, obtains the thick peptide solution of goserelin second.
In the thick peptide solution of above-mentioned goserelin second, add the hydrosulphuric acid of 100mL10g/L, stir 4.5min, color is transferred to faint yellow by redness.Drip acetic acid adjust ph to 6.3, filter, by HPLC system purifying on the thick peptide solution of goserelin second of above-mentioned process, mobile phase A is the 0.1%TFA aqueous solution, and Mobile phase B is acetonitrile, according to B% be 25% ~ 45%, the gradient elution Fractional Collections sample of 40min, detect purity, namely obtain the goserelin essence peptide solution that purity is 99.2%, in goserelin essence peptide solution, the content of goserelin essence peptide is 4.26g, and yield is 90.5%.
The preparation of embodiment 6 goserelin essence peptide
The proportions acetic acid of 4/1/5, the mixed solvent 500ml of acetonitrile and water by volume, the goserelin thick peptide 10g that Example 1 obtains is dissolved in the above-mentioned mixed solvent of 400mL, add 600mL water to dilute, obtain the thick peptide solution of goserelin first that mass body volume concentrations is 10g/L.Dripping mass percentage concentration is the sodium hydroxide solution of 5%, regulates the pH value to 7.5 of the thick peptide solution of goserelin first, obtains the thick peptide solution of goserelin second.
In the thick peptide solution of above-mentioned goserelin second, add the solution of ferrous chloride of 250mL10g/L, stir 2.5min, color is transferred to faint yellow by redness.Drip acetic acid adjust ph to 5.3, filter, by HPLC system purifying on the thick peptide solution of goserelin second of above-mentioned process, mobile phase A is the 0.1%TFA aqueous solution, and Mobile phase B is acetonitrile, according to B% be 18% ~ 38%, the gradient elution Fractional Collections sample of 40min, detect purity, namely obtain the goserelin essence peptide solution that purity is 99.2%, in goserelin essence peptide solution, the content of goserelin essence peptide is 4.27g, and yield is 90.5%.
The preparation of embodiment 7 goserelin essence peptide
The proportions acetic acid of 4/1/5, the mixed solvent 500ml of acetonitrile and water by volume, the goserelin thick peptide 10g that Example 1 obtains is dissolved in the above-mentioned mixed solvent of 400mL, add 600mL water to dilute, obtain the thick peptide solution of goserelin first that mass body volume concentrations is 10g/L.Dripping mass percentage concentration is the potassium hydroxide solution of 25%, regulates the pH value to 7.8 of the thick peptide solution of goserelin first, obtains the thick peptide solution of goserelin second.
In the thick peptide solution of above-mentioned goserelin second, add the sulfurous acid of 250mL10g/L, stir 2.5min, color disappears.Drip acetic acid adjust ph to 4.0, filter, by HPLC system purifying on the thick peptide solution of goserelin second of above-mentioned process, mobile phase A is the 0.1%TFA aqueous solution, and Mobile phase B is acetonitrile, according to B% be 25% ~ 45%, the gradient elution Fractional Collections sample of 40min, detect purity, namely obtain the goserelin essence peptide solution that purity is 99.1%, in goserelin essence peptide solution, the content of goserelin essence peptide is 4.38g, and yield is 90.9%.
The preparation of comparative example 1 goserelin essence peptide
The thick peptide 10g of goserelin that Example 1 is obtained, the method of charcoal absorption is utilized to carry out purifying to it, concrete operations are: the proportions acetic acid of 4/1/5, the mixed solvent 500ml of acetonitrile and water by volume, thick for goserelin peptide is dissolved in the above-mentioned mixed solvent of 400mL, add 600mL water to dilute, obtain the thick peptide solution of goserelin first that mass body volume concentrations is 10g/L.Add 10g gac and stir 5h, filter, get the upper HPLC system purifying of filtrate, mobile phase A is the 0.1%TFA aqueous solution, and Mobile phase B is acetonitrile, according to B% be 25% ~ 45%, the gradient elution Fractional Collections sample of 40min, detect purity, namely obtain the goserelin essence peptide solution that purity is 99.1%, in goserelin essence peptide solution, the content of goserelin essence peptide is 2.07g, and yield is 43.1%.
The preparation of comparative example 2 goserelin essence peptide
The thick peptide 10g of goserelin that Example 1 is obtained, the method of HPLC purifying is utilized to carry out purifying to it, concrete operations are: the proportions acetic acid of 4/1/5, the mixed solvent 500ml of acetonitrile and water by volume, thick for goserelin peptide is dissolved in the above-mentioned mixed solvent of 400mL, add 600mL water to dilute, obtain the thick peptide solution of goserelin first that mass body volume concentrations is 10g/L.By HPLC system purifying on thick for goserelin first peptide solution, mobile phase A is the 0.1%TFA aqueous solution, Mobile phase B is acetonitrile, be 25% ~ 45% according to B%, the gradient elution Fractional Collections sample of 40min, detect purity, the second solution is obtained after adding the water dilution of 1.5 times of volumes after the part of purity more than 99% merges, second solution is purified by this condition again and once obtains the 3rd solution, 3rd solution is purified with carrying out HPLC system again, mobile phase A is 0.2% ammonium acetate aqueous solution (ammoniacal liquor adjust pH 6.5), Mobile phase B is acetonitrile, be 26% ~ 46% according to B%, the gradient elution Fractional Collections sample of 40min, detect purity, the 4th solution is obtained after adding the water dilution of 1.5 times of volumes after the part of purity more than 99% merges, 4th solution is purified one-time detection purity by this condition again, obtain the solution without any color, namely the goserelin essence peptide solution that purity is 99.3% is obtained, in goserelin essence peptide solution, the content of goserelin essence peptide is 2.39g, yield is 49.8%.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (14)
1., containing a purification process for easy oxidized amino acid whose polypeptide, it is characterized in that, comprise the steps:
Obtain thick peptide, through pre-treatment, obtain the first thick peptide solution, through the first adjust ph to 6.0 ~ 8.0, obtain the second thick peptide solution;
Get the described second thick peptide solution to mix with reductive agent, through the second adjust ph to 4.0 ~ 7.0, after filtration, purifying, to obtain final product;
Described reductive agent is selected from the organic acid containing sulfydryl, containing sulfonium ion or the hydrionic acid of sulphur or inorganic salt, and the inorganic salt containing reducing metal ion, the inorganic salt containing inferior sulfate radical or bisulfite, or sulfurous gas;
Described easy oxidized amino acid is the amino acid that oxidizing reaction easily occurs.
2. purification process according to claim 1, is characterized in that, described easy oxidized amino acid is tryptophane.
3. purification process according to claim 1, is characterized in that, described polypeptide is goserelin.
4. purification process according to claim 1, is characterized in that, the described organic acid containing sulfydryl is halfcystine or reduced glutathion.
5. purification process according to claim 1, is characterized in that, described is hydrosulphuric acid containing sulfonium ion or the hydrionic acid of sulphur.
6. purification process according to claim 1, is characterized in that, the described inorganic salt containing sulfonium ion are sodium sulphite.
7. purification process according to claim 1, is characterized in that, described is Sodium sulfhydrate containing the hydrionic inorganic salt of sulphur.
8. purification process according to claim 1, is characterized in that, the described inorganic salt containing reducing metal ion are the inorganic salt containing ferrous ion or the inorganic salt containing cuprous ion.
9. purification process according to claim 8, is characterized in that, the described inorganic salt containing ferrous ion are ferrous sulfate or iron protochloride.
10. purification process according to claim 8, is characterized in that, the described inorganic salt containing cuprous ion are cuprous sulfate or cuprous chloride.
11. purification process according to claim 1, is characterized in that, the described inorganic salt containing inferior sulfate radical are S-WAT or potassium sulfite.
12. purification process according to claim 1, is characterized in that, the described inorganic salt containing bisulfite are sodium bisulfite or Potassium hydrogen sulfite.
13. purification process according to claim 1, is characterized in that, the mass ratio of described reductive agent and described thick peptide is (5 ~ 30): 100.
14. purification process according to claim 1, is characterized in that, the time of described mixing is 2 ~ 5min.
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| CN102863517A (en) * | 2012-09-21 | 2013-01-09 | 深圳翰宇药业股份有限公司 | Goserelin purification method |
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| CN101509022A (en) * | 2009-03-30 | 2009-08-19 | 天津科技大学 | Method for improving extract yield of L-tryptophane |
| CN101935339A (en) * | 2010-08-17 | 2011-01-05 | 深圳翰宇药业股份有限公司 | Solid-phase preparation method for buserelin |
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