CN102382178A - Separation method of sialoglycopeptide - Google Patents
Separation method of sialoglycopeptide Download PDFInfo
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- CN102382178A CN102382178A CN2011103226631A CN201110322663A CN102382178A CN 102382178 A CN102382178 A CN 102382178A CN 2011103226631 A CN2011103226631 A CN 2011103226631A CN 201110322663 A CN201110322663 A CN 201110322663A CN 102382178 A CN102382178 A CN 102382178A
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Abstract
The invention discloses a separation method of sialoglycopeptide, including the following steps: (1) taking a fresh egg yolk, treating the fresh egg yolk with phenol and then conducting rotation evaporation to obtain a sample containing the sialoglycopeptide; (2) separating the sample with sephadex G-50 and collecting eluent; (3) concentrating the eluent, absorbing the concentrated sample with active carbon and then eluting the sample in acetonitrile solution to obtain eluent containing the sialoglycopeptide; and (4) freezing and drying the eluent to obtain pure sialoglycopeptide. In the method, the steps of G-25 desalting, HPLC (High Performance Liquid Chromatography) purification, ion exchange chromatography purification and the like of the existing method are eliminated, therefore the purification period is shortened greatly, the efficiency is improved, the cost is saved and inspected by HPLC, and the purity of the prepared sialoglycopeptide can achieve 99%. The separation method is used for not only laboratories but also small-scale industrial production.
Description
Technical field
The present invention relates to a kind of separation purification method of glycopeptide compound, relate in particular to a kind of separation purification method that has natural complexity N sugar chain glycopeptide such as sialoglycopeptide.
Background technology
Sialoglycopeptide (Sialylglycopeptides) structure is:
Sialoglycopeptide (Sialylglycopeptides) is a kind of glycopeptide that has complete natural complexity N sugar chain, and relative molecular mass is 2866.Though can synthesize multiple oligosaccharides through chemical method now, present technology still can't be synthesized this complete sialylated N-glucosides.Therefore, can only be from natural product this sialoglycopeptide of separation and purification.
At present, in the world from egg yolk the method for separation and purification sialoglycopeptide mainly be that purification process with Japanese AkiraSeko report in 1997 is main.After this method is handled yolk earlier, separate through twice sephadex G-50, again through the G-25 desalination, DEAE-Toyopearl 650M IX column purification, after CM-Sephadex C-25 separated, freeze-drying obtained sample then.But the aforesaid method step is many, and the cycle is long, and sample loss is serious in purge process.Further improvement had once been carried out to purification process in the contriver laboratory, proposed to use gac and performance liquid chromatography (HPLC) to carry out purifying.Though this method has been simplified some steps, still need pass through a plurality of steps and could realize, when particularly using the HPLC purifying, because applied sample amount is few, productive rate is low, and cost is high, therefore generally is merely able to be used for a spot of separation in laboratory.
Because the complicacy of glycoprotein candy chain makes that the structure of sugar chain and the research of function are biological and the difficult point of field of medicaments research always, have the albumen of homogeneous sugar chain, be absolutely necessary for its research.And sialoglycopeptide is a kind of humanized glycopeptide, can be used as the humanization modified glucosides donor of gp.Therefore, it is low to invent a kind of cost, and the cycle is short, and step is few, and the separation method that efficient is high is very urgent and necessary.
Summary of the invention
To the deficiency that the separation method step is many, the cycle is long, cost is high that exists in the prior art, the purpose of this invention is to provide the separation purification method of the sialoglycopeptide that a kind of cycle is short, cost is low, efficient is high.
The separation method of sialoglycopeptide according to the invention comprises 1) get fresh egg yolk, after handling through phenol, obtain containing the sialoglycopeptide sample behind the rotary evaporation; 2) separate with sephadex G-50 pair last step sample, collect elutriant; 3) elutriant is concentrated, concentrating sample is adsorbed, obtain to contain the elutriant of sialoglycopeptide again with the acetonitrile solution wash-out with gac; 4) the elutriant lyophilize is obtained the pure article of sialoglycopeptide;
Wherein:
1) the said method that fresh egg yolk is handled is: get fresh unfertilized egg yolk earlier, add the dilution of isopyknic deionized water, add again its 1/5 volume phenol/water (phenol: water volume ratio 9: 1), 4 ℃ of stirrings; In mixed solution, add the deionized water of two volumes again, centrifugal behind the mixing, supernatant is concentrated with Rotary Evaporators, obtain containing the sample of sialoglycopeptide.
2) saidly go up the step sample with sephadex G-50 pair and carry out separation method and be: sephadex G-50 post is put into 23-27 ℃ thermostat container; With 1.6cm * 100cm sephadex G-50 post, flow velocity is 1ml/min when carrying out, and collects the elutriant of 110-140 milliliter;
3) said elutriant is concentrated, with gac concentrating sample is adsorbed, the method that contains the elutriant of sialoglycopeptide with the acquisition of acetonitrile solution wash-out again is:
The elutriant of collecting is put into beaker, again beaker is placed-80 ℃ freezing, then freezing back sample is put into vacuum freeze drier and carries out lyophilize, the dried frozen aquatic products that obtains containing sialoglycopeptide is a dry powder;
Freeze-drying appearance is made into the solution that concentration is the 50-150 mg/ml; Add kind ratio of 400-500 microlitre in per 500 milligrams of gacs, sample is added in the activated carbon column, absorption kept 30-40 minute; Then the 3-5 ml pure water is added in the activated carbon column; The flushing gac is removed salt and impurity, 3-5 milliliter 20-40% acetonitrile solution is added in the activated carbon column again, and wash-out obtains containing the elutriant of sialoglycopeptide.
4) saidly be: elutriant is put into container with the cryodesiccated method of elutriant; Volume does not surpass 1/2 of this container; Container is placed-80 ℃ freezing, more freezing back sample is put into vacuum freeze drier and carries out lyophilize, obtain the pure article of sialoglycopeptide at last.
In the separation method of above-mentioned sialoglycopeptide, saidly go up the step sample with sephadex G-50 pair and carry out sephadex G in the separation method-50 post and preferably put into 25 ℃ thermostat container.
In the separation method of above-mentioned sialoglycopeptide, the said concentrated appearance of step 3) is made into the solution that concentration is preferably the 100-120 mg/ml.
In the separation method of above-mentioned sialoglycopeptide, the Supelclean of the preferred SUPELCO of the said activated carbon column of step 3)
TMENVI-Carb
TMSPE Tube.
In the separation method of above-mentioned sialoglycopeptide, the said acetonitrile solution concentration of step 3) is preferably 30% in volume ratio.
Sialoglycopeptide is carried out structure to be identified and purity testing:
Utilize mass spectrum and nucleus magnetic resonance sample to be detected the result: molecular weight analyte and theoretical value are coincide, and nuclear magnetic spectrum and reference result coincide.
Utilize HPLC test sample purity, the result: substantially free of impurities, purity reaches more than 99%.
Above-mentioned during with sephadex G-50 purifying, use temperature is 23 ℃-27 ℃, is that too high or too low temperature all can't realize optimal separating efficiency because temperature variation can make experimental result unstable.Therefore our preferred temperature is 25 ℃, and under this temperature, separating effect is stable, efficient.
The present invention substitutes traditional Resorcinol salt acid system with UV-detector and detects sialoglycopeptide.Through finding that with traditional chemical method contrast 214nm test sample effect is with traditional method basically identical.This step has been saved loaded down with trivial details detection step, has reduced the sample loss that causes because of the chemical method detection simultaneously.
In the separation method of above-mentioned sialoglycopeptide, during said collection sephadex G-50 elutriant, preferably collecting the 110-140 ml sample, is because if will contain the elutriant of sialyl sample all collects, can cause sample purity to descend; If only collect the highest elutriant of samples contg, then cause the sample waste, experiment screening confirms: collect 110-140 milliliter elutriant, both guaranteed the purity of sample, at utmost reduce the loss of sample again.
In the above-mentioned activated carbon purification method, sample concentration is made into the 50-150 mg/ml, is because if sample concentration is too high, then exceed activated carbon adsorptive capacity, the waste sample; If sample concentration is low excessively, then cause separation efficiency to descend, lose time and reagent.The preferred 100-120 mg/ml of sample concentration among the present invention neither causes the sample waste to improve separation efficiency again, has practiced thrift cost.
In the above-mentioned activated carbon purification method, using 20-40% acetonitrile solution wash-out sialoglycopeptide, is because if acetonitrile concentration is too high, many impurity are together eluted, and causes sample purity to descend; If acetonitrile concentration is low excessively, then can't the wash-out sialoglycopeptide.Experiment screening confirms: acetonitrile solution concentration preferred 30% is most economical.
Sialoglycopeptide purification process according to the invention is mainly through pre-treatment, sephadex G-50 separation, three steps of activated carbon purification; Omitted steps such as the G-25 desalination in the previous methods, HPLC purifying, ion-exchange chromatogram purification, simultaneously, the condition of each purification process has been groped; Obtain the optimum condition of sialoglycopeptide separation and purification; Make purification process stable more, efficient, and do not influence product purity, thereby shortened the purifying cycle greatly; Improve purification efficiency, practiced thrift cost.The inventive method not only can be used for prepared in laboratory, also can be used for small-scale industrial production, for sialoglycopeptide purifying or exploitation provide a kind of new technology again.
Description of drawings
Fig. 1: fast protein liquid chromatography detects sephadex G-50 elutriant collecting region.
Fig. 2: mass spectrum (ESI-MS) detects sialoglycopeptide.
Wherein: positive ion mode detects sialoglycopeptide, and 956.7 is sample tricharged peak, and is identical with the molecular weight of sialoglycopeptide 2866.
Fig. 3: 300MHz
1H-NMR detects sialoglycopeptide.
Wherein: after NMR collection of illustrative plates and bibliographical information result compared, structure was coincide.
Fig. 4: HPLC detects the sialoglycopeptide sample.
Wherein: pillar: reverse C18 post; Moving phase: 10% acetonitrile; Flow velocity: 0.5ml/min; Detect wavelength: 214nm.
Embodiment
Below content according to the invention is done further to specify through embodiment.But should this be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following instance.Do not breaking away under the above-mentioned technological thought situation of the present invention, various replacements of making according to ordinary skill knowledge and customary means or more become all should comprise within the scope of the invention.
Embodiment 1
1, egg yolk pre-treatment
At first get 100 fresh unfertilized egg yolks (1.9L), add the dilution of isopyknic deionized water, (phenol: water volume ratio 9: 1), 4 ℃ continue to stir 3 hours to add the phenol/water of its 1/5 volume (380ml) again.In mixed solution, add the 4.8L deionized water, centrifugal behind the mixing (10000g, 25 minutes) are concentrated into 50 milliliters with supernatant with Rotary Evaporators, obtain containing the sample of sialoglycopeptide.
2, sephadex G-50 separates
Sephadex G-50 post (1.6cm * 100cm) be connected with the fast liquid chromatography appearance, with 0.1M NaCl as elutriant, flow velocity: 1ml/min, ultraviolet detection wavelength: 214nm.Pillar is positioned in the incubator 25 ℃ of temperature.Add the said sample 2ml of step 1, collect elutriant 110-140 ml sample, as shown in Figure 1, collect the sample that is labeled as " collecting region ".
3, elutriant is concentrated, and use the activated carbon column desalting and purifying
50 milliliters of elutriants are put into 100 ml beakers, again beaker is placed-80 ℃ freezing 1 hour, then freezing back sample is put into vacuum freeze drier and carries out lyophilize after 12 hours, the dried frozen aquatic products that obtains containing sialoglycopeptide is a dry powder.
Dried frozen aquatic products is made into the solution that concentration is 100 mg/ml, subsequent use;
Activated carbon column (Supelclean
TMENVI-Carb
TMSPE Tube, SUPELCO) use is preceding with 5 ml methanol cleaning active charcoal posts, uses pure water balance pillar then.
The ratio that adds appearance 500 microlitres in per 500 milligrams of gacs; The subsequent use sample of above-mentioned preparation is added in the activated carbon column; Absorption kept 40 minutes, then 5 ml pure waters was added in the activated carbon column, and the flushing gac is removed salt and impurity; 5 milliliter of 30% acetonitrile solution added in the activated carbon column, wash-out obtains containing the elutriant of sialoglycopeptide again.
4, lyophilize obtains the pure article of gac.
50 milliliters of elutriants are put into 100 ml beakers, beaker is placed-80 ℃ freezing 1 hour, more freezing back sample is put into vacuum freeze drier and carries out lyophilize, after dry 12 hours, obtain the pure article of sialoglycopeptide.
5, structure is identified and purity testing.
Utilize mass spectrum (result is as shown in Figure 2) and nucleus magnetic resonance (result is as shown in Figure 3) that sample is detected, molecular weight analyte and structure and theoretical value are coincide.Utilize HPLC test sample purity (result is as shown in Figure 4), substantially free of impurities, purity can reach more than 99%.
Use aforesaid method, the applicant is from 100 egg yolks, and separation of pure has dissolved the sialoglycopeptide of 640mg purity 99%, only one month consuming time.
Embodiment 2
1, egg yolk pre-treatment
At first get 100 fresh unfertilized egg yolks (1.9L), add isopyknic deionized water dilution, add phenol/water (volume ratio 9: 1) of 1/5 volume (380ml) again, 4 ℃ continue to stir 3 hours.In mixed solution, add the 4.8L deionized water, centrifugal behind the mixing (10000g, 25 minutes) are concentrated into 50 milliliters with supernatant with Rotary Evaporators, obtain containing the sample of sialoglycopeptide.
2, sephadex G-50 separates
Sephadex G-50 post (1.6cm * 100cm) be connected with the fast liquid chromatography appearance, with 0.1M NaCl as elutriant, flow velocity: 1ml/min, ultraviolet detection wavelength: 214nm.Pillar is positioned in the incubator 23 ℃ of temperature.Add above-mentioned 1 said sample 2.5ml, collect elutriant 110-140 ml sample.
3, elutriant is concentrated, and use the activated carbon column desalting and purifying
Elutriant is put into 100 ml beakers for 50 milliliters, again beaker is placed-80 ℃ freezing 1 hour, then freezing back sample is put into vacuum freeze drier and carries out lyophilize after 12 hours, the dried frozen aquatic products that obtains containing sialoglycopeptide is a dry powder.
Dried frozen aquatic products is made into the solution that concentration is 120 mg/ml, subsequent use;
Activated carbon column (Supelclean
TMENVI-Carb
TMSPE Tube, SUPELCO) use is preceding with 5 ml methanol cleaning active charcoal posts, uses pure water balance pillar then.
The ratio that adds appearance 400 microlitres in per 500 milligrams of gacs; Above-mentioned subsequent use sample is added in the activated carbon column; Absorption kept 30 minutes, then 4 ml pure waters was added in the activated carbon column, and the flushing gac is removed salt and impurity; 4 milliliter of 25% acetonitrile solution added in the activated carbon column, wash-out obtains containing the elutriant of sialoglycopeptide again.
4, lyophilize obtains the pure article of gac.
Elutriant is put into 100 ml beakers for 50 milliliters, again beaker is placed-80 ℃ freezing 1 hour, freezing back sample is put into vacuum freeze drier carries out lyophilize, after dry 12 hours, obtain the pure article of sialoglycopeptide.
5, structure is identified and purity testing.
Utilize mass spectrum and nucleus magnetic resonance that sample is detected, results sample molecular weight and structure and theoretical value are coincide.
Utilize HPLC test sample purity, substantially free of impurities as a result, purity can reach more than 99%.
Claims (5)
1. the separation method of a sialoglycopeptide comprises 1) get fresh egg yolk, after handling through phenol, obtain containing the sialoglycopeptide sample behind the rotary evaporation; 2) separate with sephadex G-50 pair last step sample, collect elutriant; 3) elutriant is concentrated, concentrating sample is adsorbed, obtain to contain the elutriant of sialoglycopeptide again with the acetonitrile solution wash-out with gac; 4) the elutriant lyophilize is obtained the pure article of sialoglycopeptide; It is characterized in that; 2) saidly go up the step sample with sephadex G-50 pair and carry out separation method and be: sephadex G-50 post is put into 23-27 ℃ thermostat container; With 1.6cm * 100cm sephadex G-50 post, flow velocity is 1ml/min when carrying out, and collects the elutriant of 110-140 milliliter; 3) saidly concentrating sample is adsorbed with gac; The method that obtains to contain the elutriant of sialoglycopeptide with the acetonitrile solution wash-out again is: earlier freeze-drying appearance is made into the solution that concentration is the 50-150 mg/ml; Add kind ratio of 400-500 microlitre in per 500 milligrams of gacs, sample is added in the activated carbon column, absorption kept 30-40 minute; Then the 3-5 ml pure water is added in the activated carbon column; The flushing gac is removed salt and impurity, again 3-5 milliliter 20-40% acetonitrile solution is added wash-out gac in the activated carbon column, obtains containing the elutriant of sialoglycopeptide.
2. the separation method of sialoglycopeptide according to claim 1 is characterized in that, saidly pair goes up the step sample with sephadex G-50 and carries out sephadex G in the separation method-50 post and put into 25 ℃ thermostat container.
3. the separation method of sialoglycopeptide according to claim 1 is characterized in that, the said concentrated appearance of step 3) is made into the solution that concentration is the 100-120 mg/ml.
4. the separation method of sialoglycopeptide according to claim 1 is characterized in that the said activated carbon column of step 3) selects the Supelclean of SUPELCO
TMENVI-Carb
TMSPE Tube.
5. the separation method of sialoglycopeptide according to claim 1 is characterized in that the said acetonitrile solution concentration of step 3) counts 30% with volume ratio.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108107141A (en) * | 2017-12-19 | 2018-06-01 | 浙江丰安生物制药有限公司 | The extracting method of polypeptide in a kind of spleen aminopeptide |
| CN109280081A (en) * | 2018-10-26 | 2019-01-29 | 浙江海洋大学 | A method of sialoglycopeptide is prepared from tuna ovum |
| CN109824762A (en) * | 2019-01-23 | 2019-05-31 | 西北大学 | A kind of method that large-scale separation, purifying prepare sialoglycopeptide (SGP) |
| CN110373443A (en) * | 2018-04-13 | 2019-10-25 | 中国科学院大连化学物理研究所 | A kind of pilose antler glycopeptide and preparation and application |
| CN117003827A (en) * | 2023-09-27 | 2023-11-07 | 山东睿鹰制药集团有限公司 | Separation and purification method of sialic acid glycopeptide |
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| CN1543471A (en) * | 2001-06-19 | 2004-11-03 | ��V��ѧ��ʽ���� | Process for producing sugar chain asparagine derivative |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108107141A (en) * | 2017-12-19 | 2018-06-01 | 浙江丰安生物制药有限公司 | The extracting method of polypeptide in a kind of spleen aminopeptide |
| CN110373443A (en) * | 2018-04-13 | 2019-10-25 | 中国科学院大连化学物理研究所 | A kind of pilose antler glycopeptide and preparation and application |
| CN109280081A (en) * | 2018-10-26 | 2019-01-29 | 浙江海洋大学 | A method of sialoglycopeptide is prepared from tuna ovum |
| CN109824762A (en) * | 2019-01-23 | 2019-05-31 | 西北大学 | A kind of method that large-scale separation, purifying prepare sialoglycopeptide (SGP) |
| CN109824762B (en) * | 2019-01-23 | 2022-11-22 | 西北大学 | Method for preparing Sialoglycopeptide (SGP) through large-scale separation and purification |
| CN117003827A (en) * | 2023-09-27 | 2023-11-07 | 山东睿鹰制药集团有限公司 | Separation and purification method of sialic acid glycopeptide |
| CN117003827B (en) * | 2023-09-27 | 2023-12-08 | 山东睿鹰制药集团有限公司 | Separation and purification method of sialic acid glycopeptide |
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