CN104480091A

CN104480091A - Method for highly purifying kallikein and drug composition containing kallikein

Info

Publication number: CN104480091A
Application number: CN201410807821.6A
Authority: CN
Inventors: 刘冠男; 林晓磊; 孙延年
Original assignee: QINGDAO KANGYUAN PHARMACEUTICAL CO Ltd
Current assignee: QINGDAO KANGYUAN PHARMACEUTICAL CO Ltd
Priority date: 2014-12-23
Filing date: 2014-12-23
Publication date: 2015-04-01

Abstract

The invention discloses a method for highly purifying kallikein and a drug composition containing kallikein. According to the technical scheme, pig pancreas taken as a raw material is extracted and settled to prepare acetone powder, extraction of acetic acid and acetone precipitation are flexibly used, conventional and modern combined protein purification technologies such as ether degreasing and dewatering, synergic impurity removal bysodium chloride, ammonium hydroxide and acetone, ion exchange resin DEAE (diethylaminoethyl)-agarose fast gel sequential elution and the like are adopted for highly purifying kallikein. The method and the drug composition have characteristics of short production cycle, good chemical and thermal stability and low cost, and are very suitable for large-scale production.

Description

A kind of method of highly purified pancreokinin proenzyme and the pharmaceutical composition containing pancreokinin proenzyme

Technical field

The present invention relates to biological technical field, specifically be that raw material extracts with pig pancreas, after precipitation pig pancreas makes acetone powder, to apply in a flexible way acetic acid extraction, acetone precipitation, ether defatting dewaters, sodium chloride-ammonia, acetone work in coordination with removal of impurities, the Protein purification techniques that the tradition and modernities such as the fast glue stepwise elution of ion exchange resin DEAE-agarose combine, prepares pancreokinin proenzyme.

Background technology

Pancreokinin proenzyme (kallikein) is the natural Enzymes medicine that separation and purification is obtained from Pancreas Sus domestica, clinically also known as kallidinogenase, have and unfold capillary vessel and arteriolar effect, be commonly used to clinically treat hyperlipidemia, prevent and treat the various diseases such as fatty liver, atherosclerosis, hypertension, stenocardia, its exploitation has important medical science and social value.In China, pancreokinin proenzyme is produced also exists very large development space.

The technique of traditional purifying pancreokinin proenzyme due to complex process, cost higher; Ion exchange chromatography is the most common method of applicable pancreokinin proenzyme suitability for industrialized production at present; But usually need to adopt multiple import Ion Exchange Medium to carry out purifying to pancreokinin proenzyme, not only complex operation, and make the rate of recovery very low because of repeatedly upper prop, for the method for the people such as the Zhou Zumeng that most is with practical value, by under pH7.0, pH5.0, pH6.7 condition, successively by DEAE-Cellulose, DEAE-Sepharosecl-6B on gained sample, obtain the pancreokinin proenzyme of about specific activity 1500u/mg, yield is 40%.In addition, mostly adopt anionresin in the world at present, in conjunction with cationic exchange, finally add a step gel permeation chromatography, and the wash-out of pancreokinin proenzyme all adopts the mode changing pH in ion exchange process, due to the shock absorption of buffered soln, pH in post is made to change slowly, cycle stretch-out, at substantial damping fluid, with the purge process of French Graham S.Bailey, although after three step purifying, pancreokinin proenzyme specific activity is up to 1254u/mg, but yield only has 28.4%; Next has employing ion exchange chromatography to combine with affinity chromatography or hydrophobic chromatography, step is more, and purification cycle is longer and with high costs, for the purge process of Britain Talal S.EI.Thaher, although yield reaches 87%, pancreokinin proenzyme specific activity only has 156.3u/mg; Again, a kind of Ion Exchange Medium is adopted to want the pancreokinin proenzyme just being obtained high purity and high yield by purifying to be almost impossible mission, utilize Amberlite-CG50 mono-step separation and purification pancreokinin proenzyme from pancreatin with Li Lihuan etc., but its specific activity and yield only has 107.4u/mg and 12.1% respectively.

And the present invention is apply in a flexible way acetic acid extraction, acetone precipitation, ether defatting dewaters, sodium chloride-ammonia, acetone work in coordination with removal of impurities, the Protein purification techniques that the tradition and modernities such as the fast glue stepwise elution of ion exchange resin DEAE-agarose combine, the fast glue of DEAE-agarose is used to have high flow rate, high carrying capacity, effectively can shorten the production cycle, and to chemistry and Heat stability is good, the yield of product maintains the leading position in similar technique; And the method for pancreokinin proenzyme stepwise elution, although international highest standard can not be reached, still exceed the requirement of clinical application, be applicable to suitability for industrialized production.

Summary of the invention

The object of the invention is the highly purified pancreokinin proenzyme extracted from qualified Pancreas Sus domestica.

Technical scheme of the present invention is: be that raw material extracts with pig pancreas, after precipitation pig pancreas makes acetone powder, to apply in a flexible way acetic acid extraction, acetone precipitation, ether defatting dewaters, sodium chloride-ammonia, acetone work in coordination with removal of impurities, the Protein purification techniques that the tradition and modernities such as the fast glue stepwise elution of ion exchange resin DEAE-agarose combine, highly purified pancreokinin proenzyme.The present invention has with short production cycle, to chemistry and Heat stability is good, feature that cost is low, is highly suitable for the large production of mass-producing.Comprise the steps:

(1) acetic acid extraction: after Pancreas Sus domestica is made acetone powder, adds acetum and stirs extraction, centrifugal, obtains supernatant liquor;

(2) acetone precipitation: collect supernatant liquor, add acetone precipitation.Precipitation acetone, ether defatting, dehydration, dry, obtain pancreokinin proenzyme crude product;

(3) sodium chloride-ammonia, acetone work in coordination with removal of impurities: dissolved by crude product sodium chloride solution, adjust PH with ammoniacal liquor, be uniformly mixed, and filter, obtain filtrate; Acetone precipitation is added by filtrate.Precipitation acetone, washed with diethylether, dry, obtain product in the middle of pancreokinin proenzyme;

(4) ion exchange chromatography: product in the middle of pancreokinin proenzyme are dissolved in 0.0005 ~ 0.0015mol/L acetate buffer solution (pH4.5) of 18 ~ 22 times amount, centrifugal, obtain supernatant liquor; Get supernatant liquor, by the fast glue post absorption of DEAE-agarose in the volume ratio of 1:8 ~ 12, after adsorption equilibrium, baseline is eluted to the 0.01mol/L acetate buffer solution containing 0.15 ~ 0.25mol/L sodium-chlor, again with the 0.01mol/L acetate buffer solution wash-out containing 0.25 ~ 0.45mol/L sodium-chlor, collect elutriant; By elutriant dialysis desalting, freeze-drying, obtains pancreokinin proenzyme fine work.

Embodiment

Below in conjunction with specific embodiment, the invention will be further described.

Embodiment 1

Described be that raw material extracts with pig pancreas, after precipitation pig pancreas makes acetone powder, to apply in a flexible way acetic acid extraction, acetone precipitation, ether defatting dewaters, sodium chloride-ammonia, acetone work in coordination with removal of impurities, the Protein purification techniques that the tradition and modernities such as the fast glue stepwise elution of ion exchange resin DEAE-agarose combine, prepare pancreokinin proenzyme, comprise the steps:

(1) acetic acid extraction: after Pancreas Sus domestica 10Kg is made acetone powder, add the acetum of 30 times amount, stirs and extracts, centrifugal, obtains supernatant liquor 29.8L.

(2) acetone precipitation: collect supernatant liquor, add acetone precipitation.Precipitation acetone, ether defatting, dehydration, dry, obtain pancreokinin proenzyme crude product 93g.

(3) sodium chloride-ammonia, acetone work in coordination with removal of impurities: dissolved by crude product 50 times amount sodium chloride solutions, adjust PH, be uniformly mixed with ammoniacal liquor, filter, obtain filtrate; , add acetone precipitation.Precipitation acetone, washed with diethylether, dry, obtain product 60g in the middle of pancreokinin proenzyme.

(4) ion exchange chromatography: product in the middle of pancreokinin proenzyme are dissolved in the 0.001mol/L acetate buffer solution (pH4.5) of 20 times amount, centrifugal, obtain supernatant liquor; Get supernatant liquor, by the fast glue post absorption of DEAE-agarose in the volume ratio of 1:10, after adsorption equilibrium, be eluted to baseline with the 0.01mol/L acetate buffer solution containing 0.2mol/L sodium-chlor, again with the 0.01mol/L acetate buffer solution wash-out containing 0.35mol/L sodium-chlor, collect elutriant; By elutriant dialysis desalting, freeze-drying, obtains pancreokinin proenzyme fine work 42g.

Embodiment 2

Pancreatic Kininogenase 6000 unit prepared by embodiment 1, pregelatinized Starch 21.7g puts in mixing machine, stir 5 minutes, add dextrin 32.5g again, Icing Sugar 20.1g, EDTA-2Na 0.162g, sodium starch glycolate 1.23g is in mixing machine, stir after 20 minutes and the tackiness agent prepared in advance (is got L-hydroxypropylcellulose 2.0g to add 75% ethanol 20ml and make dissolving, obtain) and magnesium stearate lubricant 0.0031g, slowly add equably in mixing machine and stir, formed to particle, put into nodulizer to granulate, intermediate is checked, qualified rear tabletting machine makes 1000, label is enteric coated after the assay was approved.

The above is only preferred embodiment of the present invention, and be not restriction the present invention being made to other form, any those skilled in the art may utilize the technology contents of above-mentioned announcement to be changed or be modified as the Equivalent embodiments of equivalent variations.But everyly do not depart from technical solution of the present invention content, any simple modification, equivalent variations and the remodeling done above embodiment according to technical spirit of the present invention, still belong to the protection domain of technical solution of the present invention.

Claims

1. the method for highly purified pancreokinin proenzyme and the pharmaceutical composition containing pancreokinin proenzyme, it is characterized in that, the method comprises the steps:

(2) acetone precipitation: collect supernatant liquor, add acetone precipitation;

Precipitation acetone, ether defatting, dehydration, dry, obtain pancreokinin proenzyme crude product;

(3) sodium chloride-ammonia, acetone work in coordination with removal of impurities: dissolved by crude product sodium chloride solution, adjust PH with ammoniacal liquor, be uniformly mixed, and filter, obtain filtrate; Acetone precipitation is added by filtrate;

Precipitation acetone, washed with diethylether, dry, obtain product in the middle of pancreokinin proenzyme;

(4) ion exchange chromatography: product in the middle of pancreokinin proenzyme are dissolved in acetate buffer solution, centrifugal, obtain supernatant liquor; Get supernatant liquor, the fast glue post absorption of upper DEAE-agarose, after adsorption equilibrium, is eluted to baseline with the acetate buffer solution of sodium chloride-containing, then uses the acetate buffer solution wash-out of sodium chloride-containing, collect elutriant; By elutriant dialysis desalting, freeze-drying, obtains pancreokinin proenzyme fine work.

2. method according to claim 1, is characterized in that: in step (4), and in the middle of pancreokinin proenzyme, the ratio of product and acetum is: 1Kg:18 ~ 22L, the concentration of acetum is: 0.0005 ~ 0.0015mol/L, and pH value is 4.5.

3. method according to claim 1, is characterized in that: in step (4), and the acetate buffer solution being eluted to the sodium chloride-containing of baseline is the 0.01mol/L acetate buffer solution containing 0.15 ~ 0.25mol/L sodium-chlor; The acetate buffer solution of the sodium chloride-containing of second time wash-out is the 0.01mol/L acetate buffer solution containing 0.25 ~ 0.45mol/L sodium-chlor; .

4. method according to claim 1, is characterized in that: with the absorption of novel anion-exchange column in step (4), Ion Exchange Medium used is the fast glue of DEAE-agarose, and the volume ratio of the fast glue of DEAE-agarose and supernatant liquor is: 8 ~ 12:1.

5. a pharmaceutical composition, its pancreokinin proenzyme prepared containing with good grounds claim 1-4 as activeconstituents, also containing pregelatinized Starch, dextrin, Icing Sugar, L-hydroxypropylcellulose, sodium starch glycolate, Magnesium Stearate, EDTA-2Na, 75% ethanol.