CN103073637B - Method for purifying ulinastatin by adsorption column chromatography - Google Patents

Abstract

The invention discloses a method for purifying ulinastatin by adsorption column chromatography. In specific, fresh urine of a healthy adult man is taken as a raw material to prepare high-purity ulinastatin through modern high-end protein biochemical separation technology including chitin adsorption, ammonia water elution, ammonium sulfate salting-out, and adsorption column chromatography. The total yield coefficient can be increased to over 60%, and the total titer is greater than or equal to 4000 iu/mg.

Description

A kind of method of adsorpting column chromatography purifying ulinastatin

Technical field

The present invention relates to biological technical field, relate in particular to a kind of adsorpting column chromatography method of applying and prepare that yield is high, the method for the stable ulinastatin of tiring.

Background technology

Ulinastatin (Ulinastatin) is separation and purification a kind of acid glycoprotein out from NAM's freshly voided urine, it is a kind of proteinase inhibitor of wide spectrum, mainly synthetic in liver, discharged with urine by renal metabolism, and its low molecular weight compositions being decomposed to form also has the effect of very strong inhibition lytic enzyme.Ulinastatin is made up of 143 amino acid, relative molecular mass approximately 37000～43000, belong to proteinase inhibitor, the plurality of enzymes such as the serine protease such as trypsinase, Chymetin and granulocyte elastase, Unidasa, sulfydryl enzyme, plasmin are had to restraining effect, also there is stable lysosome membrane, suppress lysosomal enzyme release, suppress myocardial depressant factor (MDF) (MDF) and produce, remove the effect that oxyradical and inflammation-inhibiting medium discharge.Ulinastatin also can improve operation stimulates the abnormal and renal function of the lower immune function, Proteometabolism that cause to reduce, and prevents that operation from stimulating the damage to internal organs and cell causing and improving recurrent state while shock etc.

First Europe in 1909 report that human urine exists trypsin inhibitor, it is found that subsequently, when human body is infected, heating, tumour, gestation, shock, when performing the operation, give glucocorticosteroid etc. and stimulating, in human urine, UTI is active raises.Within 1985, first develop listing by Japan, be widely used in clinical as the medicine of acute pancreatitis, acute circulatory failure in Japan.

The inventor started the production technique research experiment to UTI from 2008, adopt the high-end biochemical isolation technique of modern protein to produce, the total recovery of UTI is brought up to more than 60%, total titer is not less than 4000iu/mg, stable processing technique, quality controllable.

Summary of the invention

The invention provides a kind of method of adsorpting column chromatography purifying ulinastatin, that the traditional method for extracting ulinastatin yields such as kaolin absorption, the absorption of acid resistance mixed mode are too low, the not high unsettled shortcoming of tiring in order to capture, adopt adsorpting column chromatography to carry out purifying ulinastatin, thereby improve ulinastatin yield, what ensured to tire is stable.

For solving the problems of the technologies described above, the present invention is achieved by the following technical solutions:

A method for adsorpting column chromatography purifying ulinastatin, is characterized in that: select resin absorption, special elutriant desorb to carry out purifying ulinastatin, total recovery can be brought up to more than 60%, total titer is not less than 4000iu/mg.

In invention technical scheme, also there is following technical characterictic: described adsorpting column chromatography comprises the steps:

1) ulinastatin roughing

Take the clarification urine that 1T pH is less than 6.5, constantly stir, after slowly adding the absorption completely of 16.5kg chitin, use ammoniacal liquor wash-out, pass through again 3.5kg ammonium sulfate precipitation, the precipitation, centrifugal being placed on of spending the night done vacuum-drying in the vacuum drier of water-retaining agent with Vanadium Pentoxide in FLAKES, obtains ulinastatin crude product after dry.

2) adsorpting column chromatography operation (taking 0.25L cylinder as example)

2.1) take ulinastatin crude product 1kg, with elutriant A dissolving, stir 30 minutes, centrifugal 20 minutes, leave and take centrifugate;

2.2) elutriant B balance for anion-exchange column, by the above-mentioned centrifugate good anion-exchange column of balance of flowing through, flow rate control is in 120～130mL/min left and right;

2.3) with elutriant B eluant solution, flow rate control, at 120～130mL/min, obtains elutriant;

2.4) by elutriant through ultra-filtration membrane, add phosphate buffered saline buffer ultrafiltration repeatedly, ultrafiltration, to 1/20th of its volume, obtains ultrafiltrated.

3) precipitation

In ultrafiltrated, add not low 95% ethanol precipitation 12 hours in the ratio of 1: 6, centrifugal, solid is again with dehydrated alcohol dehydration twice, and centrifugal, abandoning supernatant, leaves and takes throw out.

4) freeze-drying

Throw out is packed into and coiled into Freeze Drying Equipment, and freeze-drying obtains ulinastatin.

Through adjusting, gained ulinastatin total recovery, up to more than 60%, is measured by method shown in Chinese Pharmacopoeia, and total titer is up to more than 4000iu/mg.

Compared with prior art, advantage of the present invention and positively effect are:

Select adsorpting column chromatography purifying ulinastatin, can more effective removal multiple foreign protein wherein, and make its physico-chemical property and biological activity keep stable, thus make the indices of product all meet Chinese Pharmacopoeia standard.The more important thing is, by technique update and perfect, ulinastatin total recovery is reached more than 60%, total titer is not less than 4000iu/mg, brings huge economic benefit and social benefit.

Embodiment

Below in conjunction with specific embodiment, the invention will be further described, and restriction never in any form.

Embodiment 1

1) ulinastatin roughing

Take the clarification urine that 1T pH is less than 6.5, constantly stir, slowly add 16.5kg chitin, and measure with accurate pH test paper, regulate urine pH to 6.0; After absorption completely, with ammoniacal liquor wash-out 1h, then pass through 3.5kg ammonium sulfate precipitation, the precipitation of spending the night, centrifugal be placed on to put with Vanadium Pentoxide in FLAKES do vacuum-drying in the vacuum drier of water-retaining agent, after being dried, obtain ulinastatin crude product.

2) adsorpting column chromatography operation (taking 0.25L cylinder as example)

2.1) take ulinastatin crude product 1kg, with the elutriant A dissolving of 8.5L, stir 30 minutes, centrifugal 20 minutes, leave and take centrifugate;

2.2) the elutriant B balance of 2.5L for anion-exchange column, by the above-mentioned centrifugate good anion-exchange column of balance of flowing through, flow rate control is in 120mL/min left and right;

2.3) with the elutriant B eluant solution of 33L, flow rate control, at 120mL/min, obtains elutriant;

2.4) by elutriant through ultra-filtration membrane, add phosphate buffered saline buffer ultrafiltration repeatedly, ultrafiltration, to 1/20th of its volume, obtains the about 1.65L of ultrafiltrated.

3) precipitation

In ultrafiltrated, add not low 95% ethanol precipitation 12 hours in the ratio of 1: 6, centrifugal, solid is again with dehydrated alcohol dehydration twice, and centrifugal, abandoning supernatant, leaves and takes throw out.

4) freeze-drying

Throw out is packed into and coiled into Freeze Drying Equipment, and freeze-drying obtains ulinastatin.

Through adjusting, gained ulinastatin total recovery is 66%, measures by method shown in Chinese Pharmacopoeia, and total titer is up to 4312iu/mg.

Embodiment 2

1) ulinastatin roughing

Take the clarification urine that 1T pH is less than 6.5, constantly stir, slowly add 16.5kg chitin, and measure with accurate pH test paper, regulate urine pH to 6.0; After absorption completely, with ammoniacal liquor wash-out 1h, then pass through 3.5kg ammonium sulfate precipitation, the precipitation of spending the night, centrifugal being placed on Vanadium Pentoxide in FLAKES are done vacuum-drying in the vacuum drier of water-retaining agent, after being dried, obtain ulinastatin crude product.

2) adsorpting column chromatography operation (taking 0.25L cylinder as example)

2.1) take ulinastatin crude product 1kg, with the elutriant A dissolving of 8.5L, stir 30 minutes, centrifugal 20 minutes, leave and take centrifugate;

2.2) the elutriant B balance of 3.0L for anion-exchange column, by the above-mentioned centrifugate good anion-exchange column of balance of flowing through, flow rate control is in 130mL/min left and right;

2.3) with the elutriant B eluant solution of 33L, flow rate control, at 130mL/min, obtains elutriant;

2.4) by elutriant through ultra-filtration membrane, add phosphate buffered saline buffer ultrafiltration repeatedly, ultrafiltration, to 1/20th of its volume, obtains the about 1.65L of ultrafiltrated.

3) precipitation

In ultrafiltrated, add not low 95% ethanol precipitation 12 hours in the ratio of 1: 6, centrifugal, solid is again with dehydrated alcohol dehydration twice, and centrifugal, abandoning supernatant, leaves and takes throw out.

4) freeze-drying

Pack throw out into dish and put into into Freeze Drying Equipment, freeze-drying obtains ulinastatin.

Through adjusting, gained ulinastatin total recovery is 63%, measures by method shown in Chinese Pharmacopoeia, and total titer is up to 4123iu/mg.

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

Claims (1)

1. a method for purifying ulinastatin, the method is specially:

1) ulinastatin roughing

Take the clarification urine that 1T pH is less than 6.5, constantly stir, slowly add 16.5kg chitin, and measure with accurate pH test paper, regulate urine pH to 6.0; After absorption completely, with ammoniacal liquor wash-out 1h, then pass through 3.5kg ammonium sulfate precipitation, the precipitation of spending the night, centrifugal being placed on Vanadium Pentoxide in FLAKES are done vacuum-drying in the vacuum drier of water-retaining agent, after being dried, obtain ulinastatin crude product;

2) adsorpting column chromatography operation

2.1) take ulinastatin crude product 1kg, with the elutriant A dissolving of 8.5L, stir 30 minutes, centrifugal 20 minutes, leave and take centrifugate; The acetate buffer that described elutriant A is 0.01-0.3mol/L;

2.2) the elutriant B balance of 3.0L for anion-exchange column, by the above-mentioned centrifugate good anion-exchange column of balance of flowing through, flow rate control is the damping fluid of 0.01-0.5mol/L sodium-acetate and 0.1-5mol/LNaCl at elutriant B described in 130mL/mim;

2.3) with the elutriant B eluant solution of 33L, flow rate control, at 130mL/min, obtains elutriant;

2.4) by elutriant through ultra-filtration membrane, add phosphate buffered saline buffer ultrafiltration repeatedly, ultrafiltration, to 1/20th of its volume, obtains ultrafiltrated 1.65L; The concentration of described phosphate buffered saline buffer is 0.01-0.5mol/L;

3) precipitation

In ultrafiltrated, add 95% ethanol precipitation 12 hours in the ratio of 1: 6, centrifugal, solid is again with dehydrated alcohol dehydration twice, and centrifugal, abandoning supernatant, leaves and takes throw out;

4) freeze-drying

Pack throw out into dish and put into into Freeze Drying Equipment, freeze-drying obtains ulinastatin.