CN109553679A - High-purity ulinastatin and preparation method thereof and pharmaceutical composition containing ulinastatin - Google Patents
High-purity ulinastatin and preparation method thereof and pharmaceutical composition containing ulinastatin Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
- C07K14/8114—Kunitz type inhibitors
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Abstract
The present invention provides a kind of ulinastatin and preparation method thereof, absorbance value of 100000 units/ml ulinastatin at 405nm is no more than 0.18, the content of human urokinase-type peptidase is no more than 0.0005PNAU, and content of beary metal is no more than 1.6 μ g/ml, and bacteria endotoxin content is no more than 6.0EU/ml.Urine is pre-processed by the methods of nitrite, ammonium sulfate, polyethylene glycol, adjusting pH, preliminary purifying is carried out using the method for dialysis before crossing column simultaneously, gel column is passed sequentially through again and ulinastatin sterling is prepared in cation exchange column, guarantee the purity and yield of finished product while enormously simplifying preparation process, pharmaceutical activity has very big promotion.
Description
Technical field
The present invention relates to a kind of field of biotechnology, and in particular to high-purity ulinastatin and preparation method thereof and containing crow
The pharmaceutical composition of Si Tading.
Background technique
Ulinastatin is a kind of glycoprotein for isolating and purifying out from healthy adult male freshly voided urine, by 143 ammonia
Base acid composition, relative molecular mass about 67000.1909, Beurer and Reich reported in human urine that there are this for the first time
Trypsin inhibitor, to the serine proteases such as trypsase, Chymetin and granulocyte elastase, hyaluronic acid
A variety of enzymes such as enzyme, sulfydryl enzyme, fibrinolysin have inhibiting effect.It is another to inhibit the release of lysosomal enzyme with lysosome membrane is stablized, inhibit
Myocardial depressant factor (MDF) (MDF) generates, scavenging activated oxygen and the effect for inhibiting inflammatory mediator release.Ulinastatin can also improve hand
The decline of immune function caused by art stimulates, protein metabolism exception and renal function reduce, internal internal organs caused by preventing operation from stimulating
Recurrent state the etc. when damage and improvement shock of official and cell.
The biological function of ulinastatin multiplicity is related to its unique molecular structure, and ulinastatin is mainly by 3 functional domains
It constitutes, including O connection glycosylated region (Alal-Lys21), N-terminal Kunitz type structural domain I (Lys22-Arg77) and has
The end the C Kunitz type domain II (Thr78Leul43) of trypsin inhibition activity.Two Kunitz type structural domains have
Certain homology passes through containing 6 conservative cysteines in each structural domain and forms 3 pairs of disulfide bond and maintain one
Fixed steric configuration.The enzyme inhibition activity of ulinastatin is primarily present in the Kunitz type structural domain in Bulk protein skeleton.
Patent CN100528898C discloses high-purity ulinastatin and preparation method thereof and the drug containing ulinastatin
Composition, wherein ulinastatin crude product is by the multiple column chromatography preparation such as anion-exchange column, metal chelating column, affinity column
It obtains.Patent CN100425286C discloses ulinastatin of purifying and preparation method thereof and the pharmaceutical composition containing ulinastatin
Object also passes through a variety of column chromatographies such as anion-exchange column, metal chelating column, drainage column absorption, gel filtration chromatography and is prepared.
Purification process is chromatographed by multiple column in above-mentioned patent, and complex technical process, product yield is low, and the production cycle is long, is not easy to amplify
Production.107827976 A of CN discloses a kind of ulinastatin purification process based on drainage column, only by a step drainage column
Realize purifying, and purity has reached 99.90% or more, Rate activity is at least 5100U/mg, but its drainage column using urea into
Row preservation and realize recycling, reuse the yield for leveraging ulinastatin sterling.
Summary of the invention
Based on the defect of the above-mentioned prior art, the present invention provides a kind of ulinastatin and the preparation method and application thereof, the crow
Impurity content is few in Si Tading, by changing crude product preparation method and improving purifying process, further controls ulinastatin molecule
Range is measured, pharmaceutical activity is significantly improved.
The present invention provides a kind of ulinastatin, and when concentration is 100,000 units/ml, the absorbance value at 405nm does not surpass
0.18 is crossed, the content of human urokinase-type peptidase is no more than 0.0005PNAU, and content of beary metal is no more than 1.6 μ g/ml, bacterial endotoxin
Content is no more than 6.0EU.
Further, when the ulinastatin concentration is 100,000 units/ml, the absorbance value at 405nm is no more than
0.16, the content of human urokinase-type peptidase is no more than 0.0004PNAU, and content of beary metal is no more than 1.0 μ g/ml, and bacterial endotoxin contains
Amount is no more than 5.0EU.
Further, the ulinastatin uses the molecular weight of sodium dodecyl sulfate polyacrylamide gel electrophoresis measurement
For 37,000-43,000Da or use the molecular weight of high effective liquid chromatography for measuring for 62,000-72,000Da.
The present invention further provides a kind of preparation methods of above-mentioned ulinastatin, comprising the following steps:
(1) nitrite is added in urine, adjusts pH, stirring, ammonium sulfate is eluted, and eluent is filtered, and is retained
Filter cake；
(2) filter cake is dissolved in water, and adjusts pH, and polyethylene glycol, filtering, drying precipitate is added；
(3) step (2) sediment is dissolved in water, dialysis, dry to get ulinastatin crude product；
(4) ulinastatin crude product is dissolved in water, and crosses gel filtration chromatography, distills water elution, collects component, ultrafiltration；
(5) cation-exchange chromatography post loading is added in step (4) sample, is eluted with hydrochloric acid, collects eluent concentration, and freezing is dry
It is dry to get ulinastatin sterling.
Further, urine and the weight ratio of nitrite are 1 ton: 13-16kg in the step (1).
Further, pH to 4.0-6.0 is adjusted in the step (1).
Further, the nitrite is selected from one of ammonium nilrite, sodium nitrite.
Further, the nitrite is ammonium nilrite.Nitrite ion in nitrite can be with urine
In urea reaction generate nitrogen, carbon dioxide gas, be conducive to the processing of urea in urine.
Further, ammonium sulfate concentration is 8-12% in the step (1).The addition of ammonium sulfate promotes urine
In ulinastatin protein matter occur invertibity denaturation and precipitate.
Further, filter cake adds 1-3 times of water dissolution in the step (2), adjusts pH5.0-6.0,8-12 times of poly- second is added
Glycol stirs 20-40min, stands, plate-frame filtering, sediment freeze-drying.
Further, sediment is dissolved in water in the step (3), 4 DEG C of dialysis, during which replaces 3-5 water, and freezing is dry
It is dry to get ulinastatin crude product.
Further, gel filtration chromatography filler is sephadex G -75, elution speed 0.4- in the step (4)
0.6ml/min。
Further, ultrafiltration is specially addition 0.2-0.4gEDTA-Na/ liter in eluent in the step (4), uses molecule
The ultrafiltration membrane ultrafiltration that amount is 30,000.
Further, cation exchange column is 732 type cation exchange resins in the step (5).
Further, concentration of hydrochloric acid is 1.5-3mol/L in the step (5).
The present invention also provides the combinations for the ulinastatin being prepared containing above-mentioned ulinastatin or above-mentioned preparation method
Object.
Further, the composition includes ulinastatin and mannitol.
Further, the preparation method of composition are as follows: the dissolution of mannitol water for injection, after ulinastatin mixing is added
Adjust pH6-7, membrane filtration to get.
The ulinastatin being prepared the present invention also provides the above-mentioned preparation method of above-mentioned ulinastatin is preparing protease
Application in inhibitor medicaments.
Beneficial effects of the present invention:
(1) present invention pre-processes urine using the methods of nitrite, ammonium sulfate, polyethylene glycol, adjusting pH, the Central Asia
Urea reaction in nitrite ion and urine in nitrate generates nitrogen, carbon dioxide gas, is conducive to urea in urine
Processing substantially increases the purity of ulinastatin after pretreatment, improves the efficiency of subsequent columns chromatography；Simultaneously using saturating before crossing column
The method of analysis first carries out preliminary purifying, the efficiency for avoiding excessive impurity effect column from chromatographing.If still there is a small amount of excessive nitrous
Acid ion can also be removed during dialysis.Nitrite ion in dialyzate can be complete by the way that oxidant is added
Full removal, avoids environmental pollution.
(2) method that the present invention is combined using gel filtration chromatography and cation exchange column chromatography carries out impurity more effective
Removal, not only increase pharmaceutical purity, also improve product yield and pharmaceutical activity.
Specific embodiment
1 ulinastatin of embodiment and preparation method thereof
14.7kg ammonium nilrite is added in (1) 1 ton of urine, adjusts pH to 5.0, stirs 50min, the ammonium sulfate for being 10% with concentration
Solution is eluted, and eluent is filtered, and retains filter cake；
(2) filter cake adds 2 times of water dissolutions, adjusts pH5.0-6.0,8 times of polyethylene glycol are added, stir 30min, stand, plate-frame filtering,
Sediment freeze-drying；
(3) step (2) sediment is dissolved in water, during which 4 DEG C of dialysis are replaced 4 water, are freeze-dried to get ulinastatin crude product；
(4) ulinastatin crude product is dissolved in water, and crosses sephadex G -75, distills water elution, flow velocity 0.6ml/min, and collection is washed
De- component, is added 0.3gEDTA-Na/ liter, the ultrafiltration membrane ultrafiltration for being 30,000 with molecular weight；
(5) 732 type cation-exchange chromatography post loadings are added in step (4) sample, are eluted with 1.5mol/L hydrochloric acid, flow velocity 1ml/
Min collects eluent concentration, is freeze-dried to get ulinastatin sterling.
Wherein, dialysis uses bag filter MD40(6000-8000 in step (3)), flatten width 40mm, diameter 25.5mm.
100,000,000 unit of ulinastatin sterling is taken, 30g mannitol is weighed, adds 500ml water for injection to dissolve, is adjusted after mixing
PH6-7, then add and inject water to 2000ml, filter membrane is sterile filtered, and is sub-packed in cillin bottle, is lyophilized up to ulinastatin drug
Composition.
Obtained ulinastatin sterling is diluted to every milliliter of 100,000 unit income Physico-chemical tests:
Heavy metal analysis: it is detected according to 2015 editions the 4th 0821 the second method of heavy metal inspection technique of Chinese Pharmacopoeia.
Molecular weight HPLC method:
(1) liquid-phase condition: Bio-sil exclusion chromatography column；Detection wavelength 280nm；Adjust the guarantor that flow velocity makes bovine serum albumin(BSA)
Staying the time is about 36 minutes.
(2) prepared by mobile phase: take 16.33 grams of potassium dihydrogen phosphates and 124.15 grams of ethylene glycol to dissolve and be diluted to 1000ml,
When necessary with phosphorus acid for adjusting pH to 4.0.
(3) sample preparation: taking ulinastatin, is made in every 1ml with flowing phase dilution containing about the solution of 6500 units, as
Sample solution.
(4) referring to product solution: with the flowing suitable gamma Globulin of phased soln (molecular weight 160,000), bovine serum albumin(BSA)
(molecular weight 67,000), flesh hyperglobulinemia (molecular weight 17,000) and the mixed solution that 1mg/ml is made.
(5) it measures: respectively taking sample solution and carry out high performance liquid chromatography experiment referring to product solution 50ul, referring to product point
The logarithm of son amount is ordinate, prepares standard curve by abscissa of retention time.Sample is acquired according to referring to product standard curve
The molecular weight of product.
Remaining is referring under Chinese Pharmacopoeia 2015 editions the first enlarged editions ulinastatin of page -213 of page 212 and ulinastatin solution item
Detection project is detected, as a result as follows:
| Inspection item | Inspection result |
| Color A405 | 0.15 |
| The content of human urokinase-type peptidase | 0.0003PNAU |
| Heavy metal | 1.0 μ g/ml of < |
| Bacterial endotoxin | < 5.0EU |
| SDS-PAGE molecular weight | 40,312 Da |
| HPLC molecular weight | 67,076Da |
2 ulinastatin of embodiment and preparation method thereof
16kg ammonium nilrite is added in (1) 1 ton of urine, adjusts pH to 4.0, stirs 55min, the ammonium sulfate for being 12% with concentration is molten
Liquid is eluted, and eluent is filtered, and retains filter cake；
(2) filter cake adds 1 times of water dissolution, adjusts pH5.0-6.0,10 times of polyethylene glycol are added, stir 20min, stand, sheet frame mistake
Filter, sediment freeze-drying；
(3) step (2) sediment is dissolved in water, during which 4 DEG C of dialysis are replaced 3 water, are freeze-dried to get ulinastatin crude product；
(4) ulinastatin crude product is dissolved in water, and crosses sephadex G -75, distills water elution, flow velocity 0.4ml/min, and collection is washed
De- component, is added 0.2gEDTA-Na/ liter, the ultrafiltration membrane ultrafiltration for being 30,000 with molecular weight；
(5) 732 type cation-exchange chromatography post loadings are added in step (4) sample, are eluted with 2.0mol/L hydrochloric acid, flow velocity 1ml/
Min collects eluent concentration, is freeze-dried to get ulinastatin sterling.
Wherein, dialysis uses bag filter MD40(6000-8000 in step (3)), flatten width 40mm, diameter 25.5mm.
100,000,000 unit of ulinastatin sterling is taken, 30g mannitol is weighed, adds 500ml water for injection to dissolve, is adjusted after mixing
PH6-7, then add and inject water to 2000ml, filter membrane is sterile filtered, and is sub-packed in cillin bottle, is lyophilized up to ulinastatin drug
Composition.
Obtained ulinastatin sterling is diluted to every milliliter of 100,000 unit income Physico-chemical tests:
Heavy metal analysis: it is detected according to 2015 editions the 4th 0821 the second method of heavy metal inspection technique of Chinese Pharmacopoeia.
Molecular weight HPLC method:
(1) liquid-phase condition: Bio-sil exclusion chromatography column；Detection wavelength 280nm；Adjust the guarantor that flow velocity makes bovine serum albumin(BSA)
Staying the time is about 36 minutes.
(2) prepared by mobile phase: take 16.33 grams of potassium dihydrogen phosphates and 124.15 grams of ethylene glycol to dissolve and be diluted to 1000ml,
When necessary with phosphorus acid for adjusting pH to 4.0.
(3) sample preparation: taking ulinastatin, is made in every 1ml with flowing phase dilution containing about the solution of 6500 units, as
Sample solution.
(4) referring to product solution: with the flowing suitable gamma Globulin of phased soln (molecular weight 160,000), bovine serum albumin(BSA)
(molecular weight 67,000), flesh hyperglobulinemia (molecular weight 17,000) and the mixed solution that 1mg/ml is made.
(5) it measures: respectively taking sample solution and carry out high performance liquid chromatography experiment referring to product solution 50ul, referring to product point
The logarithm of son amount is ordinate, prepares standard curve by abscissa of retention time.Sample is acquired according to referring to product standard curve
The molecular weight of product.
Remaining is referring under Chinese Pharmacopoeia 2015 editions the first enlarged editions ulinastatin of page -213 of page 212 and ulinastatin solution item
Detection project is detected, as a result as follows:
| Inspection item | Inspection result |
| Color A405 | 0.14 |
| The content of human urokinase-type peptidase | 0.0004PNAU |
| Heavy metal | 1.0 μ g/ml of < |
| Bacterial endotoxin | < 5.0EU |
| SDS-PAGE molecular weight | 38,769Da |
| HPLC molecular weight | 67,750 Da |
3 ulinastatin of embodiment and preparation method thereof
13kg ammonium nilrite is added in (1) 1 ton of urine, adjusts pH to 6.0, stirs 50min, the ammonium sulfate for being 8% with concentration
It is eluted, eluent is filtered, and filter cake is retained；
(2) filter cake adds 3 times of water dissolutions, adjusts pH5.0-6.0,12 times of polyethylene glycol are added, stir 40min, stand, sheet frame mistake
Filter, sediment freeze-drying；
(3) step (2) sediment is dissolved in water, during which 4 DEG C of dialysis are replaced 5 water, are freeze-dried to get ulinastatin crude product；
(4) ulinastatin crude product is dissolved in water, and crosses sephadex G -75, distills water elution, flow velocity 0.5ml/min, and collection is washed
De- component, is added 0.4gEDTA-Na/ liter, the ultrafiltration membrane ultrafiltration for being 30,000 with molecular weight；
(5) 732 type cation-exchange chromatography post loadings are added in step (4) sample, are eluted with 3mol/L hydrochloric acid, flow velocity 1ml/min,
Eluent concentration is collected, is freeze-dried to get ulinastatin sterling.
Wherein, dialysis uses bag filter MD40(6000-8000 in step (3)), flatten width 40mm, diameter 25.5mm.
100,000,000 unit of ulinastatin sterling is taken, 30g mannitol is weighed, adds 500ml water for injection to dissolve, is adjusted after mixing
PH6-7, then add and inject water to 2000ml, filter membrane is sterile filtered, and is sub-packed in cillin bottle, is lyophilized up to ulinastatin drug
Composition.
Obtained ulinastatin sterling is diluted to every milliliter of 100,000 unit income Physico-chemical tests:
Heavy metal analysis: it is detected according to 2015 editions the 4th 0821 the second method of heavy metal inspection technique of Chinese Pharmacopoeia.
Molecular weight HPLC method:
(1) liquid-phase condition: Bio-sil exclusion chromatography column；Detection wavelength 280nm；Adjust the guarantor that flow velocity makes bovine serum albumin(BSA)
Staying the time is about 36 minutes.
(2) prepared by mobile phase: take 16.33 grams of potassium dihydrogen phosphates and 124.15 grams of ethylene glycol to dissolve and be diluted to 1000ml,
When necessary with phosphorus acid for adjusting pH to 4.0.
(3) sample preparation: taking ulinastatin, is made in every 1ml with flowing phase dilution containing about the solution of 6500 units, as
Sample solution.
(4) referring to product solution: with the flowing suitable gamma Globulin of phased soln (molecular weight 160,000), bovine serum albumin(BSA)
(molecular weight 67,000), flesh hyperglobulinemia (molecular weight 17,000) and the mixed solution that 1mg/ml is made.
(5) it measures: respectively taking sample solution and carry out high performance liquid chromatography experiment referring to product solution 50ul, referring to product point
The logarithm of son amount is ordinate, prepares standard curve by abscissa of retention time.Sample is acquired according to referring to product standard curve
The molecular weight of product.
Remaining is referring to Chinese Pharmacopoeia 2015 editions the first enlarged editions ulinastatin of page -8213 of page 212 and ulinastatin solution item
Lower detection project is detected, as a result as follows:
| Inspection item | Inspection result |
| Color A405 | 0.16 |
| The content of human urokinase-type peptidase | 0.0003PNAU |
| Heavy metal | 1.0 μ g/ml of < |
| Bacterial endotoxin | < 5.0EU |
| SDS-PAGE molecular weight | 40,706Da |
| HPLC molecular weight | 66,439Da |
The ulinastatin and preparation method thereof that comparative example 1 replaces nitrite to be prepared using chitin
500g chitin is added in (1) 1 ton of urine, stirs 50min, is eluted with the ammonium sulfate that concentration is 10%, takes out
Filter retains filter cake；
(2) filter cake adds 2 times of water dissolutions, adjusts pH5.0-6.0,8 times of polyethylene glycol are added, stir 30min, stand, plate-frame filtering,
Sediment freeze-drying；
(3) step (2) sediment is dissolved in water, during which 4 DEG C of dialysis are replaced 4 water, are freeze-dried to get ulinastatin crude product；
(4) ulinastatin crude product is dissolved in water, and crosses sephadex G -75, distills water elution, flow velocity 0.6ml/min, and collection is washed
De- component, is added 0.3gEDTA-Na/ liter, the ultrafiltration membrane ultrafiltration for being 30,000 with molecular weight；
(5) 732 type cation-exchange chromatography post loadings are added in step (4) sample, are eluted with 1.5mol/L hydrochloric acid, flow velocity 1ml/
Min collects eluent concentration, is freeze-dried to get ulinastatin sterling.
Wherein, dialysis uses bag filter MD40(6000-8000 in step (3)), flatten width 40mm, diameter 25.5mm.
100,000,000 unit of ulinastatin sterling is taken, 30g mannitol is weighed, adds 500ml water for injection to dissolve, is adjusted after mixing
PH6-7, then add and inject water to 2000ml, filter membrane is sterile filtered, and is sub-packed in cillin bottle, is lyophilized up to ulinastatin drug
Composition.
Obtained ulinastatin sterling is diluted to every milliliter of 100,000 unit income Physico-chemical tests:
Heavy metal analysis: it is detected according to 2015 editions the 4th 0821 the second method of heavy metal inspection technique of Chinese Pharmacopoeia.
Molecular weight HPLC method:
(1) liquid-phase condition: Bio-sil exclusion chromatography column；Detection wavelength 280nm；Adjust the guarantor that flow velocity makes bovine serum albumin(BSA)
Staying the time is about 36 minutes.
(2) prepared by mobile phase: take 16.33 grams of potassium dihydrogen phosphates and 124.15 grams of ethylene glycol to dissolve and be diluted to 1000ml,
When necessary with phosphorus acid for adjusting pH to 4.0.
(3) sample preparation: taking ulinastatin, is made in every 1ml with flowing phase dilution containing about the solution of 6500 units, as
Sample solution.
(4) referring to product solution: with the flowing suitable gamma Globulin of phased soln (molecular weight 160,000), bovine serum albumin(BSA)
(molecular weight 67,000), flesh hyperglobulinemia (molecular weight 17,000) and the mixed solution that 1mg/ml is made.
(5) it measures: respectively taking sample solution and carry out high performance liquid chromatography experiment referring to product solution 50ul, referring to product point
The logarithm of son amount is ordinate, prepares standard curve by abscissa of retention time.Sample is acquired according to referring to product standard curve
The molecular weight of product.
Remaining is referring under Chinese Pharmacopoeia 2015 editions the first enlarged editions ulinastatin of page -213 of page 212 and ulinastatin solution item
Detection project is detected, as a result as follows:
| Inspection item | Inspection result |
| Color A405 | 0.19 |
| The content of human urokinase-type peptidase | 0.0005PNAU |
| Heavy metal | 2.0 μ g/ml of < |
| Bacterial endotoxin | < 6.25EU |
| SDS-PAGE molecular weight | 41,141Da |
| HPLC molecular weight | 68,413Da |
Comparative example 2 is without the ulinastatin and preparation method thereof being prepared of dialysing
14.7kg ammonium nilrite is added in (1) 1 ton of urine, adjusts pH to 5.0, stirs 50min, the ammonium sulfate for being 10% with concentration
Solution is eluted, and eluent is filtered, and retains filter cake；
(2) filter cake adds 2 times of water dissolutions, adjusts pH5.0-6.0,8 times of polyethylene glycol are added, stir 30min, stand, plate-frame filtering,
Sediment freeze-drying, obtains ulinastatin crude product；
(3) ulinastatin crude product is dissolved in water, and crosses sephadex G -75, distills water elution, flow velocity 0.6ml/min, and collection is washed
De- component, is added 0.3gEDTA-Na/ liter, the ultrafiltration membrane ultrafiltration for being 30,000 with molecular weight；
(4) 732 type cation-exchange chromatography post loadings are added in step (3) sample, are eluted with 1.5mol/L hydrochloric acid, flow velocity 1ml/
Min collects eluent concentration, is freeze-dried to get ulinastatin sterling.
Obtained ulinastatin sterling is diluted to every milliliter of 100,000 unit income Physico-chemical tests:
Heavy metal analysis: it is detected according to 2015 editions the 4th 0821 the second method of heavy metal inspection technique of Chinese Pharmacopoeia.
Molecular weight HPLC method:
(1) liquid-phase condition: Bio-sil exclusion chromatography column；Detection wavelength 280nm；Adjust the guarantor that flow velocity makes bovine serum albumin(BSA)
Staying the time is about 36 minutes.
(2) prepared by mobile phase: take 16.33 grams of potassium dihydrogen phosphates and 124.15 grams of ethylene glycol to dissolve and be diluted to 1000ml,
When necessary with phosphorus acid for adjusting pH to 4.0.
(3) sample preparation: taking ulinastatin, is made in every 1ml with flowing phase dilution containing about the solution of 6500 units, as
Sample solution.
(4) referring to product solution: with the flowing suitable gamma Globulin of phased soln (molecular weight 160,000), bovine serum albumin(BSA)
(molecular weight 67,000), flesh hyperglobulinemia (molecular weight 17,000) and the mixed solution that 1mg/ml is made.
(5) it measures: respectively taking sample solution and carry out high performance liquid chromatography experiment referring to product solution 50ul, referring to product point
The logarithm of son amount is ordinate, prepares standard curve by abscissa of retention time.Sample is acquired according to referring to product standard curve
The molecular weight of product.
Remaining is referring under Chinese Pharmacopoeia 2015 editions the first enlarged editions ulinastatin of page -213 of page 212 and ulinastatin solution item
Detection project is detected, as a result as follows:
| Inspection item | Inspection result |
| Color A405 | 0.19 |
| The content of human urokinase-type peptidase | 0.0005PNAU |
| Heavy metal | 2.0 μ g/ml of < |
| Bacterial endotoxin | < 6.25EU |
| SDS-PAGE molecular weight | 41,884Da |
| HPLC molecular weight | 68,435Da |
Ulinastatin and preparation method thereof is prepared using anion-exchange column in comparative example 3
14.7kg ammonium nilrite is added in (1) 1 ton of urine, adjusts pH to 5.0, stirs 50min, the ammonium sulfate for being 10% with concentration
Solution is eluted, and eluent is filtered, and retains filter cake；
(2) filter cake adds 2 times of water dissolutions, adjusts pH5.0-6.0,8 times of polyethylene glycol are added, stir 30min, stand, plate-frame filtering,
Sediment freeze-drying；
(3) step (2) sediment is dissolved in water, during which 4 DEG C of dialysis are replaced 4 water, are freeze-dried to get ulinastatin crude product；
(4) ulinastatin crude product is dissolved in water, and crosses sephadex G -75, distills water elution, flow velocity 0.6ml/min, and collection is washed
De- component, is added 0.3gEDTA-Na/ liter, the ultrafiltration membrane ultrafiltration for being 30,000 with molecular weight；
(5) anion exchange chromatography QAE Sephadex A-25 loading is added in step (4) sample, with containing 1mol/LNaCl and
The phosphate buffer of 0.3mol/L elutes, flow velocity 1ml/min, collects eluent concentration, is freeze-dried pure to get ulinastatin
Product.
Wherein, dialysis uses bag filter MD40(6000-8000 in step (3)), flatten width 40mm, diameter 25.5mm.
100,000,000 unit of ulinastatin sterling is taken, 30g mannitol is weighed, adds 500ml water for injection to dissolve, is adjusted after mixing
PH6-7, then add and inject water to 2000ml, filter membrane is sterile filtered, and is sub-packed in cillin bottle, is lyophilized up to ulinastatin drug
Composition.
Obtained ulinastatin sterling is diluted to every milliliter of 100,000 unit income Physico-chemical tests:
Heavy metal analysis: it is detected according to 2015 editions the 4th 0821 the second method of heavy metal inspection technique of Chinese Pharmacopoeia.
Molecular weight HPLC method:
(1) liquid-phase condition: Bio-sil exclusion chromatography column；Detection wavelength 280nm；Adjust the guarantor that flow velocity makes bovine serum albumin(BSA)
Staying the time is about 36 minutes.
(2) prepared by mobile phase: take 16.33 grams of potassium dihydrogen phosphates and 124.15 grams of ethylene glycol to dissolve and be diluted to 1000ml,
When necessary with phosphorus acid for adjusting pH to 4.0.
(3) sample preparation: taking ulinastatin, is made in every 1ml with flowing phase dilution containing about the solution of 6500 units, as
Sample solution.
(4) referring to product solution: with the flowing suitable gamma Globulin of phased soln (molecular weight 160,000), bovine serum albumin(BSA)
(molecular weight 67,000), flesh hyperglobulinemia (molecular weight 17,000) and the mixed solution that 1mg/ml is made.
(5) it measures: respectively taking sample solution and carry out high performance liquid chromatography experiment referring to product solution 50ul, referring to product point
The logarithm of son amount is ordinate, prepares standard curve by abscissa of retention time.Sample is acquired according to referring to product standard curve
The molecular weight of product.
Remaining is referring under Chinese Pharmacopoeia 2015 editions the first enlarged editions ulinastatin of page -213 of page 212 and ulinastatin solution item
Detection project is detected, as a result as follows:
| Inspection item | Inspection result |
| Color A405 | 0.20 |
| The content of human urokinase-type peptidase | 0.0005PNAU |
| Heavy metal | 2.0 μ g/ml of < |
| Bacterial endotoxin | < 6.25EU |
| SDS-PAGE molecular weight | 42,351Da |
| HPLC molecular weight | 68,759 Da |
Ulinastatin and preparation method thereof is prepared without using gel filtration chromatography in comparative example 4
14.7kg ammonium nilrite is added in (1) 1 ton of urine, adjusts pH to 5.0, stirs 50min, the ammonium sulfate for being 10% with concentration
Solution is eluted, and eluent is filtered, and retains filter cake；
(2) filter cake adds 2 times of water dissolutions, adjusts pH5.0-6.0,8 times of polyethylene glycol are added, stir 30min, stand, plate-frame filtering,
Sediment freeze-drying；
(3) step (2) sediment is dissolved in water, during which 4 DEG C of dialysis are replaced 4 water, are freeze-dried to get ulinastatin crude product；
(4) ulinastatin crude product is dissolved in water, and 0.3gEDTA-Na/ liter is added, the ultrafiltration membrane ultrafiltration for being 30,000 with molecular weight；
(5) 732 type cation-exchange chromatography post loadings are added in step (4) sample, are eluted with 2mol/L hydrochloric acid, flow velocity 1ml/min,
Collect eluent；
Wherein, dialysis uses bag filter MD40(6000-8000 in step (3)), flatten width 40mm, diameter 25.5mm.
100,000,000 unit of ulinastatin sterling is taken, 30g mannitol is weighed, adds 500ml water for injection to dissolve, is adjusted after mixing
PH6-7, then add and inject water to 2000ml, filter membrane is sterile filtered, and is sub-packed in cillin bottle, is lyophilized up to ulinastatin drug
Composition.
Obtained ulinastatin sterling is diluted to every milliliter of 100,000 unit income Physico-chemical tests:
Heavy metal analysis: it is detected according to 2015 editions the 4th 0821 the second method of heavy metal inspection technique of Chinese Pharmacopoeia.
Molecular weight HPLC method:
(1) liquid-phase condition: Bio-sil exclusion chromatography column；Detection wavelength 280nm；Adjust the guarantor that flow velocity makes bovine serum albumin(BSA)
Staying the time is about 36 minutes.
(2) prepared by mobile phase: take 16.33 grams of potassium dihydrogen phosphates and 124.15 grams of ethylene glycol to dissolve and be diluted to 1000ml,
When necessary with phosphorus acid for adjusting pH to 4.0.
(3) sample preparation: taking ulinastatin, is made in every 1ml with flowing phase dilution containing about the solution of 6500 units, as
Sample solution.
(4) referring to product solution: with the flowing suitable gamma Globulin of phased soln (molecular weight 160,000), bovine serum albumin(BSA)
(molecular weight 67,000), flesh hyperglobulinemia (molecular weight 17,000) and the mixed solution that 1mg/ml is made.
(5) it measures: respectively taking sample solution and carry out high performance liquid chromatography experiment referring to product solution 50ul, referring to product point
The logarithm of son amount is ordinate, prepares standard curve by abscissa of retention time.Sample is acquired according to referring to product standard curve
The molecular weight of product.
Remaining is referring under Chinese Pharmacopoeia 2015 editions the first enlarged editions ulinastatin of page -213 of page 212 and ulinastatin solution item
Detection project is detected, as a result as follows:
| Inspection item | Inspection result |
| Color A405 | 0.19 |
| The content of human urokinase-type peptidase | 0.0005PNAU |
| Heavy metal | 2.0 μ g/ml of < |
| Bacterial endotoxin | < 6.25EU |
| SDS-PAGE molecular weight | 37,764Da |
| HPLC molecular weight | 63,673Da |
The different ulinastatin of embodiment 4 compare
According to official method measurement ulinastatin than living, while comparing the yield and its purity of ulinastatin preparation method.
。
Above-mentioned detailed description is illustrating for one of them possible embodiments of the present invention, the embodiment not to
The scope of the patents of the invention is limited, all equivalence enforcements or change without departing from carried out by the present invention are intended to be limited solely by the technology of the present invention
In the range of scheme.
Claims (9)
1. a kind of ulinastatin, which is characterized in that when the ulinastatin concentration is 100,000 units/ml, the extinction at 405nm
Angle value is no more than 0.18, and the content of human urokinase-type peptidase is no more than 0.0005PNAU, and content of beary metal is no more than 1.6 μ g/ml, carefully
Bacterium endotoxin content is no more than 6.0EU.
2. ulinastatin according to claim 1, which is characterized in that when the ulinastatin concentration is 100,000 units/ml,
Absorbance value at 405nm is no more than 0.16, and the content of human urokinase-type peptidase is no more than 0.0004PNAU, and content of beary metal is not
More than 1.0 μ g/ml, bacteria endotoxin content is no more than 5.0EU.
3. ulinastatin according to claim 1, which is characterized in that the ulinastatin is poly- using lauryl sodium sulfate
The molecular weight of acrylamide gel electrophoresis measurement is 37,000-43,000Da or the molecular weight using high effective liquid chromatography for measuring
For 62,000-72,000Da.
4. a kind of preparation method of any one of claim 1-3 ulinastatin, comprising the following steps:
(1) nitrite is added in urine, adjusts pH, stirring, ammonium sulfate is eluted, and eluent is filtered, and is retained
Filter cake；
(2) filter cake is dissolved in water, and adjusts pH, and polyethylene glycol, filtering, drying precipitate is added；
(3) step (2) sediment is dissolved in water, dialysis, dry to get ulinastatin crude product；
(4) ulinastatin crude product is dissolved in water, and crosses gel filtration chromatography, distills water elution, collects component, ultrafiltration；
(5) cation-exchange chromatography post loading is added in step (4) sample, is eluted with hydrochloric acid, collects eluent, is freeze-dried, i.e.,
Obtain ulinastatin sterling；
Wherein, gel filtration chromatography filler is sephadex G -75, step (5) the middle-jiao yang, function of the spleen and stomach ion exchange in the step (4)
Column is 732 type cation exchange resins.
5. the preparation method according to claim 4, which is characterized in that the weight of urine and nitrite in the step (1)
Amount is than being 1 ton: 13-16kg.
6. the preparation method according to claim 4, which is characterized in that ammonium sulfate concentration is 8- in the step (1)
12%。
7. the preparation method according to claim 4, which is characterized in that filter cake adds 1-3 times of water dissolution in the step (2),
PH5.0-6.0 is adjusted, 8-12 times of polyethylene glycol is added, stirs 20-40min, is stood, plate-frame filtering, sediment freeze-drying.
8. the preparation method according to claim 4, which is characterized in that gel filtration chromatography elution speed in the step (4)
For 0.4-0.6ml/min.
9. preparation side described in a kind of pharmaceutical composition, any one of 1-3 containing claim ulinastatin or claim 4-8
The ulinastatin that method is prepared is as active constituent.
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