CN101831009A - Process for producing concentrated and purified heparin - Google Patents
Process for producing concentrated and purified heparin Download PDFInfo
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- CN101831009A CN101831009A CN201010168046A CN201010168046A CN101831009A CN 101831009 A CN101831009 A CN 101831009A CN 201010168046 A CN201010168046 A CN 201010168046A CN 201010168046 A CN201010168046 A CN 201010168046A CN 101831009 A CN101831009 A CN 101831009A
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Abstract
The invention discloses a process for producing concentrated and purified heparin, which comprises the following steps of: separating, concentrating and purifying heparin, impure protein, nucleic acid and rest sulfated polysaccharide at one time in solution of a coarse heparin product by using expansion column bed technology. The process changes the traditional process finished by multiple steps of a salt hydrolysis-ion exchange resin method. Fine heparin is separated, concentrated and purified by adopting the expansion column bed and ion exchange resin process, sedimentation is performed by adopting ethanol, dehydration is performed by adopting acetone, and the potency of the heparin is more than 180IU/mg. The expansion column bed and ion exchange resin process reduces the working procedure, shortens the time, lowers the cost, and improves the efficiency and quality.
Description
[technical field]
The present invention relates to a kind of production technique of concentrated and purified heparin, especially a kind of production technique that adopts expansion column bed ion-exchange absorption chromatographic technique concentrated and purified heparin.
[background technology]
A kind of acidic mucopolysaccharide of from liver organization, finding when heparin (heparin) is Mclean research clotting mechanism in 1916.Nineteen thirty-nine, Brinkhous etc. have proved that heparin has anticoagulant active, and from then on, heparin is subjected to the attention of countries in the world as natural anticoagulative substance.Heparin extensively is distributed in the mammiferous tissue, as liver, lung, intestinal mucosa, the heart, spleen, kidney, thymus gland, placenta, muscle and blood etc.Heparin is a kind of glycosaminoglycan, the mixture of the polysaccharide chain of forming by uronic acid and glucosamine with different chain length, and molecular weight is between 3000-30000D, about the about 12000D of molecular-weight average.
Because the heparin structure complexity, heparin mainly extracts from natural product and obtains at present, still can't carry out synthetic.
Since Charles in 1933 and Scott had set up the industrial principle of heparin of alkali salt water extraction and trysinization, the heparin production technique had had very big improvement.People keep on improving on the method for extracting, and innovation to some extent, as picric acid method and Potassium ethanoate acidic precipitation method etc.
50 to the sixties, and the heparin production patent of various countries is a lot, and is comparatively outstanding with states such as American and Britain, breast, victory, day, West Germany.Since last century the seventies, the most significant new development of heparin technology is to use aminated compounds and ion exchange technique purifying tissue extract.Heparin exists with the form of polyanion (unique critical salt concn and high charge density are arranged) in solution, therefore can utilize these characteristics or heparin is precipitated out from the aqueous solution with various positively charged ions (comprising amine) combination, thereby perhaps make heparin and anionite take place to exchange to reach to separate, cost is lower, output is higher and advantages of simple operation and the latter more has.And abroad now be devoted to the improvement of raw material treatment process and the preparation of the high heparin of tiring.
In recent years, China extensively and in depth studied and had formed several more advanced production technique with national characteristics.In China, the preparation process of heparin is divided two stages, i.e. the production of crude product and refining.The production of crude product mainly is to adopt ion-exchange-resin process, and what have carries out the removal of impurity and concentrated in conjunction with ultra-filtration technique.Heparin is fully extracted and prevents its destructurized be one of key of improving the heparin yield, so the performance of ion exchange resin and technology are the keys of decision yield.The treating process of heparin is decon and decolouring.Process for purification is divided into oxidation style and non-oxide method.Oxidation style mainly is divided into potassium permanganate oxidation method and hydrogen peroxide oxidation process.The domestic oxidation style that mostly adopts is decoloured to heparin at present, and this process for purification produces destruction to the structure of heparin more or less, and what wherein have the greatest impact is oxidation processes.The product that clearly need to propose non-oxide decolouring that abroad has at present.
At present, to be equipped with heparin method relatively commonly used be ion-exchange-resin process to non-oxide legal system.The performance of ion exchange resin and technology are the keys of decision yield, choose the treatment process and the treatment time of resin, make its exchange of carrying out heparin to greatest extent absorption.Domestic ion-exchange-resin process generally adopts the method for absorption in batches, and upper prop, removal of impurities, wash-out separately carry out.This method post is imitated low, and big to the resin infringement, operation steps is many, the expense height, and the activeconstituents extraction yield is low.
[summary of the invention]
Therefore, being devoted to the improvement of raw material treatment process and the preparation of the high heparin of tiring is a direction of the diligent research of various countries' heparin investigator in decades, along with development of biology, constantly has new technology to be found and is applied to heparin preparation technology.
Expansion column bed adsorption chromatography adapts to the demand of extensive downstream purification and the new chromatographic technique developed just.Its principle is that the adsorption medium particle expands under the effect of the flowing fluid that makes progress, the adsorption medium particle suspends in the bed body uniformly, and form gradient and continuous arrangement is arranged according to different particle sizes and density, form an equilibrium, stable absorption environment, target heparin in the liquid is adsorbed on the medium, and cell, cell debris, foreign protein, nucleic acid and mixed polysaccharide and other fragment of tissue will spill out by pillar, the other way around adsorption medium is compressed during wash-out target heparin, with elution buffer the target heparin is washed again.Expansion column bed chromatographic technique has the advantage of fluidized-bed and packed bed concurrently, does not need to remove the particle in the feed liquid in advance and can directly adsorb target product from feed liquid.Thereby play effective fractionation by adsorption effect.
Expansion column bed ion-exchange chromatography is the new technology that the expansion column bed technique is used for ion-exchange chromatography.Not only can be used for directly extracting heparin with expansion column bed ion-exchange chromatography, also can be used for the refining of crude product heparin from internal organs homogenates such as mammiferous intestinal mucosa, liver, lungs.Adopt expansion column bed ion-exchange chromatography directly to extract heparin and with crude product heparin refinement heparin, greatly reduce the complicacy of heparin purifying process, improved productive rate, reduced production cost from Mammals internal organs homogenate.Changing in the production of the refining heparin of domestic resin method in the past, all is to adopt the method for absorption in batches basically.The absorption method theoretical plate number is low in batches, and post is imitated loss greatly, and purification efficiency is low, and abundant inadequately to molecules of interest absorption, therefore loss is big.Adopt expansion column bed ion-exchange chromatography to extract heparin, heparin can be separated with impurity albumen, nucleic acid and all the other sulfated polysaccharides, concentrate, purifying is once finished.Reduce operation, the shortening time, reduce cost, raise the efficiency and quality.Adopt expansion column bed ion-exchange chromatography directly from homogenate, to extract heparin, heparin unit tires and reaches more than the 120U/mg, uses the crude product refining heparin, and tiring reaches more than the 160U/mg, (260nm 280nm) all satisfies the pharmacopeia requirement to product middle ultraviolet band impurity.
The inventor is through research repeatedly, and by repeatedly experiment, successfully expansion column bed ion exchange technique is used for the separation purifying technique of heparin, up to now, also do not find the report of the extraction process of any and the similar heparin of the present invention.
Now these contents briefly are described below:
Patent of the present invention adopts expansion column bed ion exchange resin method that the crude product heparin is carried out concentrated and purified reaching and makes with extra care.Change traditional in the past employing oxidation style refining to heparin structure destruction and cause the refined biometric activity low, it is big that poor product quality and adopt in batches ion-exchange-resin process abundant inadequately to molecules of interest absorption, post are imitated loss, complex operation step, the shortcoming that purification efficiency is low.
The present invention separates heparin in the solution of crude product heparin with impurity albumen, nucleic acid and all the other sulfated polysaccharides, concentrate, and purifying is once finished.
The present invention reduces operation, the shortening time, reduce cost, and raise the efficiency and quality.Reduced the consumption of organic solvent in producing to a great extent, and extraction yield is more stable, the crude product heparin solution adopts expansion column bed ion exchange resin method to carry out concentrated and purified and refining, the contaminating fluid of not only having eliminated oxidation style and having produced during the ion-exchange-resin process mass production in batches, and reduce cost, significantly shorten the production cycle, the production technology maturation is applicable to big production, the efficiency of investment height, cost is low, remarkable benefit.
The present invention both can be used for extracting the crude product heparin in the tissue extract of mammiferous mucous membrane of small intestine such as pig, ox, sheep or internal organs such as liver, lung, can be used for the refining of crude product heparin again.
Get the mammiferous mucous membrane of small intestine such as pig, ox, sheep of some amount or the tissue extract or the crude product heparin of internal organs such as liver, lung and be made into certain density salt alkali aqueous solution earlier, trypsin digestion, high-temperature denatured, after the treating processess such as filtration, adopt expansion column bed ion exchange resin technology to extracting solution separation, concentrated, purifying, ethanol sedimentation, acetone dehydration, cryodrying.The present technique product is crude product and elaboration raw material heparin, reaches product standard through detecting every index.
The crude product preparation:
Add water to 250L about the tissue homogenate 50g of mammiferous mucous membrane of small intestine such as pig, ox, sheep or internal organs such as liver, lung, add NaCI to 1%-8%, stir, leave standstill, filter.The tissue homogenate that intestinal mucosa filtrate adds 0.1-0.5% trypsin liver lungs adds the 0.1-0.5% papoid), at pH7.5-10.5,35-45 ℃ of following enzymolysis a few hours, NaOH transfers pH to 8.0-10.0, is warming up to 50-55 ℃, insulation 2-4h, constantly stir, be rapidly heated 92-95 ℃ then, keep 10-15min, filter with 80 mesh filter screens while hot, filtrate (I) is standby.The strongly basic anion exchange resin that expansion column Streamline50 (5cm internal diameter), (available from AmershamPharmacia Biotech (Sweden)) pack into and handled well.Controlled temperature 40-60 ℃ is 7.5-10.5 with peristaltic pump with pH, and an amount of MTris-HCl damping fluid pumps into from post bed bottom, makes the resin expansion and keeps balance.Keep the pH value constant, an amount of filtrate (I) is added the MTris-HCl damping fluid, and to be mixed with NaCI be 2-6%, pH is the solution of 7.5-10.5, with first balance resin in the logical p-adic extension p exchange column of peristaltic pump, carry out ion-exchange then repeatedly, flow velocity is about per hour 2 times of column volumes, till effluent liquid is checked no heparin, all is adsorbed on the resin.Prepare an amount of 2-6% sodium chloride solution then, the MTris-HCl damping fluid is regulated pH3-5, behind damping fluid balance/expansion column bed with 2～3 deposition bed volumes, damping fluid removal of impurities with 5-6 times of deposition bed volume, whole process control temp 15-25 ℃, flow velocity is about per hour 2.5 column volumes.Turn off peristaltic pump, make the D254 resin redeposited, regulating piston carries out wash-out with the downward liquid stream and the state of deposition bed then to just on the surface of deposition bed.Resin elder generation carries out wash-out with the sodium chloride solution of the 3-8% of about 4 times of column volumes, collects elutriant.The 2-7% sodium chloride solution (consumption earlier many back few) of using about 2 times of column volumes again is with method wash-out 2 times.Merge whole elutriants, regulating pH is 6.Press the pre-cooled ethanol that effluent volume adds 1.0 times of amounts, precipitation is spent the night.Absorb ethanol next day.Throw out dissolves with 2% sodium chloride solution, transfers pH to 6.0.Add 0.7 times of ethanol by liquor capacity, precipitation is spent the night, and siphon next day supernatant ethanol takes out throw out.Throw out is in regular turn with dehydrated alcohol, washing with acetone dehydration.Get heparin after the vacuum-drying, tire to more than the 120U/mg.
The elaboration preparation:
To tire to the heparin crude product about 60-100U/mg is dissolved in 1: 10 ratio in 3% the sodium chloride solution, and then transfer pH to 8-11 with 2% sodium hydroxide solution, leave standstill a few hours in room temperature, vacuum filtration removes throw out, and filtrate is stand-by.In above-mentioned clear liquid, add 0.01-0.1% trypsinase, at pH7-10,35-45 ℃ of enzymolysis a few hours, intensification 98-100 ℃, 10-15min, vacuum filtration while hot, filtrate for later use.The strongly basic anion exchange resin that expansion column Streamline50 (5cm internal diameter), (available from Amersham PharmaciaBiotech (Sweden)) pack into and handled well, with peristaltic pump with pH7-10, an amount of MTris-HCl damping fluid pumps into from post bed bottom, makes the resin expansion and keeps balance.Controlled temperature 40-60 ℃, it is the solution of 7.5-10.5 that an amount of filtrate is added MTris-HCl damping fluid preparation pH, with carrying out ion-exchange repeatedly in the logical p-adic extension p exchange column of peristaltic pump, keep pH value and homo(io)thermism in the exchange process, flow velocity is about per hour 2 times of column volumes, till effluent liquid is checked no heparin, all is adsorbed on the resin.Prepare the 2-6% sodium chloride solution of an amount of precooling then, the MTris-HCl damping fluid is regulated pH1-3, behind damping fluid balance/expansion column bed with 2～3 deposition bed volumes, damping fluid removal of impurities with 5-6 times of deposition bed volume, whole process control temp 2-5 ℃, flow velocity is about per hour 2.5 column volumes.Turn off peristaltic pump, make resin redeposited, regulating piston carries out wash-out with the downward liquid stream and the state of deposition bed then to just on the surface of deposition bed.Resin carries out wash-out with the 2-6% sodium chloride solution with about 4 times of column volumes earlier, collects elutriant.The 2-6% sodium chloride solution (consumption earlier many back few) of using about 2 times of column volumes again is with method wash-out 2 times.Merge whole elutriants, regulating pH is 6.Press the pre-cooled ethanol that effluent volume adds 1.0 times of amounts, precipitation is spent the night.Absorb ethanol next day.Throw out dissolves with 2% sodium chloride solution, transfers pH to 6.0.Add 0.7 times of ethanol by liquor capacity, precipitation is spent the night, and siphon next day supernatant ethanol takes out throw out.Throw out is in regular turn with dehydrated alcohol, washing with acetone dehydration.Get the elaboration heparin after the vacuum-drying, tire to more than the 150U/mg.
Expansion column bed ion exchange resin and ordinary salt are separated-comparison of ion exchange resin characteristics:
Process characteristic:
| Relative merits relatively | Salt is separated-ion exchange method | Expansion column bed ion exchange resin method |
| The post pre-treatment | Must be through acid, alkaline purification and filtration, active ingredient loss is big, and especially the acidolysis foreign protein destroys one sulfate of the N on the heparin molecule, makes the heparin inactivation, and the loss of tiring is serious. | The direct upper prop of enzymolysis processing, good impurity removing effect, step is few, and heparin activity is destroyed hardly. |
| Resins exchange is handled | Batch treatment under the post, upper prop, removal of impurities, wash-out uses a large amount of alkali salt water, and environmental pollution is big, the cost height, the rate of recovery of tiring is low, and resin is broken | Upper prop, removal of impurities, wash-out is all handled on post, and post is imitated high, and the alkali salt water consumption is few, the resin long service life. |
| Bad big. | ||
| Resin regeneration | Post is handled down, and solvent consumes big, and resin is easily broken.Treatment time is long. | Regenerate on the post, save 2/3 solvent, resin life is long.Treatment time is short. |
| Treatment step | Many, about 11 steps | Few, about 7 steps |
Quality control index:
| The crude product heparin | Product tire (IU/mg) | The rate of recovery of tiring (%) | ??OD260nm | ??OD280nm |
| Salt is separated-ion exchange method | ??150 | ??92.3 | ??0.17 | ??0.09 |
| Expansion column bed ion exchange resin method | ??187 | ??97.8 | ??0.11 | ??0.05 |
From as can be seen above, expansion column bed method than salt separate-ion exchange method technology still is the production that quality control index all helps heparin, the heparin that adopts expansion column bed ion exchange resin method to produce more has the market competitiveness.
Above-mentioned is the expansion column bed ion exchange resin technological process of production.
[embodiment 1]
A) add water to 250L about the tissue homogenate 50g of mammiferous mucous membrane of small intestine such as pig, ox, sheep, add NaCI to 3%, stir, leave standstill, filter.
B) filtrate adds 0.25% trypsinase, and at pH8.5,40 ℃ of following enzymolysis a few hours, NaOH transfers pH to 9.0, be warming up to 50 ℃, insulation 2h constantly stirs, and is rapidly heated 92-95 ℃ then, keep 10-15min, filter with 80 mesh filter screens while hot, filtrate (I) is standby.
C) get an amount of strong basicity D254 dried resin, fully soak in distilled water, filter is done after the swelling, adds equal-volume ethanol or acetone and stirs 1 hour, cleans filter with distilled water and does, and the hydrochloric acid soln that adds the 2mol/l of 4 times of amounts stirred 2 hours, was washed till neutrality with distilled water, and filter is done; The sodium hydroxide solution that adds 2 times of amount 2mol/l stirred 2 hours, and distilled water is washed till neutrality, and filter is done; Hydrochloric acid soln with 2 times of amount 2mol/l stirred 2 hours at last, was washed to neutrality.
D) get strong basicity D254 resin that 100g handled well and add expansion column Streamline50 (5cm internal diameter), be that 8.0 an amount of MTris-HCl damping fluid (II) pumps into from post bed bottom with pH with the flow velocity of 2 times of column volumes per hour, make resin expansion and maintenance balance with peristaltic pump.40 ℃ of controlled temperature (balance).
E) with an amount of filtrate (I) with damping fluid (II), be configured to feed liquid (III).Adjust feed liquid (III) NaCI concentration and reach about 3%, NaOH transfers pH to 8.0.With carrying out ion-exchange repeatedly in the logical p-adic extension p exchange column of feed liquid (III), keep constant pH value with peristaltic pump in the exchange process, temperature is 18 ℃.Flow velocity is about per hour 2.5 times of column volumes, (goes up sample) till effluent liquid is checked no heparin, all is adsorbed on the resin.，
F) 6% sodium chloride solution of 10 column volumes of preparation, the MTris-HCl damping fluid is regulated pH3-5, behind damping fluid balance/expansion column bed with 2～3 deposition bed volumes, damping fluid removal of impurities with 5-6 times of deposition bed volume, whole process control temp 15-25 ℃, flow velocity is about per hour 2.5 column volumes.(upwards flushing, removal of impurities).
G) turn off peristaltic pump, make the D254 resin redeposited, regulating piston carries out wash-out with the downward liquid stream and the state of deposition bed then to just on the surface of deposition bed.Resin carries out wash-out with 4% sodium chloride solution with about 4 times of column volumes earlier, collects elutriant.3% sodium chloride solution (consumption earlier many back few) of using about 2 times of column volumes again is with method wash-out 2 times.Merge whole elutriants, regulating pH is 6 (wash-outs).
H) press the pre-cooled ethanol that effluent volume adds 1.0 times of amounts, precipitation is spent the night.Absorb ethanol next day.Throw out dissolves with 2% sodium chloride solution, transfers pH to 6.0.Add 0.7 times of ethanol by liquor capacity, precipitation is spent the night.
I) siphon next day supernatant ethanol takes out throw out.Throw out is in regular turn with dehydrated alcohol, washing with acetone dehydration.Get the heparin finished product after the vacuum-drying.
[embodiment 2]
A) 10g heparin crude product is dissolved in 3% the 100ml sodium chloride solution, and then transfers pH to 9, left standstill 6 hours in 18 ℃ with 2% sodium hydroxide solution, vacuum filtration, filtrate is stand-by.
B) in above-mentioned clear liquid, add 0.03% trypsinase, at pH8.5,40 ℃ of following enzymolysis 4 hours, intensification 98-100 ℃, 10min, vacuum filtration while hot, filtrate (I) is standby.
C) get an amount of strong basicity D254 dried resin, fully soak in distilled water, filter is done after the swelling, adds equal-volume ethanol or acetone and stirs 1 hour, cleans filter with distilled water and does, and the hydrochloric acid soln that adds the 2mol/l of 4 times of amounts stirred 2 hours, was washed till neutrality with distilled water, and filter is done; The sodium hydroxide solution that adds 2 times of amount 2mol/l stirred 2 hours, and distilled water is washed till neutrality, and filter is done; Hydrochloric acid soln with 2 times of amount 2mol/l stirred 2 hours at last, was washed to neutrality.
D) get strong basicity D254 resin that 30g handled well and add expansion column Streamline50 (5cm internal diameter), be that 8.0 an amount of MTris-HCl damping fluid (II) pumps into from post bed bottom with pH with the tasselled of 2 times of column volumes per hour, make resin expansion and maintenance balance (balance) with peristaltic pump.
E) adjustment pH value 9 is constant, with damping fluid (II), makes the crude product sodium chloride concentration reach about 3% (III) an amount of filtrate (I).With carrying out ion-exchange repeatedly in the logical p-adic extension p exchange column of feed liquid (III), keep constant pH value with peristaltic pump in the exchange process, keep 20 ℃ of temperature.Flow velocity is about per hour 2 times of column volumes, (goes up sample) till effluent liquid is checked no heparin, all is adsorbed on the resin.
F) 6% sodium chloride solution of the precooling of about 10 column volumes of preparation, regulate pH1.2 with the MTris-HCl damping fluid, behind damping fluid balance/expansion column bed, with the damping fluid removal of impurities of 5-6 times of deposition bed volume, 5 ℃ of whole process control temps are below the degree, and flow velocity is about per hour 2.5 column volumes.(upwards flushing).
G) turn off peristaltic pump, make the D254 resin redeposited, regulating piston carries out wash-out with the downward liquid stream and the state of deposition bed then to just on the surface of deposition bed.Resin is that 8 4% sodium chloride solution carries out wash-out, the collection elutriant with the pH with about 4 times of column volumes earlier.3% sodium chloride solution (consumption earlier many back few) of using about 2 times of column volumes again is with method wash-out 2 times.Merge whole elutriants, regulating pH is 6 (wash-outs).
H) press the pre-cooled ethanol that effluent volume adds 1.0 times of amounts, precipitation is spent the night.Absorb ethanol next day.Throw out dissolves with 2% sodium chloride solution, transfers pH to 6.0.Add 0.7 times of ethanol by liquor capacity, precipitation is spent the night.
I) siphon next day supernatant ethanol takes out throw out.Throw out is in regular turn with dehydrated alcohol, washing with acetone dehydration.Get the elaboration heparin after the vacuum-drying.
[embodiment 3]
A) add water to 250L about the tissue homogenate 30g of internal organs such as mammiferous liver such as pig, ox, sheep, lung, add NaCI to 5%, stir, leave standstill, filter.
B) in above-mentioned clear liquid, add 0.4% papoid, at pH8.5,40 ℃ of following enzymolysis 4 hours, intensification 98-100 ℃, 10 minutes, vacuum filtration while hot, filtrate (I) is standby.
C) get an amount of strong basicity D254 dried resin, fully soak in distilled water, filter is done after the swelling, adds equal-volume ethanol or acetone and stirs 1 hour, cleans filter with distilled water and does, and the hydrochloric acid soln that adds the 2mol/l of 4 times of amounts stirred 2 hours, was washed till neutrality with distilled water, and filter is done; The sodium hydroxide solution that adds 2 times of amount 2mol/l stirred 2 hours, and distilled water is washed till neutrality, and filter is done; Hydrochloric acid soln with 2 times of amount 2mol/l stirred 2 hours at last, was washed to neutrality.
D) get strong basicity D254 resin that 100g handled well and add expansion column Streamline50 (5cm internal diameter), be that 8.0 an amount of MTris-HCl damping fluid (II) pumps into from post bed bottom with pH with the flow velocity of 2 times of column volumes per hour, make resin expansion and maintenance balance with peristaltic pump.40 ℃ of controlled temperature (balance).
E) with an amount of filtrate (I) with damping fluid (II), be configured to feed liquid (III).Adjust feed liquid (III) NaCI concentration and reach about 3%, NaOH transfers pH to 8.0.With carrying out ion-exchange repeatedly in the logical p-adic extension p exchange column of feed liquid (III), keep constant pH value with peristaltic pump in the exchange process, temperature is 18 ℃.Flow velocity is about per hour 2.5 times of column volumes, (goes up sample) till effluent liquid is checked no heparin, all is adsorbed on the resin.
F) 6% sodium chloride solution of 10 column volumes of preparation, the MTris-HCl damping fluid is regulated pH3-5, behind damping fluid balance/expansion column bed with 2～3 deposition bed volumes, damping fluid removal of impurities with 5-6 times of deposition bed volume, whole process control temp 15-25 ℃, flow velocity is about per hour 2.5 column volumes.(upwards flushing, removal of impurities).
G) turn off peristaltic pump, make the D254 resin redeposited, regulating piston carries out wash-out with the downward liquid stream and the state of deposition bed then to just on the surface of deposition bed.Resin carries out wash-out with 4% sodium chloride solution with about 4 times of column volumes earlier, collects elutriant.3% sodium chloride solution (consumption earlier many back few) of using about 2 times of column volumes again is with method wash-out 2 times.Merge whole elutriants, regulating pH is 6 (wash-outs).
H) press the pre-cooled ethanol that effluent volume adds 1.0 times of amounts, precipitation is spent the night.Absorb ethanol next day.Throw out dissolves with 2% sodium chloride solution, transfers pH to 6.0.Add 0.7 times of ethanol by liquor capacity, precipitation is spent the night.
I) siphon next day supernatant ethanol takes out throw out.Throw out is in regular turn with dehydrated alcohol, washing with acetone dehydration.Get the heparin finished product after the vacuum-drying.
Claims (8)
1. the production technique of a concentrated and purified heparin, its characteristics are, adopt expansion column bed ion exchange resin method the extracting solution of the tissue homogenate of internal organs such as mammiferous mucous membrane of small intestine such as pig, ox, sheep, liver, lungs can be adopted directly without pre-treatment that chromatographic technique separates, concentrates, the production technology of purified heparin, also can be used for the refining of crude product heparin, changed and used the restriction that can not contain insoluble particle in traditional ion-exchange fixed leg bed adsorption technology stock liquid.Its principle is that the adsorption medium particle expands under the effect of the flowing fluid that makes progress, the adsorption medium particle suspends in the bed body uniformly, and form gradient and continuous arrangement is arranged according to different particle sizes and density, form an equilibrium, stable absorption environment, target heparin in the liquid is adsorbed on the medium, and cell, cell debris, foreign protein, nucleic acid and mixed polysaccharide and other fragment of tissue will spill out by pillar, the other way around adsorption medium is compressed during wash-out target heparin, with elution buffer the target heparin is washed again.Expansion column bed chromatographic technique has the advantage of fluidized-bed and packed bed concurrently, does not need to remove the particle in the feed liquid in advance and can directly adsorb target product from feed liquid.Thereby play effective fractionation by adsorption effect.Expansion column bed ion-exchange chromatography not only can be used for directly extracting heparin from Mammals internal organs homogenate, also can be used for the refining of crude product heparin.Adopt expansion column bed ion-exchange chromatography directly to extract heparin and with crude product heparin refinement heparin, greatly reduce the complicacy of heparin purifying process, improved productive rate, reduced production cost from Mammals internal organs homogenate.Changing in the production of the refining heparin of domestic resin method in the past, all is to adopt the method for absorption in batches basically.The absorption method theoretical plate number is low in batches, and post is imitated loss greatly, and purification efficiency is low, and abundant inadequately to molecules of interest absorption, therefore loss is big.Adopt expansion column bed ion-exchange chromatography to extract heparin, heparin can be separated with impurity albumen, nucleic acid and all the other sulfated polysaccharides, concentrate, purifying is once finished.Reduce operation, the shortening time, reduce cost, raise the efficiency and quality.Adopt expansion column bed ion-exchange chromatography directly from homogenate, to extract heparin, heparin unit tires and reaches more than the 120U/mg, uses the crude product refining heparin, and tiring reaches more than the 160U/mg, (260nm 280nm) all satisfies the pharmacopeia requirement to product middle ultraviolet band impurity;
Technology contents:
(1) homogenate of Mammals mucous membrane of small intestine is through alkali salt hydrolysis, trypsin digestion (tissue homogenate of mammiferous liver lungs such as pig, cattle and sheep adds the 0.1-0.5% papoid), high-temperature denatured and filtration treatment, through under certain ionic concn and PH condition, carrying out the absorption of expansion column bed adsorption chromatography, concentrated, purified heparin, ethanol sedimentation, the acetone dehydration, cryodrying.
(2) the crude product heparin is through alkali salt water and trypsin treatment, high-temperature denatured and filtration treatment, after through under certain ionic concn and PH condition, carrying out the absorption of expansion column bed adsorption chromatography, concentrating, purified heparin, ethanol sedimentation, acetone dehydration, cryodrying.Technical process
The crude product preparation:
A) add water to 250L about the tissue homogenate 50g of mammiferous mucous membrane of small intestine such as pig, ox, sheep or internal organs such as liver, lung, add NaCI to 1%-8%, stir, leave standstill, filter.The tissue homogenate that intestinal mucosa filtrate adds 0.1-0.5% trypsin liver lungs adds the 0.1-0.5% papoid).
B) at pH7.5-10.5,35-45 ℃ of following enzymolysis a few hours, NaOH transfers pH to 8.0-10.0, is warming up to 50-55 ℃, and insulation 2-4h constantly stirs, and is rapidly heated 92-95 ℃ then, keeps 10-15min, and with the filtration of 80 mesh filter screens, (I) is standby for filtrate while hot.
C) expansion column Streamline50 (5cm internal diameter), (available from Amersham Pharmacia Biotech (the Sweden)) strongly basic anion exchange resin of packing into and having handled well.Controlled temperature 40-60 ℃ is 7.5-10.5 with peristaltic pump with pH, and an amount of MTris-HCl damping fluid pumps into from post bed bottom, makes the resin expansion and keeps balance.
Keep the pH value constant, an amount of filtrate (I) is added the MTris-HCl damping fluid, and to be mixed with NaCI be 2-6%, pH is the solution of 7.5-10.5, with first balance resin in the logical p-adic extension p exchange column of peristaltic pump, carry out ion-exchange then repeatedly, flow velocity is about per hour 2 times of column volumes, till effluent liquid is checked no heparin, all is adsorbed on the resin.
D) prepare an amount of 2-6% sodium chloride solution then, the MTris-HCl damping fluid is regulated pH3-5, behind damping fluid balance/expansion column bed with 2～3 deposition bed volumes, damping fluid removal of impurities with 5-6 times of deposition bed volume, whole process control temp 15-25 ℃, flow velocity is about per hour 2.5 column volumes.
E) turn off peristaltic pump, make the D254 resin redeposited, regulating piston carries out wash-out with the downward liquid stream and the state of deposition bed then to just on the surface of deposition bed.Resin elder generation carries out wash-out with the sodium chloride solution of the 3-8% of about 4 times of column volumes, collects elutriant.The 2-7% sodium chloride solution (consumption earlier many back few) of using about 2 times of column volumes again is with method wash-out 2 times.Merge whole elutriants, regulating pH is 6.
F) press the pre-cooled ethanol that effluent volume adds 1.0 times of amounts, precipitation is spent the night.
G) absorb ethanol next day.Throw out dissolves with 2% sodium chloride solution, transfers pH to 6.0.Add 0.7 times of ethanol by liquor capacity, precipitation is spent the night.
H) siphon next day supernatant ethanol takes out throw out.Throw out is in regular turn with dehydrated alcohol, washing with acetone dehydration.Get heparin after the vacuum-drying, tire to more than the 120U/mg.
The elaboration preparation:
A) will tire to the heparin crude product about 60-100U/mg is dissolved in 1: 10 ratio in 3% the sodium chloride solution, and then transfer pH to 8-11 with 2% sodium hydroxide solution, and leave standstill a few hours in room temperature, vacuum filtration removes throw out, and filtrate is stand-by.
B) in above-mentioned clear liquid, add 0.01-0.1% trypsinase, at pH7-10,35-45 ℃ of enzymolysis a few hours, intensification 98-100 ℃, 10-15min, vacuum filtration while hot, filtrate for later use.
C) expansion column Streamline50 (5cm internal diameter), (available from Amersham Pharmacia Biotech (the Sweden)) strongly basic anion exchange resin of packing into and having handled well, with peristaltic pump with pH7-10, an amount of MTris-HCl damping fluid pumps into from post bed bottom, makes the resin expansion and keeps balance.
D) controlled temperature 40-60 ℃, it is the solution of 7.5-10.5 that an amount of filtrate is added MTris-HCl damping fluid preparation pH, with carrying out ion-exchange repeatedly in the logical p-adic extension p exchange column of peristaltic pump, keep pH value and homo(io)thermism in the exchange process, flow velocity is about per hour 2 times of column volumes, till effluent liquid is checked no heparin, all is adsorbed on the resin.
E) prepare the 2-6% sodium chloride solution of an amount of precooling then, the MTris-HCl damping fluid is regulated pH1-3, behind damping fluid balance/expansion column bed with 2～3 deposition bed volumes, damping fluid removal of impurities with 5-6 times of deposition bed volume, whole process control temp 2-5 ℃, flow velocity is about per hour 2.5 column volumes.
F) turn off peristaltic pump, make resin redeposited, regulating piston carries out wash-out with the downward liquid stream and the state of deposition bed then to just on the surface of deposition bed.Resin carries out wash-out with the 2-6% sodium chloride solution with about 4 times of column volumes earlier, collects elutriant.The 2-6% sodium chloride solution (consumption earlier many back few) of using about 2 times of column volumes again is with method wash-out 2 times.Merge whole elutriants, regulating pH is 6.
G) press the pre-cooled ethanol that effluent volume adds 1.0 times of amounts, precipitation is spent the night.
H) absorb ethanol next day.Throw out dissolves with 2% sodium chloride solution, transfers pH to 6.0.Add 0.7 times of ethanol by liquor capacity, precipitation is spent the night,
I) siphon next day supernatant ethanol takes out throw out.Throw out is in regular turn with dehydrated alcohol, washing with acetone dehydration.Get the elaboration heparin after the vacuum-drying, tire to more than the 150U/mg;
According to claim 1: the raw material of expansion column bed ion-exchange chromatography is internal organs and crude product heparin such as mammiferous mucous membrane of small intestine such as pig, ox, sheep, liver, lungs;
2. the production technique of concentrated and purified heparin as claimed in claim 1 is characterized in that expansion column bed ion-exchange chromatography is used for the processing of crude product and the preparation process of elaboration.
3. the production technique of concentrated and purified heparin as claimed in claim 1 is characterized in that expansion column bed ion-exchange chromatography uses strongly basic anion exchange resin as adsorption medium.
4. the production technique of concentrated and purified heparin as claimed in claim 1 is characterized in that the heparin that expansion column bed ion-exchange chromatography is handled, and can be the salt of any heparin such as sodium salt, sylvite, calcium salt, lithium salts.
5. the production technique of concentrated and purified heparin as claimed in claim 1 is characterized in that expansion column bed chromatographic technique is adopted in the processing of this technology heparin.
6. the production technique of concentrated and purified heparin as claimed in claim 1 is characterized in that expansion column bed and ion-exchange resin technique are adopted in the processing of this technology heparin.
7. the production technique of concentrated and purified heparin as claimed in claim 1 is characterized in that this technology uses the further enzymolysis foreign protein of trypsinase in the further treating processes of heparin.
8. the production technique of concentrated and purified heparin as claimed in claim 1 is characterized in that this technology adopts the NaCl of different concns and Mtris-HCI damping fluid to be used to reach sample, removal of impurities, is eluted in the operation and finishes.
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| CN104098717A (en) * | 2014-07-21 | 2014-10-15 | 南通恒阳生物科技有限公司 | Method for extracting heparin sodium |
| CN104558248A (en) * | 2014-12-29 | 2015-04-29 | 青岛九龙生物医药有限公司 | Production technology of concentrated purified heparin sodium |
| WO2018032502A1 (en) * | 2016-08-19 | 2018-02-22 | 苏州融析生物科技有限公司 | Sheep-derived low molecular weight heparin, preparation method therefor and application thereof |
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