CN105753974A - Ulinastatin purification method based on affinity chromatography column - Google Patents
Ulinastatin purification method based on affinity chromatography column Download PDFInfo
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- C07K14/81—Protease inhibitors
- C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
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Abstract
The invention relates to a ulinastatin purification method based on an affinity chromatography column.The method comprises the following steps of urine processing, hydrophobic interaction column chromatography, affinity chromatography, gel filtration chromatography, washing and freeze-drying, and through a purification test on the finally obtained ulinastatin, it is found that the total yield of the ulinastatin is increased to 85% or above, and the total titer of the ulinastatin is 6500 iu/mg or above; the chromatography column is adopted for purification treatment, operation is easy, the production efficiency is improved through optimized packing and parameters such as the flow velocity, the production cost is greatly reduced, and further development and utilization values are achieved.
Description
Technical field
The present invention relates to biological technical field, particularly to a kind of ulinastatin purification process based on affinity column.
Background technology
Ulinastatin, have another name called human urine trypsin inhibitor (HumanUrinaryTrypsinInhibitor), being a kind of trypsin inhibitor separating purification from human urine, 1909, Beurer and Reich first time reported and there is this trypsin inhibitor in human urine.He is by a kind of glycoprotein being made up of 143 aminoacid, and N end is alanine, and C end is leucine, has sugar chain on the 10th serine and 45 aspartic acids.O-glycosides chain on serine comprises multiple chondroitin sulfate unit (5 Ch4S and 10 Ch0S).The molecular weight of ulinastatin is determined as 60～70KD through HPLC method, is determined as 40～50KD through SDS-PAGE, and isoelectric point, IP is 2.6, is a kind of heat stable acidic protein.
Multiple hydrolytic enzyme is had effective inhibitory action by ulinastatin, and cell membrane, lysosome membrane are had obvious Stabilization.Ulinastatin is not only a kind of enzyme inhibitor in human body, still participate in the important biological active substances of cell expression-secretion regulation and control simultaneously, under the morbid states such as inflammation, ulinastatin is discharged in blood by granulocyte enzyme hydrolysis, participate in the regulation and control of inflammatory reaction, limitation inflammatory reaction, helps the reparation of tissue；Meet with huge blow at body, when there is systemic inflammatory response, alleviate the damage that body tissue is caused by the inflammatory mediator of excessively release.Ulinastatin is used clinically for the treatment of acute pancreatitis, acute circulatory disorder, and the prevention etc. of systemic inflammatory reaction, multiple organ dysfunction syndrome in the Organoprotective of big-and-middle-sized average of operation periods and critical illness.
About the purification technique of ulinastatin, domestic have been reported such as:
Chinese patent CN103073638B discloses a kind of method of affinitive layer purification ulinastatin, a kind of method that it is disclosed that affinitive layer purification ulinastatin.Specifically, it is exactly with NAM's freshly voided urine for raw material, highly purified ulinastatin is prepared through the modern protein high-end bio-chemistry separation technology such as chitin absorption, ammonia eluting, ammonium sulfate precipitation, adsorpting column chromatography and affinity chromatograph, total recovery can being brought up to more than 70%, total titer is not less than 5000iu/mg.Containing part of nickel ion after affinity chromatograph.
Chinese patent CN100528898 discloses a kind of high-purity ulinastatin and preparation method thereof and the pharmaceutical composition containing ulinastatin, then through drainage column absorption and affinity chromatograph column purification after urine therein is treated, and utilize metal chelating column and metalloprotein to combine, eluting is carried out by phosphate buffer, ulinastatin is made to be able to purification, although to obtain the technology content that the ulinastatin of certain purity however it is necessary that higher.
Chinese patent CN105384812 discloses a kind of resin regeneration and improves the method for post effect purification ulinastatin and improve the pharmaceutical composition of ulinastatin solubility, the method being directed to purification ulinastatin, it is specially through the modern protein high-end bio-chemistry separation technology such as chitin absorption, ammonia eluting, ammonium sulfate precipitation, adsorpting column chromatography and affinity chromatograph to prepare highly purified ulinastatin, total recovery can be brought up to more than 70%, total titer is not less than 5000iu/mg, and post effect promotes about 50%.The technology content needed is higher.
Chinese patent CN105399819 discloses a kind of method of affinitive layer purification ulinastatin and improves the pharmaceutical composition of ulinastatin stability, highly purified ulinastatin is prepared through the modern protein high-end bio-chemistry separation technology such as chitin absorption, ammonia eluting, ammonium sulfate precipitation, adsorpting column chromatography and affinity chromatograph, total recovery can being brought up to more than 70%, total titer is not less than 5000iu/mg.Yield is relatively low.
Current Wu Sita purification process can not meet existing requirement, and therefore removing these impurity, to improve the safety of ulinastatin clinical practice further be very necessary.
Summary of the invention:
In view of the defect of prior art, a kind of method that the invention provides affinitive layer purification ulinastatin, in method based on affinity chromatograph, add hydrophobic chromatography and gel permeation chromatography carrys out purification ulinastatin crude product.The ulinastatin yield, the ratio that finally give are lived and are better than prior art, and every biochemical indicator of lyophilizing ulinastatin finished product is superior to Chinese Pharmacopoeia regulation.
For solving above-mentioned technical problem, specifically, process of the present invention is such.
A kind of ulinastatin purification process based on affinity column, affinity chromatograph is selected to carry out purification ulinastatin, described affinity chromatograph is preferably HiTraprProteinAFF or HiTraprProteinAHF chromatographic column and ProteinASepharoseCL-4B or ProteinASepharose4FF filler, finding that through purification experiment the total recovery of ulinastatin brings up to more than 80%, total titer is at more than 6000iu/mg.Especially HiTraprProteinAFF chromatographic column, the embodiment 1 of ProteinASepharoseCL-4B filler ulinastatin and the total recovery of embodiment 10 are 87.5% and 89.4, total titer is 6920iu/mg and 6987iu/mg, and every biochemical indicator of freeze-drying prods ulinastatin is superior to Chinese Pharmacopoeia requirement.
Ulinastatin purification process of the present invention, comprises the following steps:
(1) weigh the appropriate pH clarification urine less than 6.5, add appropriate chitosan, and measure with accurate pH test paper, regulate urine pH to 5.0-6.0；Adsorb with ammonia eluting 1.0-2.0h after completely, then through 3-4kg ammonium sulfate precipitation, stand, precipitate, namely obtain ulinastatin crude product after drying；
(2) hydrophobic chromatography, to ulinastatin crude product pretreatment, uses balance liquid A balance, controls loading speed and with level pad A multiple equilibria, uses eluent B eluting；
(3) affinity chromatograph, uses balance liquid A balance, controls loading speed and with level pad A multiple equilibria, uses eluent C eluting；
(4) gel permeation chromatography, uses balance liquid D balance, controls loading speed and use eluent E eluting；
(5) gel permeation chromatography eluent is through micro-filtration membrane microfiltration to 230mL, micro-filtrate adds alkaline solution 770mL, then microfiltration is to 230mL, then 770mL alkaline solution solution is added, then through ultrafilter membrane ultrafiltration to 230mL, then add 770mL alkaline solution solution, then ultrafiltration is to 57.5mL；
(6) washing: the nanofiltration liquid of step (5) is centrifuged, solid dehydrated alcohol dehydration twice, centrifugal, abandoning supernatant, leave and take precipitate；
(7) lyophilizing: the precipitate loading dish of step (6) is entered freeze dryer, and namely lyophilizing obtains ulinastatin.
Balance liquid A described in step of the present invention (2) is 10mmol/LHAc-NaAc buffer, pH6.0, and reinforcing body sodium chloride regulates cd60ms/cm；Eluent B is 10mmol/LTris-HCl buffer, pH8.5.
Balance liquid C described in step of the present invention (3) is 10mmol/LTris-HCl buffer, pH8.0.
Balance liquid D described in step of the present invention (4) is 10mmol/LHAc-NaAc, pH6.0；Eluent E is: 10mmol/LHAc-NaAc, pH6.0.
Further, ulinastatin purification process of the present invention, comprise the following steps:
(1) weigh the 1TpH clarification urine less than 6.5, be stirred continuously, be slowly added to 10kg chitosan, and measure with accurate pH test paper, use acetic acid to regulate urine pH to 6.0；With ammonia eluting 1.5h after absorption completely, then through 3.5kg ammonium sulfate precipitation, stand 24 hours, vacuum drying in precipitation, the centrifugal vacuum desiccator being placed on anhydrous magnesium sulfate water absorbing agent, namely obtain ulinastatin crude product after drying；
(2) hydrophobic chromatography (XK50/20 chromatographic column, PhenylSepharose6FastFlow (highsub) filler)
Pretreatment: weigh ulinastatin crude product 1kg, dissolves with 10mmol/LHAc-NaAc, stirs 30 minutes, centrifugal 20 minutes, leaves and takes centrifugal liquid, adjusts pH to 6.0 with acetic acid, and reinforcing body ammonium sulfate regulates cd to 60ms/cm；
Balance: using level pad (reinforcing body sodium chloride regulates cd60ms/cm for 10mmol/LHAc-NaAc buffer, pH6.0), adjustment flow rate pump is 20.0ml/min, balances 8 times of column volumes；
Loading: with 100ml/min flow velocity loading, after end of the sample, with level pad (reinforcing body ammonium sulfate regulates cd60ms/cm for 10mmol/LHAc-NaAc buffer, pH6.0) 5 times of bed volumes of multiple equilibria；
Eluting: using eluent (10mmol/LTris-HCl buffer, pH8.5) eluting, adjustment flow rate pump is 20.0ml/min, starts eluting, collects eluent.
(3) affinity chromatograph (HiTraprProteinAFF chromatographic column, ProteinASepharoseCL-4B filler) is embodied as step and is:
Balance: with level pad (the 10mmol/LHAc-NaAc buffer of 4.4L, pH6.0, reinforcing body sodium chloride regulates cd60ms/cm) balance affinity column, hydrophobic chromatography eluent is flowed through equilibrated chromatographic column, flow speed control is at 100mL/min；
Loading: with the purpose solution of 100ml/min flow velocity loading hydrophobic chromatography, after end of the sample, with level pad (reinforcing body ammonium sulfate regulates cd60ms/cm for 10mmol/LHAc-NaAc buffer, pH6.0) 5 times of column volumes of multiple equilibria；
Eluting: using eluent (10mmol/LTris-HCl buffer, pH8.0) eluting, adjustment flow rate pump is 100ml/min, starts eluting, collects eluent.
(4) gel permeation chromatography (HR16/70 chromatographic column, Sephacryls-100HR filler) is embodied as step and is:
Balance: balancing 8 column volumes with HAc-NaAc buffer (10mmol/LHAc-NaAc, pH6.0), during balance, flow rate pump is 3.0ml/min；
Loading: affinitive layer purification eluent is added appropriate alkaline solution, and supernatant loading, during loading, flow rate pump is 3.0ml/min, and sample applied sample amount is the 2% of column volume；
Eluting: after loading, with HAc-NaAc liquid eluting (10mmol/LHAc-NaAc, pH6.0), adjustment flow rate pump is 3.0ml/min, collects eluent.
(5) eluent ultrafiltration is to 57.5mL；
(6) washing: the ultrafiltrate of step (5) is centrifuged, solid dehydrated alcohol dehydration twice, centrifugal, abandoning supernatant, leave and take precipitate；
(7) lyophilizing: the precipitate loading dish of step (6) is entered freeze dryer, and namely lyophilizing obtains ulinastatin.
Alkaline solution described in above-mentioned steps (4) is the one in sodium hydroxide, potassium hydroxide, ammonia, histidine, lysine or arginine, it is preferred to the one in ammonia, histidine.
Ultrafilter membrane includes the ultrafilter membrane that molecular weight is 5000-3 ten thousand.
What the present invention obtained has the advantages that:
(1) purification process of the present invention is mainly with the purification of ulinastatin.
(2) have employed column chromatography to process, simple to operate, and preferably the parameter such as filler and flow velocity improves production efficiency, reduces production cost, has the value of exploitation further.
(3) by testing confirmation, it will be seen that the yield of the ulinastatin of embodiment 1 and embodiment 10 in the embodiment of the present invention 11, specific activity is better than other embodiments, and by dissolved freeze-dried powder, the stability of embodiment 1 and embodiment 10 is above other embodiments.
Detailed description of the invention
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, and when without departing substantially from the spirit of the present invention or basic feature, it is possible to realize the present invention in other specific forms.Therefore, no matter from which point, embodiment all should be regarded as exemplary, and be nonrestrictive, the scope of the invention rather than described above limits, it is intended that all changes in the implication of the equivalency dropping on claim and scope included in the present invention.
Embodiment 1:
(1) weigh the 1TpH clarification urine less than 6.5, be stirred continuously, be slowly added to 10kg chitosan, and measure with accurate pH test paper, use acetic acid to regulate urine pH to 6.0；With ammonia eluting 1.5h after absorption completely, then through 3.5kg ammonium sulfate precipitation, stand 24 hours, vacuum drying in precipitation, the centrifugal vacuum desiccator being placed on anhydrous magnesium sulfate water absorbing agent, namely obtain ulinastatin crude product after drying；
(2) hydrophobic chromatography (XK50/20 chromatographic column, PhenylSepharose6FastFlow (highsub) filler)
Pretreatment: weigh ulinastatin crude product 1kg, dissolves with 10mmol/LHAc-NaAc, stirs 30 minutes, centrifugal 20 minutes, leaves and takes centrifugal liquid, adjusts pH to 6.0 with acetic acid, and reinforcing body ammonium sulfate regulates cd to 60ms/cm；
Balance: using level pad (reinforcing body sodium chloride regulates cd60ms/cm for 10mmol/LHAc-NaAc buffer, pH6.0), adjustment flow rate pump is 20.0ml/min, balances 8 times of column volumes；
Loading: with 100ml/min flow velocity loading, after end of the sample, with level pad (reinforcing body ammonium sulfate regulates cd60ms/cm for 10mmol/LHAc-NaAc buffer, pH6.0) 5 times of bed volumes of multiple equilibria；
Eluting: using eluent (10mmol/LTris-HCl buffer, pH8.5) eluting, adjustment flow rate pump is 20.0ml/min, starts eluting, collects eluent.
(3) affinity chromatograph (HiTraprProteinAFF chromatographic column, ProteinASepharoseCL-4B filler) is embodied as step and is:
Balance: with level pad (the 10mmol/LHAc-NaAc buffer of 4.4L, pH6.0, reinforcing body sodium chloride regulates cd60ms/cm) balance affinity column, hydrophobic chromatography eluent is flowed through equilibrated chromatographic column, flow speed control is at 100mL/min；
Loading: with the purpose solution of 100ml/min flow velocity loading hydrophobic chromatography, after end of the sample, with 5 times of column volumes of level pad (10mmol/LHAc-NaAc buffer, pH6.0) multiple equilibria；
Eluting: using eluent (10mmol/LTris-HCl buffer, pH8.5) eluting, adjustment flow rate pump is 100ml/min, starts eluting, collects eluent.
(4) gel permeation chromatography (HR16/70 chromatographic column Sephacryls-100HR filler) is embodied as step and is:
Balance: balancing 8 column volumes with HAc-NaAc buffer (10mmol/LHAc-NaAc, pH6.0), during balance, flow rate pump is 3.0ml/min；
Loading: affinitive layer purification eluent adds the ammonia of 1ml18%, and supernatant loading, during loading, flow rate pump is 3.0ml/min, and sample applied sample amount is the 2% of column volume；
Eluting: after loading, with HAc-NaAc liquid eluting (10mmol/LHAc-NaAc, pH6.0), adjustment flow rate pump is 3.0ml/min, collects eluent.
(5) eluent is through micro-filtration membrane microfiltration to 230mL, adds sodium bicarbonate solution 770mL in micro-filtrate, and then microfiltration is to 230mL, then 770mL sodium bicarbonate solution is added, then through ultrafilter membrane ultrafiltration to 230mL, then add 770mL sodium bicarbonate solution, then ultrafiltration is to 57.5mL；
(6) washing: the nanofiltration liquid of step (5) is centrifuged, solid dehydrated alcohol dehydration twice, centrifugal, abandoning supernatant, leave and take precipitate；
(7) lyophilizing: the precipitate loading dish of step (6) is entered freeze dryer, and namely lyophilizing obtains ulinastatin.
Embodiment 2:
(1) weigh the 1TpH clarification urine less than 6.5, be stirred continuously, be slowly added to 10kg chitosan, and measure with accurate pH test paper, use acetic acid to regulate urine pH to 6.0；With ammonia eluting 1.5h after absorption completely, then through 3.5kg ammonium sulfate precipitation, stand 24 hours, vacuum drying in precipitation, the centrifugal vacuum desiccator being placed on anhydrous magnesium sulfate water absorbing agent, namely obtain ulinastatin crude product after drying；
(2) hydrophobic chromatography (XK50/20 chromatographic column, PhenylSepharose6FastFlow (highsub) filler)
Pretreatment: weigh ulinastatin crude product 1kg, dissolves with 10mmol/LHAc-NaAc, stirs 30 minutes, centrifugal 20 minutes, leaves and takes centrifugal liquid, adjusts pH to 6.0 with acetic acid, and reinforcing body ammonium sulfate regulates cd to 60ms/cm；
Balance: using level pad (reinforcing body sodium chloride regulates cd60ms/cm for 10mmol/LHAc-NaAc buffer, pH6.0), adjustment flow rate pump is 20.0ml/min, balances 8 times of column volumes；
Loading: with 100ml/min flow velocity loading, after end of the sample, with level pad (reinforcing body ammonium sulfate regulates cd60ms/cm for 10mmol/LHAc-NaAc buffer, pH6.0) 5 times of bed volumes of multiple equilibria；
Eluting: using eluent (10mmol/LTris-HCl buffer, pH8.5) eluting, adjustment flow rate pump is 20.0ml/min, starts eluting, collects eluent.
(3) gel permeation chromatography (HR16/70 chromatographic column Sephacryls-100HR filler) is embodied as step and is:
Balance: balancing 8 column volumes with HAc-NaAc buffer (10mmol/LHAc-NaAc, pH6.0), during balance, flow rate pump is 3.0ml/min；
Loading: affinitive layer purification eluent adds the ammonia of 1ml18%, and supernatant loading, during loading, flow rate pump is 3.0ml/min, and sample applied sample amount is the 2% of column volume；
Eluting: after loading, with HAc-NaAc liquid eluting (10mmol/LHAc-NaAc, pH6.0), adjustment flow rate pump is 3.0ml/min, collects eluent.
(4) eluent is through micro-filtration membrane microfiltration to 230mL, adds sodium bicarbonate solution 770mL in micro-filtrate, and then microfiltration is to 230mL, then 770mL sodium bicarbonate solution is added, then through ultrafilter membrane ultrafiltration to 230mL, then add 770mL sodium bicarbonate solution, then ultrafiltration is to 57.5mL；
(5) washing: the nanofiltration liquid of step (5) is centrifuged, solid dehydrated alcohol dehydration twice, centrifugal, abandoning supernatant, leave and take precipitate；
(6) lyophilizing: the precipitate loading dish of step (6) is entered freeze dryer, and namely lyophilizing obtains ulinastatin.
Embodiment 3:
(1) weigh the 1TpH clarification urine less than 6.5, be stirred continuously, be slowly added to 10kg chitosan, and measure with accurate pH test paper, use acetic acid to regulate urine pH to 6.0；With ammonia eluting 1.5h after absorption completely, then through 3.5kg ammonium sulfate precipitation, stand 24 hours, vacuum drying in precipitation, the centrifugal vacuum desiccator being placed on anhydrous magnesium sulfate water absorbing agent, namely obtain ulinastatin crude product after drying；
(2) hydrophobic chromatography (XK50/20 chromatographic column, PhenylSepharose6FastFlow (highsub) filler)
Pretreatment: weigh ulinastatin crude product 1kg, dissolves with 10mmol/LHAc-NaAc, stirs 30 minutes, centrifugal 20 minutes, leaves and takes centrifugal liquid, adjusts pH to 6.0 with acetic acid, and reinforcing body ammonium sulfate regulates cd to 60ms/cm；
Balance: using level pad (reinforcing body ammonium sulfate regulates cd60ms/cm for 10mmol/LHAc-NaAc buffer, pH6.0), adjustment flow rate pump is 20.0ml/min, balances 8 times of column volumes；
Loading: with 100ml/min flow velocity loading, after end of the sample, with level pad (reinforcing body ammonium sulfate regulates cd60ms/cm for 10mmol/LHAc-NaAc buffer, pH6.0) 5 times of bed volumes of multiple equilibria；
Eluting: using eluent (10mmol/LTris-HCl buffer, pH8.5) eluting, adjustment flow rate pump is 20.0ml/min, starts eluting, collects eluent.
(3) affinity chromatograph (HiTraprProteinAHF chromatographic column, ProteinASepharose4FF filler) is embodied as step and is:
Balance: with level pad (the 10mmol/LHAc-NaAc buffer of 4.4L, pH6.0, reinforcing body ammonium sulfate regulates cd60ms/cm) balance affinity column, hydrophobic chromatography eluent is flowed through equilibrated chromatographic column, flow speed control is at 100mL/min；
Loading: with the purpose solution of 100ml/min flow velocity loading hydrophobic chromatography, after end of the sample, with 5 times of column volumes of level pad (10mmol/LHAc-NaAc buffer, pH6.0) multiple equilibria；
Eluting: using eluent (10mmol/LTris-HCl buffer, pH8.5) eluting, adjustment flow rate pump is 100ml/min, starts eluting, collects eluent.
(4) gel permeation chromatography (HR16/70 chromatographic column Sephacryls-100HR filler) is embodied as step and is:
Balance: balancing 8 column volumes with HAc-NaAc buffer (10mmol/LHAc-NaAc, pH6.0), during balance, flow rate pump is 3.0ml/min；
Loading: affinitive layer purification eluent adds the ammonia of 1ml18%, and supernatant loading, during loading, flow rate pump is 3.0ml/min, and sample applied sample amount is the 2% of column volume；
Eluting: after loading, with HAc-NaAc liquid eluting (10mmol/LHAc-NaAc, pH6.0), adjustment flow rate pump is 3.0ml/min, collects eluent.
(5) eluent is through ultrafilter membrane microfiltration to 230mL, adds sodium bicarbonate solution 770mL in ultrafiltrate, and then ultrafiltration is to 230mL, then adds 770mL sodium bicarbonate solution, then ultrafiltration is to 230mL；
(6) washing: the ultrafiltrate of step (5) is centrifuged, solid dehydrated alcohol dehydration twice, centrifugal, abandoning supernatant, leave and take precipitate；
(7) lyophilizing: the precipitate loading dish of step (6) is entered freeze dryer, and namely lyophilizing obtains ulinastatin.
Embodiment 4:
(1) weigh the 1TpH clarification urine less than 6.5, be stirred continuously, be slowly added to 10kg chitosan, and measure with accurate pH test paper, use acetic acid to regulate urine pH to 6.0；With ammonia eluting 1.5h after absorption completely, then through 3.5kg ammonium sulfate precipitation, stand 24 hours, vacuum drying in precipitation, the centrifugal vacuum desiccator being placed on anhydrous magnesium sulfate water absorbing agent, namely obtain ulinastatin crude product after drying；
(2) hydrophobic chromatography (XK50/20 chromatographic column, PhenylSepharose6FastFlow (highsub) filler)
Pretreatment: weigh ulinastatin crude product 1kg, dissolves with 10mmol/LHAc-NaAc, stirs 30 minutes, centrifugal 20 minutes, leaves and takes centrifugal liquid, adjusts pH to 6.0 with acetic acid, and reinforcing body ammonium sulfate regulates cd to 60ms/cm；
Balance: using level pad (reinforcing body ammonium sulfate regulates cd60ms/cm for 10mmol/LHAc-NaAc buffer, pH6.0), adjustment flow rate pump is 20.0ml/min, balances 8 times of column volumes；
Loading: with 100ml/min flow velocity loading, after end of the sample, with level pad (reinforcing body ammonium sulfate regulates cd60ms/cm for 10mmol/LHAc-NaAc buffer, pH6.0) 5 times of bed volumes of multiple equilibria；
Eluting: using eluent (10mmol/LTris-HCl buffer, pH8.5) eluting, adjustment flow rate pump is 20.0ml/min, starts eluting, collects eluent.
(3) affinity chromatograph (HiTraprProteinAHF chromatographic column, ProteinASepharose4FF filler) is embodied as step and is:
Balance: with level pad (the 10mmol/LHAc-NaAc buffer of 4.4L, pH6.0, reinforcing body ammonium sulfate regulates cd60ms/cm) balance affinity column, hydrophobic chromatography eluent is flowed through equilibrated chromatographic column, flow speed control is at 100mL/min；
Loading: with the purpose solution of 100ml/min flow velocity loading hydrophobic chromatography, after end of the sample, with 5 times of column volumes of level pad (10mmol/LHAc-NaAc buffer, pH6.0) multiple equilibria；
Eluting: using eluent (10mmol/LTris-HCl buffer, pH8.5) eluting, adjustment flow rate pump is 100ml/min, starts eluting, collects eluent.
(4) gel permeation chromatography (HR16/70 chromatographic column Sephacryls-100HR filler) is embodied as step and is:
Balance: balancing 8 column volumes with HAc-NaAc buffer (10mmol/LHAc-NaAc, pH6.0), during balance, flow rate pump is 3.0ml/min；
Loading: affinitive layer purification eluent adds the ammonia of 1ml18%, and supernatant loading, during loading, flow rate pump is 3.0ml/min, and sample applied sample amount is the 2% of column volume；
Eluting: after loading, with HAc-NaAc liquid eluting (10mmol/LHAc-NaAc, pH6.0), adjustment flow rate pump is 3.0ml/min, collects eluent.
(5) eluent is through micro-filtration membrane microfiltration to 230mL, adds sodium bicarbonate solution 770mL in micro-filtrate, and then microfiltration is to 230mL, then 770mL sodium bicarbonate solution is added, then through ultrafilter membrane ultrafiltration to 230mL, then add 770mL sodium bicarbonate solution, then ultrafiltration is to 57.5mL；
(6) washing: the nanofiltration liquid of step (5) is centrifuged, solid dehydrated alcohol dehydration twice, centrifugal, abandoning supernatant, leave and take precipitate；
(7) lyophilizing: the precipitate loading dish of step (6) is entered freeze dryer, and namely lyophilizing obtains ulinastatin.
Embodiment 5:
(1) weigh the 1TpH clarification urine less than 6.5, be stirred continuously, be slowly added to 10kg chitosan, and measure with accurate pH test paper, use acetic acid to regulate urine pH to 6.0；With ammonia eluting 1.5h after absorption completely, then through 3.5kg ammonium sulfate precipitation, stand 24 hours, vacuum drying in precipitation, the centrifugal vacuum desiccator being placed on anhydrous magnesium sulfate water absorbing agent, namely obtain ulinastatin crude product after drying；
(2) hydrophobic chromatography (XK50/20 chromatographic column, PhenylSepharose6FastFlow (highsub) filler)
Pretreatment: weigh ulinastatin crude product 1kg, dissolves with 10mmol/LHAc-NaAc, stirs 30 minutes, centrifugal 20 minutes, leaves and takes centrifugal liquid, adjusts pH to 6.0 with acetic acid, and reinforcing body ammonium sulfate regulates cd to 60ms/cm；
Balance: using level pad (reinforcing body ammonium sulfate regulates cd60ms/cm for 10mmol/LHAc-NaAc buffer, pH6.0), adjustment flow rate pump is 20.0ml/min, balances 8 times of column volumes；
Loading: with 100ml/min flow velocity loading, after end of the sample, with level pad (reinforcing body ammonium sulfate regulates cd60ms/cm for 10mmol/LHAc-NaAc buffer, pH6.0) 5 times of bed volumes of multiple equilibria；
Eluting: using eluent (10mmol/LTris-HCl buffer, pH8.5) eluting, adjustment flow rate pump is 20.0ml/min, starts eluting, collects eluent.
(3) affinity chromatograph (HiTraprProteinAFF chromatographic column, ProteinASepharoseCL-4B filler) is embodied as step and is:
Balance: with level pad (the 10mmol/LHAc-NaAc buffer of 4.4L, pH6.0, reinforcing body ammonium sulfate regulates cd60ms/cm) balance affinity column, hydrophobic chromatography eluent is flowed through equilibrated chromatographic column, flow speed control is at 100mL/min；
Loading: with the purpose solution of 100ml/min flow velocity loading hydrophobic chromatography, after end of the sample, with 5 times of column volumes of level pad (10mmol/LHAc-NaAc buffer, pH6.0) multiple equilibria；
Eluting: using eluent (10mmol/LTris-HCl buffer, pH8.5) eluting, adjustment flow rate pump is 100ml/min, starts eluting, collects eluent.
(4) Cationic column chromatography (XK50/30 chromatographic column, Source30S filler) is embodied as step and is:
Sample pretreatment: protein solution after gained hydrophobic chromatography, adjusts pH to 5.0 with acetic acid, and being diluted with water to cd is 4ms/cm；
Balance: balancing 8 times of column volumes with level pad (10mmol/LHAc-NaAc, pH6.0), during balance, flow rate pump is 80.0ml/min；
Loading: during loading, flow rate pump is 25ml/min, with 5 bed volumes of level pad (10mmol/LHAc-NaAc, pH6.0) multiple equilibria after end of the sample；
Eluting: after loading, with elution buffer eluting (10mmol/LTris-HCl buffer, pH8.5), adjustment flow rate pump is 30ml/min, collects eluent.
(5) gel permeation chromatography (HR16/70 chromatographic column Sephacryls-100HR filler) is embodied as step and is:
Balance: balancing 8 column volumes with HAc-NaAc buffer (10mmol/LHAc-NaAc, pH6.0), during balance, flow rate pump is 3.0ml/min；
Loading: affinitive layer purification eluent adds the ammonia of 1ml18%, and supernatant loading, during loading, flow rate pump is 3.0ml/min, and sample applied sample amount is the 2% of column volume；
Eluting: after loading, with HAc-NaAc liquid eluting (10mmol/LHAc-NaAc, pH6.0), adjustment flow rate pump is 3.0ml/min, collects eluent；
(6) eluent is through micro-filtration membrane microfiltration to 230mL, micro-filtrate adds sodium bicarbonate solution 770mL, then microfiltration is to 230mL, then 770mL sodium bicarbonate solution is added, then through ultrafilter membrane ultrafiltration to 230mL, then 770mL sodium bicarbonate solution is added, then through NF membrane ultrafiltration to 57.5mL；
(7) washing: the nanofiltration liquid of step (5) is centrifuged, solid dehydrated alcohol dehydration twice, centrifugal, abandoning supernatant, leave and take precipitate；
(8) lyophilizing: the precipitate loading dish of step (6) is entered freeze dryer, and namely lyophilizing obtains ulinastatin.
Embodiment 6:
1) ulinastatin roughing
Weigh the 1TpH clarification urine less than 6.5, be stirred continuously, be slowly added to 16.5kg chitin, and measure with accurate pH test paper, regulate urine pH to 6.0；With ammonia eluting 1h after absorption completely, then through 3.5kg ammonium sulfate precipitation, overnight precipitation, centrifugal it is placed on vacuum drying in the vacuum desiccator making water absorbing agent with phosphorus pentoxide, namely obtains ulinastatin crude product after drying.
2) adsorpting column chromatography (for 0.25L cylinder)
2.1) weigh ulinastatin crude product 1kg, dissolve with the eluent A of 8.5L, stir 30 minutes, centrifugal 20 minutes, leave and take centrifugal liquid；
2.2) the eluent A balance of anion-exchange column 2.5L, flows through the anion-exchange column balanced by above-mentioned centrifugal liquid, and flow speed control is at about 120mL/min；
2.3) with the eluent B solution eluting of 33L, flow speed control, at 120mL/min, obtains eluent；
2.4) by eluent through ultrafilter membrane, ultrafiltration, to 1st/20th of its volume, obtains ultrafiltrate and is about 1.65L.
3) affinity chromatograph (for 0.25L cylinder)
3.1) balancing affinity column with the eluent C of 3.0L, ultrafiltrate flows through equilibrated resin column, flow speed control is at 120mL/min；
3.2), after end of the sample, with the eluent C eluting of 10L, flow velocity about 120mL/min, eluent is collected；
3.3) the affine resin in post is after eluent C eluting, washs to remove with the eluent D of 5.0L and adsorbs superincumbent impurity, and flow speed control, at 120mL/min, seals stand-by；
3.4) eluent is through ultrafilter membrane ultrafiltration to 170mL, adds disodium phosphate soln 830mL in ultrafiltrate, continues ultrafiltration to 170mL, then adds 830mL disodium phosphate soln, then through ultrafilter membrane ultrafiltration to 170mL.
4) precipitation
Precipitating 12 hours in the ethanol of the ratio addition not low 95% of 1:6 in ultrafiltrate, centrifugal, solid is again with dehydrated alcohol dehydration twice, centrifugal, and abandoning supernatant leaves and takes precipitate.
5) lyophilizing
Precipitate loading dish and enters freeze dryer, namely lyophilizing obtains ulinastatin.
Embodiment 7:
1) ulinastatin roughing
Weigh the 1TpH clarification urine less than 6.5, be stirred continuously, be slowly added to 16.5kg chitin, and measure with accurate pH test paper, regulate urine pH to 6.0；With ammonia eluting 1h after absorption completely, then through 3.5kg ammonium sulfate precipitation, overnight precipitation, centrifugal it is placed on vacuum drying in the vacuum desiccator making water absorbing agent with phosphorus pentoxide, namely obtains ulinastatin crude product after drying.
2) adsorpting column chromatography (for 0.25L cylinder)
2.1) weigh ulinastatin crude product 1kg, dissolve with the eluent A of 8.5L, stir 30 minutes, centrifugal 20 minutes, leave and take centrifugal liquid；
2.2) the eluent A balance of anion-exchange column 3.0L, flows through the resin column balanced by above-mentioned centrifugal liquid, and flow speed control is at about 130mL/min；
2.3) with the eluent B solution eluting of 33L, flow speed control, at 130mL/min, obtains eluent；
2.4) by eluent through ultrafilter membrane, ultrafiltration, to 1st/20th of its volume, obtains ultrafiltrate and is about 1.65L.
3) affinity chromatograph (for 0.25L cylinder)
3.1) balancing affinity column with the eluent C of 3.0L, ultrafiltrate flows through equilibrated resin column, flow speed control is at 130mL/min；
3.2), after end of the sample, with the eluent C eluting of 10L, flow velocity about 130mL/min, eluent is collected；
3.3) the affine resin in post is after eluent C eluting, washs to remove with the eluent D of 7.5L and adsorbs superincumbent impurity, and flow speed control, at 130mL/min, seals stand-by；
3.4) eluent is through ultrafilter membrane ultrafiltration to 170mL, adds disodium phosphate soln 830mL in ultrafiltrate, continues ultrafiltration to 170mL, then adds 830mL disodium phosphate soln, then through ultrafilter membrane ultrafiltration to 170mL.
4) precipitation
Precipitating 12 hours in the ethanol of the ratio addition not low 95% of 1:6 in ultrafiltrate, centrifugal, solid is again with dehydrated alcohol dehydration twice, centrifugal, and abandoning supernatant leaves and takes precipitate.
5) lyophilizing
Precipitate loading dish and enters freeze dryer, namely lyophilizing obtains ulinastatin.
Embodiment 8:
A 1.1 tons of urine are pumped in stirring pool by (), regulate pH to 5.5, add the absorption of 500g chitin, with ammonium sulfate (pH7.0) eluting that concentration is 10%, eluent kieselguhr makes filter medium, carries out sucking filtration, drains out ulinastatin semifinished product 100g；B () takes ulinastatin semifinished product 100g, add 300ml water stirring and dissolving, plate-and-frame filtration, filtrate cycle 5～10 minutes, leaching supernatant is crossed after clarification, regulate pH6.5 ± 0.2 with sodium hydroxide solution, add 1.1 times of 95% ethanol and carry out first time precipitation, take supernatant, add 2.5 times of 95% ethanol and carry out second time precipitation, take precipitation, add 5 times of water and fully dissolve, filter；Take filtrate, by anion-exchange column QAESephadexA-25 on the flow velocity of 400cm/h, with containing the phosphatic wash buffer pillar of 0.5mol/LNaCl and 0.1mol/L to effluent OD280< 0.2, with containing the phosphatic buffer solution elution pillar of 0.5mol/LNaCl and 0.1mol/L, collecting OD280The eluting peak of > 0.2, therefrom samples as sample A；
C () regulates eluent pH to 7.0, upper metal-chelating adsorption column CoSepharoseFF, carry out eluting with the phosphatic buffer containing 0.5mol/LNaCl and 0.1mol/L, collects eluent, therefrom samples as sample B；
D () adjusts eluent pH to 7.0, then go up and prick, with phenyl-carrier-benzene, the affinity column that pyrimidine cross-linked composite is chromatographic material, rinse with the phosphate buffer containing 0.1mol/LNaCl and 0.1mol/L, collect flushing liquor, add 0.3gEDTA-Na/ liter, with the ultrafilter membrane ultrafiltration that molecular weight is 30,000, therefrom sample as sample C；
E () regulates ultrafiltrate pH to 4.0, upper drainage column OctylSepharoseCL-4B absorption, carry out gradient elution with the buffer containing 0.5mol/LNaCl and 0.1mol/L sodium acetate, collects eluent；
F () adjusts eluent pH to 5.0 after, 60 DEG C are heated 10 hours, upper anion-exchange column DEAESephadexA-25, eluting is carried out with the phosphatic buffer containing 0.5mol/LNaCl and 0.1mol/L, the eluent ultrafilter membrane ultrafiltration of molecular weight 10,000, obtain high-purity ulinastatin.
Embodiment 9:
(1) weigh the 1TpH clarification urine less than 6.5, be stirred continuously, be slowly added to 10kg chitosan, and measure with accurate pH test paper, use acetic acid to regulate urine pH to 6.0；With ammonia eluting 1.5h after absorption completely, then through 3.5kg ammonium sulfate precipitation, stand 24 hours, vacuum drying in precipitation, the centrifugal vacuum desiccator being placed on anhydrous magnesium sulfate water absorbing agent, namely obtain ulinastatin crude product after drying；
(2) hydrophobic chromatography (XK50/20 chromatographic column, PhenylSepharose6FastFlow (highsub) filler)
Pretreatment: weigh ulinastatin crude product 1kg, dissolves with 10mmol/LHAc-NaAc, stirs 30 minutes, centrifugal 20 minutes, leaves and takes centrifugal liquid, adjusts pH to 6.0 with acetic acid, and reinforcing body ammonium sulfate regulates cd to 60ms/cm；
Balance: using level pad (reinforcing body ammonium sulfate regulates cd60ms/cm for 10mmol/LHAc-NaAc buffer, pH6.0), adjustment flow rate pump is 40.0ml/min, balances 8 times of column volumes；
Loading: with 100ml/min flow velocity loading, after end of the sample, with level pad (reinforcing body ammonium sulfate regulates cd60ms/cm for 10mmol/LHAc-NaAc buffer, pH6.0) 5 times of bed volumes of multiple equilibria；
Eluting: using eluent (10mmol/LTris-HCl buffer, pH8.5) eluting, adjustment flow rate pump is 20.0ml/min, starts eluting, collects eluent.
(3) affinity chromatograph (HiTraprProteinAFF chromatographic column, ProteinASepharoseCL-4B filler) is embodied as step and is:
Balance: with level pad (the 10mmol/LHAc-NaAc buffer of 4.4L, pH6.0, reinforcing body ammonium sulfate regulates cd60ms/cm) balance affinity column, hydrophobic chromatography eluent is flowed through equilibrated chromatographic column, flow speed control is at 230mL/min；
Loading: with the purpose solution of 230ml/min flow velocity loading hydrophobic chromatography, after end of the sample, with 5 times of column volumes of level pad (10mmol/LHAc-NaAc buffer, pH6.0) multiple equilibria；
Eluting: using eluent (10mmol/LTris-HCl buffer, pH8.5) eluting, adjustment flow rate pump is 100ml/min, starts eluting, collects eluent.
(4) gel permeation chromatography (HR16/70 chromatographic column Sephacryls-100HR filler) is embodied as step and is:
Balance: balancing 8 column volumes with HAc-NaAc buffer (10mmol/LHAc-NaAc, pH6.0), during balance, flow rate pump is 5.0ml/min；
Loading: affinitive layer purification eluent adds the ammonia of 1ml18%, and supernatant loading, during loading, flow rate pump is 3.0ml/min, and sample applied sample amount is the 2% of column volume；
Eluting: after loading, with HAc-NaAc liquid eluting (10mmol/LHAc-NaAc, pH6.0), adjustment flow rate pump is 3.0ml/min, collects eluent；
(5) eluent is through micro-filtration membrane microfiltration to 230mL, adds sodium bicarbonate solution 770mL in micro-filtrate, and then microfiltration is to 230mL, then 770mL sodium bicarbonate solution is added, then through ultrafilter membrane ultrafiltration to 230mL, then add 770mL sodium bicarbonate solution, then ultrafiltration is to 57.5mL；
(6) washing: the nanofiltration liquid of step (5) is centrifuged, solid dehydrated alcohol dehydration twice, centrifugal, abandoning supernatant, leave and take precipitate；
(7) lyophilizing: the precipitate loading dish of step (6) is entered freeze dryer, and namely lyophilizing obtains ulinastatin.
Embodiment 10:
(1) weigh the 1TpH clarification urine less than 6.5, be stirred continuously, be slowly added to 10kg chitosan, and measure with accurate pH test paper, use acetic acid to regulate urine pH to 6.0；With ammonia eluting 1.5h after absorption completely, then through 3.5kg ammonium sulfate precipitation, stand 24 hours, vacuum drying in precipitation, the centrifugal vacuum desiccator being placed on anhydrous magnesium sulfate water absorbing agent, namely obtain ulinastatin crude product after drying；
(2) hydrophobic chromatography (XK50/20 chromatographic column, PhenylSepharose6FastFlow (highsub) filler)
Pretreatment: weigh ulinastatin crude product 1kg, dissolves with 10mmol/LHAc-NaAc, stirs 30 minutes, centrifugal 20 minutes, leaves and takes centrifugal liquid, adjusts pH to 6.0 with acetic acid, and reinforcing body ammonium sulfate regulates cd to 60ms/cm；
Balance: using level pad (reinforcing body ammonium sulfate regulates cd60ms/cm for 10mmol/LHAc-NaAc buffer, pH6.0), adjustment flow rate pump is 20.0ml/min, balances 8 times of column volumes；
Loading: with 100ml/min flow velocity loading, after end of the sample, with level pad (reinforcing body ammonium sulfate regulates cd60ms/cm for 10mmol/LHAc-NaAc buffer, pH6.0) 5 times of bed volumes of multiple equilibria；
Eluting: using eluent (10mmol/LTris-HCl buffer, pH8.5) eluting, adjustment flow rate pump is 20.0ml/min, starts eluting, collects eluent.
(3) affinity chromatograph (HiTraprProteinAFF chromatographic column, ProteinASepharoseCL-4B filler) is embodied as step and is:
Balance: with level pad (the 10mmol/LHAc-NaAc buffer of 4.4L, pH6.0, reinforcing body sodium chloride regulates cd60ms/cm) balance affinity column, hydrophobic chromatography eluent is flowed through equilibrated chromatographic column, flow speed control is at 100mL/min；
Loading: with the purpose solution of 100ml/min flow velocity loading hydrophobic chromatography, after end of the sample, with 5 times of column volumes of level pad (10mmol/LHAc-NaAc buffer, pH6.0) multiple equilibria；
Eluting: using eluent (10mmol/LTris-HCl buffer, pH8.5) eluting, adjustment flow rate pump is 100ml/min, starts eluting, collects eluent.
(4) gel permeation chromatography (HR16/70 chromatographic column Sephacryls-100HR filler) is embodied as step and is:
Balance: balancing 8 column volumes with HAc-NaAc buffer (10mmol/LHAc-NaAc, pH6.0), during balance, flow rate pump is 3.0ml/min；
Loading: affinitive layer purification eluent is added 0.5g histidine, and supernatant loading, during loading, flow rate pump is 3.0ml/min, and sample applied sample amount is the 2% of column volume；
Eluting: after loading, with HAc-NaAc liquid eluting (10mmol/LHAc-NaAc, pH6.0), adjustment flow rate pump is 3.0ml/min, collects eluent.
(5) eluent is through micro-filtration membrane microfiltration to 230mL, adds sodium bicarbonate solution 770mL in micro-filtrate, and then microfiltration is to 230mL, then 770mL sodium bicarbonate solution is added, then through ultrafilter membrane ultrafiltration to 230mL, then add 770mL sodium bicarbonate solution, then ultrafiltration is to 57.5mL；
(6) washing: the nanofiltration liquid of step (5) is centrifuged, solid dehydrated alcohol dehydration twice, centrifugal, abandoning supernatant, leave and take precipitate；
(7) lyophilizing: the precipitate loading dish of step (6) is entered freeze dryer, and namely lyophilizing obtains ulinastatin.
Embodiment 11: index observing
(1) to ulinastatin yield obtained for embodiment 1-10, being measured than work, electrophoresis purity, HPLC purity, result is table 1 such as.
Table 1 embodiment group indices investigates result
| Yield (%) | Electrophoresis purity (%) | HPLC purity (%) | Endotoxin | Than living (iu/mg) | |
| Embodiment 1 | 87.5 | 100 | 99.8 | Meet regulation | 6920 |
| Embodiment 2 | 87.7 | 91.5 | 98.5 | Against regulation | 5104 |
| Embodiment 3 | 75.5 | 99.8 | 99.4 | Meet regulation | 5221 |
| Embodiment 4 | 80.1 | 99.8 | 99.5 | Meet regulation | 6021 |
| Embodiment 5 | 63.3 | 100 | 99.9 | Meet regulation | 6311 |
| Embodiment 6 | 72.5 | 98.5 | 97.9 | Meet regulation | 5209 |
| Embodiment 7 | 71.9 | 98.8 | 98.2 | Meet regulation | 5222 |
| Embodiment 8 | 63.7 | 98.9 | 99.2 | Meet regulation | 4044 |
| Embodiment 9 | 77.7 | 97.5 | 98.3 | Meet regulation | 6218 |
| Embodiment 10 | 89.4 | 100 | 99.9 | Meet regulation | 6987 |
Be can be seen that by table 1, the yield of embodiment 1 and 10, electrophoresis purity, HPLC purity, endotoxin, higher than stability alive, from yield, embodiment 2 and 4 close to embodiment 1, ratio embodiment 1 weak effect but its electrophoresis purity, HPLC purity, ratio are lived, especially embodiment 2 endotoxin is against regulation, has certain clinical application dangerous, and embodiment 3-9 indices is below embodiment 1 and embodiment 10.
(2) by the embodiment 1-10 lyophilized powder obtained, add 5ml distilled water and dissolve, observe biochemical indicator as shown in table 2.
The every biochemical indicator of table 2 embodiment group investigates result
By 2 it can be seen that after the ulinastatin of each embodiment is dissolved in distilled water, it has been found that only every biochemical indicator of embodiment 1 and 10 is best.
Consolidated statement 1 and table 2 be not it will be seen that embodiment 2 has hydrophilic chromatography, and outward appearance is yellow powder, and purity is only 79.65%, and endotoxin is against regulation；After the filtration step (5) of embodiment 3 changes, yield is subject to certain impact；Embodiment 4 uses the pillar of different affinity chromatographs, and effect is better than other embodiments outside other embodiments 1；Adding cation exchange column in embodiment 5, yield declines, lower than other embodiments；Embodiment 6-8 employs different purification process, and effect is also poor than embodiment 1；Embodiment 9 changes parameter, the change of flow velocity, causes the reduction in various degree alive of yield, electrophoresis purity, HPLC purity, endotoxin, ratio；Embodiment 1 and 10 is desirable purification process, has further Development volue.
Claims (6)
1. the ulinastatin purification process based on affinity column, it is characterised in that comprise the following steps:
(1) weigh the appropriate pH value clarification urine less than 6.5, add appropriate chitosan, and measure with accurate pH test paper, regulate urine ph values to 5.0-6.0；Adsorb with ammonia eluting 1.0-2.0h after completely, then through 3-4kg ammonium sulfate precipitation, stand, precipitate, namely obtain ulinastatin crude product after drying；
(2) hydrophobic chromatography, to ulinastatin crude product pretreatment, uses balance liquid A balance, controls loading speed and with level pad A multiple equilibria, uses eluent B eluting；
(3) affinity chromatograph, uses balance liquid A balance, controls loading speed and with level pad A multiple equilibria, uses eluent C eluting；
(4) gel permeation chromatography, uses balance liquid D balance, controls loading speed and use eluent E eluting；
(5) ultrafiltration of gel permeation chromatography eluent is to 57.5mL；
(6) washing: the ultrafiltrate of step (5) is centrifuged, solid dehydrated alcohol dehydration twice, centrifugal, abandoning supernatant, leave and take precipitate；
(7) lyophilizing: the precipitate loading dish of step (6) is entered freeze dryer, and namely lyophilizing obtains ulinastatin.
2. purification process according to claim 1, it is characterised in that the balance liquid A described in step (2) is 10mmol/LHAc-NaAc buffer, pH value is 6.0, and reinforcing body sodium chloride regulates cd60ms/cm；Eluent B is 10mmol/LTris-HCl buffer, and pH value is 8.5.
3. purification process according to claim 1, it is characterised in that the balance liquid C described in step (3) is 10mmol/LTris-HCl buffer, pH value is 8.0；Described affinity chromatograph is HiTraprProteinAFF chromatographic column ProteinASepharoseCL-4B filler or HiTraprProteinAHF chromatographic column ProteinASepharose4FF filler.
4. purification process according to claim 1, it is characterised in that the balance liquid D described in step (4) is 10mmol/LHAc-NaAc, pH value is 6.0；Eluent E is: 10mmol/LHAc-NaAc, and pH value is 6.0.
5. purification process according to claim 1, it is characterised in that comprise the following steps:
(1) weigh the 1TpH clarification urine less than 6.5, be stirred continuously, be slowly added to 10kg chitosan, and measure with accurate pH test paper, use acetic acid to regulate urine pH to 6.0；With ammonia eluting 1.5h after absorption completely, then through 3.5kg ammonium sulfate precipitation, stand 24 hours, vacuum drying in precipitation, the centrifugal vacuum desiccator being placed on anhydrous magnesium sulfate water absorbing agent, namely obtain ulinastatin crude product after drying；
(2) hydrophobic chromatography, uses XK50/20 chromatographic column, PhenylSepharose6FastFlow filler
Pretreatment: weigh ulinastatin crude product 1kg, dissolves with 10mmol/LHAc-NaAc, stirs 30 minutes, centrifugal 20 minutes, leaves and takes centrifugal liquid, adjusts pH to 6.0 with acetic acid, and reinforcing body sodium chloride regulates cd to 60ms/cm；
Balance: use level pad, is specially 10mmol/LHAc-NaAc buffer, pH6.0, and reinforcing body sodium chloride regulates cd60ms/cm；Adjustment flow rate pump is 20.0ml/min, balances 8 times of column volumes；
Loading: with 100ml/min flow velocity loading, after end of the sample, with level pad 10mmol/LHAc-NaAc buffer, pH6.0, reinforcing body sodium chloride regulates cd60ms/cm, 5 times of bed volumes of multiple equilibria；
Eluting: using eluent 10mmol/LTris-HCl buffer, pH8.5, eluting, adjustment flow rate pump is 20.0ml/min, starts eluting, collects eluent；
(3) affinity chromatograph, selects HiTraprProteinAFF chromatographic column, ProteinASepharoseCL-4B filler, concretely comprises the following steps:
Balance: with the level pad 10mmol/LHAc-NaAc buffer of 4.4L, pH6.0, reinforcing body sodium chloride regulates cd60ms/cm, balances affinity column, hydrophobic chromatography eluent is flowed through equilibrated chromatographic column, and flow speed control is at 100mL/min；
Loading: with the purpose solution of 100ml/min flow velocity loading hydrophobic chromatography, after end of the sample, with level pad 10mmol/LHAc-NaAc buffer, pH6.0, reinforcing body sodium chloride regulates cd60ms/cm, 5 times of column volumes of multiple equilibria；
Eluting: using eluent 10mmol/LTris-HCl buffer, pH8.0, eluting, adjustment flow rate pump is 100ml/min, starts eluting, collects eluent；
(4) gel permeation chromatography, selects HR16/70 chromatographic column Sephacryls-100HR filler, concretely comprises the following steps:
Balance: with HAc-NaAc buffer 10mmol/LHAc-NaAc, pH6.0, balancing 8 column volumes, during balance, flow rate pump is 3.0ml/min；
Loading: affinitive layer purification eluent is added appropriate alkaline solution, and supernatant loading, during loading, flow rate pump is 3.0ml/min, and sample applied sample amount is the 2% of column volume；
Eluting: after loading, with HAc-NaAc liquid eluting 10mmol/LHAc-NaAc, pH6.0, adjustment flow rate pump is 3.0ml/min, collects eluent；
(5) eluent is through ultrafilter membrane ultrafiltration to 57.5mL；
(6) washing: the ultrafiltrate of step (5) is centrifuged, solid dehydrated alcohol dehydration twice, centrifugal, abandoning supernatant, leave and take precipitate；
(7) lyophilizing: the precipitate loading dish of step (6) is entered freeze dryer, and namely lyophilizing obtains ulinastatin.
6. purification process according to claim 5, it is characterised in that the ultrafilter membrane described in step (5) includes the ultrafilter membrane that molecular weight is 5000-3 ten thousand.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107827976A (en) * | 2017-11-08 | 2018-03-23 | 广东天普生化医药股份有限公司 | A kind of UTI purification process based on drainage column |
| CN108059670A (en) * | 2017-11-01 | 2018-05-22 | 广东天普生化医药股份有限公司 | A kind of ulinastatin purification process based on affinity column |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1824301A (en) * | 2006-01-09 | 2006-08-30 | 广东天普生化医药股份有限公司 | Purified ustading and its preparation method and medicinal composition containing said ustading |
| CN1931875A (en) * | 2006-01-09 | 2007-03-21 | 广东天普生化医药股份有限公司 | High purity ulinastatin and its prepn process and medicine composition |
| CN104497133A (en) * | 2014-12-23 | 2015-04-08 | 青岛康原药业有限公司 | Method for purifying ulinastatin by using adsorption column chromatography and pharmaceutical composition containing ulinastatin |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1824301A (en) * | 2006-01-09 | 2006-08-30 | 广东天普生化医药股份有限公司 | Purified ustading and its preparation method and medicinal composition containing said ustading |
| CN1931875A (en) * | 2006-01-09 | 2007-03-21 | 广东天普生化医药股份有限公司 | High purity ulinastatin and its prepn process and medicine composition |
| CN104497133A (en) * | 2014-12-23 | 2015-04-08 | 青岛康原药业有限公司 | Method for purifying ulinastatin by using adsorption column chromatography and pharmaceutical composition containing ulinastatin |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108059670A (en) * | 2017-11-01 | 2018-05-22 | 广东天普生化医药股份有限公司 | A kind of ulinastatin purification process based on affinity column |
| CN107827976A (en) * | 2017-11-08 | 2018-03-23 | 广东天普生化医药股份有限公司 | A kind of UTI purification process based on drainage column |
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