CN104498460A - Method for purifying kallikrein by hydrophobic interaction chromatography - Google Patents

Method for purifying kallikrein by hydrophobic interaction chromatography Download PDF

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Publication number
CN104498460A
CN104498460A CN201410807827.3A CN201410807827A CN104498460A CN 104498460 A CN104498460 A CN 104498460A CN 201410807827 A CN201410807827 A CN 201410807827A CN 104498460 A CN104498460 A CN 104498460A
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Prior art keywords
kallikrein
hydrophobic chromatography
solution
sepharose
butyl
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刘冠男
林晓磊
孙延年
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QINGDAO KANGYUAN PHARMACEUTICAL CO Ltd
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QINGDAO KANGYUAN PHARMACEUTICAL CO Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6445—Kallikreins (3.4.21.34; 3.4.21.35)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21034—Plasma kallikrein (3.4.21.34)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21035—Tissue kallikrein (3.4.21.35)

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  • Health & Medical Sciences (AREA)
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Abstract

The invention discloses a method for purifying kallikrein by hydrophobic interaction chromatography. The method comprises the following steps: (1) ion exchange chromatography; (2) salting out; (3) ultrafiltration; (4) pyrogen treatment; (5) dialysis; (6) hydrophobic interaction chromatography; (7) freeze-drying to prepare kallikrein. According to the method, the production process of kallikrein is continuously improved, especially each process parameter is repeatedly tested and studied and continuously optimized, a set of more scientific large-scale production process is finally determined, the titer of kallikrein prepared by hydrophobic interaction chromatography is 1500-1800 iu/mg, and each index of kallikrein accords with the specification of the Chinese pharmacopoeia.

Description

A kind of method of hydrophobic chromatography purifying kallikrein
Technical field
The present invention relates to biological technical field, relate in particular to a kind of method of using hydrophobic chromatogram purification kallikrein.
Background technology
Kallikrein belongs to serine protease, it comprises blood plasma type kallikrein and tissue-type kallikrein two type, molecular weight is respectively 25200 dalton and 33800 dalton, the equal prokinin that can make discharges a peptide species---kassinin kinin, and kallikrein kinin system (kallikrein kinin system, KKS) be one of depressurizing system main in body, as a complicated endogenous multienzyme system, participate in regulation and control cardiovascular, kidney, the physiological function of neural system etc., with heart trouble, ephrosis, inflammatory reaction, the generation close relation of the diseases such as cancer.
Progress in recent years in cardiovascular systems is very fast, and many clinical studyes and infrastest have confirmed that the generations of disease such as diabetes, hypertension, heart failure, myocardial infarction and left ventricular hypertrophy reduce relevant with the activity of KKS.Thus the pathogenesis acting as Research on Cardiovascular relative disease and the treatment means of furtheing investigate KKS provide another new approach.
The present inventor started the production technique research experiment to pancreatin from 2008, particularly in the production process of chymotrypsinogen and trypsinogen, continuous exploration, make every effort to optimize processing parameter, and look for another way on the basis of original technique, be successfully completed the technical study extracting prekallikrein, its yield is 0.2% ~ 0.4%, stable processing technique, quality controllable.
Summary of the invention
The invention provides a kind of method of hydrophobic chromatography purifying kallikrein, by bioseparation engineering purifying kallikreins such as hydrophobic chromatography, keep protein-active preferably.
A method for hydrophobic chromatography purifying kallikrein, is characterized in that: adopt the modern biotechnology isolation technique such as hydrophobic chromatography, tired by kallikrein and bring up to 1500 ~ 1800iu/mg, better maintain the activity of albumen.In invention technical scheme, be further characterized in that described extraction process comprises the steps:
1) ion exchange chromatography
1.1) dissolve centrifugal by the acetate buffer solution of prekallikrein 0.001-0.01mol/L, PH3-6, supernatant liquor adds ion exchange resin, and stir, adsorb, collect resin, rinsing is until non-foam;
1.2) use 0.01-0.3mol/L again, the NaCL solution stirring wash-out resin of the 0.1-5mol/L of PH3-8, PH5-10, separation resin obtains elutriant;
2) saltout
Regulate elutriant pH value to 3.0 ~ 5.0, add solid ammonium sulfate, hold over night, next day filters, and collects filter cake;
3) ultrafiltration
Add (w/v) phosphate buffered saline buffer of filter cake weight 15 times amount, stir, adjust ph to 7.0 ~ 9.0, treat liquid ultrafiltration, fixing fabric structure is 3/10;
4) pyrogen process
4.1) get ultrafiltrated, in the ratio of 50:5:3, successively add sodium radio-phosphate,P-32 solution and calcium acetate solution;
4.2) adjust ph to 8.0 ~ 10.0, stir, centrifugal, discard throw out, get filtrate, prepare dialysis Zha Bao;
5) dialysis operation
Get filtrate, dialyzate Zha Bao, puts into purified water, carries out dialysis and exchanges, dialyse 4 ~ 6 days;
6) hydrophobic chromatography
6.1) 0.5-3mol/L (NH is contained by appropriate 4) 2sO 40.01-0.3mol/L, the NaAc-HAc damping fluid of PH3-8 balances the fast glue post of hydrophobic chromatography;
6.2) dialyzate is flowed through this post, flow rate control is at about 1 ~ 5mL/min;
6.3) after balance, with (the NH of 0.5-3mol/L 4) 2sO 4eluant solution is to the display of Protein Detection instrument to baseline;
6.4) again with not containing (NH 4) 2sO 4level pad wash-out, collects the elutriant containing activity of kallikrein peak ,-20 DEG C of preservations;
7) freeze-drying
Adjust ph to 5.5 ~ 6.5, sabot, freeze-drying, obtain kallikrein.
After testing, gained kallikrein is tired and is about 1500 ~ 1800iu/mg.
Compared with prior art, advantage of the present invention and positively effect are:
Select the modern biotechnology isolation technique purifying kallikreins such as hydrophobic chromatography, can make its physico-chemical property and biological activity in subsequent production process, keep stable, finally make the indices of kallikrein all meet Chinese Pharmacopoeia standard.The more important thing is, by technique update and perfect, kallikrein is tired and brings up to 1500 ~ 1800iu/mg, cost-saving, improve economic benefit.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described, and limit never in any form.
Embodiment 1
1) ion exchange chromatography
1.1) dissolve centrifugal by the acetate buffer solution 100L of prekallikrein 20kg 0.005mol/L, PH4, supernatant liquor adds ion exchange resin, whip attachment 2h, and collect resin, rinsing is until non-foam;
1.2) use 0.03mol/L again, the NaCL solution 150L of PH5 stirs wash-out resin 1h, and separation resin obtains elutriant 138L;
2) saltout
Regulate elutriant pH value to 3.0 ~ 5.0, add solid ammonium sulfate, hold over night, next day filters, and collects filter cake 13.5kg;
4) ultrafiltration
Dropped in retort by gained filter cake, add phosphate buffer 1 63L, stir 10 minutes, by 5mol/L NaOH adjust ph to 8.5, treat liquid ultrafiltration, fixing fabric structure, 3/10, obtains ultrafiltrated 165L;
5) pyrogen process
5.1) get above-mentioned ultrafiltrated, in the ratio of 50:5:3, first add 0.4mol/L sodium radio-phosphate,P-32 solution 16.5L, under constantly stirring, slowly add 1mol/L calcium acetate solution 9.90L;
5.2) by 2mol/L NaOH adjust ph to 9.0, continue stirring 1.5 hours after stable, after terminating, centrifugal 20 minutes, discard throw out, obtain filtrate 162L;
6) dialysis operation
Get above-mentioned filtrate, dialyzate Zha Bao (100mL/ bag), puts into 4 DEG C of purified water, carries out dialysis exchange 5 days by dialysis tubing, within every 24 hours, change a purified water;
7) hydrophobic chromatography
7.1) that uses 44L contains 1.5mol/L (NH 4) 2sO 40.2mol/L, the NaAc-HAc damping fluid of PH3-8 balances the fast glue post of hydrophobic chromatography;
7.2) dialyzate is flowed through this post, flow rate control is at about 3mL/min;
7.3) after balance, with (the NH of 1.5mol/L 4) 2sO 4eluant solution is to the display of Protein Detection instrument to baseline;
7.4) again with not containing (NH 4) 2sO 4level pad wash-out, collects the elutriant containing activity of kallikrein peak ,-20 DEG C of preservations;
8) freeze-drying
By 2mol/L NaOH solution adjust ph to 6.0, sabot, freeze-drying, obtain kallikrein 6.5kg.
After testing, gained kallikrein is tired and is about 1725iu/mg.
Embodiment 2
1) ion exchange chromatography
1.1) dissolve centrifugal by the acetate buffer solution 90L of prekallikrein 1.8kg 0.005mol/L, PH4, supernatant liquor adds ion exchange resin, whip attachment 2h, and collect resin, rinsing is until non-foam;
1.2) use 0.03mol/L again, the NaCL solution 140L of PH5 stirs wash-out resin 1h, and separation resin obtains elutriant 126L;
2) saltout
Regulate elutriant pH value to 3.0 ~ 5.0, add solid ammonium sulfate, hold over night, next day filters, and collects filter cake 12.9kg;
4) ultrafiltration
Dropped in retort by gained filter cake, add phosphate buffer 1 56L, stir 10 minutes, by 5mol/L NaOH adjust ph to 8.5, treat liquid ultrafiltration, fixing fabric structure, 3/10, obtains ultrafiltrated 158L;
5) pyrogen process
5.1) get above-mentioned ultrafiltrated, in the ratio of 50:5:3, first add 0.4mol/L sodium radio-phosphate,P-32 solution 15.8L, under constantly stirring, slowly add 1mol/L calcium acetate solution 9.48 L;
5.2) by 2mol/L NaOH adjust ph to 9.0, continue stirring 1.5 hours after stable, after terminating, centrifugal 20 minutes, discard throw out, obtain filtrate 155L;
6) dialysis operation
Get above-mentioned filtrate, dialyzate Zha Bao (100mL/ bag), puts into 4 DEG C of purified water, carries out dialysis exchange 5 days by dialysis tubing, within every 24 hours, change a purified water;
7) hydrophobic chromatography
7.1) that uses 42L contains 2mol/L (NH 4) 2sO 40.15mol/L, the NaAc-HAc damping fluid balance hydrophobic chromatography fast glue post of PH3-8;
7.2) dialyzate is flowed through this post, flow rate control is at about 2mL/min;
7.3) after balance, with (the NH of 1mol/L 4) 2sO 4eluant solution is to the display of Protein Detection instrument to baseline;
7.4) again with not containing (NH 4) 2sO 4level pad wash-out, collects the elutriant containing activity of kallikrein peak ,-20 DEG C of preservations;
8) freeze-drying
By 2mol/L NaOH solution adjust ph to 6.5, sabot, freeze-drying, obtain kallikrein 6.2kg.
After testing, gained kallikrein is tired and is about 1688iu/mg.
The above is only preferred embodiment of the present invention, and be not restriction the present invention being made to other form, any those skilled in the art may utilize the technology contents of above-mentioned announcement to be changed or be modified as the Equivalent embodiments of equivalent variations.But everyly do not depart from technical solution of the present invention content, any simple modification, equivalent variations and the remodeling done above embodiment according to technical spirit of the present invention, still belong to the protection domain of technical solution of the present invention.

Claims (9)

1. a method for hydrophobic chromatography purifying kallikrein, the method, by modern biotechnology isolation technique such as hydrophobic chromatography, improves the efficiency of purifying kallikrein, keeps the titer plateaus of kallikrein.
2. the method for claim 1, is characterized in that: take prekallikrein as raw material, successively through ion exchange chromatography, saltout, ultrafiltration, pyrogen process, dialysis, hydrophobic chromatography, the operation purifying such as freeze-drying obtain kallikrein.
3. method as claimed in claim 1 or 2, is characterized in that described purifying process comprises the steps:
1) dissolved by prekallikrein acetate buffer solution, centrifugal, supernatant liquor adds ion exchange resin, stirring, absorption, rinsing, wash-out;
2) add solid ammonium sulfate after adjust ph to saltout, hold over night, filter, collect filter cake;
3) phosphate buffered saline buffer stirring and adjusting pH value is added, ultrafiltration;
4), after ultrafiltrated adds sodium radio-phosphate,P-32 solution and calcium acetate solution, pH is regulated to stir, centrifugal, abandon throw out;
5) getting above-mentioned filtrate is placed in dialysis tubing, dialyses 4 ~ 6 days;
6) hydrophobic chromatography, carries out wash-out with 2 kinds of elutriants, the final elutriant collected containing activity of kallikrein peak ,-20 DEG C of preservations;
7) adjust ph, sabot, freeze-drying, obtain kallikrein.
4. method as claimed in claim 3, is characterized in that ion exchange resin selects AmheriteCG-50, and m (resin): m(raw product)=50:1.
5. method as claimed in claim 3, is characterized in that ion exchange chromatography selects elutriant to be the NaCL solution of 0.01-0.3mol/L, PH3-8.
6. method as claimed in claim 3, is characterized in that adding 500g ammonium sulfate in often liter of extracting solution in salting-out procedures.
7. method as claimed in claim 3; it is characterized in that: the fast glue post of described hydrophobic chromatography comprises: Butyl-Sepharose 2B FF; Butyl-Sepharose 4B FF; Butyl-Sepharose 6B FF; Butyl-Sepharose CL-2B FF; Butyl-Sepharose CL-4B FF, Butyl-Sepharose CL-6B FF; .
8. method as claimed in claim 3, is characterized in that: described hydrophobic chromatography condition is: balance liquid is selected containing 0.5-3mol/L (NH 4) 2sO 40.01-0.3mol/L, the NaAc-HAc damping fluid of PH3-8, flow rate control is at about 1 ~ 5mL/min, and (the NH of 0.5-3mol/L selected by elutriant 4) 2sO 4solution and not containing (NH 4) 2sO 4level pad.
9. the method as described in claim 1 ~ 7, is characterized in that: hydrophobic chromatography purifying gained kallikrein is tired up to 1500 ~ 1800iu/mg.
CN201410807827.3A 2014-12-23 2014-12-23 Method for purifying kallikrein by hydrophobic interaction chromatography Pending CN104498460A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105400757A (en) * 2015-11-26 2016-03-16 青岛康原药业有限公司 Method for purifying kallikrein based on hydrophobic interaction chromatography and pharmaceutical composition capable of improving stability of kallikrein

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CN103667224A (en) * 2013-12-02 2014-03-26 青岛康原药业有限公司 Extraction method for kallikreinogen through secondary affinity chromatography
CN103667226A (en) * 2013-11-23 2014-03-26 青岛康原药业有限公司 Method for purifying chymotrypsin through affinity chromatography and stepwise elution
CN103725664A (en) * 2013-11-28 2014-04-16 青岛康原药业有限公司 Method for highly purifying kallikrein
CN103740689A (en) * 2013-11-30 2014-04-23 青岛康原药业有限公司 Method for affinity chromatography fractional elution purification of chymotrypsin

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CN1336434A (en) * 2000-08-01 2002-02-20 上海惠海生化制品厂 Prepn. and affinity chromatographic purification process of kallidinogen enzyme
CN103667226A (en) * 2013-11-23 2014-03-26 青岛康原药业有限公司 Method for purifying chymotrypsin through affinity chromatography and stepwise elution
CN103725664A (en) * 2013-11-28 2014-04-16 青岛康原药业有限公司 Method for highly purifying kallikrein
CN103740689A (en) * 2013-11-30 2014-04-23 青岛康原药业有限公司 Method for affinity chromatography fractional elution purification of chymotrypsin
CN103667224A (en) * 2013-12-02 2014-03-26 青岛康原药业有限公司 Extraction method for kallikreinogen through secondary affinity chromatography

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105400757A (en) * 2015-11-26 2016-03-16 青岛康原药业有限公司 Method for purifying kallikrein based on hydrophobic interaction chromatography and pharmaceutical composition capable of improving stability of kallikrein

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Application publication date: 20150408