CN106349387A - Method for purifying alpha1-antitrypsin from Cohn component IV precipitate - Google Patents
Method for purifying alpha1-antitrypsin from Cohn component IV precipitate Download PDFInfo
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- CN106349387A CN106349387A CN201611021105.0A CN201611021105A CN106349387A CN 106349387 A CN106349387 A CN 106349387A CN 201611021105 A CN201611021105 A CN 201611021105A CN 106349387 A CN106349387 A CN 106349387A
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- aat
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
- C07K14/8121—Serpins
- C07K14/8125—Alpha-1-antitrypsin
Abstract
The invention discloses a method for purifying alpha1-antitrypsin from a Cohn component IV precipitate. The method comprises the following steps of sequentially carrying out dissolution and concentration, PEG precipitation, primary viral inactivation, primary ultrafiltration and concentration, thrice chromatography refining, freeze-drying, and secondary viral inactivation on the Cohn component IV precipitate, thus finally obtaining the alpha1-antitrypsin. According to the method provided by the invention, the Cohn component IV precipitate which is a waste of albumin produced by a low-temperature alcohol method is adopted as a starting material for preparing AAT, so that the comprehensive utilization ratio of raw blood plasma is favorably improved. During the whole process of the method, PEG precipitation is combined with anion exchange chromatography and twice blue dyestuff affinity chromatography, so that the impurity protein can be effectively removed, an AAT preparation with the purity being larger than 95 percent is finally obtained, and the specific activity is larger than 3,900U/mg.
Description
Technical field
The present invention relates to Protein purification techniques field, more particularly to one kind purification of alpha 1- from cohn components precipitate resists
Tryptic method.
Background technology
Alpha1-antitrypsin (alpha 1-antitrypsin, aat) is by 394 aminoacid and 3 oligosaccharide side chains structures
The single chain glycoprotein becoming, molecular weight is 52kda, and isoelectric point, IP is 4.8, and avtive spot is located at the met-ser region of 358～359.
70～80% aat by hepatocyte synthesis secretion, additionally, mononuclear cell, macrophage, alveolar cell also can produce a small amount of
aat.
The main physiological function of aat is the balance maintaining protease-anti-protease system, protects normal structure not digested
Damage.In human normal plasma, the concentration of aat is 1.0～2.0mg/ml, during less than 0.5mg/ml, is then difficult to opposing neutrophilic granulocyte
The elastoser (neutrophil elastase, ne) of retting conditions release, the elasticity in ne degradable alveolar connective tissue is fine
Dimension, develops as one pleases and will lead to emphysema, chronic obstructive pulmonary disease.Aat lacks for autosomal dominant inherited disease, by Sweden
Doctor laurell and eriksso found in 1963, and was named as aat deficiency disease (alpha-1antitrypsin
Defiency, aatd).Common mutant is s type and Z-shaped, and Z-shaped aat also can form polymer, be gathered in hepatocellular endoplasm
On the net, the diseases such as hepatitis, liver cirrhosis, hepatocarcinoma are finally caused.Clinically adopt exogenous aat replacement therapy to correct aat-ne's
Unbalance, venoclysises aat (60mg/kg/week) can make the level of aat in blood plasma reach rotection thresholds, and serum anti-ne ability is bright
Aobvious raising.
In recent years, increasing research shows that aat is not only a kind of protease inhibitor moreover it is possible to adjusting immunity, controlling
Inflammation and cause pathogeny imcrobe infection.Wherein, aat is passed through to suppress inflammatory reaction, delay the development of i patients with type Ⅰ DM, reduce transplanting
Islets of langerhans death toll quantifier elimination has been enter into clinical experimental stage.
1987, fda have approved aat lyophilized formulations (prolastin) listing in first blood plasma source, for treating elder generation
The emphysema patient that nature aat lack.At present, have 6 kinds of aat products (prolastin, aralast, zemaira,
Prolastin-c, aralast np, glassia) on sale.Above-mentioned aat product is produced albuminous discarded with cold ethanol method
Thing cohn fraction (cohn component) is precipitated as initiation material and is prepared, in addition to zemaira, other 5 kinds of aat
Product all first adopts Polyethylene Glycol (polyethylene glycol, the peg) sedimentation method or peg to combine zncl2The sedimentation method are slightly pure；
Refined by anion-exchange chromatography or anions and canons displacement chromatography again；Pasteur's viral inactivation method, nano-film filtration, s/d method are effective
Inactivation or remove virus it is ensured that the safety of product Clinical practice.
Wherein, the prototype of prolastin preparation technology is the purification process that coan mh et al. announced in 1985
(preparation and properties of alpha 1-proteinase inhibitor concentrate from
human plasma.vox sang.1985；48 (6): 333-42.), cohn component -1 precipitation is resuspended in ph by the method
In 8.2 tris-hcl buffer, deae-sepharose cl-6b anion is combined using peg 3350 precipitation classification and hands over
Change chromatography preparation aat.Coan mh et al. from molecular weight is in the patent (us4379087, us4439358) of announcement
The peg of 3000-4000 is precipitant, and the consumption of peg increases with the rising of solution ph.After peg precipitation, foreign protein is gone
Remove, aat is retained in supernatant, anion-exchange chromatography alternative eluting aat.Above-mentioned purifying process is with sucrose and sodium citrate
Carry out Pasteur's inactivation of virus for protective agent, the purity of aat finished product is 60%.The product purity that above-mentioned purifying process obtains is not
Height, and only include a step virus inactivation technology it is impossible to ensure the safety of finished product Clinical practice.
In the disclosed patent (us6462180) such as lebing w combining the peg sedimentation method with cation and anion exchange chromatography can
Improve the purity of aat, eluted by selectivity in conjunction with the aat on anion-exchange chromatography, by adjusting ph value and conductance
Can make what aat was retained in cation-exchange chromatography to penetrate in peak, finally adopt detergent (tween 20), nano-film filtration to carry out
Viral inactivation treatment, the purity of aat finished product is 95%.Show in patent application cn102993298a to tie using the peg sedimentation method
Close cation and anion exchange chromatography, virus inactivation technology is s/d method and 15nm membrane filtration, the aat that purity is 99-100% can be obtained
Preparation (purity that cellulose-acetate membrane electrophoresis obtains).Cohn component -1 resolution of precipitate is shown in patent application cn102180966a
Liquid priority carries out cold ethanol precipitation, s/d method inactivation of virus, and peg precipitates, two step anion-exchange chromatographies, Pasteur's inactivation of virus,
Obtain the aat finished product that purity is 96-99% eventually.Above purifying process all refines aat using ion-exchange chromatography, but ion
Displacement chromatography is that under the conditions of different ph values, charge number is different using albumen, and the combination with chromatography media has differences and carries out
Detached.The charge number of albumen depends on the size of isoelectric point, IP after all, and albuminous isoelectric point, IP is 4.7～4.9, with
Aat (4.8) is close, therefore, is very difficult to except the very high albumin of content in cohn components precipitate using ion-exchange chromatography,
Have a strong impact on the purity of aat and than work.Additionally, sds-page collection of illustrative plates shows that albumin and the electrophoresis of aat are closely located to (Fig. 3),
This makes to assume a protein band containing albuminous aat sample when carrying out sds-page analysis, the sample of a swimming lane in Fig. 3
It is mixed into 20% human albumin, this sample and reference substance aat for the aat after purifiedPosition on electrophoretogram
Put essentially identical, this is also that above-mentioned purifying process is very difficult to except albumin, that is to say, that three kinds the reason but obtain higher degree
The purity that the aat that purifying process obtains records is inaccurate.
Zhu Wei etc. publishes an article, and (preparation of alpha1-antitrypsin preparation and its inactivation of virus, Products in China is miscellaneous
Will, 2001,14 (2): 97-101) peg, organic solvent, isoelectric precipitation are carried out to cohn components precipitate extract, by the moon
Ion-exchange chromatography and gel filtration obtain the aat preparation that purity is 91.3 ± 1.52%, about 27.42 times of purification, bar
Family name's viral inactivation method can effective inactivation of viruses.The aat system that two patents (cn101274956a, cn101747432a) provide below
Preparation Method equally adopts anion-exchange chromatography and gel filtration to refine aat, and virus inactivating method is respectively Pasteur's inactivation of virus
Method combines dry heating method, and s/d method combines d20 nano-film filtration.All using gel-filtration purified aat in said method, but gel
Filtration be carried out according to molecular weight of albumen size detached, albuminous molecular weight be 66.5kda, still close with aat
(52kda), therefore, still it is very difficult to except albumin using gel filtration.
Travis j etc. publishes an article (selective removal of albumin from plasma by
affinity chromatography.clin chim acta.1973nov 23；49 (1): 49-52.) find a kind of blue dye
Material gibacron blue f3ga can be coupled at, with albumin bound, the white egg that can remove in blood plasma 96% on agarose
In vain, and not other albumen are lost.Pannell r etc. publishes an article (isolation and properties of human
plasma alpha-1-proteinase inhibitor.biochemistry.1974dec17；13 (26): 5439-45.) adopt
Adsorb albumin with sepharose-blue dextran, in conjunction with ammonium sulfate precipitation, two step anion-exchange chromatographies from whole blood
Extract aat.Yu Jian etc. publishes an article (application in Purification of Human serum α1 antitrypsin for the blue sepharose 4B, Chinese biological
Learn and molecular biosciences journal, 1991,7 (1): 122-4.), four step chromatography (blue sepharose 4Bs slightly pure using a step ammonium sulfate precipitation
Affinity chromatograph, deae cellulose chromatography, con a sepharose 4B affinity chromatograph, sephacryl s-200 gel filtration) refine
aat.Though above-mentioned aat purification process introduces affinity chromatograph and can effectively remove albumin, preparation method is loaded down with trivial details, is unsuitable for technique life
Produce.
Content of the invention
The purpose of the present invention is for technological deficiency present in prior art, provides one kind from cohn components precipitate
The method being purified into highly purified alpha1-antitrypsin, by cohn components precipitate sequentially pass through dissolving concentrate, peg precipitation, one
Secondary inactivation of virus, be once concentrated by ultrafiltration, three chromatographies are refined, lyophilizing, secondary inactivation of virus, finally give alpha1-antitrypsin
(aat).
Described three chromatographies refine to refine for an anion-exchange chromatography and refine with blue dyess affinity chromatograph twice.
In being refined with described anion-exchange chromatography, the aat eluent balance blue dyess affinity chromatograph of eluting aat refines
In blue dyess affinity column, and be used for eluting aat；Preferably, balance anion in being refined with anion-exchange chromatography
The balance liquid eluting of displacement chromatography post removes and is not associated with the transferrinss on post and albumin.
Described aat eluent is ph 5.0-5.4,10-50mm acetate buffer containing 45-65mm nacl；Preferably,
Described balance liquid is ph 5.0-5.4,10-50mm acetate buffer.
Described anion-exchange chromatography refine in first use balance liquid balance anion displacement chromatography post, then loading, it
First rinse anion exchange chromatography removing the transferrinss being not associated with post and albumin with same balance liquid afterwards, then use
Albumin on post for ph4.6-4.8,10-50mm acetate buffer elution of bound and vitamin d associated proteins, finally use
Aat elution aat, obtains aat elution samples, the flow velocity of whole process of purification is 150-350cm/h；
The medium preferred ion cation exchange groups of anion exchange chromatography are diethylamino ethyl
The weak anionic displacement chromatography medium of (diethylaminoethyl, deae) or ion-exchange group are quaternary amine base
(quaternary ammonium group, strong anion displacement chromatography medium q).
Described blue dyess affinity chromatograph balances blue dyess affinity column with described aat eluent in refining first,
Then loading, is rinsed blue dyess affinity column with described aat eluent afterwards and is not combined with blue dyess with removing
Aat, obtains aat and penetrates sample, the flow velocity of whole process of purification is 70-120cm/h；
The preferred affinity ligand of medium of blue dyess affinity column is the affinity chromatography medium of blue dyess, such as blue
sepharose 6fast flow；
Preferably, secondary blue dyess affinity chromatograph and once blue dye during described blue dyess affinity chromatograph refines twice
The column volume ratio of material affinity chromatograph is for 1:(15-20).
Blue dyess affinity chromatograph refines and can use same blue dyess affinity column twice；Particularly as follows: it is once blue
After dye affinity chromatography terminates, used blue dyess affinity column ph 5.0-8.5,10- containing 0.5-1.0m kscn
50mm acetate buffer, phosphate buffer or tris-hcl buffer solution elution combine macromole (the molecular weight ＞ on post
180kda) foreign protein, transferrinss, albumin, vitamin d associated proteins.
The ph value in a described inactivation of virus, peg being precipitated gained supernatant is adjusted to 6.5-7.5,0.22 μm of filter membrane mistake
Filter, adds TRI N BUTYL PHOSPHATE and tween 80 to make its final concentration be respectively 0.3% (w/v, g/100ml), 1% (w/v, g/
100ml), 23-25 DEG C of water bath with thermostatic control 6h carries out s/d method viral inactivation treatment, obtains inactivateing supernatant.
In described secondary inactivation of virus, lyophilizing gained aat dried frozen aquatic productses are carried out dry heating method virus in 99-100 DEG C of water-bath
Inactivation treatment 30min, to inactivate togavirus and non-togavirus, obtains final alpha1-antitrypsin.
Specifically include following steps:
A), the dissolving of cohn components precipitate, concentration: the cohn components precipitate water dissolution of 8-10 times of weight, 0-10 DEG C
Stirring 2-4h, adjusts ph value to 7.5-8.0, solid-liquid separation, abandons solid, obtain cohn component lysate, by cohn component
Lysate is concentrated by ultrafiltration 4-10 times of volume and obtains cohn component concentrated solution, and the protein content in cohn component concentrated solution is
20-40mg/ml；
B), peg precipitation: the ph value of regulating step a) gained cohn component concentrated solution to 5.0-5.4,0-10 DEG C of addition
Peg 4000 makes peg 4000 be completely dissolved to its final concentration of 10-15% (w/v, g/100ml), stirring, after 0-10 DEG C of standing
It is centrifuged 20-60min under 4000-6000g, discard and comprise macromole (molecular weight ＞ 180kda) foreign protein, transferrinss, albumin
Protein-bonded precipitation, takes supernatant with vitamin d；
C) a, inactivation of virus: the ph value of step b) gained supernatant is adjusted to 6.5-7.5,0.22 μm of membrane filtration,
TRI N BUTYL PHOSPHATE (tri-n-butyl phosphate, tnbp) and tween 80 (tween 80) is added to make its final concentration respectively
For 0.3% (w/v, g/100ml), 1% (w/v, g/100ml), 23-25 DEG C of water bath with thermostatic control 6h carries out s/d method viral inactivation treatment,
Obtain inactivateing supernatant；
D), once it is concentrated by ultrafiltration: with the inactivation of ph 5.0-5.4,10-50mm acetate buffer ultrafiltration step c) gained
Clear liquid, obtains ultrafiltrate, the molecular cut off of ultrafilter membrane is 20-30kda；
E), anion-exchange chromatography refines: using anion-exchange chromatography purification step d) gained ultrafiltrate；Anion is handed over
Change chromatography media can be selected for ion-exchange group be diethylamino ethyl (diethylaminoethyl, deae) weak cloudy from
Sub- displacement chromatography medium or ion-exchange group are that quaternary amine base (quaternary ammonium group, hand over by strong anion q)
Change chromatography media；Use ph 5.0-5.4,10-50mm acetate buffer first as balance liquid balance anion displacement chromatography
Post, then by step d) gained ultrafiltrate loading, first rinsed with same balance liquid afterwards be not associated with transferrinss on post and
Albumin, then combine egg with albumin on post for ph4.6-4.8,10-50mm acetate buffer elution of bound and vitamin d
In vain, finally with the balance liquid containing 45-65mm nacl as aat elution aat, obtain aat elution samples, whole purification
The flow velocity of process is 150-350cm/h；
F), a blue dyess affinity chromatograph refines: using blue dyess affinitive layer purification step e) gained aat eluting
Sample；Blue dyess affinity chromatography medium can be selected for the affinity chromatography medium that affinity ligand is blue dyess, such as blue
sepharose 6fast flow；Aat eluent is used to balance blue dyess affinity column, then by step e) gained first
Aat elution samples loading, the aat using the flushing of aat eluent not to be combined with blue dyess afterwards, obtain an aat and penetrate sample,
The flow velocity of whole process of purification is 70-120cm/h；Used blue dyess affinity column can use containing 0.5-1.0m kscn's
Ph 5.0-8.5,10-50mm acetate buffer, phosphate buffer or tris-hcl buffer solution elution combine big on post
Molecule (molecular weight ＞ 180kda) foreign protein, transferrinss, albumin, vitamin d associated proteins, then reuse；
G), secondary blue dyess affinity chromatograph refines: use aat eluent to balance blue dyess affinity column first, blue
The same step f) of color dye affinity chromatography medium；Then aat of step f) gained is penetrated sample loading, use aat eluting afterwards
The aat that liquid flushing is not combined with blue dyess, obtains secondary aat and penetrates sample；In step g) and step f), blue dyess are affine
The column volume of chromatographic column is than for 1:(15-20)；
H), lyophilizing: the secondary aat of step g) gained is concentrated by ultrafiltration and penetrates sample, make the protein content of this sample reach 10-
20mg/ml, is subsequently adding protective agent histidine, the final concentration of 0.05-0.1m of histidine, and 0.22 μm of membrane filtration is degerming, subpackage,
Lyophilizing, obtains aat dried frozen aquatic productses；
I), secondary inactivation of virus: step h) gained aat dried frozen aquatic productses are carried out dry heating method virus in 99-100 DEG C of water-bath
Inactivation treatment 30min, obtains final alpha1-antitrypsin (aat).
Compared with prior art, the invention has the beneficial effects as follows:
It is initial former that the method for the present invention produces albuminous garbage cohn components precipitate with cold ethanol method
Material preparation aat, is conducive to improving the comprehensive utilization ratio of raw blood plasma.In whole procedure, anion is combined using peg precipitation
Displacement chromatography, twice blue dyess affinity chromatograph, can effectively remove macromole (molecular weight ＞ 180kda) foreign protein, turn ferrum egg
In vain, albumin, vitamin d associated proteins, finally obtain highly purified aat preparation, and the finished product purity obtaining is more than 95%, with
Trypsin is suppression target enzyme, is more than 3900u/mg than living.In the method, blue dyess affinity chromatograph chromatography condition is identical twice,
And balance liquid is identical with the eluent of anion-exchange chromatography aat, it is to avoid the inconvenience that multiple solution preparations bring.Additionally, this
Decrease ultrafiltration concentration step in inventive method, simplify production process, be suitable to prepare with scale aat.Last the inventive method
Using the s/d method that can effectively inactivate togavirus, and can effectively inactivate dry heating method two step of togavirus and non-togavirus not
With principle virus inactivation technology it is ensured that the Viral safety of aat preparation.
Brief description
The flow chart that Fig. 1 show the inventive method；
Fig. 2 show cohn components precipitate lysate electrophoretogram, and wherein: m is albumen marker, a is cohn component
Resolution of precipitate liquid；
Fig. 3 show aat and albumin electrophoresis position versus figure, and wherein: m is albumen marker, a obtains for embodiment 3
Aat in be mixed into 20% human albumin mixture, b be human albumin (csl behring), a carries for sigma company
For aat standard substance；
Obtain after peg supernatant, peg precipitation and ultrafiltration concentration that Fig. 4 obtains after showing peg precipitation in embodiment 1
The electrophoretogram of ultrafiltrate, wherein: m is albumen marker, a is cohn components precipitate lysate, and b is peg supernatant, and c is peg
Precipitation, d is ultrafiltrate；
What Fig. 5 showed that anion-exchange chromatography in embodiment 2 obtains after purification penetrate sample, ph 4.7 elution samples,
The electrophoretogram of 50mm nacl aat elution samples, wherein: m is albumen marker, a is cohn f resolution of precipitate liquid, and b is peg
Supernatant, c precipitates for peg, and d is ultrafiltrate, and e is to penetrate sample, and f is ph 4.7 elution samples, and g washes for 50mm nacl aat
De- sample, h is 100m nacl elution samples；
The ph 5.4aat that Fig. 6 obtains after showing in embodiment 3 blue dyess affinitive layer purification twice penetrate sample,
The electrophoretogram of 0.5m kscn elution samples, wherein: m is albumen marker, a is 45mm nacl aat elution samples ultrafiltrate, b
Penetrate sample for first time ph 5.4aat, c is first time 0.5m kscn elution samples, d penetrates sample for second ph 5.4aat
Product, e is second 0.5m kscn elution samples, the aat standard substance that a provides for sigma company, and b is human albumin (csl
behring)；
Fig. 7 show the peg supernatant ultrafiltrate that in embodiment 2, whole isolation and purification method obtains, anion-exchange chromatography
50mm nacl aat elution samples, first time blue dyess affinity chromatograph ph 5.2aat penetrate sample, second blue dyes
Affinity chromatograph ph 5.2aat penetrates the electrophoretogram of sample, and wherein: m is albumen marker, a is cohn f resolution of precipitate liquid, b
For peg supernatant ultrafiltrate, c is anion-exchange chromatography 50mm nacl aat elution samples, and d is that first time blue dyess are affine
Chromatography ph 5.2aat penetrates sample, and e penetrates sample for second blue dyess affinity chromatograph ph5.2aat, and a is sigma company
The aat standard substance providing；
Fig. 8 show high-efficient liquid phase analysis collection of illustrative plates, and sample introduction sample is that in embodiment 3, second blue dyess affinity chromatograph is pure
Change gained ph 5.4aat and penetrate sample；
Fig. 9 show high-efficient liquid phase analysis collection of illustrative plates, the aat standard substance that sample introduction sample provides for sigma company；
Figure 10 show anion-exchange chromatography purification gained 50mm nacl in comparative example 1 and penetrates sample, 100mm nacl
The electrophoretogram of aat elution samples, wherein: m is albumen marker, a is cohn components precipitate lysate, and b is peg supernatant, c
For peg precipitation, d is ultrafiltrate, and e penetrates sample for 50mm nacl, and f is 100mm nacl aat elution samples；
Figure 11 show anion-exchange chromatography purification gained 25mm nacl elution samples, 50mm nacl in comparative example 2
Elution samples, 100mm nacl aat elution samples, 150mm nacl elution samples, the electrophoresis of 200mm nacl elution samples
Figure, wherein: m is albumen marker, a is cohn components precipitate lysate, and b is peg supernatant ultrafiltrate, and c is to penetrate sample, d
For 25mm nacl elution samples, e is 50mm nacl elution samples, and f is 100mm nacl aat elution samples, and g is 150mm
Nacl elution samples, h is 200mm nacl elution samples；
Figure 12 show that in comparative example 3, blue dyess affinity chromatograph gained ph 6.5aat penetrates sample, 0.5m kscn washes
The electrophoretogram of de- sample, wherein: m is albumen marker, a is cohn f resolution of precipitate liquid, and b is peg supernatant, and c sinks for peg
Form sediment, d is ultrafiltrate, e penetrates sample for anion-exchange chromatography, f is ph 4.7 elution samples, g is 55mm nacl aat eluting
Sample, h be 100m nacl elution samples, i be aat elution samples ultrafiltrate, g be ph 6.5aat penetrates sample, h be 0.5m
Kscn elution samples.
Specific embodiment
The antitryptic method of purification of alpha 1- from cohn components precipitate that the present invention provides, is shown in Fig. 1, including following
Step:
A), the dissolving of cohn components precipitate, concentration: the cohn components precipitate water dissolution of 8-10 times of weight, 0-10 DEG C
Stirring 2-4h, adjusts ph value to 7.5-8.0, solid-liquid separation, abandons solid, obtain cohn component lysate, by cohn component
Lysate is concentrated by ultrafiltration 4-10 times of volume and obtains cohn component concentrated solution, and the protein content in cohn component concentrated solution is
20-40mg/ml；
B), peg precipitation: the ph value of regulating step a) gained cohn component concentrated solution to 5.0-5.4,0-10 DEG C of addition
Peg 4000 makes peg 4000 be completely dissolved to its final concentration of 10-15% (w/v, g/100ml), stirring, after 0-10 DEG C of standing
It is centrifuged 20-60min under 4000-6000g, discard and comprise macromole (molecular weight ＞ 180kda) foreign protein, transferrinss, albumin
Protein-bonded precipitation, takes supernatant with vitamin d；
C) a, inactivation of virus: the ph value of step b) gained supernatant is adjusted to 6.5-7.5,0.22 μm of membrane filtration,
TRI N BUTYL PHOSPHATE (tri-n-butyl phosphate, tnbp) and tween 80 (tween 80) is added to make its final concentration respectively
For 0.3% (w/v, g/100ml), 1% (w/v, g/100ml), 23-25 DEG C of water bath with thermostatic control 6h carries out s/d method viral inactivation treatment,
Obtain inactivateing supernatant；
D), once it is concentrated by ultrafiltration: with the inactivation of ph 5.0-5.4,10-50mm acetate buffer ultrafiltration step c) gained
Clear liquid, obtains ultrafiltrate, the molecular cut off of ultrafilter membrane is 20-30kda；
E), anion-exchange chromatography refines: using anion-exchange chromatography purification step d) gained ultrafiltrate；Anion is handed over
Change chromatography media can be selected for ion-exchange group be diethylamino ethyl (diethylaminoethyl, deae) weak cloudy from
Sub- displacement chromatography medium or ion-exchange group are that quaternary amine base (quaternary ammonium group, hand over by strong anion q)
Change chromatography media；Use ph 5.0-5.4,10-50mm acetate buffer first as balance liquid balance anion displacement chromatography
Post, then by step d) gained ultrafiltrate loading, is first rinsed with balance liquid afterwards and is not associated with the transferrinss on post and white egg
In vain, then with albumin on post for ph4.6-4.8,10-50mm acetate buffer elution of bound and vitamin d associated proteins,
Finally with the balance liquid eluting aat containing 45-65mm nacl, obtain aat elution samples, the flow velocity of whole process of purification is 150-
350cm/h.
F), a blue dyess affinity chromatograph refines: using blue dyess affinitive layer purification step e) gained aat eluting
Sample；Blue dyess affinity chromatography medium can be selected for the affinity chromatography medium that affinity ligand is blue dyess, such as blue
sepharose 6fast flow；First with the balance liquid balance blue dyess affinity column containing 45-65mm nacl, then
By step e) gained aat elution samples loading, rinsed with the balance liquid containing 45-65mm nacl afterwards and be not combined with blue dyess
Aat, obtain an aat and penetrate sample, the flow velocity of whole process of purification is 70-120cm/h；Used blue dyess are affine layer
Analysis post can use ph 5.0-8.5,10-50mm acetate buffer containing 0.5-1.0m kscn, phosphate buffer or tris-
Hcl buffer solution elution is with reference to macromole (the molecular weight ＞ 180kda) foreign protein on post, transferrinss, albumin, vitamin d
Associated proteins, then reuse；
G), secondary blue dyess affinity chromatograph refines: first with the balance liquid balance blue dyess containing 45-65mm nacl
Affinity column, the same step f) of blue dyess affinity chromatography medium；Then aat of step f) gained is penetrated sample loading,
The aat not being combined with blue dyess with the balance liquid flushing containing 45-65mm nacl afterwards, is obtained secondary aat and penetrates sample；Step
Rapid g) with the column volume of blue dyess affinity column in step f) ratio for 1:(15-20).
H), lyophilizing: the secondary aat of step g) gained is concentrated by ultrafiltration and penetrates sample, make the protein content of this sample reach 10-
20mg/ml, is subsequently adding protective agent histidine, the final concentration of 0.05-0.1m of histidine, and 0.22 μm of membrane filtration is degerming, subpackage,
Lyophilizing, obtains aat dried frozen aquatic productses.
I), secondary inactivation of virus: step h) gained aat dried frozen aquatic productses are carried out dry heating method virus in 99-100 DEG C of water-bath
Inactivation treatment 30min, obtains final alpha1-antitrypsin (aat) product.
Below in conjunction with specific embodiment, further illustrate present disclosure, and the present invention is further elaborated, but
These embodiments limit the invention absolutely not.
Embodiment 1
A), the dissolving of cohn components precipitate, concentration: 200g cohn components precipitate 2000ml water dissolution, 4 DEG C of stirrings
3h, adjusts ph value to 7.5, is centrifuged and removes kieselguhr, obtain cohn component lysate (electrophoretogram is shown in Fig. 2).By cohn component
Lysate is concentrated by ultrafiltration 4 times of volumes to 500ml, obtains cohn component concentrated solution, and protein content is 20-22mg/ml.
B), peg precipitation: adjust cohn component concentrated solution ph value and add 50g peg while stirring under the conditions of 5.0,4 DEG C
4000 to its final concentration of 10% (w/v, g/100ml), and continuing stirring 30-45min after adding makes peg4000 fully dissolve；4℃
Standing 30-45min, 6000g are centrifuged 20min, discard precipitation, take supernatant；Precipitation includes macromole (molecular weight ＞
180kda) foreign protein, transferrinss, albumin and vitamin d associated proteins (see Fig. 4).
C) a, inactivation of virus: s/d method inactivation of virus, supernatant 0.5m naoh adjusts ph value to 6.5, through 0.22 μm
After membrane filtration, s/d reagent (being made up of 3%tnbp and 10%tween 80) is added so that the final concentration of tnbp, tween 80 is divided
Not Wei 0.3%, 1% (w/v, g/100ml), 24 DEG C of water bath with thermostatic control 6h, obtain inactivate supernatant.
D), once it is concentrated by ultrafiltration: inactivation supernatant ph 5.0 10mm 5-10 volume of acetate buffer ultrafiltration, obtain
To ultrafiltrate, the molecular cut off of ultrafilter membrane is 30kda.
E), anion-exchange chromatography refines: anion-exchange chromatography purification, the weak the moon of deae sepharose fast flow
Ion exchange column (column volume 30ml) uses ph 5.0 10mm acetate buffer to balance 8-10 cylinder as balance liquid
Long-pending, then loading ultrafiltrate, first rinsed with balance liquid afterwards and be not associated with the transferrinss on post and albumin, then with ph 4.6
Albumin on post for the 10mm acetate buffer elution of bound and vitamin d associated proteins, finally with flat containing 65mm nacl
Weighing apparatus aat on post for the liquid elution of bound, obtains aat elution samples, the flow velocity of whole process of purification is 150cm/h.
F), a blue dyess affinity chromatograph refines: blue dyess affinitive layer purification, blue sepharose 6fast
Flow blue dyess affinity column (column volume 20ml) balances 8-10 column volume with the balance liquid containing 65mm nacl, then
Loading aat elution samples, are rinsed with the balance liquid containing 65mm nacl afterwards and are not associated with the aat on post, obtain an aat and wear
Sample thoroughly, the flow velocity of whole process of purification is 70cm/h；Finally washed with the ph 5.010mm acetate buffer containing 0.5m kscn
De- macromole (the molecular weight ＞ 180kda) foreign protein combining on post, transferrinss, albumin and vitamin d associated proteins
Afterwards, this chromatographic column repeats and utilizes.
G), secondary blue dyess affinity chromatograph refine: same to step f), be only column volume be 1ml, loading sample be once
Aat penetrates sample, obtains secondary aat and penetrates sample.
H), lyophilizing: secondary aat penetrates sample and is concentrated by ultrafiltration to protein content is 10-11mg/ml, and the retention of ultrafilter membrane divides
Son is measured as 30kda；Add protective agent histidine so as to final concentration of 0.05m, 0.22 μm of membrane filtration is degerming, subpackage, lyophilizing,
Obtain aat dried frozen aquatic productses.
I), secondary inactivation of virus: aat dried frozen aquatic productses carry out dry heating method viral inactivation treatment in 99-100 DEG C of water-bath
30min, obtains final alpha1-antitrypsin (aat) product.
It is shown in Table 1 with the index of gained sample in the antitryptic each step of embodiment 1 method purification of alpha 1-.
The each step of table 1 obtain in sample aat activity, protein content and than live
Sds-page gray scale scanning shows that the purity of the aat product that embodiment 1 obtains is 99.08%, with trypsin is
The target enzyme of aat suppression, is 42638.28u/ml using the activity that Chromogenic assay measures finished product, and bca method measures protein content
For 10.17mg/ml, it is 4192.55u/mg that the ratio of aat product is lived, and purification is 68.90 times.
Embodiment 2
A), the dissolving of cohn components precipitate, concentration: 1.2kg cohn components precipitate 10.8l water dissolution, 0 DEG C of stirring
4h, adjusts ph value to 7.8 with 0.5m naoh, carries out solid-liquid separation using pressure filter and remove kieselguhr, obtain cohn component molten
Solution liquid.Cohn component lysate is concentrated by ultrafiltration 7 times of volumes to 2l, obtains cohn component concentrated solution, protein content is 29-
31mg/ml.
B), peg precipitation: cohn component concentrated solution with 0.5m hcl adjust ph value under the conditions of 5.2,0 DEG C while stirring
Add 300g peg 4000 to its final concentration of 15% (w/v, g/100ml), continuing stirring 30-45min after adding makes peg
4000 fully dissolve；0 DEG C of standing 30-45min, 4000g are centrifuged 30min, discard precipitation, take supernatant.
C) a, inactivation of virus: s/d method inactivation of virus, supernatant 0.5m naoh adjusts ph value to 7.0, through 0.22 μm
After membrane filtration, s/d reagent (being made up of 3%tnbp and 10%tween 80) is added so that the final concentration of tnbp, tween 80 is divided
Not Wei 0.3%, 1% (w/v, g/100ml), 25 DEG C of water bath with thermostatic control 6h, obtain inactivate supernatant.
D), once it is concentrated by ultrafiltration: inactivation supernatant ph 5.2 20mm 5-10 volume of acetate buffer ultrafiltration, obtain
To ultrafiltrate, the molecular cut off of ultrafilter membrane is 30kda.
E), anion-exchange chromatography refine: anion-exchange chromatography purification, q sepharose fast flow reinforcing YIN-essence from
Sub- displacement chromatography post (column volume 150ml) uses ph 5.2 20mm acetate buffer to balance 8-10 column volume as balance liquid,
Then loading ultrafiltrate, is first rinsed with balance liquid afterwards and is not associated with the transferrinss on post and albumin, then with ph 4.7
Albumin on post for the 20mm acetate buffer elution of bound and vitamin d associated proteins, finally with flat containing 50mm nacl
Weighing apparatus aat on post for the liquid elution of bound, obtains aat elution samples (Fig. 5), the flow velocity of whole process of purification is 350cm/h.
F), a blue dyess affinity chromatograph refines: blue dyess affinitive layer purification, blue sepharose 6fast
Flow blue dyess affinity column (column volume 80ml) balances 8-10 column volume with the balance liquid containing 50mm nacl, then
Loading aat elution samples, are rinsed with the balance liquid containing 50mm nacl afterwards and are not associated with the aat on post, obtain an aat and wear
Sample thoroughly, the flow velocity of whole process of purification is 100cm/h；Finally washed with the ph 7.020mm phosphate buffer containing 1m kscn
De- macromole (the molecular weight ＞ 180kda) foreign protein combining on post, transferrinss, albumin and vitamin d associated proteins
Afterwards, this chromatographic column repeats and utilizes.
G), secondary blue dyess affinity chromatograph refine: same to step f), be only column volume be 4ml, loading sample be once
Aat penetrates sample, obtains secondary aat and penetrates sample.
H), lyophilizing: secondary aat penetrates sample and is concentrated by ultrafiltration to protein content is 15-16mg/ml, and the retention of ultrafilter membrane divides
Son is measured as 30kda；Add protective agent histidine so as to final concentration of 0.07m, 0.22 μm of membrane filtration is degerming, subpackage, lyophilizing,
Obtain aat dried frozen aquatic productses.
I), secondary inactivation of virus: aat dried frozen aquatic productses carry out dry heating method viral inactivation treatment in 99-100 DEG C of water-bath
30min, obtains final alpha1-antitrypsin (aat) product.
It is shown in Table 2 with the index of gained sample in the antitryptic each step of embodiment 2 method purification of alpha 1-.Embodiment 2 side
The aat that aat elution samples that supernatant that in method, step b) obtains, step e) obtain, step f) obtain penetrates sample, step
The electrophoretogram that rapid secondary aat g) obtaining penetrates sample is shown in Fig. 7.
The each step of table 2 obtain in sample aat activity, protein content and than live
Sds-page gray scale scanning shows that the purity of aat finished product is 98.17%, the target being suppressed for aat with trypsin
Enzyme, is 61940.16u/ml using the activity that Chromogenic assay measures finished product, and it is 15.55mg/ that bca method measures protein content
It is 3983.29iu/mg that the ratio of ml, aat finished product is lived, and purification is 66.05 times.
Embodiment 3
A), the dissolving of cohn components precipitate, concentration: 4kg cohn components precipitate 32l water dissolution, 10 DEG C of stirring 2h,
Adjust ph value to 8.0 with 0.5m naoh, carry out solid-liquid separation using pressure filter and remove kieselguhr, obtain the dissolving of cohn component
Liquid.Cohn component lysate is concentrated by ultrafiltration 10 times of volumes to 3.6l, obtains cohn component concentrated solution, protein content is
38-40mg/ml.
B), peg precipitation: cohn component concentrated solution with 0.5m hcl adjust ph value under the conditions of 5.5,10 DEG C while stirring
Add 432g peg 4000 to its final concentration of 12% (w/v, g/100ml), continuing stirring 3-4h after adding makes peg 4000 fill
Divide dissolving；10 DEG C stand overnight, and 4000g is centrifuged 60min, discards precipitation, takes supernatant.
C) a, inactivation of virus: s/d method inactivation of virus, supernatant 0.5m naoh adjusts ph value to 7.5, through 0.22 μm
After membrane filtration, s/d reagent (being made up of 3%tnbp and 10%tween 80) is added so that the final concentration of tnbp, tween 80 is divided
Not Wei 0.3%, 1% (w/v, g/100ml), 23 DEG C of water bath with thermostatic control 6h, obtain inactivate supernatant.
D), once it is concentrated by ultrafiltration: inactivation supernatant ph 5.4 50mm 5-10 volume of acetate buffer ultrafiltration, obtain
To ultrafiltrate, the molecular cut off of ultrafilter membrane is 20kda.
E), anion-exchange chromatography refine: anion-exchange chromatography purification, q sepharose fast flow reinforcing YIN-essence from
Sub- displacement chromatography post (column volume 500ml) uses ph 5.4 50mm acetate buffer to balance 8-10 column volume as balance liquid,
Then loading ultrafiltrate, is first rinsed with balance liquid afterwards and is not associated with the transferrinss on post and albumin, then with ph 4.8
Albumin on post for the 50mm acetate buffer elution of bound and vitamin d associated proteins, finally with flat containing 45mm nacl
Weighing apparatus aat on post for the liquid elution of bound, obtains aat elution samples, the flow velocity of whole process of purification is 250cm/h.For the ease of
The chromatography purification of next step, aat elution samples are concentrated by ultrafiltration after 10 times of volumes as next with the balance liquid containing 45mm nacl
The loading sample of step affinity chromatograph.
F), a blue dyess affinity chromatograph refines: blue dyess affinitive layer purification, blue sepharose 6fast
Flow blue dyess affinity column (column volume 300ml) balances 8-10 column volume with the balance liquid containing 45mm nacl, then
Loading aat elution samples, are rinsed with the balance liquid containing 45mm nacl afterwards and are not associated with the aat on post, obtain an aat and wear
Sample thoroughly, the flow velocity of whole process of purification is 120cm/h；Finally buffered with the ph 8.550mm tris-hcl containing 0.5m kscn
Macromole (molecular weight ＞ 180kda) foreign protein on post for the liquid elution of bound, transferrinss, albumin and vitamin d combine egg
Bai Hou, this chromatographic column repeats and utilizes.
G), secondary blue dyess affinity chromatograph refine: same to step f), be only column volume be 20ml, loading sample be once
Aat penetrates sample, obtains secondary aat and penetrates sample；With the ph 8.5 50mm tris-hcl buffer solution elution containing 0.5m kscn
In conjunction with after macromole (the molecular weight ＞ 180kda) foreign protein on post, transferrinss, albumin and vitamin d associated proteins
(Fig. 6), this chromatographic column repeats and utilizes.
H), lyophilizing: secondary aat penetrates sample and is concentrated by ultrafiltration to protein content is 20-21mg/ml, and the retention of ultrafilter membrane divides
Son is measured as 20kda；Add protective agent histidine so as to final concentration of 0.1m, 0.22 μm of membrane filtration is degerming, subpackage, lyophilizing obtains
To aat dried frozen aquatic productses.
I), secondary inactivation of virus: aat dried frozen aquatic productses carry out dry heating method viral inactivation treatment in 99-100 DEG C of water-bath
30min, obtains final alpha1-antitrypsin (aat) product.
It is shown in Table 3 with the index of gained sample in the antitryptic each step of embodiment 3 method purification of alpha 1-.
The each step of table 3 obtain in sample aat activity, protein content and than live
Sds-page gray scale scanning shows that the purity of aat finished product is 95.54%, the target being suppressed for aat with trypsin
Enzyme, is 80206.92iu/ml using the activity that Chromogenic assay measures finished product, and it is 20.38mg/ that bca method measures protein content
It is 3935.57iu/mg that the ratio of ml, aat finished product is lived, and purification is 66.34 times.
Mass Spectrometric Identification:
Secondary aat is penetrated sample and first carries out 10%sds-page analysis, after dyeing, target protein band is cut into
1mm2Micelle, by film dosim, extract peptide fragment；Combined using thermo easy-nlc liquid phase-q exactive hf
Quadrupole rod orbitrap mass spectrograph detects protein sequence, carries out data inspection using mascot (version: 2.1.0) search engine
Rope, data base is uniport-human storehouse, one-level error 15ppm, two grades of errors 20mmu.Fixing modification is as follows: cysteine
It is modified to urea methyl cysteine (carbamidomethyl-cys), variable modification is as follows: methionine oxidized
(oxidation-m), albumen n end acetylation (protein n-term)；Trypsin enzyme action, enzymatic fragment allows be up to 2
Leakage enzyme site.
Mascot retrieves 74 kinds of albumen, and the albumen that ranking is first 6 is as shown in table 4.
Table 4 aat Mass Spectrometric Identification result
As shown in Table 4, alpha-1-antitrypsin ranks the first, and peak area is maximum, reaches 1.40e+11,
Mascot marking value highest, is 35070.4.Transferrinss, albumin, vitamin d combination do not occur in the albumen that ranking is first 6
Albumen, illustrates that this purification process can effectively remove above-mentioned foreign protein, obtains aat.
Efficient liquid phase chromatographic analysis:
Penetrate whether sample is one-component using the secondary aat of rigol l-3000 high-efficient liquid phase analysis, reference substance is
The aat standard substance that sigma company buys, loading 30 μ g, chromatographic condition is as follows:
Chromatographic column: c4-st2 post (4.6mm × 50mm, 5um c4 filler)
Mobile phase: a:2% acetonitrile/98%h2O/0.1% trifluoroacetic acid；
B:2%h2O/98% acetonitrile/0.1% trifluoroacetic acid；
Flow velocity: 0.7 μ l/min
Detection wavelength: 280nm
Elution requirement:
| time | 0 | 5 | 37 | 42 | 45 | 50 |
| %b | 5 | 5 | 95 | 95 | 5 | 5 |
Result is shown in Fig. 8 and Fig. 9.The HPLC-UV detection of Fig. 8 and Fig. 9 shows that secondary aat penetrates sample in retention time
At 28.5385min, a single main peak occurs, reference substance aat standard substance are in retention time 24.273min, 28.7635min
Two peaks in place, illustrates to be higher than reference substance aat standard substance using the purity of the inventive method purification gained aat.
Comparative example 1
A) dissolving of cohn components precipitate, concentration: with embodiment 1.
B), peg precipitation: with embodiment 1.
C) a, inactivation of virus: no.
D), once it is concentrated by ultrafiltration: with embodiment 1.
E), anion-exchange chromatography refines: anion-exchange chromatography purification, the weak the moon of deae sepharose fast flow
Ion exchange column (column volume 30ml) is put down as balance liquid with the ph 6.5 25mm phosphate buffer containing 50mm nacl
8-10 column volume of weighing apparatus, then loading ultrafiltrate, is first rinsed with the balance liquid containing 50mm nacl afterwards and is not associated with turning on post
Ferritin and albumin and vitamin d associated proteins, finally with the balance liquid elution of bound containing 100mm nacl on post
Aat, obtains aat elution samples, the flow velocity of whole process of purification is 150cm/h.
F), a blue dyess affinity chromatograph refines: no.
G), secondary blue dyess affinity chromatograph refines: no.
H), lyophilizing: no.
I), secondary inactivation of virus: no.
The target enzyme being suppressed for aat with trypsin, measures the activity of finished product using Chromogenic assay, and bca method measures egg
Bai Hanliang, calculates ratio and lives, in each step of comparative example 1, the index of gained sample is shown in Table 5.
The each step of table 5 obtain in sample aat activity, protein content and than live
From the determination of activity result of table 5, under the conditions of ph 6.5, balance deae sepharose fast flow is weak
Anion exchange chromatography, aat needs just can be eluted with the buffer containing 100mm nacl, and the present invention is with containing 65mm
The buffer of nacl just can elute, and in the eluent of comparative example 1, salinity is more much higher than the present invention, and the aat obtaining
Although protein active, content and ratio work are above the present invention in elution samples, sds-page shows in the albumen eluting
Miscellaneous band is many, and that is, foreign protein content is high, and aat purity is low.
From sds-page analysis result (see Figure 10), in aat elution samples, contain transferrinss, albumin and Wei Sheng
Plain d associated proteins, also part macromole (the molecular weight ＞ under eluting while eluting aat of the buffer containing 100mm nacl
180kda) foreign protein, and the residual of transferrinss is more, purification effect is not so good as first to carry out low ph value (ph 4.6-ph in the present invention
4.8) eluting foreign protein, is further added by salinity (45-65mm nacl) eluting aat (see Fig. 5).
Comparative example 2
A) dissolving of cohn components precipitate, concentration: with embodiment 1.
B), peg precipitation: with embodiment 1.
C) a, inactivation of virus: no.
D), once it is concentrated by ultrafiltration: with embodiment 1.
E), anion-exchange chromatography refine: anion-exchange chromatography purification, q sepharose fast flow reinforcing YIN-essence from
Sub- displacement chromatography post (column volume 30ml) uses ph 5.2 25mm acetate buffer to balance 8-10 column volume as balance liquid,
Then loading ultrafiltrate, is first rinsed with balance liquid afterwards and is not associated with the transferrinss on post and albumin, more respectively with containing
The balance liquid of 25mm nacl, the balance liquid containing 50mm nacl, the balance liquid containing 100mm nacl, the balance containing 150mm nacl
Liquid, the balance liquid stepwise elution containing 200mm nacl combine the albumen on post, collect each elution samples, obtain aat eluting sample
Product, the flow velocity of whole process of purification is 150cm/h.
F)-i), blue dyess affinity chromatograph refines, secondary blue dyess affinity chromatograph refines, lyophilizing, secondary virus
Inactivation: no.
The target enzyme being suppressed for aat with trypsin, measures the activity of finished product using Chromogenic assay, and bca method measures egg
Bai Hanliang, calculates ratio and lives, in each step of comparative example 2, the index of gained sample is shown in Table 6.
The each step of table 6 obtain in sample aat activity, protein content and than live
From the determination of activity result of table 6, under the conditions of ph 5.2 balance q sepharose fast flow reinforcing YIN-essence from
Sub- displacement chromatography post, aat needs just can be eluted with the buffer containing 100mm nacl, and the present invention is with containing 65mm nacl's
Buffer just can elute, and in the eluent of comparative example 2, salinity is more much higher than the present invention, and the aat eluting sample obtaining
Although protein active, content and ratio work are above the present invention in product, sds-page shows that in the albumen eluting, miscellaneous band is many,
I.e. foreign protein content is high, and aat purity is low.
From sds-page analysis result (see Figure 11), the balance liquid eluting containing 25mm nacl remove transferrinss and
Albumin, the balance liquid eluting containing 50mm nacl removes albumin and vitamin d associated proteins, the balance containing 100mm nacl
Liquid also part macromole (molecular weight ＞ 180kda) foreign protein under eluting while eluting aat, and the residual of transferrinss is relatively
Many, purification effect is not so good as first to carry out low ph value (ph 4.6-ph 4.8) eluting foreign protein in the present invention, is further added by salinity (45-
65mm nacl) eluting aat (Fig. 5).
Comparative example 3
A) dissolving of cohn components precipitate, concentration: with embodiment 1.
B), peg precipitation: with embodiment 1.
Inactivation of virus: no.
C), once it is concentrated by ultrafiltration: with embodiment 1.
D), anion-exchange chromatography refine: anion-exchange chromatography purification, q sepharose fast flow reinforcing YIN-essence from
Sub- displacement chromatography post (column volume 30ml) uses ph 5.3 20mm acetate buffer to balance 8-10 column volume as balance liquid,
Then loading ultrafiltrate, is first rinsed with balance liquid afterwards and is not associated with the transferrinss on post and albumin, then with ph 4.7
Albumin on post for the 20mm acetate buffer elution of bound and vitamin d associated proteins, finally with the ph containing 55mm nacl
Aat on post for the 5.3 20mm acetate buffer elution of bound, obtains aat elution samples, the flow velocity of whole process of purification is
150cm/h.
E), second ultrafiltration concentrates: aat elution samples ph 6.5 20mm 5-10 volume of phosphate buffer ultrafiltration, obtains
To second ultrafiltration liquid, the molecular cut off of ultrafilter membrane is 30kda.
F), blue dyess affinity chromatograph refines: blue dyess affinitive layer purification, blue sepharose 6fast
Flow blue dyess affinity column (column volume 20ml) uses ph 6.5 20mm phosphate buffer to balance 8-10 column volume,
Then loading second ultrafiltration liquid, is rinsed with balance liquid afterwards and is not associated with the aat on post, obtain aat and penetrate sample, whole purification
The flow velocity of process is 40cm/h；Finally with the ph 6.5 20mm phosphate buffer elution of bound containing 0.5m kscn on post
After albumin, this chromatographic column repeats and utilizes.
G), secondary blue dyess affinity chromatograph refines: no.
H), lyophilizing: no.
I), secondary inactivation of virus: no.
The target enzyme being suppressed for aat with trypsin, measures the activity of finished product using Chromogenic assay, and bca method measures egg
Bai Hanliang, calculates ratio and lives, in each step of comparative example 3, the index of gained sample is shown in Table 7.
The each step of table 7 obtain in sample aat activity, protein content and than live
From the determination of activity result of table 7, under the conditions of ph6.5, balance blue sepharose 6fast flow is blue
Color dye affinity chromatography post, aat is not combined with blue dyess, is retained in and penetrates in sample, and the ratio work that this penetrates sample is
1329.65u/mg, is lived less than the ratio being penetrated sample using the aat that balance affinity column under the conditions of ph 5.0-5.4 obtains.
From sds-page analysis result (see Figure 12), under ph6.5 equilibrium condition, aat penetrates in sample and contains in a large number
Vitamin d associated proteins, only have albumin and lack in the ph 6.5 20mm phosphate buffer elution samples containing 0.5m kscn
Amount transferrinss, illustrate under ph 6.5 equilibrium condition, blue dyess more albumin-binding, and the combination to other albumen
Less.Under ph 5.0-5.4 equilibrium condition of the present invention, comprise macromole in the buffer solution elution sample containing 0.5m kscn and (divide
Son amount ＞ 180kda) foreign protein, transferrinss, albumin, vitamin d associated proteins, illustrate in ph 5.0-5.4 equilibrium condition
Lower blue dyess not only albumin-binding, herein in connection with other albumen (Fig. 6).Therefore, the affine condition adopting in the present invention is favourable
In aat is separated with other foreign proteins.
The above is only the preferred embodiment of the present invention it is noted that common skill for the art
For art personnel, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications
Also should be regarded as present disclosure.
Claims (10)
1. one kind from cohn components precipitate the antitryptic method of purification of alpha 1- it is characterised in that cohn component is sunk
Shallow lake sequentially pass through dissolving concentrate, peg precipitation, inactivation of virus, be once concentrated by ultrafiltration, three chromatographies are refined, lyophilizing, secondary disease
Poison inactivation, finally gives alpha1-antitrypsin (aat).
2. according to claim 1 method it is characterised in that described three chromatographies refine as an anion-exchange chromatography essence
System and twice blue dyess affinity chromatograph refine.
3. according to claim 2 method it is characterised in that with described anion-exchange chromatography refine in eluting aat aat
Eluent balances the blue dyess affinity column during blue dyess affinity chromatograph refines, and is used for eluting aat；Preferably, use
During anion-exchange chromatography refines, the balance liquid eluting of balance anion displacement chromatography post removes and is not associated with turning ferrum egg on post
White and albumin.
4. according to claim 3 method it is characterised in that described aat eluent is the ph 5.0- containing 45-65mm nacl
5.4th, 10-50mm acetate buffer；Preferably, described balance liquid is ph 5.0-5.4,10-50mm acetate buffer.
5. according to the arbitrary methods described of claim 2-4 it is characterised in that using flat during described anion-exchange chromatography is refined first
Weighing apparatus liquid balance anion displacement chromatography post, then loading, first rinse anion exchange chromatography to go with same balance liquid afterwards
Remove and be not associated with the transferrinss on post and albumin, then existed with ph4.6-4.8,10-50mm acetate buffer elution of bound
Albumin on post and vitamin d associated proteins, finally use aat elution aat, obtain aat elution samples, whole purification
The flow velocity of process is 150-350cm/h；
The medium preferred ion cation exchange groups of anion exchange chromatography be diethylamino ethyl (diethylaminoethyl,
Deae weak anionic displacement chromatography medium) or ion-exchange group be quaternary amine base (quaternary ammonium group,
Q) strong anion displacement chromatography medium.
6. according to the arbitrary methods described of claim 2-5 it is characterised in that described blue dyess affinity chromatograph is used in refining first
Described aat eluent balances blue dyess affinity column, then loading, rinses blue dyess with described aat eluent afterwards
Affinity column, to remove the aat not being combined with blue dyess, is obtained aat and penetrates sample, the flow velocity of whole process of purification is 70-
120cm/h；
The preferred affinity ligand of medium of blue dyess affinity column is the affinity chromatography medium of blue dyess, such as blue
sepharose 6fast flow；
Preferably, secondary blue dyess affinity chromatograph and a blue dyess parent during described blue dyess affinity chromatograph refines twice
With the column volume ratio of chromatography for 1:(15-20).
7. according to the arbitrary methods described of claim 2-6 it is characterised in that twice blue dyess affinity chromatograph refine can using with
One blue dyess affinity column；Particularly as follows: after a blue dyess affinity chromatograph terminates, used blue dyess affinity chromatograph
Ph 5.0-8.5,10-50mm acetate buffer containing 0.5-1.0m kscn for the post, phosphate buffer or tris-hcl delay
Rush macromole (molecular weight ＞ 180kda) foreign protein on post for the liquid elution of bound, transferrinss, albumin, vitamin d combination
Albumen.
8. according to the arbitrary methods described of claim 1-7 it is characterised in that peg is precipitated gained in a described inactivation of virus
The ph value of supernatant is adjusted to 6.5-7.5,0.22 μm of membrane filtration, adds TRI N BUTYL PHOSPHATE and tween 80 to make its final concentration
It is respectively 0.3% (w/v, g/100ml), 1% (w/v, g/100ml), 23-25 DEG C of water bath with thermostatic control 6h carries out s/d method inactivation of virus
Process, obtain inactivateing supernatant.
9. according to the arbitrary methods described of claim 1-8 it is characterised in that by lyophilizing gained aat in described secondary inactivation of virus
Dried frozen aquatic productses carry out dry heating method viral inactivation treatment 30min to inactivate togavirus and non-cyst membrane disease in 99-100 DEG C of water-bath
Poison, obtains final alpha1-antitrypsin.
10. according to the arbitrary methods described of claim 1-9 it is characterised in that specifically including following steps:
A), the dissolving of cohn components precipitate, concentration: the cohn components precipitate water dissolution of 8-10 times of weight, 0-10 DEG C of stirring
2-4h, adjusts ph value to 7.5-8.0, solid-liquid separation, abandons solid, obtain cohn component lysate, cohn component is dissolved
Liquid is concentrated by ultrafiltration 4-10 times of volume and obtains cohn component concentrated solution, and the protein content in cohn component concentrated solution is 20-
40mg/ml；
B), peg precipitation: the ph value of regulating step a) gained cohn component concentrated solution to 5.0-5.4,0-10 DEG C of addition peg
4000 make peg 4000 be completely dissolved to its final concentration of 10-15% (w/v, g/100ml), stirring, 4000- after 0-10 DEG C of standing
It is centrifuged 20-60min under 6000g, discard and comprise macromole (molecular weight ＞ 180kda) foreign protein, transferrinss, albumin and dimension
The raw element protein-bonded precipitation of d, takes supernatant；
C) a, inactivation of virus: the ph value of step b) gained supernatant is adjusted to 6.5-7.5,0.22 μm of membrane filtration, add
TRI N BUTYL PHOSPHATE (tri-n-butyl phosphate, tnbp) and tween 80 (tween 80) make its final concentration be respectively
0.3% (w/v, g/100ml), 1% (w/v, g/100ml), 23-25 DEG C of water bath with thermostatic control 6h carries out s/d method viral inactivation treatment, obtains
To inactivation supernatant；
D), once it is concentrated by ultrafiltration: inactivate supernatant with ph 5.0-5.4,10-50mm acetate buffer ultrafiltration step c) gained,
Obtain ultrafiltrate, the molecular cut off of ultrafilter membrane is 20-30kda；
E), anion-exchange chromatography refines: using anion-exchange chromatography purification step d) gained ultrafiltrate；Anion exchange layer
Analysis medium can be selected for the weak anionic that ion-exchange group is diethylamino ethyl (diethylaminoethyl, deae) and hands over
Changing chromatography media or ion-exchange group is quaternary amine base (quaternary ammonium group, strong anion switching layer q)
Analysis medium；Use ph 5.0-5.4,10-50mm acetate buffer first as balance liquid balance anion displacement chromatography post, so
Afterwards by step d) gained ultrafiltrate loading, first rinsed with same balance liquid afterwards and be not associated with the transferrinss on post and white egg
In vain, then with albumin on post for ph4.6-4.8,10-50mm acetate buffer elution of bound and vitamin d associated proteins,
Finally with the balance liquid containing 45-65mm nacl as aat elution aat, obtain aat elution samples, whole process of purification
Flow velocity be 150-350cm/h；
F), a blue dyess affinity chromatograph refines: using blue dyess affinitive layer purification step e) gained aat eluting sample
Product；Blue dyess affinity chromatography medium can be selected for the affinity chromatography medium that affinity ligand is blue dyess, such as blue
sepharose 6fast flow；Aat eluent is used to balance blue dyess affinity column, then by step e) gained first
Aat elution samples loading, the aat using the flushing of aat eluent not to be combined with blue dyess afterwards, obtain an aat and penetrate sample,
The flow velocity of whole process of purification is 70-120cm/h；Used blue dyess affinity column can use containing 0.5-1.0m kscn's
Ph 5.0-8.5,10-50mm acetate buffer, phosphate buffer or tris-hcl buffer solution elution combine big on post
Molecule (molecular weight ＞ 180kda) foreign protein, transferrinss, albumin, vitamin d associated proteins, then reuse；
G), secondary blue dyess affinity chromatograph refines: uses aat eluent to balance blue dyess affinity column first, blue dye
The material same step f) of affinity chromatography medium；Then aat of step f) gained is penetrated sample loading, use aat eluent to rush afterwards
Wash the aat not being combined with blue dyess, obtain secondary aat and penetrate sample；Blue dyess affinity chromatograph in step g) and step f)
The column volume of post is than for 1:(15-20)；
H), lyophilizing: the secondary aat of step g) gained is concentrated by ultrafiltration and penetrates sample, make the protein content of this sample reach 10-20mg/
Ml, is subsequently adding protective agent histidine, the final concentration of 0.05-0.1m of histidine, and 0.22 μm of membrane filtration is degerming, subpackage, lyophilizing,
Obtain aat dried frozen aquatic productses；
I), secondary inactivation of virus: step h) gained aat dried frozen aquatic productses are carried out dry heating method inactivation of virus in 99-100 DEG C of water-bath
Process 30min, obtain final alpha1-antitrypsin (aat).
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107163138A (en) * | 2017-03-28 | 2017-09-15 | 深圳市卫光生物制品股份有限公司 | A kind of antitryptic isolation and purification methods of human plasma protein fraction α 1 |
| CN107216384A (en) * | 2017-05-11 | 2017-09-29 | 深圳市卫光生物制品股份有限公司 | A kind of method that human plasma transferrins is isolated and purified |
| CN111269307A (en) * | 2020-02-26 | 2020-06-12 | 国药集团武汉血液制品有限公司 | Method for removing hybrid protein IgM in raw material for preparing C1 esterase inhibitor |
| CN112409476A (en) * | 2020-08-13 | 2021-02-26 | 重庆中元汇吉生物技术有限公司 | Purification method of four blood-derived proteins |
| CN113176133A (en) * | 2021-03-15 | 2021-07-27 | 广州邦德盛生物科技有限公司 | Method for separating protein and lipid in blood plasma or blood serum and matrix blood serum |
| WO2021147857A1 (en) * | 2020-01-20 | 2021-07-29 | Wuxi Biologics (Shanghai) Co., Ltd | A novel wash buffer solution for affinity chromatography |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107163138A (en) * | 2017-03-28 | 2017-09-15 | 深圳市卫光生物制品股份有限公司 | A kind of antitryptic isolation and purification methods of human plasma protein fraction α 1 |
| CN107216384A (en) * | 2017-05-11 | 2017-09-29 | 深圳市卫光生物制品股份有限公司 | A kind of method that human plasma transferrins is isolated and purified |
| WO2021147857A1 (en) * | 2020-01-20 | 2021-07-29 | Wuxi Biologics (Shanghai) Co., Ltd | A novel wash buffer solution for affinity chromatography |
| CN114901671A (en) * | 2020-01-20 | 2022-08-12 | 上海药明生物技术有限公司 | Novel wash buffer solution for affinity chromatography |
| CN114901671B (en) * | 2020-01-20 | 2024-02-20 | 上海药明生物技术有限公司 | Novel wash buffer solutions for affinity chromatography |
| CN111269307A (en) * | 2020-02-26 | 2020-06-12 | 国药集团武汉血液制品有限公司 | Method for removing hybrid protein IgM in raw material for preparing C1 esterase inhibitor |
| CN111269307B (en) * | 2020-02-26 | 2021-12-21 | 国药集团武汉血液制品有限公司 | Method for removing hybrid protein IgM in raw material for preparing C1 esterase inhibitor |
| CN112409476A (en) * | 2020-08-13 | 2021-02-26 | 重庆中元汇吉生物技术有限公司 | Purification method of four blood-derived proteins |
| CN113176133A (en) * | 2021-03-15 | 2021-07-27 | 广州邦德盛生物科技有限公司 | Method for separating protein and lipid in blood plasma or blood serum and matrix blood serum |
| CN113176133B (en) * | 2021-03-15 | 2024-02-13 | 广州邦德盛生物科技有限公司 | Method for separating protein and lipid in blood plasma or serum and matrix serum |
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