CN106349387B - Method for purifying α 1-antitrypsin from Cohn component IV precipitate - Google Patents

Abstract

The invention discloses a method for purifying α 1-antitrypsin from Cohn component IV precipitate, which comprises the steps of sequentially dissolving and concentrating the Cohn component IV precipitate, PEG precipitating, primary virus inactivating, primary ultrafiltration concentrating, refining by three-time chromatography, freeze-drying and secondary virus inactivating to finally obtain α 1-antitrypsin, wherein the Cohn component IV precipitate which is the waste of albumin production by a low-temperature ethanol method is used as a starting material to prepare AAT, so that the comprehensive utilization rate of raw material plasma is improved.

Description

Method for purifying α 1-antitrypsin from Cohn component IV precipitate

Technical Field

The invention relates to the technical field of protein purification, in particular to a method for purifying α 1-antitrypsin from Cohn component IV precipitate.

Background

α 1-antitrypsin (alpha 1-antitrypsin, AAT) is a single-chain glycoprotein composed of 394 amino acids and 3 oligosaccharide side chains, and has molecular weight of 52kDa, isoelectric point of 4.8, active site located at 358-359 Met-Ser region, 70-80% of AAT synthesized and secreted by liver cells, and in addition, small amount of AAT can be produced by monocyte, macrophage and alveolus.

The main physiological function of AAT is to maintain the balance of the protease-antipain system and protect normal tissues from enzymatic damage. When the concentration of AAT in normal human plasma is 1.0-2.0 mg/ml and is lower than 0.5mg/ml, elastase (NE) released by degranulation of neutrophils is difficult to resist, and NE can degrade elastic fibers in alveolar connective tissue, so that emphysema and chronic obstructive pulmonary disease can be caused by development of NE. AAT deficiency is an autosomal dominant genetic disease, discovered by Swedish physicians Laurell and Eriksso in 1963, and named AAT deficiency (alpha-1 antistrypsindence, AATD). The common mutants are S type and Z type, and Z type AAT can also form polymers, and the polymers are gathered on the endoplasmic reticulum of liver cells to finally cause diseases such as hepatitis, liver cirrhosis, liver cancer and the like. Exogenous AAT supplementation therapy is clinically adopted to correct the unbalance of AAT-NE, and the level of AAT in plasma can reach a protective threshold by intravenous infusion of AAT (60mg/kg/week), so that the anti-NE capability of serum is obviously improved.

In recent years, more and more studies have shown that AAT is not only a protease inhibitor, but also regulates immunity, controls inflammation and infection by pathogenic microorganisms. Among them, studies on the delay of development of type I diabetes and reduction of the number of transplanted islets by inhibiting inflammatory response by AAT have entered the clinical trial stage.

In 1987, the FDA approved the first plasma-derived freeze-dried preparation of AAT (prolactin) for the treatment of patients with emphysema with congenital AAT deficiency. Currently, there are 6 AAT products (prolactin, Araliast, Zemaira, prolactin-C, Araliast NP, Glasia) on the market. The above-mentioned AAT product is prepared by using Cohn Fraction IV precipitate as waste material produced by low-temp. alcohol method to produce albumin as initial raw material, except Zemaira, other 5AAT products are all firstly made up by adopting polyethylene glycol (PEG) precipitation method or PEG-conjugated ZnCl₂Coarse purification by a precipitation method; refining by anion exchange chromatography or anion and cation exchange chromatography; the pasteur virus inactivation method, the nano-membrane filtration and the S/D method effectively inactivate or remove viruses, and ensure the safety of the clinical use of the product.

Among them, the prototype of the process for preparing prolactin is the purification method (Preparation and properties of alpha 1-protease inhibitor concentrate from human plasma. Vox Sang.1985; 48(6):333-42.) published by Coan MH et al in 1985, in which the Cohn component IV-1 precipitate is resuspended in Tris-HCl buffer solution with pH8.2, and AAT is prepared by PEG 3350 fractional precipitation combined with DEAE-Sepharose CL-6B anion exchange chromatography. In the published patents (U.S. Pat. No. 4,4379087,4439358) Coan MH et al selected PEG with a molecular weight of 3000-4000 as a precipitant, the amount of PEG increased with increasing pH of the solution. After PEG precipitation, the impure protein is removed, AAT is retained in the supernatant, and anion exchange chromatography can selectively elute the AAT. The purification process uses sucrose and sodium citrate as protective agents to inactivate the pasteurellosis virus, and the purity of the AAT final product is 60 percent. The product obtained by the purification process has low purity, only comprises one-step virus inactivation process, and cannot ensure the safety of clinical use of the final product.

In the patent of LebingW et al (US6462180), the purity of AAT is improved by combining PEG precipitation with anion-cation exchange chromatography, the AAT bound to anion exchange chromatography is selectively eluted, the AAT is retained in the penetration peak of cation exchange chromatography by adjusting pH value and electric conductivity, and finally virus inactivation treatment is carried out by using detergent (Tween 20) and nano-membrane filtration, and the purity of the final product of AAT is 95%. Patent application CN102993298A shows that by adopting PEG precipitation method combined with anion-cation exchange chromatography, and virus inactivation process including S/D method and 15nm membrane filtration, AAT preparation with purity of 99-100% (purity obtained by cellulose acetate membrane electrophoresis) can be obtained. Patent application CN102180966A shows that cold ethanol precipitation, virus inactivation by an S/D method, PEG precipitation, two-step anion exchange chromatography and pasteur virus inactivation are successively carried out on a Cohn component IV-1 precipitation dissolving solution, and finally an AAT final product with the purity of 96-99% is obtained. The purification process adopts ion exchange chromatography to refine AAT, however, the ion exchange chromatography is used for separating the protein by utilizing the difference of the charge number of the protein under the condition of different pH values and the difference of the combination with a chromatography medium. The number of charges of the protein is determined by the size of the isoelectric point, the isoelectric point of the albumin is 4.7-4.9, and the albumin is connected with AAT (4.8)Recently, it is therefore difficult to remove albumin in high content in the Cohn fraction iv precipitate using ion exchange chromatography, which seriously affects the purity and specific activity of AAT. In addition, SDS-PAGE shows that the electrophoresis positions of albumin and AAT are close (FIG. 3), which makes the AAT sample containing albumin present a protein band when being analyzed by SDS-PAGE, and the sample in lane a in FIG. 3 is the AAT mixed with 20% of human serum albumin after being purified, and the sample and the control AAT

The positions on the electrophoretogram are basically the same, which is why the purification process is difficult to remove albumin, but higher purity is obtained, that is, the purity measured by the AAT obtained by the three purification processes is not accurate.

Zhuwei et al (α 1-preparation of antitrypsin preparation and its virus inactivation, journal of China biologics, 2001, 14 (2): 97-101) performed PEG, organic solvent, isoelectric point precipitation on Cohn fraction IV precipitation extract, obtained AAT preparation with purity of 91.3 + -1.52% by anion exchange chromatography and gel filtration with purification multiple of about 27.42 times, and Pasteur virus inactivation method effectively inactivated virus.A method for preparing AAT provided in the following two patents (CN101274956A, CN101747432A) also refined AAT by anion exchange chromatography and gel filtration, and virus inactivation methods were respectively Papanicur virus inactivation method combined with dry heat method, and S/D method combined with D20 nano-membrane filtration.

Travis J et al (Selective removal of albumin from plasma by yaffinity chromatography. Clin Chim acta.1973Nov 23; 49(1):49-52.) found that a Blue dye Gibacron Blue F3GA could bind to albumin and be coupled to agarose to remove 96% of the albumin in plasma without losing other proteins Pannel R et al (Isolation and properties of human plasma alpha-1-protease inhibitor. biochemistry. 1974Dec17; 13(26):5439-45.) Sepharose-Blue was not suitable for albumin adsorption, ammonium sulfate precipitation binding, two-step anion exchange chromatography extraction of pure AAT from whole blood and the like (purification of Blue agarose beads α 1, anti-trypsin preparation using Sepharose-Sepharose chromatography, purification of albumin from whole blood using Sepharose-Blue Sepharose chromatography, purification of crude agarose gel, purification of albumin by Sepharose gel chromatography, purification of Sepharose-Sepharose affinity chromatography, purification of albumin by Sepharose chromatography, purification of Sepharose affinity chromatography, purification of albumin by Sepharose chromatography, purification of Sepharose-Sepharose gel chromatography, purification of albumin by Sepharose chromatography, purification of Sepharose gel chromatography, purification of albumin by Sepharose chromatography, purification of albumin, purification of.

Disclosure of Invention

The invention aims to provide a method for purifying high-purity α 1-antitrypsin from Cohn component IV precipitate, which comprises the steps of dissolving and concentrating the Cohn component IV precipitate, precipitating with PEG, inactivating viruses for the first time, performing ultrafiltration concentration for the first time, performing chromatography refining for the third time, freeze-drying and inactivating viruses for the second time to finally obtain α 1-antitrypsin (AAT).

The three times of chromatographic refining are one time of anionic exchange chromatographic refining and two times of blue dye affinity chromatographic refining.

Balancing the blue dye affinity chromatographic column in the blue dye affinity chromatographic refining with the AAT eluent for eluting AAT in the anion exchange chromatographic refining, and eluting AAT; preferably, transferrin and albumin not bound to the column are removed by elution with the equilibration solution from an equilibrium anion exchange chromatography column in anion exchange chromatography purification.

The AAT eluent is acetate buffer solution with pH of 5.0-5.4 and 10-50mM, which contains 45-65mM NaCl; preferably, the equilibration solution is pH 5.0-5.4, 10-50mM acetate buffer solution.

In the anion exchange chromatography refining, firstly, balancing an anion exchange chromatography column by using a balancing solution, then loading, then washing the anion exchange chromatography column by using the same balancing solution to remove transferrin and albumin which are not bound on the column, then eluting albumin and vitamin D binding protein which are bound on the column by using acetate buffer solution with pH4.6-4.8 and 10-50mM, and finally eluting AAT by using AAT eluent to obtain an AAT elution sample, wherein the flow rate in the whole purification process is 150-350 cm/h;

the medium of the anion exchange chromatography column is preferably a weak anion exchange chromatography medium whose ion exchange group is Diethylaminoethyl (DEAE) or a strong anion exchange chromatography medium whose ion exchange group is quaternary ammonium group (Q).

In the blue dye affinity chromatography refining, firstly, the AAT eluent is used for balancing a blue dye affinity chromatography column, then, the sample is loaded, and then, the AAT eluent is used for washing the blue dye affinity chromatography column to remove AAT which is not combined with the blue dye, so that an AAT penetrating sample is obtained, wherein the flow rate in the whole purification process is 70-120 cm/h;

the medium of the blue dye affinity chromatographic column preferably has affinity chromatography medium whose affinity ligand is blue dye, such as blue Sepharose 6Fast Flow;

preferably, the column volume ratio of the secondary blue dye affinity chromatography to the primary blue dye affinity chromatography in the two blue dye affinity chromatography purifications is 1 (15-20).

The same blue dye affinity chromatographic column can be used for the two times of blue dye affinity chromatographic refining; the method specifically comprises the following steps: after one blue dye affinity chromatography is finished, the used blue dye affinity chromatography column uses 0.5-1.0M KSCN containing pH 5.0-8.5, 10-50mM acetate buffer solution, phosphate buffer solution or Tris-HCl buffer solution to elute macromolecular (molecular weight is more than 180kDa) hybrid protein, transferrin, albumin and vitamin D binding protein which are combined on the column.

And (3) adjusting the pH value of the supernatant obtained by PEG precipitation to 6.5-7.5 in the primary virus inactivation, filtering with a 0.22-micron filter membrane, adding tri-n-butyl phosphate and Tween-80 to make the final concentrations of the three-n-butyl phosphate and the Tween-80 respectively be 0.3% (w/v, g/100mL) and 1% (w/v, g/100mL), and carrying out S/D virus inactivation treatment in a thermostatic water bath at 23-25 ℃ for 6h to obtain an inactivated supernatant.

And in the secondary virus inactivation, carrying out dry heat virus inactivation treatment on the AAT freeze-dried product obtained by freeze drying in a water bath at 99-100 ℃ for 30min to inactivate the enveloped virus and the non-enveloped virus, thus obtaining the final α 1-antitrypsin.

The method specifically comprises the following steps:

a) and dissolving and concentrating Cohn component IV precipitate: dissolving the Cohn component IV precipitate with 8-10 times of water by weight, stirring for 2-4h at 0-10 ℃, adjusting the pH value to 7.5-8.0, performing solid-liquid separation, discarding solids to obtain a Cohn component IV dissolved solution, and performing ultrafiltration concentration on the Cohn component IV dissolved solution by 4-10 times of volume to obtain a Cohn component IV concentrated solution, wherein the protein content in the Cohn component IV concentrated solution is 20-40 mg/ml;

b) and PEG precipitation: adjusting the pH value of the Cohn component IV concentrated solution obtained in the step a) to 5.0-5.4, adding PEG4000 at 0-10 ℃ until the final concentration is 10-15% (w/v, g/100ml), stirring to completely dissolve the PEG4000, standing at 0-10 ℃, centrifuging for 20-60min at 4000-6000g, discarding the precipitate containing macromolecular (molecular weight is more than 180kDa) hybrid protein, transferrin, albumin and vitamin D binding protein, and taking the supernatant;

c) and primary virus inactivation: adjusting the pH value of the supernatant obtained in the step b) to 6.5-7.5, filtering with a 0.22 mu m filter membrane, adding tri-n-butyl phosphate (TNBP) and Tween-80 (Tween 80) to make the final concentrations of the tri-n-butyl phosphate and the Tween-80 respectively be 0.3% (w/v, g/100mL) and 1% (w/v, g/100mL), and carrying out S/D virus inactivation treatment in a constant-temperature water bath at 23-25 ℃ for 6h to obtain an inactivated supernatant;

d) and primary ultrafiltration concentration: ultrafiltering the inactivated supernatant of step c) with 10-50mM acetate buffer solution at pH 5.0-5.4 to obtain ultrafiltrate, wherein the molecular weight cut-off of the ultrafiltration membrane is 20-30 kDa;

e) and anion exchange chromatography refining: purifying the ultrafiltrate obtained in step d) by anion exchange chromatography; the anion exchange chromatography medium can be weak anion exchange chromatography medium with Diethylaminoethyl (DEAE) ion exchange group or strong anion exchange chromatography medium with quaternary ammonium group (Q); balancing an anion exchange chromatography column by using an acetate buffer solution with pH of 5.0-5.4 and 10-50mM as a balancing solution, then loading the ultrafiltrate obtained in the step D), washing transferrin and albumin which are not bound on the column by using the same balancing solution, eluting albumin and vitamin D binding protein which are bound on the column by using the acetate buffer solution with pH of 4.6-4.8 and 10-50mM, and finally eluting AAT by using a balancing solution containing 45-65mM NaCl as an AAT eluent to obtain an AAT elution sample, wherein the flow rate in the whole purification process is 150-350 cm/h;

f) and one-time blue dye affinity chromatography refining: purifying the AAT elution sample obtained in the step e) by adopting blue dye affinity chromatography; the blue dye affinity chromatography medium can be affinity chromatography medium with blue dye as affinity ligand, such as blue Sepharose 6Fast Flow; firstly, using AAT eluent to balance a blue dye affinity chromatographic column, then loading the AAT eluted sample obtained in the step e), and then using the AAT eluent to wash the AAT which is not combined with the blue dye to obtain a primary AAT penetrating sample, wherein the flow rate of the whole purification process is 70-120 cm/h; the used blue dye affinity chromatographic column can use 0.5-1.0M KSCN pH 5.0-8.5, 10-50mM acetate buffer, phosphate buffer or Tris-HCl buffer to elute macromolecular (molecular weight > 180kDa) hetero protein, transferrin, albumin and vitamin D binding protein bound on the column, and then the macromolecular hetero protein, the transferrin, the albumin and the vitamin D binding protein are repeatedly used;

g) and secondary blue dye affinity chromatography refining: firstly, using AAT eluent to balance a blue dye affinity chromatographic column, wherein the blue dye affinity chromatographic medium is the same as that in the step f); then loading the primary AAT penetrating sample obtained in the step f), and then washing the AAT which is not combined with the blue dye by using an AAT eluent to obtain a secondary AAT penetrating sample; the column volume ratio of the blue dye affinity chromatographic column in the step g) to the blue dye affinity chromatographic column in the step f) is 1 (15-20);

h) and freeze-drying: ultrafiltering and concentrating the secondary AAT obtained in the step g) to penetrate a sample to enable the protein content of the sample to reach 10-20mg/ml, then adding a protective agent histidine, wherein the final concentration of the histidine is 0.05-0.1M, filtering and sterilizing with a 0.22-micron filter membrane, subpackaging, and freeze-drying to obtain an AAT freeze-dried product;

i) and secondary virus inactivation, namely performing virus inactivation treatment on the AAT freeze-dried product obtained in the step h) for 30min by a dry heat method in a water bath at the temperature of 99-100 ℃ to obtain the final α 1-antitrypsin (AAT).

Compared with the prior art, the invention has the beneficial effects that:

the method of the invention takes the waste Cohn component IV precipitate which is the albumin production waste produced by the low-temperature ethanol method as the initial raw material to prepare AAT, which is beneficial to improving the comprehensive utilization rate of the raw material plasma. In the whole process, PEG precipitation, anion exchange chromatography and two-time blue dye affinity chromatography are adopted, so that macromolecular (molecular weight is more than 180kDa) hybrid protein, transferrin, albumin and vitamin D binding protein can be effectively removed, the high-purity AAT preparation is finally obtained, the purity of the obtained final product is more than 95 percent, trypsin is used as an inhibition target enzyme, and the specific activity is more than 3900U/mg. In the method, the conditions of the affinity chromatography of the blue dye are the same in two times, and the equilibrium solution is the same as the eluent of the anion exchange chromatography AAT, so that the inconvenience caused by the preparation of various solutions is avoided. In addition, the method of the invention reduces the step of ultrafiltration concentration, simplifies the production process and is suitable for large-scale preparation of AAT. Finally, the method adopts two virus inactivation processes with different principles, namely an S/D method capable of effectively inactivating the enveloped viruses and a dry heat method capable of effectively inactivating the enveloped viruses and the non-enveloped viruses, so that the virus safety of the AAT preparation is ensured.

Drawings

FIG. 1 is a flow chart of the method of the present invention;

FIG. 2 shows an electrophoretogram of a solution of a Cohn fraction IV precipitate, wherein: m is a protein Marker, and a is a Cohn component IV precipitation solution;

FIG. 3 is a graph showing the comparison of the electrophoresis positions of AAT and albumin, wherein: m is a protein Marker, a is a mixture of 20% of human serum albumin mixed in the AAT obtained in example 3, B is human serum albumin (CSL Behring), and A is an AAT standard product provided by Sigma company;

FIG. 4 is an electrophoretogram of PEG supernatant obtained after PEG precipitation, PEG precipitation and ultrafiltrate obtained after ultrafiltration concentration in example 1, wherein: m is a protein Marker, a is a solution of a Cohn component IV precipitate, b is PEG supernatant, c is PEG precipitate, and d is ultrafiltrate;

FIG. 5 is an electrophoretogram showing a penetration sample, a pH 4.7 elution sample, and a 50mM NaCl AAT elution sample obtained after anion exchange chromatography purification in example 2, wherein: m is a protein Marker, a is a Cohn FV precipitation dissolving solution, b is PEG supernatant, c is PEG precipitation, d is ultrafiltrate, e is a penetration sample, F is a pH 4.7 elution sample, g is a 50mM NaCl AAT elution sample, and h is a 100M NaCl elution sample;

FIG. 6 is an electrophoretogram of a pH 5.4AAT breakthrough sample, a 0.5M KSCN elution sample obtained after two blue dye affinity chromatography purifications of example 3, wherein: m is protein Marker, a is 45mM NaCl AAT elution sample ultrafiltrate, B is a first pH 5.4AAT penetration sample, c is a first 0.5M KSCN elution sample, d is a second pH 5.4AAT penetration sample, e is a second 0.5M KSCN elution sample, A is an AAT standard product provided by Sigma company, and B is human blood albumin (CSLBehring);

FIG. 7 is an electrophoretogram of PEG supernatant ultrafiltrate, anion exchange chromatography 50mM NaCl AAT elution sample, first blue dye affinity chromatography pH5.2AAT breakthrough sample, and second blue dye affinity chromatography pH5.2AAT breakthrough sample obtained by the entire separation and purification method of example 2, wherein: m is a protein Marker, a is a Cohn FV precipitation dissolving solution, b is PEG supernatant ultrafiltrate, c is an anion exchange chromatography 50mM NaCl AAT elution sample, d is a first blue dye affinity chromatography pH5.2AAT penetration sample, e is a second blue dye affinity chromatography pH5.2AAT penetration sample, and A is an AAT standard product provided by Sigma company;

FIG. 8 is a High Performance Liquid Chromatography (HPLC) profile, wherein the sample is pH 5.4 AAT-penetrating sample obtained by the second blue dye affinity chromatography purification of example 3;

FIG. 9 shows a High Performance Liquid Chromatography (HPLC) profile, wherein the sample is an AAT standard provided by Sigma;

FIG. 10 is a diagram showing an electrophoresis chart of a 50mM NaCl-permeated sample and a 100mM NaClAAT-eluted sample obtained by anion exchange chromatography purification in comparative example 1, wherein: m is a protein Marker, a is a Cohn component IV precipitation dissolving solution, b is PEG supernatant, c is PEG precipitation, d is ultrafiltrate, e is a 50mM NaCl penetration sample, and f is a 100mM NaCl AAT elution sample;

FIG. 11 is an electrophoretic image of 25mM NaCl eluate, 50mM NaCl eluate, 100mM NaCl AAT eluate, 150mM NaCl eluate, 200mM NaCl eluate obtained by anion exchange chromatography purification in comparative example 2, in which: m is a protein Marker, a is a Cohn component IV precipitation dissolving solution, b is PEG supernatant ultrafiltrate, c is a penetration sample, d is a 25mM NaCl elution sample, e is a 50mM NaCl elution sample, f is a 100mM NaCl AAT elution sample, g is a 150mM NaCl elution sample, and h is a 200mM NaCl elution sample;

FIG. 12 is a graph showing the electrophoresis of a pH6.5 AAT-permeated sample, a 0.5M KSCN eluted sample obtained by blue dye affinity chromatography in comparative example 3, wherein: m is a protein Marker, a is a Cohn FV precipitation dissolving solution, b is PEG supernatant, c is PEG precipitation, d is ultrafiltrate, e is an anion exchange chromatography penetration sample, F is a pH 4.7 elution sample, g is a 55mM NaCl AAT elution sample, h is a 100M NaCl elution sample, i is an AAT elution sample ultrafiltrate, g is a pH6.5 AAT penetration sample, and h is a 0.5MKSCN elution sample.

Detailed Description

The present invention provides a method for purifying α 1-antitrypsin from a Cohn fraction IV precipitate, see FIG. 1, comprising the steps of:

a) and dissolving and concentrating Cohn component IV precipitate: dissolving the Cohn component IV precipitate with 8-10 times of water by weight, stirring for 2-4h at 0-10 ℃, adjusting the pH value to 7.5-8.0, performing solid-liquid separation, discarding solids to obtain a Cohn component IV dissolved solution, and performing ultrafiltration concentration on the Cohn component IV dissolved solution by 4-10 times of volume to obtain a Cohn component IV concentrated solution, wherein the protein content in the Cohn component IV concentrated solution is 20-40 mg/ml;

b) and PEG precipitation: adjusting the pH value of the Cohn component IV concentrated solution obtained in the step a) to 5.0-5.4, adding PEG4000 at 0-10 ℃ until the final concentration is 10-15% (w/v, g/100ml), stirring to completely dissolve the PEG4000, standing at 0-10 ℃, centrifuging for 20-60min at 4000-6000g, discarding the precipitate containing macromolecular (molecular weight is more than 180kDa) hybrid protein, transferrin, albumin and vitamin D binding protein, and taking the supernatant;

c) and primary virus inactivation: adjusting the pH value of the supernatant obtained in the step b) to 6.5-7.5, filtering with a 0.22 mu m filter membrane, adding tri-n-butyl phosphate (TNBP) and Tween-80 (Tween 80) to make the final concentrations of the tri-n-butyl phosphate and the Tween-80 respectively be 0.3% (w/v, g/100mL) and 1% (w/v, g/100mL), and carrying out S/D virus inactivation treatment in a constant-temperature water bath at 23-25 ℃ for 6h to obtain an inactivated supernatant;

d) and primary ultrafiltration concentration: ultrafiltering the inactivated supernatant of step c) with 10-50mM acetate buffer solution at pH 5.0-5.4 to obtain ultrafiltrate, wherein the molecular weight cut-off of the ultrafiltration membrane is 20-30 kDa;

e) and anion exchange chromatography refining: purifying the ultrafiltrate obtained in step d) by anion exchange chromatography; the anion exchange chromatography medium can be weak anion exchange chromatography medium with Diethylaminoethyl (DEAE) ion exchange group or strong anion exchange chromatography medium with quaternary ammonium group (Q); firstly, balancing an anion exchange chromatography column by using 10-50mM acetate buffer solution with pH of 5.0-5.4 as balancing solution, then loading the ultrafiltrate obtained in the step D), washing transferrin and albumin which are not bound on the column by using the balancing solution, then eluting albumin and vitamin D binding protein which are bound on the column by using 10-50mM acetate buffer solution with pH of 4.6-4.8, and finally eluting AAT by using the balancing solution containing 45-65mM NaCl to obtain an AAT elution sample, wherein the flow rate of the whole purification process is 150-350 cm/h.

f) And one-time blue dye affinity chromatography refining: purifying the AAT elution sample obtained in the step e) by adopting blue dye affinity chromatography; the blue dye affinity chromatography medium can be affinity chromatography medium with blue dye as affinity ligand, such as blue Sepharose 6Fast Flow; firstly, balancing a blue dye affinity chromatographic column by using a balance liquid containing 45-65mM NaCl, then loading the AAT elution sample obtained in the step e), and then washing the AAT which is not combined with the blue dye by using the balance liquid containing 45-65mM NaCl to obtain a primary AAT penetration sample, wherein the flow rate of the whole purification process is 70-120 cm/h; the used blue dye affinity chromatographic column can use 0.5-1.0M KSCN pH 5.0-8.5, 10-50mM acetate buffer, phosphate buffer or Tris-HCl buffer to elute macromolecular (molecular weight > 180kDa) hetero protein, transferrin, albumin and vitamin D binding protein bound on the column, and then the macromolecular hetero protein, the transferrin, the albumin and the vitamin D binding protein are repeatedly used;

g) and secondary blue dye affinity chromatography refining: balancing a blue dye affinity chromatographic column by using a balancing solution containing 45-65mM NaCl, wherein the blue dye affinity chromatographic medium is the same as the step f); then loading the primary AAT penetrating sample obtained in the step f), and then washing the AAT which is not combined with the blue dye by using an equilibrium solution containing 45-65mM NaCl to obtain a secondary AAT penetrating sample; the column volume ratio of the blue dye affinity chromatographic column in the step g) to the blue dye affinity chromatographic column in the step f) is 1 (15-20).

h) And freeze-drying: ultrafiltering and concentrating the secondary AAT obtained in the step g) to penetrate a sample to enable the protein content of the sample to reach 10-20mg/ml, then adding a protective agent histidine, wherein the final concentration of the histidine is 0.05-0.1M, filtering and sterilizing by a 0.22 mu M filter membrane, subpackaging, and freeze-drying to obtain an AAT freeze-dried product.

i) And secondary virus inactivation, namely performing dry heat virus inactivation treatment on the AAT freeze-dried product obtained in the step h) in a water bath at 99-100 ℃ for 30min to obtain a final α 1-antitrypsin (AAT) product.

The present invention will be described more specifically and further illustrated with reference to specific examples, which are by no means intended to limit the scope of the present invention.

Example 1

a) And dissolving and concentrating Cohn component IV precipitate: 200g of the Cohn fraction IV precipitate was dissolved in 2000ml of water, stirred at 4 ℃ for 3 hours, adjusted to pH 7.5, and centrifuged to remove the diatomaceous earth, to obtain a Cohn fraction IV dissolved solution (see FIG. 2 for an electrophoretogram). And (3) carrying out ultrafiltration concentration on the Cohn component IV dissolved solution by 4 times to 500ml to obtain a Cohn component IV concentrated solution, wherein the protein content is 20-22 mg/ml.

b) And PEG precipitation: adjusting the pH value of the Cohn component IV concentrated solution to 5.0, adding 50g of PEG4000 to a final concentration of 10% (w/v, g/100ml) while stirring at 4 ℃, and continuously stirring for 30-45min to fully dissolve the PEG4000 after the addition is finished; standing at 4 deg.C for 30-45min, centrifuging at 6000g for 20min, discarding precipitate, and collecting supernatant; the precipitate contains large molecules (molecular weight > 180kDa), hetero-proteins, transferrin, albumin and vitamin D binding protein (see FIG. 4).

c) And primary virus inactivation: and (2) inactivating the virus by an S/D method, adjusting the pH value of the supernatant to 6.5 by using 0.5M NaOH, filtering by using a 0.22-micron filter membrane, adding an S/D reagent (consisting of 3% of TNBP and 10% of Tween 80) to ensure that the final concentrations of the TNBP and the Tween80 are 0.3% and 1% (w/v, g/100ml) respectively, and carrying out thermostatic water bath at 24 ℃ for 6h to obtain an inactivated supernatant.

d) And primary ultrafiltration concentration: the inactivated supernatant was ultrafiltered 5-10 volumes with pH 5.010mM acetate buffer to give an ultrafiltrate with a molecular weight cut-off of 30 kDa.

e) And anion exchange chromatography refining: purifying by anion exchange chromatography, using DEAE Sepharose Fast Flow weak anion exchange chromatography column (30 ml of column volume) as balance solution to balance 8-10 column volumes, using pH 5.010mM acetate buffer solution as balance solution, loading ultrafiltrate, washing transferrin and albumin which are not bound on the column by the balance solution, then using pH 4.610mM acetate buffer solution to elute albumin and vitamin D binding protein which are bound on the column, finally using balance solution containing 65mM NaCl to elute AAT which is bound on the column to obtain AAT elution sample, wherein the Flow rate of the whole purification process is 150 cm/h.

f) And one-time blue dye affinity chromatography refining: blue dye affinity chromatography purification, Blue Sepharose 6Fastflow Blue dye affinity chromatography column (column volume 20ml) with 65mM NaCl equilibrium liquid balance 8-10 column volume, then load AAT elution sample, after using 65mM NaCl equilibrium liquid washing not bound to the column AAT, get once AAT permeate sample, the whole purification process flow rate is 70 cm/h; finally, after eluting the macromolecular (molecular weight > 180kDa) hetero-protein, transferrin, albumin and vitamin D binding protein bound on the column with 0.5M KSCN in pH 5.010mM acetate buffer, the chromatographic column can be reused.

g) And secondary blue dye affinity chromatography refining: as in step f), the column volume was only 1ml and the sample loaded was the primary AAT breakthrough sample, resulting in a secondary AAT breakthrough sample.

h) And freeze-drying: ultrafiltering and concentrating the secondary AAT penetrating sample until the protein content is 10-11mg/ml and the molecular weight cut-off of the ultrafiltration membrane is 30 kDa; adding histidine as protective agent to make the final concentration of histidine 0.05M, filtering with 0.22 μ M filter membrane for sterilization, packaging, and lyophilizing to obtain AAT lyophilized product.

i) And performing secondary virus inactivation, namely performing virus inactivation treatment on the AAT freeze-dried product for 30min by a dry heat method in a water bath at the temperature of 99-100 ℃ to obtain a final α 1-antitrypsin (AAT) product.

The indices of the samples obtained in the various steps for the purification α 1-antitrypsin by the method of example 1 are given in Table 1.

Table 1 the AAT activity, protein content and specific activity in the samples obtained in the steps

SDS-PAGE gray scale scanning shows that the purity of the AAT product obtained in example 1 is 99.08%, trypsin is used as a target enzyme for AAT inhibition, the activity of the final product is 42638.28U/ml by using a chromogenic substrate method, the protein content is 10.17mg/ml by using a BCA method, the specific activity of the AAT product is 4192.55U/mg, and the purification multiple is 68.90 times.

Example 2

a) And dissolving and concentrating Cohn component IV precipitate: 1.2kg of Cohn fraction IV precipitate was dissolved in 10.8L of water, stirred at 0 ℃ for 4 hours, adjusted to pH 7.8 with 0.5M NaOH, and subjected to solid-liquid separation using a filter press to remove diatomaceous earth, thereby obtaining a Cohn fraction IV dissolved solution. And (3) carrying out ultrafiltration concentration on the Cohn component IV dissolved solution by 7 times to 2L to obtain a Cohn component IV concentrated solution, wherein the protein content is 29-31 mg/ml.

b) And PEG precipitation: adjusting the pH value of the Cohn component IV concentrated solution to 5.2 by using 0.5M HCl, adding 300g of PEG4000 to the final concentration of 15% (w/v, g/100ml) while stirring at the temperature of 0 ℃, and continuously stirring for 30-45min after the addition is finished to fully dissolve the PEG 4000; standing at 0 deg.C for 30-45min, centrifuging at 4000g for 30min, discarding precipitate, and collecting supernatant.

c) And primary virus inactivation: and (2) inactivating the virus by an S/D method, adjusting the pH value of the supernatant to 7.0 by using 0.5M NaOH, filtering by using a 0.22-micron filter membrane, adding an S/D reagent (consisting of 3% of TNBP and 10% of Tween 80) to ensure that the final concentrations of the TNBP and the Tween80 are 0.3% and 1% (w/v, g/100ml) respectively, and carrying out constant-temperature water bath for 6h at 25 ℃ to obtain an inactivated supernatant.

d) And primary ultrafiltration concentration: the inactivated supernatant was ultrafiltered 5-10 volumes with pH 5.220 mM acetate buffer to give an ultrafiltrate having a molecular weight cut-off of 30 kDa.

e) And anion exchange chromatography refining: anion exchange chromatography purification, Q Sepharose Fast Flow strong anion exchange chromatography column (column volume 150ml) using pH 5.220 mM acetate buffer solution as balance solution balance 8-10 column volumes, then the ultrafiltrate loading, then using balance solution to wash the transferrin and albumin not bound to the column, then using pH 4.720mM acetate buffer solution to elute albumin and vitamin D binding protein bound to the column, finally using balance solution containing 50mM NaCl to elute AAT bound to the column, obtaining AAT elution sample (figure 5), the Flow rate of the whole purification process is 350 cm/h.

f) And one-time blue dye affinity chromatography refining: blue dye affinity chromatography purification, Blue Sepharose 6Fastflow Blue dye affinity chromatography column (column volume 80ml) with 50mM NaCl equilibrium liquid balance 8-10 column volume, then sample AAT elution sample, after using 50mM NaCl equilibrium liquid washing not bound to the column AAT, get once AAT penetration sample, the whole purification process flow rate is 100 cm/h; finally, after eluting macromolecular (molecular weight > 180kDa) hetero-protein, transferrin, albumin and vitamin D binding protein bound on the column with 1M KSCN in phosphate buffer solution with pH 7.020mM, the chromatographic column can be reused.

g) And secondary blue dye affinity chromatography refining: as in step f), the column volume was only 4ml and the sample loaded was the primary AAT breakthrough sample, resulting in a secondary AAT breakthrough sample.

h) And freeze-drying: ultrafiltering and concentrating the secondary AAT penetrating sample until the protein content is 15-16mg/ml, and the molecular weight cut-off of the ultrafiltration membrane is 30 kDa; adding histidine as protective agent to make the final concentration of histidine 0.07M, filtering with 0.22 μ M filter membrane for sterilization, packaging, and lyophilizing to obtain AAT lyophilized product.

i) And performing secondary virus inactivation, namely performing virus inactivation treatment on the AAT freeze-dried product for 30min by a dry heat method in a water bath at the temperature of 99-100 ℃ to obtain a final α 1-antitrypsin (AAT) product.

The indices of the samples obtained in the various steps of α 1-antitrypsin purification by the method of example 2 are shown in Table 2. the electrophoretic patterns of the supernatant obtained in step b) of the method of example 2, the AAT-eluted sample obtained in step e), the primary AAT-permeabilized sample obtained in step f), and the secondary AAT-permeabilized sample obtained in step g) are shown in FIG. 7.

Table 2 the AAT activity, protein content and specific activity in the samples obtained in the steps

SDS-PAGE gray scanning shows that the purity of the AAT final product is 98.17 percent, trypsin is used as a target enzyme for AAT inhibition, the activity of the final product is 61940.16U/ml by using a chromogenic substrate method, the protein content is 15.55mg/ml by using a BCA method, the specific activity of the AAT final product is 3983.29IU/mg, and the purification multiple is 66.05 times.

Example 3

a) And dissolving and concentrating Cohn component IV precipitate: 4kg of Cohn fraction IV precipitate was dissolved in 32L of water, stirred at 10 ℃ for 2 hours, adjusted to pH 8.0 with 0.5M NaOH, and subjected to solid-liquid separation using a filter press to remove diatomaceous earth, to obtain a solution of Cohn fraction IV. And (3) carrying out ultrafiltration concentration on the Cohn component IV dissolved solution by 10 times to 3.6L to obtain a Cohn component IV concentrated solution, wherein the protein content is 38-40 mg/ml.

b) And PEG precipitation: adjusting the pH value of the Cohn component IV concentrated solution to 5.5 by using 0.5M HCl, adding 432g of PEG4000 to the final concentration of 12% (w/v, g/100ml) while stirring at 10 ℃, and continuously stirring for 3-4h after the addition is finished to fully dissolve the PEG 4000; standing at 10 deg.C overnight, centrifuging at 4000g for 60min, discarding precipitate, and collecting supernatant.

c) And primary virus inactivation: and (2) inactivating the virus by an S/D method, adjusting the pH value of the supernatant to 7.5 by using 0.5M NaOH, filtering by using a 0.22 mu M filter membrane, adding an S/D reagent (consisting of 3% of TNBP and 10% of Tween 80) to ensure that the final concentrations of the TNBP and the Tween80 are 0.3% and 1% (w/v, g/100ml) respectively, and carrying out constant-temperature water bath for 6h at 23 ℃ to obtain an inactivated supernatant.

d) And primary ultrafiltration concentration: the inactivated supernatant was ultrafiltered 5-10 volumes with pH 5.450 mM acetate buffer to give an ultrafiltrate having a molecular weight cut-off of 20 kDa.

e) And anion exchange chromatography refining: purifying by anion exchange chromatography, wherein a Q Sepharose Fast Flow strong anion exchange chromatography column (column volume 500ml) is used for balancing 8-10 column volumes by using pH 5.450 mM acetate buffer solution as a balancing solution, then an ultrafiltrate is loaded, then transferrin and albumin which are not bound on the column are washed by the balancing solution, then the albumin and vitamin D binding protein which are bound on the column are eluted by using pH 4.850mM acetate buffer solution, finally AAT which is bound on the column is eluted by using the balancing solution containing 45mM NaCl, and an AAT eluted sample is obtained, wherein the Flow rate of the whole purification process is 250 cm/h. For further chromatographic purification, AAT eluted sample is ultrafiltered and concentrated by 10 times volume with equilibrium solution containing 45mM NaCl and then used as the sample for next affinity chromatography.

f) And one-time blue dye affinity chromatography refining: blue dye affinity chromatography purification, Blue Sepharose 6Fastflow Blue dye affinity chromatography column (column volume 300ml) with 45mM NaCl balance solution balance 8-10 column volume, then load AAT elution sample, later with 45mM NaCl balance solution washing not bound to the column AAT, get once AAT permeate sample, the whole purification process flow rate is 120 cm/h; finally, after eluting the macromolecular (molecular weight > 180kDa) hetero-protein, transferrin, albumin and vitamin D binding protein bound to the column with 0.5M KSCN in a Tris-HCl buffer solution (pH 8.550 mM), the column can be reused.

g) And secondary blue dye affinity chromatography refining: the same step f) is carried out, only the column volume is 20ml, and the sample loading is a primary AAT penetration sample to obtain a secondary AAT penetration sample; after elution of the large molecules (> 180kDa molecular weight) hetero-protein, transferrin, albumin and vitamin D binding protein bound to the column with 0.5M KSCN in Tris-HCl buffer pH 8.550mM (FIG. 6), the column was reusable.

h) And freeze-drying: ultrafiltering and concentrating the secondary AAT penetrating sample until the protein content is 20-21mg/ml and the molecular weight cut-off of the ultrafiltration membrane is 20 kDa; adding protective agent histidine to make its final concentration be 0.1M, filtering with 0.22 μ M filter membrane for sterilization, packaging, and lyophilizing to obtain AAT lyophilized product.

i) And performing secondary virus inactivation, namely performing virus inactivation treatment on the AAT freeze-dried product for 30min by a dry heat method in a water bath at the temperature of 99-100 ℃ to obtain a final α 1-antitrypsin (AAT) product.

The indices of the samples obtained in the various steps for the purification α 1-antitrypsin by the method of example 3 are given in Table 3.

Table 3 the AAT activity, protein content and specific activity in the samples obtained in the steps

SDS-PAGE gray scanning shows that the purity of the AAT final product is 95.54 percent, trypsin is used as a target enzyme for AAT inhibition, the activity of the final product is 80206.92IU/ml by using a chromogenic substrate method, the protein content is 20.38mg/ml by using a BCA method, the specific activity of the AAT final product is 3935.57IU/mg, and the purification multiple is 66.34 times.

Mass spectrum identification:

the secondary AAT penetration sample is firstly analyzed by 10% SDS-PAGE, and after dyeing, the target protein band is cut into 1mm²The colloidal particles are subjected to in-gel enzymolysis to extract peptide fragments; a Thermo EASY-nLC liquid phase-Q active HF combined quadrupole rod Orbitrap mass spectrometer is adopted to detect a protein sequence, a Mascot (version: 2.1.0) search engine is utilized to carry out data retrieval, and a database is a uniport-human library, the first-order error is 15ppm, and the second-order error is 20 mmu. The fixation modification is as follows: cysteine was modified to urea methyl cysteine (Carbamidomethyl-Cys) with the following variable modifications: methionine Oxidation (Oxidation-M), Protein N-terminal acetylation (Protein N-term); trypsin digestion, and the enzymolysis fragment allows 2 missed cutting sites at most.

74 proteins were retrieved by Mascot, and the first 6 proteins are shown in Table 4.

TABLE 4AAT Mass Spectrometry identification results

As shown in Table 4, the results show that Alpha-1-antiprypsin is ranked first, the peak area is the largest, and reaches 1.40E +11, and the Mascot score is the highest and is 35070.4. Transferrin, albumin and vitamin D binding protein do not appear in the protein at the 6 th position, which shows that the purification method can effectively remove the foreign protein to obtain AAT.

High performance liquid chromatography analysis:

the secondary AAT-permeated sample was analyzed by high performance liquid chromatography using RIGOL L-3000 for single fractions, the control was an AAT standard purchased from Sigma, 30 μ g loaded, and the chromatographic conditions were as follows:

a chromatographic column: C4-ST2 column (4.6mm X50 mm, 5um C4 packing)

Mobile phase: a: 2% acetonitrile/98% H₂O/0.1% trifluoroacetic acid;

B:2％H₂o/98% acetonitrile/0.1% trifluoroacetic acid;

flow rate: 0.7. mu.l/min

Detection wavelength: 280nm

Elution conditions:

time	0	5	37	42	45	50
							％B	5	5	95	95	5	5

the results are shown in FIGS. 8 and 9. The HPLC charts in FIG. 8 and FIG. 9 show that a single main peak appears at the retention time of 28.5385min for the secondary AAT sample and two peaks appear at the retention times of 24.273min and 28.7635min for the control AAT standard, indicating that the purity of AAT purified by the method of the present invention is higher than that of the control AAT standard.

Comparative example 1

a) Dissolving and concentrating a Cohn component IV precipitate: the same as in example 1.

b) And PEG precipitation: the same as in example 1.

c) And primary virus inactivation: none.

d) And primary ultrafiltration concentration: the same as in example 1.

e) And anion exchange chromatography refining: purifying by anion exchange chromatography, using DEAE Sepharose Fast Flow weak anion exchange chromatography column (30 ml column volume) to balance 8-10 column volumes by using pH 6.525 mM phosphate buffer solution containing 50mM NaCl as balance solution, then loading ultrafiltrate, then washing transferrin and albumin and vitamin D binding protein which are not bound on the column by using the balance solution containing 50mM NaCl, and finally eluting AAT bound on the column by using the balance solution containing 100mM NaCl to obtain AAT elution sample, wherein the Flow rate of the whole purification process is 150 cm/h.

f) And one-time blue dye affinity chromatography refining: none.

g) And secondary blue dye affinity chromatography refining: none.

h) And freeze-drying: none.

i) And secondary virus inactivation: none.

Trypsin is used as a target enzyme for AAT inhibition, the activity of a final product is determined by a chromogenic substrate method, the protein content is determined by a BCA method, the specific activity is calculated, and indexes of samples obtained in the steps of comparative example 1 are shown in Table 5.

Table 5 the AAT activity, protein content and specific activity in the samples obtained in the steps

As can be seen from the results of activity measurement in Table 5, when the DEAE Sepharose Fast Flow weak anion exchange chromatography column was equilibrated at pH6.5, AAT was eluted with a buffer solution containing 100mM NaCl, the present invention was eluted with a buffer solution containing 65mM NaCl, the salt content of the eluate used in comparative example 1 was much higher than that of the present invention, and the obtained AAT eluate sample had higher protein activity, content and specific activity than that of the present invention, but SDS-PAGE showed more impurity bands in the eluted protein, i.e., high impurity protein content and low AAT purity.

From the results of SDS-PAGE analysis (see FIG. 10), it can be seen that the AAT elution sample contains transferrin, albumin and vitamin D binding protein, the buffer containing 100mM NaCl elutes part of the large molecular (molecular weight > 180kDa) of the hetero-protein while eluting AAT, and transferrin remains more, the purification effect is not as good as the present invention which elutes hetero-protein at a low pH (pH 4.6-pH4.8) and then elutes AAT at an increased salt concentration (45-65mM NaCl) (see FIG. 5).

Comparative example 2

a) Dissolving and concentrating a Cohn component IV precipitate: the same as in example 1.

b) And PEG precipitation: the same as in example 1.

c) And primary virus inactivation: none.

d) And primary ultrafiltration concentration: the same as in example 1.

e) And anion exchange chromatography refining: purifying by anion exchange chromatography, wherein a Q Sepharose Fast Flow strong anion exchange chromatography column (30 ml of column volume) is used for balancing 8-10 column volumes by using pH 5.225 mM acetate buffer solution as a balancing solution, then ultrafiltrate is loaded, then transferrin and albumin which are not bound on the column are washed by the balancing solution, then the proteins bound on the column are eluted step by using the balancing solution containing 25mM NaCl, the balancing solution containing 50mM NaCl, the balancing solution containing 100mM NaCl, the balancing solution containing 150mM NaCl and the balancing solution containing 200mM NaCl, and each eluted sample is collected to obtain an AAT eluted sample, and the Flow rate of the whole purification process is 150 cm/h.

f) -i), primary blue dye affinity chromatography refining, secondary blue dye affinity chromatography refining, freeze-drying, secondary virus inactivation: none.

Trypsin is used as a target enzyme for AAT inhibition, the activity of a final product is determined by a chromogenic substrate method, the protein content is determined by a BCA method, the specific activity is calculated, and indexes of samples obtained in the steps of comparative example 2 are shown in Table 6.

Table 6 the AAT activity, protein content and specific activity in the samples obtained in the steps

From the activity measurement results in table 6, it is known that, when Q Sepharose Fast Flow strong anion exchange chromatography column is equilibrated at pH5.2, AAT needs to be eluted with a buffer solution containing 100mM NaCl, the present invention can be eluted with a buffer solution containing 65mM NaCl, the salt content of the eluent used in comparative example 2 is much higher than that of the present invention, and the obtained AAT elution sample has higher protein activity, content and specific activity than that of the present invention, but SDS-PAGE shows that the eluted protein has more impurity bands, i.e., the impurity protein content is high, and the AAT purity is low.

From the results of SDS-PAGE analysis (see FIG. 11), transferrin and albumin were eluted from the 25mM NaCl-containing equilibrium solution, albumin and vitamin D binding protein were eluted from the 50mM NaCl-containing equilibrium solution, and a portion of large molecular (molecular weight > 180kDa) of the hetero protein was also eluted from the 100mM NaCl-containing equilibrium solution simultaneously with the elution of AAT, and transferrin was more remained, and the purification effect was not as good as that of the present invention in which hetero protein was eluted at a low pH (pH 4.6-pH4.8) and AAT was eluted at an increased salt concentration (45-65mM NaCl) (FIG. 5).

Comparative example 3

a) Dissolving and concentrating a Cohn component IV precipitate: the same as in example 1.

b) And PEG precipitation: the same as in example 1.

Primary virus inactivation: none.

c) And primary ultrafiltration concentration: the same as in example 1.

d) And anion exchange chromatography refining: anion exchange chromatography purification, Q Sepharose Fast Flow strong anion exchange chromatography column (column volume 30ml) using pH 5.320 mM acetate buffer solution as the balance solution balance 8-10 column volumes, then the ultrafiltrate loading, then using the balance solution to wash the not-bound transferrin and albumin on the column, then using pH 4.720mM acetate buffer solution to elute the albumin and vitamin D binding protein bound on the column, finally using pH 5.320mM acetate buffer solution containing 55mM NaCl to elute the AAT bound on the column, get AAT elution sample, the whole purification process Flow rate is 150 cm/h.

e) And secondary ultrafiltration concentration: the AAT eluted sample was ultrafiltered 5-10 volumes with pH 6.520 mM phosphate buffer to obtain a secondary ultrafiltrate, the molecular weight cut-off of the ultrafiltration membrane was 30 kDa.

f) And blue dye affinity chromatography refining: blue dye affinity chromatography purification, Blue Sepharose 6Fastflow Blue dye affinity chromatography column (column volume 20ml) with pH 6.520 mM phosphate buffer balanced 8-10 column volumes, then the sample loading of secondary ultrafiltrate, then use the balanced solution to wash the AAT not binding on the column, get AAT to penetrate the sample, the flow rate of the whole purification process is 40 cm/h; after final elution of the albumin bound to the column with 0.5M KSCN in pH 6.520 mM phosphate buffer, the column was reusable.

g) And secondary blue dye affinity chromatography refining: none.

h) And freeze-drying: none.

i) And secondary virus inactivation: none.

Trypsin is used as a target enzyme for AAT inhibition, the activity of a final product is determined by a chromogenic substrate method, the protein content is determined by a BCA method, the specific activity is calculated, and indexes of samples obtained in the steps of comparative example 3 are shown in Table 7.

Table 7 the AAT activity, protein content and specific activity in the samples obtained in the respective steps

From the results of activity measurement in Table 7, it was found that when Blue Sepharose 6Fast Flow Blue dye affinity column was equilibrated at pH6.5, AAT was not bound to the Blue dye and remained in the permeation sample having a specific activity of 1329.65U/mg, which was lower than that of the AAT permeation sample obtained by equilibrating the affinity column at pH 5.0 to 5.4.

From the SDS-PAGE analysis (see FIG. 12), it can be seen that the AAT penetration sample contains a large amount of vitamin D binding protein under the pH6.5 equilibrium condition, and only albumin and a small amount of transferrin are eluted from the pH 6.520 mM phosphate buffer containing 0.5M KSCN, indicating that the blue dye binds albumin more and less to other proteins under the pH6.5 equilibrium condition. Under the pH 5.0-5.4 equilibrium conditions of the present invention, the buffer elution sample containing 0.5M KSCN contained large molecules (molecular weight > 180kDa) of hetero-protein, transferrin, albumin, vitamin D binding protein, indicating that the blue dye bound not only albumin but also other proteins under the pH 5.0-5.4 equilibrium conditions (FIG. 6). Thus, the affinity conditions employed in the present invention facilitate the separation of AAT from other heteroproteins.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the content of the present invention.

Claims (9)

1. A method for purifying α 1-antitrypsin from Cohn component IV precipitate is characterized in that the Cohn component IV precipitate is subjected to dissolving concentration, PEG precipitation, primary virus inactivation, primary ultrafiltration concentration, tertiary chromatography refining, freeze drying and secondary virus inactivation in sequence to finally obtain α 1-antitrypsin (AAT);

the third chromatographic refining is one-time anion exchange chromatographic refining and two-time blue dye affinity chromatographic refining;

balancing the blue dye affinity chromatographic column in the blue dye affinity chromatographic refining with the AAT eluent for eluting AAT in the anion exchange chromatographic refining, and eluting AAT;

the AAT eluent is acetate buffer solution with pH of 5.0-5.4 and 10-50mM, which contains 45-65mM NaCl;

performing dry heat virus inactivation treatment on the AAT freeze-dried product obtained by freeze drying in water bath at 99-100 ℃ for 30 min;

in the blue dye affinity chromatography refining, firstly, the AAT eluent is used for balancing a blue dye affinity chromatography column, then, the sample is loaded, and then, the AAT eluent is used for washing the blue dye affinity chromatography column to remove AAT which is not combined with the blue dye, so that an AAT penetrating sample is obtained, wherein the flow rate in the whole purification process is 70-120 cm/h;

the medium of the blue dye affinity chromatographic column is selected from affinity chromatographic medium BlueSepharose 6Fast Flow with blue dye as affinity ligand;

the column volume ratio of the secondary blue dye affinity chromatography to the primary blue dye affinity chromatography in the two blue dye affinity chromatography refinements is 1 (15-20).

2. The method of claim 1, wherein transferrin and albumin not bound to the column are removed by eluting with the equilibration solution from an equilibrium anion exchange chromatography column in an anion exchange chromatography purification.

3. The method of claim 2, wherein the equilibration fluid is a 10-50mM acetate buffer at pH 5.0-5.4.

4. The method as claimed in claim 2, wherein the anion exchange chromatography purification comprises first balancing the anion exchange chromatography column with a balancing solution, then loading, then washing the anion exchange chromatography column with the same balancing solution to remove transferrin and albumin not bound to the column, then eluting albumin and vitamin D binding protein bound to the column with an acetate buffer solution of pH4.6-4.8 and 10-50mM, and finally eluting AAT with an AAT eluent to obtain an AAT elution sample, wherein the flow rate of the whole purification process is 150-350 cm/h.

5. The method of claim 4, wherein the medium of the anion exchange chromatography column is selected from a weak anion exchange chromatography medium having Diethylaminoethyl (DEAE) as an ion exchange group and a strong anion exchange chromatography medium having a quaternary ammonium group as an ion exchange group.

6. The method of any one of claims 1 to 5, wherein two blue dye affinity chromatography purifications use the same blue dye affinity chromatography column; the method specifically comprises the following steps: after one blue dye affinity chromatography is finished, the used blue dye affinity chromatography column uses 0.5-1.0M KSCN, pH 5.0-8.5, 10-50mM acetate buffer solution, phosphate buffer solution or Tris-HCl buffer solution to elute macromolecular hetero protein, transferrin, albumin and vitamin D binding protein with molecular weight more than 180kDad which are combined on the column.

7. The method according to claim 6, wherein the pH of the supernatant obtained by PEG precipitation is adjusted to 6.5-7.5 in the first virus inactivation, the supernatant is filtered through a 0.22 μm filter membrane, tri-n-butyl phosphate and Tween-80 are added to the final concentration of 0.3% and 1% in g/100mL, and the virus inactivation treatment is carried out in a thermostatic water bath at 23-25 ℃ for 6h to obtain an inactivated supernatant.

8. The method of claim 7, wherein in the second virus inactivation, the freeze-dried AAT freeze-dried product is subjected to dry-heat virus inactivation treatment in a water bath at 99-100 ℃ for 30min to inactivate both enveloped viruses and non-enveloped viruses, thereby obtaining the final α 1-antitrypsin.

9. The method according to claim 8, characterized in that it comprises in particular the steps of:

a) and dissolving and concentrating Cohn component IV precipitate: dissolving the Cohn component IV precipitate with 8-10 times of water by weight, stirring for 2-4h at 0-10 ℃, adjusting the pH value to 7.5-8.0, performing solid-liquid separation, discarding solids to obtain a Cohn component IV dissolved solution, and performing ultrafiltration concentration on the Cohn component IV dissolved solution by 4-10 times of volume to obtain a Cohn component IV concentrated solution, wherein the protein content in the Cohn component IV concentrated solution is 20-40 mg/ml;

b) and PEG precipitation: adjusting the pH value of the Cohn component IV concentrated solution obtained in the step a) to 5.0-5.4, adding PEG4000 at 0-10 ℃ until the final concentration is 10-15% in terms of g/100mL, stirring to completely dissolve the PEG4000, standing at 0-10 ℃ and then centrifuging for 20-60min at 4000-6000g, discarding the precipitate containing macromolecular hetero protein with the molecular weight of more than 180kDa, transferrin, albumin and vitamin D binding protein, and taking the supernatant;

c) and primary virus inactivation: adjusting the pH value of the supernatant obtained in the step b) to 6.5-7.5, filtering with a 0.22 mu m filter membrane, adding tri-n-butyl phosphate (TNBP) and Tween-80 (Tween 80) to make the final concentration of the tri-n-butyl phosphate and the Tween-80 (TNBP) be 0.3% and 1% respectively according to g/100mL, and carrying out S/D virus inactivation treatment in a thermostatic water bath at 23-25 ℃ for 6h to obtain an inactivated supernatant;

d) and primary ultrafiltration concentration: ultrafiltering the inactivated supernatant of step c) with 10-50mM acetate buffer solution at pH 5.0-5.4 to obtain ultrafiltrate, wherein the molecular weight cut-off of the ultrafiltration membrane is 20-30 kDa;

e) and anion exchange chromatography refining: purifying the ultrafiltrate obtained in step d) by anion exchange chromatography; the anion exchange chromatography medium is weak anion exchange chromatography medium whose ion exchange group is Diethylaminoethyl (DEAE) or strong anion exchange chromatography medium whose ion exchange group is quaternary ammonium group; balancing an anion exchange chromatography column by using an acetate buffer solution with pH of 5.0-5.4 and 10-50mM as a balancing solution, then loading the ultrafiltrate obtained in the step D), washing transferrin and albumin which are not bound on the column by using the same balancing solution, eluting albumin and vitamin D binding protein which are bound on the column by using the acetate buffer solution with pH of 4.6-4.8 and 10-50mM, and finally eluting AAT by using a balancing solution containing 45-65mM NaCl as an AAT eluent to obtain an AAT elution sample, wherein the flow rate in the whole purification process is 150-350 cm/h;

f) and one-time blue dye affinity chromatography refining: purifying the AAT elution sample obtained in the step e) by adopting blue dye affinity chromatography; the blue dye affinity chromatography medium adopts an affinity chromatography medium with affinity ligand as blue dye; firstly, using AAT eluent to balance a blue dye affinity chromatographic column, then loading the AAT eluted sample obtained in the step e), and then using the AAT eluent to wash the AAT which is not combined with the blue dye to obtain a primary AAT penetrating sample, wherein the flow rate of the whole purification process is 70-120 cm/h; eluting the used blue dye affinity chromatographic column with 0.5-1.0M KSCN-containing pH 5.0-8.5, 10-50mM acetate buffer, phosphate buffer or Tris-HCl buffer solution to obtain macromolecular hetero protein, transferrin, albumin and vitamin D binding protein with molecular weight more than 180kDa, which are bound on the column, and then repeatedly using the macromolecular hetero protein, the transferrin, the albumin and the vitamin D binding protein;

g) and secondary blue dye affinity chromatography refining: firstly, using AAT eluent to balance a blue dye affinity chromatographic column, wherein the blue dye affinity chromatographic medium is the same as that in the step f); then loading the primary AAT penetrating sample obtained in the step f), and then washing the AAT which is not combined with the blue dye by using an AAT eluent to obtain a secondary AAT penetrating sample; the column volume ratio of the blue dye affinity chromatographic column in the step g) to the blue dye affinity chromatographic column in the step f) is 1 (15-20);

h) and freeze-drying: ultrafiltering and concentrating the secondary AAT obtained in the step g) to penetrate a sample to enable the protein content of the sample to reach 10-20mg/ml, then adding a protective agent histidine, wherein the final concentration of the histidine is 0.05-0.1M, filtering and sterilizing with a 0.22-micron filter membrane, subpackaging, and freeze-drying to obtain an AAT freeze-dried product;

i) and secondary virus inactivation, namely performing virus inactivation treatment on the AAT freeze-dried product obtained in the step h) for 30min by a dry heat method in a water bath at the temperature of 99-100 ℃ to obtain the final α 1-antitrypsin (AAT).

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