CN107163138A - A kind of antitryptic isolation and purification methods of human plasma protein fraction α 1 - Google Patents

Abstract

The invention discloses a kind of antitryptic isolation and purification methods of human plasma protein fraction α 1, comprise the following steps：(1) albumen precipitations of FIV 1 for producing cold ethanol Cohn method separated plasmas protein Process are dissolved in buffer solution, treat that albumen has fully dissolved, and are filtered, are taken filtrate standby；(2) filtrate passes through anion-exchange chromatography, collects the eluent containing AAT, the filler of the anion-exchange chromatography is DEAE Sepharose High Performance；(3) eluent containing AAT carries out xeothermic inactivation and S/D inactivations, the eluent containing AAT after being inactivated；(4) eluent containing AAT after inactivating passes through cation-exchange chromatography, and the AAT for obtaining high-purity high-activity penetrates liquid, and the filler of the cation-exchange chromatography is Macro Prep High S；(5) AAT penetrates liquid and freezed by ultrafiltration concentration, aseptic filtration and packing, you can obtain AAT preparations.The antitrypsins of α 1 that the present invention is prepared have high-purity, high activity, high-recovery, step simple, reproducible and the characteristics of easy large-scale production, solve problem present in its current production technology.

Description

A kind of isolation and purification method of human plasma protein fraction alpha1-antitrypsin

Technical field

The present invention relates to separating and purifying technology field, in particular it relates to a kind of point of human plasma protein fraction alpha1-antitrypsin From purification process.

Background technology

The 21 century that science and technology is developed rapidly, blood product industry turns into the important component of Bio-pharmaceutical Industry.Mesh The blood product in preceding blood plasma source（Plasma protein medicine）Mainly there are albumin, immunoglobulin, clotting factor, albumen enzyme level The major class of agent four, American-European blood product enterprise can obtain the plasma protein medicine more than 20 kinds from blood plasma.However, by people , the factor such as technology, clinical cognitive level is restricted, and domestic blood product enterprise can only at most obtain 7-8 kinds, and mainly base In the product of chilled alcohol precipitation technique separating-purifying, including human serum albumin, immunoglobulin and platelet cofactor Ⅰ product etc.. Main purifying blood plasma protein drug domestic at present is still based on the cold ethanol method invented Cohn et al. nineteen forties, leads to Concentration of alcohol, salinity, temperature and pH are overregulated, is separated by the different solubility of plasma protein, although the method program Simply, solvent is inexpensive, it is adaptable to industrialized production, and can effectively keep the activity of albumen, but has larger limitation, mainly may be used The higher albumen of content in separated plasma（Such as albumin and immunoglobulin）, the albumen less to content（Such as the anti-tryptoses of α 1- Enzyme etc.）Then it is difficult to purify.

Alpha1-antitrypsin (α 1-antitrypsin, AAT), also known as α 1- albumen inhibitory enzyme (α 1-protease inhibitor, α1-PI).It is most important protein inhibitor in human plasma, accounts for Total plasma protein enzyme level ability More than 90%.It can suppress a variety of serine endopeptidases, such as neutrophil elastase, trypsase, fibrin ferment, Its main function is to protect organism normal cell and organ not to be damaged by protease, suppresses infection and inflammation, is maintained in body The balance of environment.AAT in blood plasma is mainly the single chain glycoprotein synthesized by liver, and peptide chain is made up of 394 amino acid, always contained Sugar amount 12 ~ 14%, its molecular weight is~52KD, and the content that isoelectric point (PI) is AAT in 4.7~5.0, human normal plasma is (2.9 ± 0.45) g/L, half-life period in vivo is 3 ~ 5 days.AAT has stronger vasopermeability, the concentration in lung tissue Higher and stronger to the selectivity of elastoser, therefore, AAT major function is the activity for suppressing lung elastase, Lung is not damaged by the enzymolysis of elastoser, protect lung.Clinically, AAT preparations can for treat the congenital or day after tomorrow Property AAT lack caused by the disease such as pulmonary emphysema, ALI, cystic fibrosis, the urgent syndrome of adult's breathing, clinically have There is higher application value.

Congenital AAT deficiency diseases patient 15%-25% needs replacement therapy, it is meant that the people of the U.S. about 20,000 needs to treat, and Europe The people of continent at least about 40,000 4 to 70,000 3 thousand needs treatment.Surrogate therapeutic patient needs to enter row vein by 60 mg AAT/kg body weight weekly Injection, annual everyone needs about 208 g AAT.But be entirely to derive from human plasma, the limitation in source for the AAT for the treatment of Causing global AAT, supply falls short of demand, and China does not possess the AAT separating and purifying technologies of maturation also.Therefore, exploitation AAT separation is pure Change technology, improves yield, protein active and purity and is particularly important.

The method that AAT is isolated and purified from human plasma is various, mainly chromatography with precipitation etc. other method be used in conjunction with, Chromatography method therein includes ion-exchange chromatography, gel permeation chromatography, hydrophobic interaction chromatography and affinity chromatography^[11-14].Under Several representative AAT purification process methods are enumerated in face.Glaser et al. eighties of last century utilizes AAT half Guang ammonia the eighties Sour residue forms disulfide bond this characteristic not in peptide bond, and Cohn methods FIV-1 is precipitated into lysate through Isosorbide-5-Nitrae-dithiothreitol (DTT) Processing, then after 50% ammonium sulfate precipitation, remove ammonium sulfate with the buffer solution dialysis containing cysteine, use DEAE-cellulose Obtained AAT is further purified in ion-exchange chromatography, AAT concentrate formulations is obtained through dialysis is lyophilized, without inactivation of virus.Its purity About 70%, yield is about 45% (Glaser, C. B.et al. Anal. Biochem. 1982, 124, 364-371.) .Hein and Coan et al. are set out using Cohn method FIV-1 precipitate waste materials, are obtained by PEG precipitations and weak anionic displacement chromatography To AAT, but the AAT purity that this method is obtained only has 60% or so, and the rate of recovery is 45% (Hein, R. H.et al. Eur. Respir. J. Suppl.1990, 9, 16-20. Coan, M. H. et al. Vox. Sang, 1985, 48, 333- 342.) .The AAT purity and activity that both approaches are obtained is not high, and the rate of recovery is relatively low. Alpha Therapeutic Corporation is from Cohn FIV-1 suspension, by PEG precipitations and ZnCl₂The moon is carried out again after precipitation, inactivation of virus Ion-exchange chromatography, AAT eluents are by ultrafiltration, being sterile filtered and freezing can obtain AAT preparations, and this is Aralast preparations Production technology (Hwang, D. S.et al.1997, US 5616693.), α 1-AT specific activities are every milligram in product Total protein feature AAT >=0.55mg, purity >=90%, and on being approved in November, 2003.CSL Behring companies are first with also The gentle dissolubility silica gel treatment Cohn FIV-1 suspension of former agent Isosorbide-5-Nitrae-second-rate threitol (DTT), then by anion exchange resin Chromatography and hydrophobic resin chromatography, 60 DEG C of 10 h inactivation of viruses and the continuous nano-film filtration of two steps remove virus, aseptic filtration point The step such as lyophilized is filled, the α-AT specific activities produced are every milligram of total protein feature AAT >=0.70mg, and purity >=90% is single Body (HPLC)>95%, it is approved to list (Cook, P. I. in August, 2003et al. 2011, EP 2295126 A1.)。 Kumpalume etc. has reported for work from the component A+1 supernatants of Nitchman and Kistler cold ethanol methods, uses an anion Exchange column and two metal-chelating column separating purification people α 1-AT, as a result the α 1-AT rate of recovery is more than 60%, and purity is more than 90%, And in the case of without any stabilizer, the α 1-AT of production can stablize 12 months (Kumpalume, P. at 4 DEG Cet al. J. Chromatogr. A 2007, 1148, 31-37.).China Shanghai biological products postgraduate Zhu Wei et al. is to Cohn FIV extracts make viral inactivation treatment, then use ion-exchange gel after PEG, isoelectric precipitation and organic solvent deposit Adsorbed, then by sieve chromatography, AAT (Zhu Wei etc., Products in China magazine, 2001,14 can be prepared (2), 97-101.).There is problems with the preparation method that AAT preparation methods in summary can be seen that current AAT： (1) purity and the active low, rate of recovery are low, although purity and activity can reach requirement, be also to sacrifice the rate of recovery as cost, Three can not be optimal simultaneously.(2) early stage precipitation or adsorption-edulcoration all are carried out to FIV-1 lysates in most of method Albumen processing, adds operating procedure and cost, reduces the rate of recovery.(3) some methods are using costliness or are difficult scale The chromatography scheme of method.Metal chelate chromatography post is expensive in being chromatographed such as metal ion-chelant, and easily occurs metal leakage.Gel Then there is certain shortcoming in treating capacity and follow-up large-scale production in filtration chromatography.Therefore, high work can be obtained by seeking one kind Property high-purity and high-recovery AAT high efficiency separation and purification method seem extremely important.

The content of the invention

The invention aims to the above-mentioned deficiency for overcoming prior art, there is provided a kind of anti-pancreas eggs of human plasma protein fraction α 1- The isolation and purification method of white enzyme.The FIV-1 albumen precipitations that the present invention is produced from cold ethanol-Cohn method separated plasmas protein Process Discarded object sets out, main to pass through a step anion-exchange chromatography and a step cation-exchange chromatography, you can to isolate and purify out high-purity Spend the alpha1-antitrypsin of high activity high-recovery.The alpha1-antitrypsin that the present invention is prepared have high-purity, high activity, High-recovery, step be simple, reproducible and the characteristics of easy large-scale production, solves present in its current production technology Problem.

To achieve these goals, the present invention is achieved by the following technical programs：

A kind of isolation and purification method of human plasma protein fraction alpha1-antitrypsin, comprises the following steps：

(1) the FIV-1 albumen precipitations for producing cold ethanol-Cohn method separated plasmas protein Process are dissolved in buffer solution, are treated Albumen has fully dissolved, filtering, takes filtrate standby；

(2) filtrate passes through anion-exchange chromatography, collects the eluent containing AAT, and the filler of the anion-exchange chromatography is DEAE Sepharose High Performance；

(3) eluent containing AAT carries out xeothermic inactivation and S/D inactivations, the eluent containing AAT after being inactivated；

(4) eluent containing AAT after inactivating passes through cation-exchange chromatography, and the AAT for obtaining high-purity high-activity penetrates liquid, described The filler of cation-exchange chromatography is Macro-Prep High S；

(5) AAT penetrates liquid and freezed by ultrafiltration concentration, aseptic filtration and packing, you can obtain AAT preparations.

Preferably, the buffer solution described in step (1) is slow for pH 7.0-9.0,10-100 mM phosphate Fliud flushing or Tris-HCl buffer solutions, solution temperature are 20-45 DEG C, and dissolution time is 5-10h, and AAT's answers in being precipitated beneficial to FIV-1 Property.

Preferably, the pH of buffer that step (2) described anion-exchange chromatography is used is 6.5-9.0, is delayed Fliud flushing type is 10-100 mM Tris-HCl buffer solutions or phosphate buffer.

Preferably, the pH of buffer that step (4) described cation-exchange chromatography is used is 4.5-6.0, is delayed Fliud flushing type is 10-100 mM citric acids or phosphate buffer.

A kind of isolation and purification method of human plasma protein fraction alpha1-antitrypsin, comprises the following steps：

(1) dissolving of FIV-1 precipitations：The FIV-1 albumen precipitations that cold ethanol-Cohn method separated plasmas protein Process is produced are molten Solution stirs 5-10 h, temperature range in pH 7.0-9.0,10-100 mM Tris-HCl buffer solutions or phosphate buffer For 20-45 DEG C, treat that albumen has fully dissolved, filter, take filtrate standby；

(2) anion-exchange chromatography：Anion-exchange gel suspension is taken, room temperature, which is placed, to be reached post is filled after equalized temperature, with flat The buffer solution that weighs balances 5-8 column volume, the filtrate pH of FIV-1 precipitation lysates is adjusted to identical with level pad, then carries out Loading, loading volume is 1-5 column volume, is eluted with elution buffer, collects the eluent containing AAT, after chromatography terminates, Pillar is cleaned with the 0.5M NaOH of 5-8 column volume, it is whole then with the 20% ethanol water balance pillar of 5-8 column volume Individual chromatography process is carried out on the protein purification instrument of GE kta Explore 100；The filler of the anion-exchange chromatography is DEAE Sepharose High Performance；

(3) inactivation of virus：Anion-exchange chromatography eluent containing AAT is freezed, lyophilized process is as follows：First in air - 25 DEG C of holding 2-4 h during atmosphere is total, then -40 DEG C of 2-4 h, then under vacuum, -10 DEG C of holding 30-40 h are warming up to 35 DEG C keep 20-30 h again, and the sample freezed is then at 80 DEG C of xeothermic h of inactivation of virus 72, and the sample after xeothermic inactivation is molten again In Xie Yushui, the stable AAT of sucrose is added, ultimate density is 30-40%, then add TRI N BUTYL PHOSPHATE and Tween-80 carries out S/ D is inactivated, and ultimate density is respectively 0.2-0.4% and 0.1-0.3%, and 30 DEG C inactivate after 3-5 h, are filtered and are dialysed, after dialysis Adjust pH value of solution identical with the pH of the cation-exchange chromatography level pad of next step；

(4) cation-exchange chromatography：Cation exchange gel suspension is taken, room temperature, which is placed, to be reached post is filled after equalized temperature, with flat The buffer solution that weighs balances 5-8 column volume, the dialyzate pH after inactivation of virus is adjusted to identical with originating level pad, then carries out Loading, loading volume is 1-5 column volume, is eluted with buffer solution, collects the AAT liquid that flows through, after chromatography terminates, uses 5-8 The 0.5 M NaOH cleaning pillars of individual column volume, then with the 20% ethanol water balance pillar of 5-8 column volume, whole layer Analysis process is carried out on the protein purification instrument of GE kta Explore 100；The filler of the cation-exchange chromatography is Macro- Prep High S；

(5) ultrafiltration, aseptic filtration, packing, lyophilized：The AAT of cation-exchange chromatography is flowed through into liquid to be concentrated by ultrafiltration, protein concentration is 10-50 mg/ml, carry out aseptic filtration, packing and freeze afterwards；Freeze-drying process：First -25 DEG C of holding 2-4 in air atmosphere is total H, then -40 DEG C of 2-4 h, then under vacuum, -10 DEG C of holding 30-40 h are warming up to 35 DEG C and keep 20-30 h again, freeze After the completion of dry, that is, prepare AAT preparations.

Compared with prior art, the present invention has the advantages that：

First, the alpha1-antitrypsin that the present invention is prepared has high-purity, high activity and high-recovery.Purity >=95%, than Living >=0.9, often the step chromatography rate of recovery >=90%, solves current AAT preparation methods moderate purity, activity and the low shortcoming of the rate of recovery. Secondly, the step of present invention prepares alpha1-antitrypsin is simple, mainly there is two-step solution chromatography, without major part method In the step such as early stage precipitation or the processing of adsorption-edulcoration albumen is carried out to FIV-1 lysates, it is low with simple to operate and cost Advantage.Again, the chromatography scheme in the present invention is anion-exchange chromatography and cation-exchange chromatography, with easy amplification, and is filled out The advantages of material is easy to get.

Brief description of the drawings

Fig. 1 is the route map that AAT of the present invention is isolated and purified.

Fig. 2 is the anion-exchange chromatography figure that FIV-1 dissolves filtrate.

Fig. 3 is the cation-exchange chromatography figure after inactivation of virus.

Fig. 4 is the SDS-PAGE figures that AAT isolates and purifies process.

Embodiment

The present invention is made with reference to Figure of description and specific embodiment and further being elaborated, the embodiment It is served only for explaining the present invention, is not intended to limit the scope of the present invention.Test method used in following embodiments is such as without spy Different explanation, is conventional method；Used material, reagent etc., are the reagent commercially obtained unless otherwise specified And material.

Embodiment 1

The dissolving of 1.FIV-1 precipitations：Weigh 1g FIV-1 to be precipitated and dissolved in 10m l20mM pH7.5 phosphate buffers, fill Divide mixing, in the h of room-temperature dissolution on shaking table 5.After dissolving terminates, filtered with 0.45 μm of needle cylinder type filter membrane, take filtrate standby.

2. anion-exchange chromatography：The DEAE Sepharose High Performance for taking 35m l20% ethanol to preserve Gel, is replaced repeatedly with water, removes ethanol, and placement is reached after equalized temperature with the dress posts of XK 16/20.Following chromatography process all exists Carried out on the protein purification instrument of GE kta Explore 100.Fill after the completion of post, pillar height is 13.2 cm, its post effect is surveyed with 1% acetone For 4865 N/m.Surveyed post effect after, 5 column volumes of pillar watering balance, then with originate the phosphorus of 20 mM pH of level pad 7.0 Phthalate buffer balances 5 column volumes.The FIV-1 ml of filtrate 15 for precipitating lysate are adjusted into pH to 7.0.Reached with 10 column volumes Linear gradient to the M NaCl of 20 mM pH, 7.0 phosphate buffers+0.1 is eluted, and collects the eluent containing AAT.Wash After de- end, pillar is cleaned with 0.5 M NaOH of 5 column volumes, the associated proteins not eluted are washed away.Again with 5 column volumes 20% ethanol solution balance pillar is preserved.The AAT eluents purity that this step anion-exchange chromatography is obtained is 65%, is than work 0.59, the rate of recovery is 98%.

3. inactivation of virus：Before second step chromatography is carried out, the eluent containing AAT need to pass through two step inactivation of virus, xeothermic to go out Living and S/D inactivations.First, the anion-exchange chromatography eluent containing AAT is freezed, lyophilized process is as follows：First in sky - 25 DEG C of holding 2-4 h during atmosphere is enclosed always, then -40 DEG C of 2-4 h.Then under vacuum, -10 DEG C of holding 30-40 h, heating 20-30 h are kept again to 35 DEG C.The sample freezed is then at 80 DEG C of xeothermic h of inactivation of virus 72.Sample after xeothermic inactivation is again It is dissolved in the water, adds the stable AAT of sucrose, ultimate density is 35%.Then add TRI N BUTYL PHOSPHATE and Tween-80 carries out S/D Inactivation, ultimate density is respectively 0.3% and 0.2%, and 30 DEG C inactivate after 3-5 h, are filtered and are dialysed.Solution is adjusted after dialysis PH is identical with the pH of the cation-exchange chromatography level pad of next step.

4. cation-exchange chromatography：The Macro-Prep High S gels for taking the ethanol of 30 ml 20% to preserve, it is anti-with water Preset is changed, and removes ethanol, and placement is reached after equalized temperature with the dress posts of XK 16/20.Following chromatography process is all in GE kta Carried out on the protein purification instrument of Explore 100.Fill after the completion of post, pillar height is 12 cm, it is 4102 N/ that its post effect is surveyed with 1% acetone m.Surveyed post effect after, 5 column volumes of pillar watering balance, then with originate the lemon acid bufferings of 20 mM pH of level pad 5.5 Liquid balances 5 column volumes.AAT eluents after inactivation of virus are adjusted into pH to 5.5.Applied sample amount is 15 ml, is reached with 10 column volumes Linear gradient to the M NaCl of 20 mM pH, 5.5 citrate buffer solutions+0.1 is eluted, and wherein AAT is received in liquid is penetrated Collection penetrates liquid.After elution terminates, pillar is cleaned with 0.5 M NaOH of 5 column volumes, the associated proteins not eluted are washed away.Use again The 20% ethanol solution balance pillar of 5 column volumes is preserved.The AAT that this step cation-exchange chromatography is obtained penetrates AAT purity in liquid For 96%, than it is living be 0.93, the rate of recovery is 91%.

5. it is ultrafiltration, aseptic filtration, packing, lyophilized：The AAT of cation-exchange chromatography is flowed through into liquid to be concentrated by ultrafiltration, albumen is dense Spend for 15 mg/ml, aseptic filtration, packing are carried out afterwards lyophilized.Freeze-drying process：First -25 DEG C of holding 2-4 in air atmosphere is total H, then -40 DEG C of 2-4 h.Then under vacuum, -10 DEG C of holding 30-40 h, are warming up to 35 DEG C and keep 20-30 h again.Freeze After the completion of dry, that is, prepare AAT preparations.

Embodiment 2

1. the dissolving of FIV-1 precipitations：Weigh 1 g FIV-1 and be precipitated and dissolved in the phosphate buffers of 10 ml, 20 mM pH 8.0 In, it is sufficiently mixed, in 35 DEG C of 5 h of dissolving on shaking table.After dissolving terminates, put to room temperature, filtered with 0.45 μm of needle cylinder type filter membrane, Take filtrate standby.

2. anion-exchange chromatography：The DEAE Sepharose High for taking the ethanol of 35 ml 20% to preserve Performance gels, are replaced repeatedly with water, remove ethanol, and placement is reached after equalized temperature with the dress posts of XK 16/20.With lower floor Analysis process is carried out all on the protein purification instrument of GE kta Explore 100.Fill after the completion of post, pillar height is 13.2 cm, with 1% the third It is 4865 N/m that ketone, which surveys its post effect,.Surveyed post effect after, 5 column volumes of pillar watering balance, then with originate the mM of level pad 20 The phosphate buffers of pH 8.0 balance 5 column volumes.The phosphate buffers+0.1 of 20 mM pH 8.0 are reached with 10 column volumes M NaCl linear gradient is eluted, and collects the eluent containing AAT.After elution terminates, with 0.5 M NaOH of 5 column volumes Pillar is cleaned, the associated proteins not eluted are washed away.Preserved again with the 20% ethanol solution balance pillar of 5 column volumes.This step it is cloudy from The AAT eluents purity that sub- displacement chromatography is obtained is 63%, is 0.57 than living, the rate of recovery is 99%.

3. inactivation of virus：Before second step chromatography is carried out, the eluent containing AAT need to pass through two step inactivation of virus, xeothermic to go out Living and S/D inactivations.First, the anion-exchange chromatography eluent containing AAT is freezed, lyophilized process is as follows：First in sky - 25 DEG C of holding 2-4 h during atmosphere is enclosed always, then -40 DEG C of 2-4 h.Then under vacuum, -10 DEG C of holding 30-40 h, heating 20-30 h are kept again to 35 DEG C.The sample freezed is then at 80 DEG C of xeothermic h of inactivation of virus 72.Sample after xeothermic inactivation is again It is dissolved in the water, adds the stable AAT of sucrose, ultimate density is 35%.Then add TRI N BUTYL PHOSPHATE and Tween-80 carries out S/D Inactivation, ultimate density is respectively 0.3% and 0.2%, and 30 DEG C inactivate after 3-5 h, are filtered and are dialysed.Solution is adjusted after dialysis PH is identical with the pH of the cation-exchange chromatography level pad of next step.

4. cation-exchange chromatography：The Macro-Prep High S gels for taking the ethanol of 30ml 20% to preserve, with water repeatedly Displacement, removes ethanol, and placement is reached after equalized temperature with the dress posts of XK 16/20.Following chromatography process is all in GE kta Carried out on the protein purification instrument of Explore 100.Fill after the completion of post, pillar height is 12 cm, it is 4102 N/ that its post effect is surveyed with 1% acetone m.Surveyed post effect after, 5 column volumes of pillar watering balance, then with originate the lemon acid bufferings of 20 mM pH of level pad 5.5 Liquid balances 5 column volumes.AAT eluents after inactivation of virus are adjusted into pH to 5.5.Applied sample amount is 30 ml, is reached with 10 column volumes Linear gradient to the M NaCl of 20 mM pH, 5.5 citrate buffer solutions+0.1 is eluted, and wherein AAT is received in liquid is penetrated Collection penetrates liquid.After elution terminates, pillar is cleaned with 0.5 M NaOH of 5 column volumes, the associated proteins not eluted are washed away.Use again The 20% ethanol solution balance pillar of 5 column volumes is preserved.The AAT that this step cation-exchange chromatography is obtained penetrates AAT purity in liquid For 97%, than it is living be 0.93, the rate of recovery is 90%.

5. it is ultrafiltration, aseptic filtration, packing, lyophilized：The AAT of cation-exchange chromatography is flowed through into liquid to be concentrated by ultrafiltration, albumen is dense Spend for 15 mg/ml, aseptic filtration, packing are carried out afterwards lyophilized.Freeze-drying process：First -25 DEG C of holding 2-4 in air atmosphere is total H, then -40 DEG C of 2-4 h.Then under vacuum, -10 DEG C of holding 30-40 h, are warming up to 35 DEG C and keep 20-30 h again.Freeze After the completion of dry, that is, prepare AAT preparations.

Embodiment 3

1. the dissolving of FIV-1 precipitations：Weigh 1 g FIV-1 and be precipitated and dissolved in the Tris-HCl of 10 ml, 20 mM pH 8.0 bufferings In liquid, it is sufficiently mixed, in the h of room-temperature dissolution on shaking table 5.After dissolving terminates, put to room temperature, with 0.45 μm of needle cylinder type filter membrane mistake Filter, takes filtrate standby.

2. anion-exchange chromatography：The DEAE Sepharose High for taking the ethanol of 35 ml 20% to preserve Performance gels, are replaced repeatedly with water, remove ethanol, and placement is reached after equalized temperature with the dress posts of XK 16/20.Below Chromatography process is carried out all on the protein purification instrument of GE kta Explore 100.Fill after the completion of post, pillar height is 13.5 cm, with 1% It is 4562 N/m that acetone, which surveys its post effect,.Surveyed post effect after, 5 column volumes of pillar watering balance, then with originate level pad 20 MM pH 8.0Tris-HCl buffer solutions balance 5 column volumes.Reach that the Tris-HCl of 20 mM pH 8.0 are rushed with 10 column volumes The M of liquid+0.1 NaCl linear gradient is eluted, and collects the eluent containing AAT.After elution terminates, with the 0.5 of 5 column volumes M NaOH clean pillar, wash away the associated proteins not eluted.Preserved again with the 20% ethanol solution balance pillar of 5 column volumes.This The AAT eluents purity that step anion-exchange chromatography is obtained is 67%, is 0.58 than living, the rate of recovery is 99%.

3. inactivation of virus：Before second step chromatography is carried out, the eluent containing AAT need to pass through two step inactivation of virus, xeothermic to go out Living and S/D inactivations.First, the anion-exchange chromatography eluent containing AAT is freezed, lyophilized process is as follows：First in sky - 25 DEG C of holding 2-4 h during atmosphere is enclosed always, then -40 DEG C of 2-4 h.Then under vacuum, -10 DEG C of holding 30-40 h, heating 20-30 h are kept again to 35 DEG C.The sample freezed is then at 80 DEG C of xeothermic h of inactivation of virus 72.Sample after xeothermic inactivation is again It is dissolved in the water, adds the stable AAT of sucrose, ultimate density is 35%.Then add TRI N BUTYL PHOSPHATE and Tween-80 carries out S/D Inactivation, ultimate density is respectively 0.3% and 0.2%, and 30 DEG C inactivate after 3-5 h, are filtered and are dialysed.Solution is adjusted after dialysis PH is identical with the pH of the cation-exchange chromatography level pad of next step.

4. cation-exchange chromatography：The Macro-Prep High S gels for taking the ethanol of 30ml 20% to preserve, with water repeatedly Displacement, removes ethanol, and placement is reached after equalized temperature with the dress posts of XK 16/20.Following chromatography process is all in GE kta Carried out on the protein purification instrument of Explore 100.Fill after the completion of post, pillar height is 12 cm, it is 4102 N/ that its post effect is surveyed with 1% acetone m.Surveyed post effect after, 5 column volumes of pillar watering balance, then with originate the phosphate-buffereds of 20 mM pH of level pad 5.5 Liquid balances 5 column volumes.AAT eluents after inactivation of virus are adjusted into pH to 5.5.Applied sample amount is 30 ml, is reached with 10 column volumes Linear gradient to the M NaCl of 20 mM pH, 5.5 phosphate buffers+0.1 is eluted, and wherein AAT is received in liquid is penetrated Collection penetrates liquid.After elution terminates, pillar is cleaned with 0.5 M NaOH of 5 column volumes, the associated proteins not eluted are washed away.Use again The 20% ethanol solution balance pillar of 5 column volumes is preserved.The AAT that this step cation-exchange chromatography is obtained penetrates AAT purity in liquid For 99%, than it is living be 0.93, the rate of recovery is 95%.

5. it is ultrafiltration, aseptic filtration, packing, lyophilized：The AAT of cation-exchange chromatography is flowed through into liquid to be concentrated by ultrafiltration, albumen is dense Spend for 15 mg/ml, aseptic filtration, packing are carried out afterwards lyophilized.Freeze-drying process：First -25 DEG C of holding 2-4 in air atmosphere is total H, then -40 DEG C of 2-4 h.Then under vacuum, -10 DEG C of holding 30-40 h, are warming up to 35 DEG C and keep 20-30 h again.Freeze After the completion of dry, that is, prepare AAT preparations.

Comparative example 1

1. the dissolving of FIV-1 precipitations：Weigh 1 g FIV-1 and be precipitated and dissolved in the Tris-HCl of 10 ml, 20 mM pH 8.0 bufferings In liquid, it is sufficiently mixed, in the h of room-temperature dissolution on shaking table 5.After dissolving terminates, put to room temperature, with 0.45 μm of needle cylinder type filter membrane mistake Filter, takes filtrate standby.

2. anion-exchange chromatography：The DEAE Sepharose Fast Flow for taking the ethanol of 35 ml 20% to preserve coagulate Glue, is replaced repeatedly with water, removes ethanol, and placement is reached after equalized temperature with the dress posts of XK 16/20.Following chromatography process is all in GE Carried out on the protein purification instrument of kta Explore 100.Fill after the completion of post, pillar height is 13.5 cm, its post effect is surveyed with 1% acetone is 4562 N/m.Surveyed post effect after, 5 column volumes of pillar watering balance, then with originate the mM pH 8.0 of level pad 20 Tris-HCl buffer solutions balance 5 column volumes.The M of 20 mM pH, 8.0 phosphate buffers+0.1 is reached with 10 column volumes NaCl linear gradient is eluted, and collects the eluent containing AAT.After elution terminates, the 0.5 M NaOH with 5 column volumes are clear Pillar is washed, the associated proteins not eluted are washed away.Preserved again with the 20% ethanol solution balance pillar of 5 column volumes.This step anion The AAT eluents purity that displacement chromatography is obtained is 55%, is 0.39 than living, the rate of recovery is 90%.

3. inactivation of virus：Before second step chromatography is carried out, the eluent containing AAT need to pass through two step inactivation of virus, xeothermic to go out Living and S/D inactivations.First, the anion-exchange chromatography eluent containing AAT is freezed, lyophilized process is as follows：First in sky - 25 DEG C of holding 2-4 h during atmosphere is enclosed always, then -40 DEG C of 2-4 h.Then under vacuum, -10 DEG C of holding 30-40 h, heating 20-30 h are kept again to 35 DEG C.The sample freezed is then at 80 DEG C of xeothermic h of inactivation of virus 72.Sample after xeothermic inactivation is again It is dissolved in the water, adds the stable AAT of sucrose, ultimate density is 35%.Then add TRI N BUTYL PHOSPHATE and Tween-80 carries out S/D Inactivation, ultimate density is respectively 0.3% and 0.2%, and 30 DEG C inactivate after 3-5 h, are filtered and are dialysed.Solution is adjusted after dialysis PH is identical with the pH of the cation-exchange chromatography level pad of next step.

4. cation-exchange chromatography：The SP Sepharose Fast Flow gels for taking the ethanol of 30ml 20% to preserve, are used Water is replaced repeatedly, removes ethanol, and placement is reached after equalized temperature with the dress posts of XK 16/20.Following chromatography process is all in GE kta Carried out on the protein purification instrument of Explore 100.Fill after the completion of post, pillar height is 12 cm, it is 4102 N/ that its post effect is surveyed with 1% acetone m.Surveyed post effect after, 5 column volumes of pillar watering balance, then with originate the phosphate-buffereds of 20 mM pH of level pad 5.5 Liquid balances 5 column volumes.AAT eluents after inactivation of virus are adjusted into pH to 5.5.Applied sample amount is 30 ml, is reached with 10 column volumes Linear gradient to the M NaCl of 20 mM pH, 5.5 phosphate buffers+0.1 is eluted, and wherein AAT is received in liquid is penetrated Collection penetrates liquid.After elution terminates, pillar is cleaned with 0.5 M NaOH of 5 column volumes, the associated proteins not eluted are washed away.Use again The 20% ethanol solution balance pillar of 5 column volumes is preserved.The AAT that this step cation-exchange chromatography is obtained penetrates AAT purity in liquid For 83%, than it is living be 0.79, the rate of recovery is 70%.The rate of recovery and purity are significantly lower than embodiment 3.

5. it is ultrafiltration, aseptic filtration, packing, lyophilized：The AAT of cation-exchange chromatography is flowed through into liquid to be concentrated by ultrafiltration, albumen is dense Spend for 15 mg/ml, aseptic filtration, packing are carried out afterwards lyophilized.Freeze-drying process：First -25 DEG C of holding 2-4 in air atmosphere is total H, then -40 DEG C of 2-4 h.Then under vacuum, -10 DEG C of holding 30-40 h, are warming up to 35 DEG C and keep 20-30 h again.Freeze After the completion of dry, that is, prepare AAT preparations.

Comparative example 2

1. the dissolving of FIV-1 precipitations：Weigh 1 g FIV-1 and be precipitated and dissolved in the phosphate buffers of 10 ml, 20 mM pH 7.5 In, it is sufficiently mixed, in the h of room-temperature dissolution on shaking table 5.After dissolving terminates, filtered with 0.45 μm of needle cylinder type filter membrane, take filtrate standby With.

2. cation-exchange chromatography：The Macro-Prep High S gels for taking the ethanol of 30 ml 20% to preserve, it is anti-with water Preset is changed, and removes ethanol, and placement is reached after equalized temperature with the dress posts of XK 16/20.Following chromatography process is all in GE kta Carried out on the protein purification instrument of Explore 100.Fill after the completion of post, pillar height is 12 cm, it is 4102 N/ that its post effect is surveyed with 1% acetone m.Surveyed post effect after, 5 column volumes of pillar watering balance, then with originate the lemon acid bufferings of 20 mM pH of level pad 5.5 Liquid balances 5 column volumes.The FIV-1 ml of filtrate 15 for precipitating lysate are adjusted into pH to 5.5.Applied sample amount is 15 ml, with 10 posts Volume reaches that the M NaCl of 20 mM pH, 5.5 citrate buffer solutions+0.1 linear gradient is eluted, and wherein AAT is penetrating liquid In, collection penetrates liquid.After elution terminates, pillar is cleaned with 0.5 M NaOH of 5 column volumes, the combination egg not eluted is washed away In vain.Preserved again with the 20% ethanol solution balance pillar of 5 column volumes.The AAT that this step cation-exchange chromatography is obtained is penetrated in liquid AAT purity is 65%, is 0.60 than living, the rate of recovery is 81%.

3. inactivation of virus：Before second step chromatography is carried out, the liquid that penetrates containing AAT need to be xeothermic to go out by two step inactivation of virus Living and S/D inactivations.First, the anion-exchange chromatography eluent containing AAT is freezed, lyophilized process is as follows：First in sky - 25 DEG C of holding 2-4 h during atmosphere is enclosed always, then -40 DEG C of 2-4 h.Then under vacuum, -10 DEG C of holding 30-40 h, heating 20-30 h are kept again to 35 DEG C.The sample freezed is then at 80 DEG C of xeothermic h of inactivation of virus 72.Sample after xeothermic inactivation is again It is dissolved in the water, adds the stable AAT of sucrose, ultimate density is 35%.Then add TRI N BUTYL PHOSPHATE and Tween-80 carries out S/D Inactivation, ultimate density is respectively 0.3% and 0.2%, and 30 DEG C inactivate after 3-5 h, are filtered and are dialysed.Solution is adjusted after dialysis PH is identical with the pH of the cation-exchange chromatography level pad of next step.

4. anion-exchange chromatography：The DEAE Sepharose High for taking the ethanol of 35 ml 20% to preserve Performance gels, are replaced repeatedly with water, remove ethanol, and placement is reached after equalized temperature with the dress posts of XK 16/20.With lower floor Analysis process is carried out all on the protein purification instrument of GE kta Explore 100.Fill after the completion of post, pillar height is 13.2 cm, with 1% the third It is 4865 N/m that ketone, which surveys its post effect,.Surveyed post effect after, 5 column volumes of pillar watering balance, then with originate the mM of level pad 20 The phosphate buffers of pH 7.0 balance 5 column volumes.AAT eluents after inactivation of virus are adjusted into pH to 7.0.With 10 cylinders Product reaches that the M NaCl of 20 mM pH, 7.0 phosphate buffers+0.1 linear gradient is eluted, and collects the elution containing AAT Liquid.After elution terminates, pillar is cleaned with 0.5 M NaOH of 5 column volumes, the associated proteins not eluted are washed away.Again with 5 posts The 20% ethanol solution balance pillar of volume is preserved.The AAT eluents purity that this step anion-exchange chromatography is obtained is 85%, than work For 0.81, the rate of recovery is 89%.The rate of recovery and purity are significantly lower than embodiment 1,2 and 3.

5. it is ultrafiltration, aseptic filtration, packing, lyophilized：The AAT of anion-exchange chromatography is flowed through into liquid to be concentrated by ultrafiltration, albumen is dense Spend for 15 mg/ml, aseptic filtration, packing are carried out afterwards lyophilized.Freeze-drying process：First -25 DEG C of holding 2-4 in air atmosphere is total H, then -40 DEG C of 2-4 h.Then under vacuum, -10 DEG C of holding 30-40 h, are warming up to 35 DEG C and keep 20-30 h again.Freeze After the completion of dry, that is, prepare AAT preparations.

Claims (5)

1. a kind of isolation and purification method of human plasma protein fraction alpha1-antitrypsin, it is characterised in that comprise the following steps：

(1) the FIV-1 albumen precipitations for producing cold ethanol-Cohn method separated plasmas protein Process are dissolved in buffer solution, are treated Albumen has fully dissolved, filtering, takes filtrate standby；

(2) filtrate passes through anion-exchange chromatography, collects the eluent containing AAT, and the filler of the anion-exchange chromatography is DEAE Sepharose High Performance；

(3) eluent containing AAT carries out xeothermic inactivation and S/D inactivations, the eluent containing AAT after being inactivated；

(4) eluent containing AAT after inactivating passes through cation-exchange chromatography, and the AAT for obtaining high-purity high-activity penetrates liquid, described The filler of cation-exchange chromatography is Macro-Prep High S；

(5) AAT penetrates liquid and freezed by ultrafiltration concentration, aseptic filtration and packing, you can obtain AAT preparations.

2. the isolation and purification method of human plasma protein fraction alpha1-antitrypsin according to claim 1, it is characterised in that step Suddenly the buffer solution described in (1) is pH 7.0-9.0,10-100 mM phosphate buffers or Tris-HCl buffer solutions, dissolving temperature Spend for 20-45 DEG C, dissolution time is 5-10h.

3. the isolation and purification method of human plasma protein fraction alpha1-antitrypsin according to claim 1, it is characterised in that step Suddenly the pH of buffer that (2) described anion-exchange chromatography is used is 6.5-9.0, and buffer type is 10-100 mM Tris-HCl Buffer solution or phosphate buffer.

4. the isolation and purification method of human plasma protein fraction alpha1-antitrypsin according to claim 1, it is characterised in that step Suddenly the pH of buffer that (4) described cation-exchange chromatography is used be 4.5-6.0, buffer type be 10-100 mM citric acids or Phosphate buffer.

5. the isolation and purification method of human plasma protein fraction alpha1-antitrypsin according to claim 1, it is characterised in that bag Include following steps：

(1) dissolving of FIV-1 precipitations：The FIV-1 albumen precipitations that cold ethanol-Cohn method separated plasmas protein Process is produced are molten Solution stirs 5-10 h, temperature range in pH 7.0-9.0,10-100 mM Tris-HCl buffer solutions or phosphate buffer For 20-45 DEG C, treat that albumen has fully dissolved, filter, take filtrate standby；

(2) anion-exchange chromatography：Anion-exchange gel suspension is taken, room temperature, which is placed, to be reached post is filled after equalized temperature, with flat The buffer solution that weighs balances 5-8 column volume, the filtrate pH of FIV-1 precipitation lysates is adjusted to identical with level pad, then carries out Loading, loading volume is 1-5 column volume, is eluted with elution buffer, collects the eluent containing AAT, after chromatography terminates, Pillar is cleaned with the 0.5M NaOH of 5-8 column volume, it is whole then with the 20% ethanol water balance pillar of 5-8 column volume Individual chromatography process is carried out on the protein purification instrument of GE kta Explore 100；The filler of the anion-exchange chromatography is DEAE Sepharose High Performance；

(3) inactivation of virus：Anion-exchange chromatography eluent containing AAT is freezed, lyophilized process is as follows：First in air - 25 DEG C of holding 2-4 h during atmosphere is total, then -40 DEG C of 2-4 h, then under vacuum, -10 DEG C of holding 30-40 h are warming up to 35 DEG C keep 20-30 h again, and the sample freezed is then at 80 DEG C of xeothermic h of inactivation of virus 72, and the sample after xeothermic inactivation is molten again In Xie Yushui, the stable AAT of sucrose is added, ultimate density is 30-40%, then add TRI N BUTYL PHOSPHATE and Tween-80 carries out S/ D is inactivated, and ultimate density is respectively 0.2-0.4% and 0.1-0.3%, and 30 DEG C inactivate after 3-5 h, are filtered and are dialysed, after dialysis Adjust pH value of solution identical with the pH of the cation-exchange chromatography level pad of next step；

(4) cation-exchange chromatography：Cation exchange gel suspension is taken, room temperature, which is placed, to be reached post is filled after equalized temperature, with flat The buffer solution that weighs balances 5-8 column volume, the dialyzate pH after inactivation of virus is adjusted to identical with originating level pad, then carries out Loading, loading volume is 1-5 column volume, is eluted with buffer solution, collects the AAT liquid that flows through, after chromatography terminates, uses 5-8 The 0.5 M NaOH cleaning pillars of individual column volume, then with the 20% ethanol water balance pillar of 5-8 column volume, whole layer Analysis process is carried out on the protein purification instrument of GE kta Explore 100；The filler of the cation-exchange chromatography be or Macro-Prep High S；

(5) ultrafiltration, aseptic filtration, packing, lyophilized：The AAT of cation-exchange chromatography is flowed through into liquid to be concentrated by ultrafiltration, protein concentration is 10-50 mg/ml, carry out aseptic filtration, packing and freeze afterwards；Freeze-drying process：First -25 DEG C of holding 2-4 in air atmosphere is total H, then -40 DEG C of 2-4 h, then under vacuum, -10 DEG C of holding 30-40 h are warming up to 35 DEG C and keep 20-30 h again, freeze After the completion of dry, that is, prepare AAT preparations.