CN102180966B - Method for producing human alpha1-antitrypsin in large scale - Google Patents
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 10
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Abstract
The invention discloses a method for producing human alpha1-antitrypsin in large scale, and particularly relates to alpha1-antitrypsin purified from a component IV-1 separated from human plasma. The method comprises the following steps of: preparing plasma sediment IV-1 by adopting a low-temperature ethanol method; dissolving the sediment IV-1 into buffer solution, adding cold ethanol, settling, filtering, and taking supernate A; performing S/D virus inactivation on the supernate A; and then adding polyethylene glycol (PEG), settling to obtain supernate B, filtering, performing diethylaminoethyl (DEAE) Sepharose fast flow ion exchange chromatography and DEAE Sepharose CL 6B chromatography respectively, washing, eluting, collecting eluent, performing ultra-filtration and dialysis, performing Pasteur inactivation, sterilization and filtration, packing, and performing freeze-drying to obtain alpha1-antitrypsin powder. The human alpha1-antitrypsin produced by the method has high yield, high purity and high specific activity; and the method can also be used for industrialized and large-scale production.
Description
Technical field
The present invention relates to a kind of method of large-scale production people alpha1-antitrypsin.
Background technology
People's alpha1-antitrypsin is the glycoprotein that a kind of about molecular weight is 52000-55000 dalton (Da).This albumen is by the covalently bound single chain protein of several oligonucleotide, it is a kind of endogenous protein enzyme inhibitors, for example trypsinase, Chymotrypsin, pancreatic elastase, renin, skin collagen enzyme, urea kinases and leaflet karyolymph leukoprotease.
Lacking directly related disease with alpha1-antitrypsin mainly is lung and hepatic diseases, and chronic obstructive pulmonary disease (COPD) is a kind of common chronic respiratory system diseases, and number of patients is many, and case fatality rate is high.Its principal character is chronic airflow obstruction, and is and carries out sexual development, has a strong impact on patient's work capacity and quality of life.
Recently begin to answer the treatment of employment alpha1-antitrypsin because the heredopathia that the alpha1-antitrypsin defective causes and the day after tomorrow acquired alpha1-antitrypsin defective disease, obtained preferably result for the treatment of.Give alpha1-antitrypsin and can effectively suppress lung medium size lymphocyte elastoser.The lymphocyte elastoser can be destroyed the foreign protein of lung.When alpha1-antitrypsin lacks, the activity of adjustable elastic proteolytic enzyme well, make elastoser just destroy lung tissue and cause lung injury to cause pulmonary emphysema, alpha1-antitrypsin can be successfully used in treating pulmonary emphysema, chronic tracheitis and trachitis etc.
Because the alpha1-antitrypsin that is used for the treatment of is the source human plasma basically, raw material sources are limited, in order to satisfy the demand of clinical patient, should improve most possibly the alpha1-antitrypsin yield rate in blood plasma source, also will strictly control its purity simultaneously.The production method of existing published people's alpha1-antitrypsin; all propose the yield height, but the rate of recovery of unexposed its blood plasma per ton, therefore can't know the output of goods after the large-scale production; and embodiment shows and to be laboratory scale production, can't verify that can it implement scale production.The method that a kind of large-scale production people alpha1-antitrypsin has been invented through research by my company; the method is precipitated as raw material with blood products production waste component I V-1; precipitate first most of foreign protein with alcohol and PEG; make the purity of alpha1-antitrypsin reach 60%～70%; adopt again two step chromatography techniques; produce highly purified alpha1-antitrypsin; this production technique operation is relatively simple; production cost is lower; the rate of recovery of goods is high; purity is high, and specific activity is also higher, is fit to industrially scalable production.And the inactivation of virus of goods adopts S/D deactivation and pasteurization, and is all effective to lipid-coated virus and non-lipid-coated virus, guaranteed the Viral safety of product.
Summary of the invention
The method that the purpose of this invention is to provide a kind of large-scale production people alpha1-antitrypsin, the rate of recovery of product is high, purity is high, specific activity is high, can industrialization, large-scale production.
The method of production alpha1-antitrypsin provided by the present invention comprises the steps:
Blood plasma adds physiological saline and dilutes, and the pH value of adjusting solution is 7.0～7.4, and adding ethanol to alcohol concn is 8%～10%, holding temperature-3～-1 ℃, and ionic strength 0.12～0.14 stirs, and is centrifugal, obtains supernatant I and precipitates I;
It is 6.5～6.9 that supernatant I adjusts the pH value, and adding ethanol to alcohol concn is 20%～25%, and holding temperature is-5～-3 ℃, and ionic strength 0.08～0.10 stirs, and is centrifugal, obtains supernatant II+III and precipitation II+III;
It is 5.0～5.4 that supernatant II+III adjusts the pH value, and adjusting alcohol concn is 16%～19%, and holding temperature is-5～-3 ℃, and ionic strength 0.07～0.09 stirs, and is centrifugal, obtains supernatant IV-1 and precipitation IV-1;
Precipitation IV-1 dissolve with 5～8 times of damping fluids, temperature-3～-1 ℃, and adding cold ethanol to alcohol concn is 8%～12%, pH5.2～5.6, and stirring is centrifugal, and centrifugate is got supernatant A with the filter filtration of 0.45 μ m;
Supernatant A in 10: 1 ratio add the tributyl phosphate for preparing and tween-80 solution (concentration of tributyl phosphate be 3.3% and the concentration of tween-80 be 11%), the ultimate density that makes tributyl phosphate is 0.3% (g/ml), the ultimate density of tween-80 is 1% (g/ml), under 24～26 ℃ of conditions, hatched 6 hours; Then adding PEG4000 is 10%～15% (g/ml) to its concentration, and the pH value of adjusting solution is 5.0～5.5, and temperature is-1～1 ℃, and is centrifugal, gets supernatant B;
Supernatant B filters with the filter of 0.45 μ m, filtered liquid DEAE Sepharose fast flow weak anionic displacement chromatography, and chromatography column is first 6.8～7.2 phosphate buffered saline buffer balance with pH, loading is collected and is flowed out protein peak; Chromatography column regeneration is washed with the NaCl solution of 0.5M.
The protein liquid of collecting is again through DEAE Sepharose CL 6B chromatography; use first 0.01M pH6.2～6.8 phosphate buffered saline buffer balance chromatography columns; with 0.01M～0.03M pH6.2～6.8 phosphate buffered saline buffers washing; use again 0.04M～0.06MpH6.2～6.8 phosphate buffered saline buffer wash-outs; the elutriant molecular weight cut-off is the ultra-fine filter ultrafiltration of 10KD; dialysis; add 3%～5% (g/ml) glycine and 3%～5% (g/ml) glucose and cook protective material; pasteurization; Sterile Filtration; packing, freeze-drying obtains people's alpha1-antitrypsin powder.
The method of a kind of large-scale production people of the present invention alpha1-antitrypsin, wherein: described damping fluid is that the pH value is 8.0～8.6 Tris damping fluid.
The method of a kind of large-scale production people of the present invention alpha1-antitrypsin, recyclable people's alpha1-antitrypsin 300～400 grams of every 1000L blood plasma, the purity of goods reaches 96%～99%, and the specific activity of goods reaches 0.7～1.0PEU/mg albumen.
Description of drawings
The present invention is further detailed explanation below in conjunction with the drawings and specific embodiments.
Fig. 1 is technical process block diagram of the present invention.
Embodiment
Embodiment 1,
Blood plasma 1000L adds 1 times of physiological saline and dilutes, and the acetic acid of usefulness pH4.0-sodium-acetate buffer is adjusted pH7.2, adding ethanol to alcohol concn under the agitation condition is 8%, and holding temperature is-1 ℃, ionic strength 0.14, continue to stir 2 hours, centrifugal, obtain supernatant I and precipitation I;
Supernatant I adjusts pH6.7 with the acetic acid of pH4.0-sodium-acetate buffer, and adding ethanol to alcohol concn under the agitation condition is that 25% holding temperature be-5 ℃, and ionic strength 0.09 continues to stir 2 hours, and is centrifugal, obtains supernatant II+III and precipitates II+III;
Supernatant II+III adjusts pH5.2 with the acetic acid of pH4.0-sodium-acetate buffer, and adding physiological saline under the agitation condition, to adjust alcohol concn be 18%, and holding temperature be-4 ℃, and ionic strength 0.09 continues to stir 2 hours, and is centrifugal, obtains supernatant IV-1 and precipitates IV-1;
Precipitation IV-1 is that 8.0 Tris damping fluid dissolves with 6 times of pH values, temperature-2 ℃, and the pH value of regulator solution is 5.4, adds cold ethanol to alcohol concn and is 12%, and is centrifugal, centrifugate is got supernatant A with the filter filtration of 0.45 μ m;
Supernatant A in 10: 1 ratio add the tributyl phosphate for preparing and tween-80 solution (concentration of tributyl phosphate be 3.3% and the concentration of tween-80 be 11%), the ultimate density that makes tributyl phosphate is 0.3% (g/ml), the ultimate density of tween-80 is 1% (g/ml), under 24～26 ℃ of conditions, hatched 6 hours; Then adding PEG4000 is 12% (g/ml) to its concentration, and the pH value of adjusting solution is 5.1, and temperature is 0 ℃, and is centrifugal, gets supernatant B;
Supernatant B filters with the filter of 0.45 μ m, and filtered liquid is collected and flowed out protein peak with DEAE Sepharose Fast Flow weak anionic displacement chromatography.
The protein liquid of collecting is again through DEAE Sepharose CL 6B chromatography; be the washing of 6.4 phosphate buffered saline buffer with 0.02M pH value; be 6.4 phosphate buffered saline buffer wash-out again with 0.05M pH value, the elutriant molecular weight cut-off is the ultra-fine filter ultrafiltration of 10KD, dialysis; add 4% (g/ml) glycine and 4% (g/ml) glucose and cook protective material; carry out pasteurization, Sterile Filtration after the deactivation, packing; freeze-drying obtains people's alpha1-antitrypsin powder.Detected result sees Table 1.
Table 1: interpretation of result
Embodiment 2,
Blood plasma 1000L adds 1 times of physiological saline and dilutes, and the pH value of adjusting solution with the acetic acid of pH4.0-sodium-acetate buffer is 7.0, adding ethanol to alcohol concn under the agitation condition is 9%, and holding temperature is-2 ℃, ionic strength 0.14, continue to stir 2 hours, centrifugal, obtain supernatant I and precipitation I;
It is 6.5 that supernatant I adjusts pH value with the acetic acid of pH4.0-sodium-acetate buffer, and adding ethanol to alcohol concn under the agitation condition is that 21% holding temperature be-3 ℃, and ionic strength 0.08 continues to stir 2 hours, and is centrifugal, obtains supernatant II+III and precipitates II+III;
It is 5.0 that supernatant II+III adjusts pH value with the acetic acid of pH4.0-sodium-acetate buffer, and adding physiological saline adjustment alcohol concn under the agitation condition is 16%, and holding temperature is-3 ℃, ionic strength 0.08, continue to stir 2 hours, centrifugal, obtain supernatant IV-1 and precipitation IV-1;
Precipitation IV-1 is that 8.2 Tris damping fluid dissolves with 5 times of pH values, temperature-1 ℃, and the pH value of regulator solution is 5.2, adds cold ethanol to alcohol concn and is 10%, and is centrifugal, centrifugate is got supernatant A with the filter filtration of 0.45 μ m;
Supernatant A in 10: 1 ratio add the tributyl phosphate for preparing and tween-80 solution (concentration of tributyl phosphate be 3.3% and the concentration of tween-80 be 11%), the ultimate density that makes tributyl phosphate is 0.3% (g/ml), the ultimate density of tween-80 is 1% (g/ml), under 24～26 ℃ of conditions, hatched 6 hours; Then adding PEG4000 is 10% (g/ml) to its concentration, and the pH value of adjusting solution is 5.3, and temperature is-1 ℃, and is centrifugal, gets supernatant B;
Supernatant B filters with the filter of 0.45 μ m, and filtered liquid is collected and flowed out protein peak with DEAE Sepharose Fast Flow weak anionic displacement chromatography.
The protein liquid of collecting is again through DEAE Sepharose CL 6B chromatography; be the washing of 6.2 phosphate buffered saline buffer with 0.01M pH value; be 6.2 phosphate buffered saline buffer wash-out again with 0.04M pH value, the elutriant molecular weight cut-off is the ultra-fine filter ultrafiltration of 10KD, dialysis; add 3% (g/ml) glycine and 3% (g/ml) glucose and cook protective material; carry out pasteurization, Sterile Filtration after the deactivation, packing; freeze-drying obtains people's alpha1-antitrypsin powder.Detected result sees Table 2.
Table 2: interpretation of result
Embodiment 3,
Blood plasma 1000L adds 1 times of physiological saline and dilutes, and the pH value of adjusting solution with the acetic acid of pH4.0-sodium-acetate buffer is 7.4, and adding ethanol to alcohol concn is 10% under the agitation condition, holding temperature is-3 ℃, and ionic strength 0.12 continues to stir 2 hours, centrifugal, obtain supernatant I and precipitation I;
The pH value that supernatant I adjusts solution with the acetic acid of pH4.0-sodium-acetate buffer is 6.9, adding ethanol to alcohol concn under the agitation condition is that 20% holding temperature is-4 ℃, and ionic strength 0.10 continues to stir 2 hours, centrifugal, obtain supernatant II+III and precipitation II+III;
The pH value that supernatant II+III adjusts solution with the acetic acid of pH4.0-sodium-acetate buffer is 5.4, and adding physiological saline adjustment alcohol concn under the agitation condition is 19%, and holding temperature is-5 ℃, ionic strength 0.07, continue to stir 2 hours, centrifugal, obtain supernatant IV-1 and precipitation IV-1;
Precipitation IV-1 is that 8.4 Tris damping fluid dissolves with 8 times of pH values, 0 ℃ of temperature, and the pH value of regulator solution is 5.6, adds cold ethanol to alcohol concn and is 8%, and is centrifugal, centrifugate is got supernatant A with the filter filtration of 0.45 μ m;
Supernatant A in 10: 1 ratio add the tributyl phosphate for preparing and tween-80 solution (concentration of tributyl phosphate be 3.3% and the concentration of tween-80 be 11%), the ultimate density that makes tributyl phosphate is 0.3% (g/ml), the ultimate density of tween-80 is 1% (g/ml), under 24～26 ℃ of conditions, hatched 6 hours; Then adding PEG4000 is 15% (g/ml) to its concentration, and the pH value of adjusting solution is 5.3, and temperature is 1 ℃, and is centrifugal, gets supernatant B;
Supernatant B filters with the filter of 0.45 μ m, and filtered liquid is collected and flowed out protein peak with DEAE Sepharose Fast Flow weak anionic displacement chromatography.
The protein liquid of collecting is again through DEAE Sepharose CL 6B chromatography; be the washing of 6.8 phosphate buffered saline buffer with 0.03M pH value; be 6.8 phosphate buffered saline buffer wash-out again with 0.06M pH value, the elutriant molecular weight cut-off is the ultra-fine filter ultrafiltration of 10KD, dialysis; add 5% (g/ml) glycine and 5% (g/ml) glucose and cook protective material; carry out pasteurization, Sterile Filtration after the deactivation, packing; freeze-drying obtains people's alpha1-antitrypsin powder.Detected result sees Table 3
Table 3: interpretation of result
Above embodiment is described preferred implementation of the present invention; be not that scope of the present invention is limited; design under the prerequisite of spirit not breaking away from the present invention; various distortion and improvement that the common engineering technical personnel in this area make technical scheme of the present invention all should fall in the definite protection domain of claims of the present invention.
Claims (8)
1. the method for a large-scale production people alpha1-antitrypsin comprises the steps:
(a) adopt cold ethanol method to make blood plasma precipitation IV-1;
(b) precipitation IV-1 dissolves with damping fluid;
(c) add cold ethanol precipitation, added cold ethanol to alcohol concn is 8%～12%, adjusts pH5.2～5.6, stirs, and centrifugal, centrifugate is filtered with filter, gets supernatant A;
(d) supernatant A solution carries out the S/D inactivation of virus, then with the PEG precipitation, gets supernatant B;
(e) supernatant B filters with filter, filtered liquid DEAE Sepharose Fast Flow weak anionic displacement chromatography;
(f) protein liquid of collecting is again through DEAE Sepharose CL 6B ion exchange chromatography;
(g) collect elutriant, ultrafiltration, dialysis, pasteurization, Sterile Filtration, packing, freeze-drying obtains the alpha1-antitrypsin powder.
2. the method for a kind of large-scale production people alpha1-antitrypsin according to claim 1, it is characterized in that in the step (a), described precipitation IV-1 gets by following steps: blood plasma adds physiological saline and dilutes, adjust pH 7.0～7.4, adding ethanol to alcohol concn is 8%～10%, holding temperature-3～-1 ℃, ionic strength 0.12～0.14, stir, centrifugal, obtain supernatant I and precipitation I; Supernatant I adjusts pH6.5～6.9, and adding ethanol to alcohol concn is 20%～25%, and holding temperature is-5～-3 ℃, and ionic strength 0.08～0.10 stirs, and is centrifugal, obtains supernatant II+III and precipitation II+III; Supernatant II+III adjusts pH5.0～5.4, and adjusting alcohol concn is 16%～19%, and holding temperature is-5～-3 ℃, and ionic strength 0.07～0.09 stirs, and is centrifugal, obtains supernatant IV-1 and precipitation IV-1.
3. the method for a kind of large-scale production people alpha1-antitrypsin according to claim 1 and 2 is characterized in that the damping fluid described in the step (b) is that the pH value is 8.0～8.6 Tris damping fluid.
4. the method for a kind of large-scale production people alpha1-antitrypsin according to claim 3 is characterized in that described filter, and its pore size is 0.45 μ m.
5. the method for a kind of large-scale production people alpha1-antitrypsin according to claim 3 is characterized in that the PEG molecular weight described in the step (d) is 4000, and concentration is 10%～15% (g/ml).
6. the method for a kind of large-scale production people alpha1-antitrypsin according to claim 3; it is characterized in that the PEG precipitation described in the step (d) is to add PEG concentration to 10%～15% (g/ml) in solution; the pH value of solution is 5.0～5.5, and temperature precipitates for-1～1 ℃.
7. the method for a kind of large-scale production people alpha1-antitrypsin according to claim 1; it is characterized in that the S/D inactivation of virus described in the step (d) is that the ratio in 10: 1 adds tributyl phosphate and the tween-80 solution for preparing in supernatant A; the ultimate density that makes tributyl phosphate is 0.3% (g/ml); the ultimate density of tween-80 is 1% (g/ml); under 24～26 ℃ of conditions, hatched 6 hours.
8. the method for a kind of large-scale production people alpha1-antitrypsin according to claim 1 when it is characterized in that the pasteurization described in the step (g), adds 3%～5% glycine and 3%～5% glucose is cooked protective material in elutriant.
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