CN102190722B - Purifying method for recombinant human serum albumin - Google Patents
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Abstract
The invention relates to a purifying method for recombinant human serum albumin (rHSA), which comprises the following steps of: directly carrying out one-step chromatography on a fermentation liquid with an expanded bed anion column to realize the purification actions of solid-liquid separation, concentration and primary purification; and carrying out heating, hydrophobic chromatography, cationic exchange, ultrafiltration, chelating, precipitation and the like to obtain ultrahigh-purity recombinant human serum albumin.
Description
Technical field
The invention belongs to biological technical field; More specifically, the present invention relates to the method for a kind of purification of Recombinant human serum albumin (rHSA).
Background technology
Human serum albumin is the major protein composition in blood plasma, 585 amino acid, consists of, and molecular weight is 66kD.Human serum albumin Main Function in blood is to maintain normal osmotic pressure, combines, and play the effect of transport vehicle with calcium ion, lipid acid, amino acid, bilirubin and various medicine.Human serum albumin is usually used in the treatment of the diseases such as albumin deficiency disease that surgical operation, hemorrhagic shock, scald, nephrotic syndrome cause, tumour liver ascites clinically.
All the time, human serum albumin purifying from human blood obtains.Due to reasons such as deficient and viral (hepatitis, the AIDS) pollutions in blood source, gene recombination human serum albumin has obtained the attention of domestic and international big drug firm in nearly ten years.In recent years, adopt the method for gene recombination, at yeast (USP5,330,901, JP11-509525, JP6-100592), intestinal bacteria (Lawn, R.M.Construction of DNA sequencesand their use for microbial production of proteins, in particular human serumalbumin " European Patent Appl. " (1983) 73, p646), express and be purified into human serum albumin in transgenosis milk (WO9602573A1SEPARATION OF HUMAN SERUM ALBUMIN).
Yet the dosage in the clinical treatment of human serum albumin is larger, be generally between 5～10g/ dosage, so compare with the conventional reconstituted drug of micro-administration, the side effect that impurity produces becomes very serious problems.Therefore, for gene recombination albumin, than the purity of the preparation of human serum albumin in the market and other gene recombination medicines, want height many.The rHSA of the up to the present pichia spp of Jin You Mitsubishi Pharmaceutical Co., Ltd fermentation went on the market in 2008, trade(brand)name:
solve the Major Difficulties of rhSA, set up exactly the preparation method of ultra-high purity (purity is higher than 99%) and good economy (cost is not higher than the albumin of blood purifying) rHSA.
The people such as wild Tian Zonghong have described the method that steps such as utilizing heating fermented liquid, dilution, acid adjustment, expanding bed positively charged ion (STREAMLINE SP-GE Healthcare company aims at the commercialization resin of expanding bed design) chromatography, heating, hydrophobic chromatography, chelating absorption, anionresin, lime borate precipitation after yeast fermentation obtains high purity rHSA in patent CN1127299A and CN1364643A.In the method, the object of the first step heating is inactivated proteases, otherwise the acidic conditions that Zeo-karb needs is the activity of activator enzyme, thereby in purge process, degrades rHSA.The object of the method dilution is to reduce the effect that ionic strength can be adsorbed to reach Streamline SP.But the shortcoming of the method is that heat inactivation extends process; Acid adjustment activator enzymic activity causes rHSA degraded risk; Large volume dilution increases the liquor capacity of purifying, thereby need to expand the scale of purifier apparatus, and purge process extends.To cause like this yield of rHSA product to reduce, cost increases, and the quality of product likely declines, risk increase of product microbiological contamination etc. problem.
Bavin field is stretched first-class people and in Chinese patent CN1934128A, is disclosed a kind of method of preparing human serum albumin under divalent positively charged ion exists by heat treated, wild in person of outstanding talent the people such as stretch and in Chinese patent CN1406246A, disclose the manufacture method of the human serum albumin that comprises heat treatment step.
Yet all there is the shortcomings such as treatment condition are complicated, and solid-liquid separation efficiency is not high, and the product purity of acquisition is not high in aforesaid method.Therefore, also need at present further to develop the more preferably sero-abluminous method of purification of recombinant human, required and clinical required to meet production.
Summary of the invention
The object of the present invention is to provide the method for a kind of purification of Recombinant human serum albumin (rHSA).
In a first aspect of the present invention, provide a kind of purification of recombinant human sero-abluminous method, described method comprises:
(a) fermented liquid that contains recombination human serum albumin is loaded to the fluidized-bed that contains sorbent material, makes recombination human serum albumin described in sorbent material selective adsorption; Wherein, described sorbent material is anionite-exchange resin; And
(b) reclaim the recombination human serum albumin being adsorbed.
In a preference, before entering the fluidized-bed that contains sorbent material, the described fermented liquid that contains recombination human serum albumin is without heat treated.
In another preference, before loading, the described fluidized-bed that contains sorbent material is regulated to pH7.0-8.0; Preferably, use damping fluid balance to pH7.0-8.0; Or
After loading, the fluidized-bed that contains sorbent material that has adsorbed described recombination human serum albumin is regulated to pH7.0-8.0; Preferably, use the buffer solution for cleaning of pH7.0-8.0.
In another preference, before loading, the described fluidized-bed that contains sorbent material regulates pH7.0-7.5; Or after loading, the fluidized-bed that contains sorbent material that has adsorbed described recombination human serum albumin regulates pH7.0-7.5.
In another preference, described anionite-exchange resin is Streamline anionite-exchange resin.
In another preference, described Streamline anionite-exchange resin is selected from: Streamline Q, Streamline Q XL, or Streamline DEAE.
In another preference, in step (b), also comprise: adopt successively following methods to be further purified: (1) hydrophobic chromatography; (2) decolouring; (3) dextran gel filtration; (4) anion exchange resin layer is analysed.
In another preference, step (2) comprising:
(i) adopt boric acid or borate and divalent-metal ion to process the sample liquid through hydrophobic chromatography, be then heated to 60 ± 20 ℃; Preferably 60 ± 10 ℃; More preferably 60 ± 5 ℃.Preferably, heat 2 ± 1 hours, filter results filtered solution;
(ii) filtered solution is carried out to macroporous resin decolouring, obtain the sample liquid through decolouring.
In another preference, at (i) with (ii), also comprise step: filtered solution is carried out to ultrafiltration.
In another preference, divalent-metal ion is selected from: Ca
2+or Mg
2+; Or
Boric acid or boratory concentration are 1～500mM; Be preferably 10～200mM; Be more preferably 50 ± 20mM; Be 50 ± 10mM best; Or
The concentration of divalent-metal ion is between 1～500mM; Be preferably 10～80mM; Be more preferably 30 ± 20mM; Be 30 ± 10mM best.
In another preference, described macroporous resin is the copolymerized macromolecule porous resin that contains amino and hydroxyl.
In another preference, the aglucon of hydrophobic chromatography is selected from: phenyl, aliphatics or heterocycle.
In another preference, the purifying mode of hydrophobic chromatography is that stream is worn.
In another preference, described dextran gel filtration adopts Superdex series or Sephadex G series.More preferably, described dextrane gel Sephadex G75.
In another preference, the resin that anion exchange resin layer is analysed comprises: DEAE, QAE, Q aglucon.
Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.
Accompanying drawing explanation
The sample peak of rHSA after Fig. 1, expanding bed Streamline Q XL process.
The sample peak of rHSA after Fig. 2, Phenyl Sepharose (H Sub) hydrophobic chromatography.
The sample peak of rHSA after Fig. 3, macropore decolorizing resin XSC700 process.
Fig. 4, DEAE Sepharose FF anion chromatography are processed the sample peak of rear rHSA.
Embodiment
The inventor is through research widely, develop a kind of by fermented liquid without heat treated, directly with the fluidized-bed that contains sorbent material (resin anion(R.A)), carry out solid-liquid separation, then through a plurality of purification steps, obtain the method for the recombinant human serum albumin of ultra-high purity.
As used herein, described recombination human serum albumin (rHSA) refers to the human serum albumin producing by recombinant technology.Be about to the sero-abluminous DNA of encoding human and import in suitable host cell, be suitable for cultivating this cell under the condition of expressing, thereby producing recombination human serum albumin.Described host cell is selected from: bacterium (as Escherichia coli and Bacillus subtilis), yeast (as cereuisiae fermentum, pichia pastoris phaff and Crewe Vickers etc.), vegetable cell, zooblast.Preferred microorganism is pichia pastoris phaff.Preferably, described recombination human serum albumin is secreted into outside born of the same parents.
The invention provides a kind of sero-abluminous method of purification of recombinant human, described method comprises: (a) fermented liquid that contains recombination human serum albumin is loaded to the fluidized-bed that contains sorbent material, makes recombination human serum albumin described in sorbent material selective adsorption; Wherein, described sorbent material is anionite-exchange resin; And the recombination human serum albumin that (b) recovery is adsorbed.
The inventor finds, carries out heat treated before solid-liquid separation, can cause impurity aggegation, is unfavorable for follow-up separation, and the step of removal agglutinator is also very complicated, directly has influence on extraction yield and purification effect.In addition, before solid-liquid separation, carry out enzyme inhibitors processing, can cause cost greatly to improve (enzyme inhibitors price is high), and the result of introducing external contamination thing.And method of the present invention is different from purification process conventional in prior art, before entering the fluidized-bed that contains sorbent material, the described fermented liquid that contains recombination human serum albumin, without heat treated, is also processed without enzyme inhibitors.
In the described fluidized-bed that contains sorbent material, sorbent material is anionite-exchange resin, is preferably Streamline resin anion(R.A).It has good absorption and separation performance to rHSA under alkaline condition, does not need weakly alkaline fermented liquid to carry out acid adjustment, thereby can not cause the degraded to target protein by activator enzyme, has also avoided the polyreaction of rHSA under acidic conditions simultaneously.The step that depolymerizes after adopting alkaline condition to save in purge process.Therefore,, before loading, the described fluidized-bed that contains sorbent material is regulated to pH7.0-8.0; Preferably, use damping fluid balance to pH7.0-8.0; More preferably regulate pH7.0-7.5; Or after loading, the fluidized-bed that contains sorbent material that has adsorbed described recombination human serum albumin is regulated to pH7.0-8.0; Preferably, use the buffer solution for cleaning of pH7.0-8.0; More preferably regulate pH7.0-7.5.
Described Streamline anionite-exchange resin is selected from: Streamline Q, Streamline Q XL, or Streamline DEAE.More preferably, the described preferred Sreamline XL of Streamline resin anion(R.A) resin anion(R.A) (GE Healthcare company), nearly 5～10 times of improving of the albumen carrying capacity of this resin have good absorption property within the scope of wider pH He under high ionic strength.
After solid-liquid separation, can also carry out further purifying to the recombination human serum albumin obtaining, can adopt multiple protein purification process well known to those skilled in the art to carry out.
The inventor also conducts in-depth research and optimizes the protein purification after solid-liquid separation, has found preferably purification process.Therefore, as optimal way of the present invention, after solid-liquid separation, also comprise: adopt successively following methods to be further purified: (1) hydrophobic chromatography; (2) decolouring; (3) dextran gel filtration; (4) anion exchange resin layer is analysed.According to above-mentioned purifying order, carry out being further purified of recombination human serum albumin, purification effect is ideal, and yield is high.
The present invention has no particular limits for the method for hydrophobic chromatography.Preferably, the aglucon of hydrophobic chromatography is selected from: phenyl, aliphatics or heterocycle.Preferably, the purifying mode of hydrophobic chromatography is that stream is worn.For example, can adopt PhenylSepharose (H Sub) post (GE company) to carry out.
Decolouring also can adopt method well known to those skilled in the art, as long as the method can effectively be removed the various pigments in fermented liquid and contain coloured material.Yet, inventor's discovery, under boric acid or boratory existence, the metal ion of divalence has the ability of better removal pigment albumin-binding after heating.Therefore, as optimal way of the present invention, decoloring method comprises: (i) adopt boric acid or borate and divalent-metal ion to process the sample liquid through hydrophobic chromatography, be then heated to 60 ± 20 ℃, preferably 60 ± 10 ℃; More preferably 60 ± 5 ℃; Preferably heat 2 ± 1 hours, filter results filtered solution; (ii) filtered solution is carried out to macroporous resin decolouring, obtain the sample liquid through decolouring.Adopt described preferred decoloring method, A350/A280 value is low, and decolorizing effect is even more ideal.
As more preferred mode of the present invention, at (i) with (ii), also comprise step: filtered solution is carried out to ultrafiltration.
As more preferred mode of the present invention, described divalent-metal ion is selected from: Ca
2+or Mg
2+.
As more preferred mode of the present invention, boric acid or boratory concentration are 1～500mM; Be preferably 10～200mM; Be more preferably 50 ± 20mM; Be 50 ± 10mM best; Or the concentration of divalent-metal ion is between 1～500mM; Be preferably 10～80mM; Be more preferably 30 ± 20mM; Be 30 ± 10mM best.Mesoboric acid salt of the present invention comprises: salt and the hydrate thereof of ortho-boric acid, metaboric acid, tetraboric acid or their generations, and such as four water Sodium Tetraboratees and six water Sodium Tetraboratees etc.
As more preferred mode of the present invention, described macroporous resin is the copolymerized macromolecule porous resin that contains amino and hydroxyl.
In the heat-processed of decolouring step, also can add Sodium octoate as protective material; Maybe can add the reductive agents such as acetylcysteine, halfcystine, place rHSA polymerization; Also allow to add the cracking agents such as Guanidinium hydrochloride, amino Guanidinium hydrochloride, the rHSA of polymerization of depolymerization.Described boric acid or borate allow in rHSA purifying flow process from divalent-metal ion heat treatment step to carry out in the different stages.
Dextran gel filtration also can adopt technology well known to those skilled in the art.As optimal way of the present invention, described dextran gel filtration adopts Superdex series or Sephadex G series.More preferably, described dextrane gel Sephadex G75.
Anion exchange resin layer is analysed also can adopt technology well known to those skilled in the art.As optimal way of the present invention, the resin that anion exchange resin layer is analysed comprises: DEAE, QAE, Q aglucon.
After results rHSA, can adopt technology well known in the art to carry out the analysis of purity of protein, described method includes but not limited to: HPLC, pillar G3000SW
xL(7.8mm IDx30cm, Tosoh company), UV 280nm.
The analysis of colourity can adopt ultraviolet spectrophotometer.Recombinant human serum albumin is diluted with ultrapure water, make its absorbance at 280nm between 0.3～0.7.Take ultrapure water as blank, measure 280nm absorbance.Recombinant human serum albumin is diluted with ultrapure water, make its at 350nm absorbance between 0.3～0.7.Take ultrapure water as blank, in the interscan of 330-520nm scope.Choose 350nm absorbance.Research A350/A280 value=(absorbance * extension rate of 350nm)/(absorbance * extension rate of 280nm).
Can adopt technology well known in the art to carry out the analysis of protein concentration, for example, can adopt BCA detection kit (BCA Protien Assay Kit, Thermo).
Major advantage of the present invention is:
(1) the invention provides and a kind ofly in short period and less technical process, rHSA is purified to the simple method of a ultra-high purity level.Before solid-liquid separation, fermented liquid does not need heating, does not dilute or synchronous a small amount of dilution can be carried out the solid-liquid separation of Streamline resin anion(R.A), concentrated and preliminary purification fast before upper prop.The present invention has overcome in prior art must just can carry out the technological deficiency of solid-liquid separation by heat inactivation proteolytic enzyme.
(2) the present invention is still optimized the step that is further purified after solid-liquid separation, has found preferably purifying order.
(3) the present invention has also optimized decoloring method, makes protein decolouring effect ideal.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition as people such as Sambrook, molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
Unless otherwise defined, the same meaning that all specialties of using in literary composition and scientific words and one skilled in the art are familiar.In addition, any method similar or impartial to described content and material all can be applicable in the present invention.The use that better implementation method described in literary composition and material only present a demonstration.
embodiment 1, recombinant bacterium structure and expression
GS115 Pichi strain (from ATCC), the HSA cDNA fragment of inserting total length by pPIC9K (purchased from Invitrogen) multiple clone site is (referring to GenBank accession number: AF190168.The rHSA expression plasmid of linearization of Sal I enzyme is transformed to GS115 bacterial strain with electroporation, be coated with MD dull and stereotyped, get colony inoculation containing in 96 orifice plates of 200 microlitre YPD substratum, cultivate 2 days for 30 ℃, get 10 microlitres and transfer in containing the 96 new plates in hole of 190 microlitre YPD, incubated overnight.After the same dilution once, get the 1 microlitre bacterium liquid YPD that point sample is 0.5～8mg/ml in G418 concentration respectively dull and stereotyped.Choose resistance clone in BMGY substratum 30 ℃ of shaking culture to D
600be 2～6 o'clock, centrifugal receipts thalline is also suspended from 1/5～1/10 BMMY substratum and continues to cultivate, and mends methyl alcohol every day and carries out abduction delivering.Culture supernatant is carried out to SDS-PAGE and filter out high expression level bacterial strain.(referring to 202nd～204 pages of institute of < < Military Medical Science Institute periodical > > the 25th volume third phases of September calendar year 2001).
One pipe is filtered out to the rear frozen restructuring rHSA Pichi strain of amplification preparation after 30 ℃ of water-baths are melted, the aseptic triangle shaking flask that contains 200mL YPD substratum of transferring to.Sealing shaking flask is also placed in the shaking table of 30 ℃, shakes concussion and cultivates 24 hours.Then, by the aseptic 10L fermentor tank of transferring to containing 5LYPD substratum of seed culture.Ventilation, stirring start to ferment 30 ℃, 24 hours.Then by the aseptic 1.2m that transfers to of nutrient solution
3fermentor tank in, add in advance glycerinated YPD culture medium culturing 48 hours, after glycerine is exhausted, adds containing the YPM substratum of methyl alcohol and the induction of SM substratum and produce rHSA, cultivate 250 hours, obtain containing the nutrient solution of rHSA 8.5g/L.
embodiment 2, solid-liquid separation
The Pichia Pastoris containing rHSA gene of restructuring, by high density fermentation, obtaining expression amount is the nutrient solution of 8.5g (rHSA)/L.Get this nutrient solution of 20L, use distilled water diluting 20% (24L) left and right (; The object of dilution is to increase mobility), direct injection is to Streamline Q XL post (Direct95/1.7 post, 95mm * 170cm, gel volume 2500mL, GE company), 400cm/h flow velocity, this post is used 20mM sodium phosphate buffer (pH 7.4) balance in advance, after sample introduction completes, with same 20mM sodium phosphate buffer (pH 7.0), cleans.Then use 20mM sodium phosphate buffer+1.0M NaCl eluant solution, flow velocity 200cm/h, collects the sample peak (6.2L) containing rHSA.See Fig. 1.
embodiment 3, hydrophobic purifying
The rHSA direct injection of the preliminary purification obtaining in embodiment 2 is arrived to PhenylSepharose (H Sub) post (500mm * 25cm, GE company), this post is used 50mM phosphoric acid-150mM sodium-chlor damping fluid (pH 6.9) balance in advance, after completion of the sample, by identical balance, change and rush liquid washing, when wear liquid appearance containing rHSA stream after, start to collect sample (12.7L).See Fig. 2.
embodiment 4, Sodium Tetraborate+calcium chloride thermal precipitation decolouring purifying
The solution containing rHSA by obtaining in embodiment 3, adds final concentration to 10mM Sodium octoate, and 50mM Sodium Tetraborate regulates pH to 9.5 with 20% (w/v) sodium hydroxide, places 4 hours.Then add calcium chloride to final concentration 30mM, with 20% sodium hydroxide, regulate pH to 9.5,60 ℃ are heated 2 hours.With press filtration, obtain clear soln (13.5L).
By the solution obtaining in embodiment 4 containing rHSA, with the ultra-filtration membrane ultrafiltration of the molecular weight 10kDa that dams, concentrated, then to the ultrafiltration of distilled water desalination, obtain the solution of 2.1L.
embodiment 6, decolouring
With macropore decolorizing resin XSC700 (Xi-an Electric Power Resin Factory) post (300mm * 300mm), decolour, this decolorizing column is used the acetum balance of pH 3 in advance, the solution containing rHSA that embodiment 5 is obtained (adjusting pH to 4.5 with 50% (v/v) acetic acid) pumps into, and circulates 16 hours.After circulation completes, with the rHSA albumen of distilled water wash-out absorption, obtain the decolouring rHSA solution of 3.9L.See Fig. 3.
embodiment 7, except intracellular toxin, nucleic acid, small-molecule substance and desalination
By in embodiment 6, obtain containing rHSA solution (adjusting pH to 6.9 with the NaOH of 2M), pump in Sephadex G75 (200mmx300mm, GE company) post, distilled water wash-out, obtains the rHSA solution of 7.8L.
embodiment 8, negatively charged ion purifying
By the solution containing rHSA obtaining in embodiment 7, be loaded into DEAE Sepharose FF XK 50/20 post (GE company) of using in advance 100mM phosphate buffered saline buffer pH7.2 balance good, be written into and with balance liquid, washed afterwards, then use 100mM phosphoric acid salt+0.5M NaCl (0 → 100%) gradient elution, obtain the rHSA solution of 17.2L.See Fig. 4.
Adopt document (Wataru Ohtani, * Toyoo Ohda, Akinori Sumi, Kaoru Kobayashi, andTakao Ohmura.Analysis of Pichia pastoris Components in Recombinant HumanSerum Albumin by Immunological Assays and by HPLC with PulsedAmperometric Detection, Anal.Chem.1998,70,425-429) method records pichia spp host's content < 1ng/ml (250mg/ml).
The purity of the rHSA obtaining reaches and is greater than 99.999999%
embodiment 9, solid-liquid separation 2
The Pichia Pastoris (GS115) containing rHSA gene of restructuring, by high density fermentation, obtaining expression amount is the nutrient solution of 9.2g (rHSA)/L.Get this nutrient solution of 20L, direct injection is to Streamline QXL post (Direct95/1.7 post, 95mmx170cm, gel volume 2500mL), 400cm/h flow velocity, this post is used 20mM sodium phosphate buffer (pH 7.4) balance in advance, after sample introduction completes, with same 20mM sodium phosphate buffer (pH 7.0), cleans.Then use 20mM sodium phosphate buffer+1.0M NaCl eluant solution, flow velocity 200cm/h, collects the sample peak (8.8L) containing rHSA.
The solution 8.8L containing rHSA by obtaining in embodiment 9, is divided into four parts.
First part adds final concentration to 10mM Sodium octoate, and 50mM Sodium Tetraborate regulates pH to 9.5 with 20% (w/v) sodium hydroxide, places 4 hours.Then add calcium chloride to final concentration 30mM, with 20% sodium hydroxide, regulate pH to 9.5,60 ℃ are heated 2 hours.Filtration obtains clear soln.
Second part adds final concentration to 10mM Sodium octoate, and 50mM Sodium Tetraborate regulates pH to 9.5 with 20% sodium hydroxide, places 4 hours.Then add calcium chloride to final concentration 30mM, with 20% sodium hydroxide, regulate pH to 9.5, normal temperature is placed 2 hours.Filtration obtains clear soln.
The 3rd part adds final concentration to 10mM Sodium octoate, and 50mM Sodium Tetraborate regulates pH to 9.5 with 20% sodium hydroxide, place 4 hours, and then 60 ℃ is heated 2 hours.Filtration obtains clear soln.
The 4th part adds calcium chloride to final concentration 30mM, with 20% sodium hydroxide, regulates pH to 9.5, and 60 ℃ are heated 2 hours.Filtration obtains clear soln.
Calculate the A350/A280 value of the solution after above-mentioned processing, be worth lower expression decolorizing effect better.Concrete different purification result are in Table 1.
Purification result under table 1 different condition
| Sodium octoate (10mM) | Sodium Tetraborate (50mM) | Calcium chloride (30mM) | Heating (60 ℃) | A350/A280 | |
| Before processing | 0.172 | ||||
| First part | + | + | + | + | 0.021 |
| Second part | + | + | + | - | 0.038 |
| The 3rd part | + | + | - | - | 0.079 |
| The 4th part | - | - | + | + | 0.075 |
(note :+represent to contain ,-represent not have)
embodiment 11, solid-liquid separation comparative example
The Pichia Pastoris (GS115) containing rHSA gene of restructuring, by high density fermentation, obtaining expression amount is the nutrient solution of 8.5g (rHSA)/L.Get this nutrient solution of 20L, 56 ℃ are heated 30 minutes, by proteolytic enzyme deactivation.Use again 4 times of left and right of distilled water diluting, HAC adjusts pH to 4.5, sample introduction is to Streamline SP (resin cation (R.C.)) post (Direct95/1.7 post, 95mm * 170cm, gel volume 2500mL, GE company), 300cm/h flow velocity, this post is used phosphoric acid buffer (pH 4.5) balance in advance, after sample introduction completes, with same 20mM sodium phosphate buffer (pH 4.5), cleans.Then use 50mM sodium phosphate buffer+150mM NaCl solution (pH 9.0) wash-out, flow velocity 200cm/h, collects the sample peak (5.8L) containing rHSA.After according to the step of embodiment 2 to 7, process, obtain the rHSA solution of 12.3L, the total recovery of this embodiment method is lower, is 32.2%.
Adopt document (Wataru Ohtani, Toyoo Ohda, Akinori Sumi, Kaoru Kobayashi, andTakao Ohmura.Analysis of Pichia pastoris Components in Recombinant HumanSerum Albumin by Immunological Assays and by HPLC with PulsedAmperometric Detection, Anal.Chem.1998,70,425-429) method records pichia spp host's content 4.3ng/ml (250mg/ml), and the clearance of its host protein is lower as seen.
All documents of mentioning in the present invention are all quoted as a reference in this application, just as each piece of document, are quoted as a reference separately.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (1)
1. the sero-abluminous method of purification of recombinant human, is characterized in that, described method comprises:
(a) fermented liquid that contains recombination human serum albumin is loaded to the fluidized-bed that contains sorbent material, makes recombination human serum albumin described in sorbent material selective adsorption; Wherein, described sorbent material is Streamline anionite-exchange resin; Before loading, the described fluidized-bed that contains sorbent material is regulated to pH7.0-8.0; After loading, the fluidized-bed that contains sorbent material that has adsorbed described recombination human serum albumin is regulated to pH7.0-8.0; Described Streamline anionite-exchange resin is selected from: Streamline Q, Streamline Q XL, or Streamline DEAE; And
(b) reclaim the recombination human serum albumin being adsorbed;
In step (b), also comprise: adopt successively following methods to be further purified: (1) hydrophobic chromatography; (2) decolouring; (3) dextran gel filtration; (4) anion exchange resin layer is analysed; In step (1), the aglucon of hydrophobic chromatography is selected from: phenyl, aliphatics or heterocycle;
Step (2) comprising: (i) adopt boric acid or borate and divalent-metal ion to process the sample liquid through hydrophobic chromatography, be then heated to 60 ± 20 ℃, filter results filtered solution; (ii) filtered solution is carried out to macroporous resin decolouring, obtain the sample liquid through decolouring; And at (i) with (ii), also comprise step: filtered solution is carried out to ultrafiltration; Wherein divalent-metal ion is selected from: Ca
2+or Mg
2+; Boric acid or boratory concentration are 1～500mM; The concentration of divalent-metal ion is between 1～500mM; Described macroporous resin is the copolymerized macromolecule porous resin that contains amino and hydroxyl.
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| CN102127164B (en) | 2010-12-20 | 2013-01-30 | 武汉禾元生物科技有限公司 | Method for extracting recombinant human serum albumin from rice seeds |
| CN102532254B (en) | 2010-12-24 | 2015-06-24 | 武汉禾元生物科技股份有限公司 | Method for separating and purifying recombinant human serum albumin (rHSA) from rice seeds |
| CN103773793B (en) * | 2012-10-19 | 2018-02-13 | 上海安睿特生物医药科技有限公司 | A kind of method of high efficient expression human serum albumins |
| CN103880947B (en) * | 2012-12-21 | 2016-01-13 | 武汉禾元生物科技股份有限公司 | A kind of chromatography method of separating and purifying high-purity recombination human serum albumin |
| CN112210002B (en) * | 2020-10-15 | 2022-06-10 | 楚天源创生物技术(长沙)有限公司 | Purification method of recombinant human serum albumin |
| JP2024501601A (en) | 2020-12-08 | 2024-01-15 | 通化安睿特生物製薬股▲フェン▼有限公司 | How to purify recombinant proteins |
| KR20240038049A (en) * | 2021-07-23 | 2024-03-22 | 클라라 푸드즈 컴퍼니 | Purified protein composition and preparation method |
| CN113735962B (en) * | 2021-08-27 | 2023-11-10 | 常熟纳微生物科技有限公司 | Method for purifying recombinant human serum albumin from pig blood and application thereof |
| CN117088962B (en) * | 2023-10-20 | 2024-01-09 | 健通(济南)生物科技有限公司 | Endotoxin removal process in recombinant human serum albumin purification process |
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