CN102234332A - Process for separating and purifying recombinant human serum albumin and fusion protein thereof - Google Patents
Process for separating and purifying recombinant human serum albumin and fusion protein thereof Download PDFInfo
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- CN102234332A CN102234332A CN2010101549874A CN201010154987A CN102234332A CN 102234332 A CN102234332 A CN 102234332A CN 2010101549874 A CN2010101549874 A CN 2010101549874A CN 201010154987 A CN201010154987 A CN 201010154987A CN 102234332 A CN102234332 A CN 102234332A
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Abstract
Different from the conventional method for purifying human serum albumin from human plasma, the invention provides a simple method for separating and purifying the human serum albumin from fermentation liquor or a mammal cell culture solution. By an immobilized metal affinity chromatography (IMAC) step, the method is suitable for purifying recombinant proteins at various salt concentrations, fermentation supernate at high salt concentration is allowed to be directly loaded, the total volume of loading buffer is effectively reduced and the cost is reduced. The method is stable and high-efficiency, and is suitable for laboratories, pilot scale tests and industrial production scale-up.
Description
Technical field
The invention belongs to the protein separation technical field, particularly to the purifying of human serum albumin of reorganization.
Background technology
Human serum albumin (HSA) is the maximum protein of content in the blood plasma, accounts for the 40%-60% of blood plasma total protein, and it is a carrier very important in the blood plasma, and in the environment of body fluid pH7.4, albumin is a negative ion, and per molecule can have 200 above negative charges.The material of many poorly water-solubles can be by betransporteding with albuminous the combination.These materials comprise that bilirubin, longer chain fatty acid (per molecule can in conjunction with 4-6 molecule), bile salt, prostaglandin(PG), steroid hormone, metal ion be (as Cu
2+, Ni
2+, Ca
2+) medicine (as acetylsalicylic acid, penicillin etc.).Another act as keeps osmotic pressure.
Albuminous molecular structure was illustrated in 1975, and for containing the globular proteins of 585 amino-acid residues, molecular weight is 66458Da, contains 17 disulfide linkage in the molecule, did not contain the component of sugar.The human serum albumin gene is positioned on No. 4 karyomit(e)s 16961 pairs of base pairs, is divided into 15 transcribed spacers.The m RNA that expresses behind the process RNA processing and splicing can encode one and have 585 amino acid whose protein.
Human serum albumin has very big application and clinical value.For example: can be used for treating because of serum albumin dyssynthesis and the wound hypoproteinemia that the albumin content that causes descends and cause of losing blood as medicine; Can be used as the interpolation composition of cell culture medium, can be used as the assistant agent of medicine, excipient; Have active hormone or medicine when the time, can not show its activity, and be considered as its storage form, because this bonded reversibility and be in running balance is therefore in the metabolism of regulating these hormones and medicine, significant with albumin bound.
Present mankind's separation and purification human serum albumin from human blood.Yet there is huge problem in this method, and for example: the shortage in blood source, be subjected to the pollution of pathogenic micro-organisms such as hepatitis B virus, AIDS virus, therefore the several different methods of producing recombinant human serum albumin (rHA) based on the technological method of recombinant DNA obtains research and development.Utilize the scale operation and the purification process of the recombinant expressed human serum albumin of microbe feasible.This method has obtained exploitation recently.Recombination method quite a lot can use, but purification rHA is still step crucial and that have much room for improvement at any time from fermented liquid.
Separation and purification human serum albumin from human blood, antigenicity substance is few, and is relatively low to the technical requirements of purifying.By comparison, the albumin of utilizing the genetically engineered recombinant technology to express, exogenous material is more, and because the human serum albumin injected dose is bigger, the antigenicity substance of trace all may cause allergic reaction, even harm patient's life security, therefore higher to the purity requirement of genetically engineered recombinant human serum albumin, the purification technique difficulty is bigger.
EP0612761 discloses a kind of method of production high purity recombinant human serum albumin, does not contain nonantigenic impurity freely in its product.This method has been used hydrophobic chromatography chromatogram (HIC) under specific environment, also have other step as: ion-exchange chromatography, boric acid or borate are handled, ultrafiltration and heat treated.Yet this method is how too complicated because of step, thereby is difficult to satisfy the requirement of large-scale commercial production.
EP0570916 discloses the method for producing rHA with gene manipulation techniques, this method comprises: ultrafiltration fermented supernatant fluid, heat treated, acid treatment and ultrafiltration once more, a series of purification steps such as use cationic exchange, hydrophobic chromatography and anionresin subsequently and saltout.Yet because of having similar problem to EP0612761, purge process is too complicated, the cycle is long, cost is high and can not become the effective ways of scale operation.
The disclosed rHA purification process of EP0699687 is: the heating cell culture fluid is with inactivated proteases, and through the fluidized bed processing of cationic exchange particulate, elutriant obtains pure rHA through ultrafiltration, HIC and anion-exchange chromatography more again.Yet fluidized-bed is different with the tomography devices of conventional filling to the requirement of equipment, thereby, still need more economical effective means purifying rHA from fermented liquid.
So, develop a kind of stable, efficiently, the separation purification method that is used for recombinant human serum albumin and fusion rotein thereof is extremely important.
Summary of the invention
One of purpose of the present invention provide a kind of be different from present commonly used from human plasma the method for purification human serum albumin, the present invention is a kind of method of separating the purification recombinant human serum albumin from fermented liquid or Mammals nutrient solution of suitable large-scale operation.
The present invention can reach by following chromatographic step:
(1) fermented supernatant fluid that will contain recombinant human serum albumin carries out immobilization metal chelating affinity chromatography,
(2) product with step (1) passes through hydrophobic chromatography,
(3) product with step (2) passes through anion-exchange chromatography (hydrophobic type anion-exchange chromatography).
1975, Poroth proposed the notion of " immobilization metal chelating affinity chromatography (Immobilized Metal-ChelatedAffinity Chromatography) " first, first successfully on agarose coupling iminodiethanoic acid (IDA) sodium.IDA sodium salt and metal ion such as Cu
++Behind the chelating, can with biomolecules such as protein bound, different protein is different with the metal ion bonding force, thus with protein separation.Studies show that through the fixed metal ion afterwards not only combines with chelated forms with matrix, can also ionic linkage, the combination of covalent linkage form, or be dispersed in the solid substrate and exist with colloidal.Nineteen eighty-three Poroth is defined as " immobilized metal affinity chromatography (IMAC) " to the every metal (no matter whether existing with ionic condition) and affinity interaction of solute that is fixed on the matrix.At present, immobilization metal chelating affinity chromatography technology has become protein, particularly gene recombinant protein and polypeptide separation and purification one of the most effective instrument.
The ultimate principle of immobilization metal chelating affinity chromatography is: transition metal ion can combine with coordinate bond with atoms such as electron donor nitrogen, sulphur, oxygen, remaining unoccupied orbital is the hapto of electron donor on the metal ion, is occupied by water molecules or negatively charged ion in solution.When the bonding force of protein surface amino-acid residue and metal ion was stronger than them, then protein replaced they and metal ion formation mixture, thereby biomolecules is adsorbed.Compete bonded solute wash-out affinity matrix with certain and adsorbed proteins, the range protein with absorption elutes respectively specifically, as: imidazoles.
The matrix of IMAC filler can be selected from: the derivative and the multipolymer of agarose, Mierocrystalline cellulose, dextran, polymeric amide, polycarbonate, poly-hydroxyethyl methacrylate, polysulfones, polyvinyl alcohol, polyacrylic acid oxirane, resin and above-mentioned substance also have inorganic materials such as macroporous silica gel, sintered glass, phosphatic rock in addition.
Contain aglucon on the matrix of IMAC filler, aglucon commonly used has iminodiethanoic acid (IDA), trishydroxymethyl quadrol (TED), also has complexon I (NTA), carboxymethyl to replace aspartic acid (TEPA) and CMASP, CMDASA.
The IMAC filler that has added aglucon has commercially available, wherein aglucon is that the filler trade name of trishydroxymethyl quadrol (TED) is IMAC Sepharose 6Fast Flow, and aglucon is that the filler trade name of iminodiethanoic acid (IDA) is Chelating Sepharose Fast Flow.
With IMAC filler chelated metal ion Cu is arranged
2+, Ni
2+, Zn
2+, Co
2+Deng, with Cu
2+For best.
Step of the present invention (1) is operated according to following technology:
We buy IMAC filler (1 liter of IMAC Sepharose 6Fast Flow) from GE company, load the XK50/30 chromatography column, then, and chelated metal ions on the IMAC filler.Method is: use the 0.2M metal ion solution of 0.5 times of chromatography column volume, be preferably copper ion solution, flow through chromatography column, cupric ion can combine with aglucon, use again the purified water flush away not with aglucon bonded metal ion.
Then with the buffer A balance chromatography column that contains 10-200mmol/L phosphoric acid salt, 10-1500mmol/L sodium-chlor, pH6-8, the buffer A that preferably contains 20mmol/L phosphoric acid salt, 600mmol/L sodium-chlor, pH7.5, identical up to the damping fluid that flows into chromatography column with electric conductivity value, the pH value of the damping fluid of outflow chromatography column, can be considered that balance is good.
The sample that will contain recombinant human serum albumin is then adjusted to the condition identical with buffer A, preferably contains 10-100mmol/L phosphoric acid salt, 10-1500mmol/L sodium-chlor, pH6-8.
The liquid that contains the recombinant human serum albumin sample that to adjust then is by chromatography column, the material that is not adsorbed by chromatography column with buffered soln A flush away.
Then with containing 10-200mmol/L phosphoric acid salt, 10-1500mmol/L sodium-chlor, 0.1-1.0M imidazoles, the buffer B of pH6-8 is come the wash-out target protein, be preferably the buffer B of 20mmol/L phosphoric acid salt, 600mmol/L sodium-chlor, 0.3M imidazoles, pH7.5, collect the elution peak of buffered soln B.The purity of its recombinant human serum albumin can reach 65%--70%.
Step (1) is preceding entering, and can under the condition of stablizer and reductive agent existence fermented supernatant fluid be heat-treated.Wherein stablizer is a Sodium octoate, and reductive agent is a halfcystine.
Step of the present invention (1) has been used the IMAC column chromatography, is applicable to the purifying recombinant proteins under the various salt concn, and the fermented supernatant fluid that allows to contain higher salt concentrations directly goes up sample, even also can directly go up sample up to the sample of 1.5M salt concn.This advantage can effectively reduce the sample solution cumulative volume or save the step of ultrafiltration desalination and reduce cost.Can also under the condition of pH6-8, adsorb recombinant albumin, avoid under low pH value condition (pH3-5) recombinant albumin by proteasome degradation.The IMAC column chromatography also is applicable to the effective separation and the purifying work of the recombinant human serum albumin of laboratory, pilot scale and suitability for industrialized production.
Another object of the present invention is to remove the proteolytic degradation product in the recombinant human serum albumin and reduce pigment content, so that further improve the purity of finished product.The target protein that step (1) is obtained further passes through the hydrophobic chromatography of step (2), can achieve the above object.
Hydrophobic chromatography (HIC) is well-known chromatographic technique, and its separation principle is based on the hydrophobic difference of protein surface.Manyly be considered to hydrophilic biomacromolecule, still have abundant hydrophobic grouping to be exposed to the outside, itself and the hydrophobic grouping that is connected in chromatography matrix are interacted.For example in patent EP0699687, hydrophobic chromatography early is proposed to be used in the purifying of recombinant human serum albumin.
The hydrophobic chromatography of step of the present invention (2) can be removed the degraded product of the recombinant human serum albumin in the recombinant human serum albumin, and these degraded product molecular weight are usually at 10-50Kda.The degraded product of using hydrophobic chromatography absorption recombinant human serum albumin, and the recombinant human serum albumin of total length at bound fraction not by wash-out.Hydrophobic interaction intensity between the degraded product of recombinant human serum albumin and the medium hydrophobic grouping can be strengthened by the ionic strength that improves employed damping fluid by a small margin.
Many commercially available hydrophobic mediums all are operable at present, and there are AM General company (GE company) and Japanese eastern Cao Da company (TOSOH company) etc. in manufacturer, and the present invention does not limit any specific matrix and/or the aglucon on the matrix to hydrophobic medium.The example of hydrophobic medium: the Phenyl Sepharose 6FF that is such as but not limited to AM General company
TMHigh sub, Phenyl Sepharose 6FF
TMLow sub, ButylSepharose FF
TMButyl-650M, the Phenyl-650M of Japan TOSOH company, Ether-650S or the like.
Used matrix can be organic materials or inorganic materials.Organic materials is natural polymer such as agarose, dextran, Mierocrystalline cellulose, starch etc. for example, or synthetic polymer such as divinylbenzene, vinylbenzene.Inorganic materials has macroporous silica gel, sintered glass etc., and wherein silica gel is well-known widely used a kind of.
Aglucon on the matrix can be phenyl, butyl, octyl group, ether, sec.-propyl etc.
Between the hydrophobic medium of step (2) and the recombinant human serum albumin one or more action sites can be arranged.Be the medium of matrix with the agarose preferably, preferably contain the aglucon of phenyl, butyl such as normal-butyl, octyl group such as n-octyl etc.Also preferred be matrix with the divinylbenzene, be the medium of aglucon with ether, sec.-propyl or phenyl, the Source of GE company for example
TM
Step (2) is optimum for using the medium that Sepharose links the phenyl aglucon, the Phenyl Sepharose 6FF that trade name is produced for AM General company
TMHigh sub.
The hydrophobic chromatography of step (2) can be at pH4-8, and preferred pH6.5-7 carries out between the salt concn 0.1-0.5M.Process is: come balance hydrophobic chromatography post with the damping fluid C that contains 10-100mmol/L phosphoric acid salt, 10-500mmol/L sodium-chlor, pH6-8 earlier, preferably contain the damping fluid C of 50mmol/L phosphoric acid salt, 0.5M sodium-chlor, pH6.5; The sample that step (1) is obtained is adjusted to the condition of damping fluid C then; Then sample flow is crossed the hydrophobic chromatography post, and with damping fluid C wash-out not with the part of chromatography column absorption; At last, collect that stream is worn and not with the part of chromatography column absorption.
In step (2) before, can be at stablizer, reductive agent and metal chelator exist down, and the product that step (1) obtains is heat-treated.Wherein stablizer is a Sodium octoate, and reductive agent is a halfcystine, and metal chelator is EDTA.
Compare with the reverse-phase chromatography that the hydrophobic interaction separation principle is similar, the aglucon concentration that hydrophobic chromatography uses is much lower.These characteristics have improved its selectivity, and can use gentle elution requirement to help the biological activity that keeps target protein.
Another object of the present invention is to remove the rHA aggressiveness effectively, removes intracellular toxin simultaneously.The target protein that step (2) is obtained further passes through the anion-exchange chromatography of step (3), can achieve the above object.
The anion-exchange chromatography medium can use the Q Sepharose of GE company
TMFast Flow, QSepharose
TMHigh preformance, DEAE Sepahrose
TMFast Flow, Q Sepharose
TMHighpreformance or Capto adhere; Or the TOYOPEARL resin of TOSOH company: QAE, SuperQ, DEAE; Or the UNOSPHERE Q of BIO-RAD company; Or fillers such as the Q CeramicHyperD 20 of PALL company, Q Ceramic HyperF, DEAE Ceramic HyperD F.The Q medium is except having the function of removing foreign protein and pigment, also has the function of removing polymkeric substance and concentrating sample in the fusion protein sample.
Compound filler Capto adhere has the double properties of anionresin and hydrophobic interaction, and it has comprised two sites with the object effect at least, and one provides coulombic interaction, and one provides based on hydrogen bond and/or hydrophobic interaction.This medium can be removed nucleic acid, host protein, dimer, polymer, virus effectively.
In step (3) before, the product that step (2) can be obtained is taken off the salt in the solution through the ultra-filtration membrane desalination of 10K.
In another embodiment of the present invention, before step (3) anion-exchange chromatography, use borate and calcium chloride, the chromatographic solution 0.5-24 that treatment step under the pH9.0 condition (2) obtains hour, can reduce the residual quantity of sugar and host protein effectively, can impel the aggressiveness of recombinant human serum albumin to the recombinant human serum albumin conversion of monomer simultaneously.
The invention provides a kind of method of from fermented liquid or Mammals nutrient solution, separating purification recombinant human serum albumin and fusion rotein thereof.In this application, fermented supernatant fluid is to contain the tired pichia spp of human serum albumin base by the clone technology structure to express, and obtains fermented liquid, uses Hitachi high-capacity and high-speed refrigerated centrifuge (model C R21G) then under the 10000g condition, centrifugal 20 minutes, obtain after getting the Sterile Filtration of supernatant liquid.Compare with previous methods, operation steps is few, stable operation, purification process simple, have uniqueness.Each step is based upon on the basis of isocratic elution, therefore is suitable for laboratory, pilot scale and suitability for industrialized production and amplifies.Particularly the IMAC column chromatography of step (1) use is applicable to the purifying recombinant proteins under the various salt concn, and the fermented supernatant fluid that allows to contain higher salt concentrations is directly gone up sample.Effectively reduce the sample solution cumulative volume, saved the cost of ultrafiltration desalination.Can also under the condition of pH6-8, adsorb recombinant albumin, avoid under low pH value condition (pH3-5) recombinant albumin by proteasome degradation.
Description of drawings
Fermentation broth sample before accompanying drawing 1, the purifying is analyzed collection of illustrative plates
Fermentation broth sample is analyzed through TSK-SW3000, shows purity 12%
Equipment: Agilent 1200 liquid chromatographs, liquid-phase chromatographic column TSK-GEL G3000SW
XL7.8 * 300mm chromatographic condition:
Moving phase: the 0.2mol/L phosphate buffered saline buffer that contains the pH7.0 of 1% Virahol (is got 0.5mol/L SODIUM PHOSPHATE, MONOBASIC 200ml, 0.5mol/L Sodium phosphate dibasic 420ml, Virahol 15.5ml and water 914.5ml, mixing) flow velocity: 0.6ml/min, detect wavelength: 280nm, sample size: 20 μ l, working time: operation such as degree of grade 30 minutes, column temperature: room temperature
The sample that sample analysis collection of illustrative plates behind accompanying drawing 2, the process this patent technology purifying obtains through embodiment 1 method
Sample is analyzed through TSK-SW3000 behind the purifying, shows that purity is more than 99%
Accompanying drawing 3, non-reduced electrophoresis SDS-PAGE collection of illustrative plates be resolving gel concentration (12%), applied sample amount (8ug) wherein
From left to right
From left to right
1Maker
The human serum albumin sample of 2 references
3 fermented liquids
4 metal chelate chromatography target proteins are collected liquid
5 hydrophobic chromatography samples
Sample behind 6 anion chromatographies
Sample behind the 7Capto adhere chromatography
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are common to explanation the present invention and be not restricted to scope of the present invention.
Embodiment 1:
Step (1) is caught sample with immobilization metal chelating affinity chromatography (IMAC)
1 liter of filler IMAC Sepharose 6Fast Flow is available from GE company, and oneself loads chromatography column, the XK50/30 pillar, and column diameter 5cm, packed height 15cm washes out with purified water and to preserve liquid (20% ethanol).
0.2M copper-bath with 0.5 times of column volume is crossed post, washes out the cupric ion that is not adsorbed with purified water.
Use buffer A then: 20mmol/L phosphoric acid salt, 600mmol/L sodium-chlor, pH7.5, balance chromatography column.
The fermented supernatant fluid that contains human serum albumin is added phosphoric acid salt, sodium-chlor, reaching of making it: 20mmol/L phosphoric acid salt, 600mmol/L sodium-chlor, pH7.5.
Use AKTA then
TMSample on the Purify chromatographic system, flow velocity 50ml/min.Behind the last sample,, reach basic point to absorption value with buffer A washing.
Use buffer B: 20mmol/L phosphoric acid salt, 600mmol/L sodium-chlor, 0.3M imidazoles, PH7.5 come the wash-out target protein, collect the elution peak of buffered soln B.
Step (2) is carried out purifying with hydrophobic chromatography (HIC)
The B buffered soln elution peak that step (1) is collected carries out the hydrophobic chromatography purifying.
With phenyl hydrophobic chromatoghaphy medium Phenyl Sepharose
TM6Fast Flow (high sub) (GE company) loads the chromatography column of diameter 5cm, the high 15cm of post.
Utilize other hydrophobic medium also to have identical separating effect, but the hydrophobicity of its hydrophobicity and phenyl medium is different, the trier can according to the hydrophobicity of isolating target protein select.
Medium before use need be with damping fluid C:50mmol/L phosphoric acid salt, 0.5M sodium-chlor, the PH6.5 balance of 3 times of column volumes.
In step (1) obtains containing the elutriant of B buffered soln of target protein, add deionized water, be diluted to the concentration of 0.5MNaCl, with phosphoric acid adjust pH to 6.5.
Use AKTA
TMSample on the Purify chromatographic system, flow velocity 50ml/min behind the end of the sample, continues wash-out with damping fluid C:50mmol/L phosphoric acid salt, 0.5M sodium-chlor, the PH6.5 of 2 times of column volumes.Collect that stream is worn the peak and with the elution peak of damping fluid C.With hydrophobic chromatography post bonded part, with the deionized water wash-out of 2 times of column volumes, effluent liquid abandons.
The hydrophobic chromatography post is collected liquid, and adding final concentration is sodium tetraborate and the calcium chloride solution of 0.1M, regulates pH to 9.0, handles 0.5-24 hour, and the centrifugal 20min of 10000rpm gets supernatant liquor then, with the 10K ultra-filtration membrane desalination of MIPPORE company.
Step (3): use the refining sample of anion-exchange chromatography
Dress Q Sepharose
TMHigh Preformance filler in the chromatography pillar of diameter 2.6cm, a high 15cm, 80 milliliters of packing volumes.With the deionized water wash of 2 times of column volumes, use damping fluid E (5OmM PB, pH7.0) balance of 5 times of column volumes then.Use the damping fluid E washing of 2 column volumes behind the last sample.Use 0-0.5M NaCl through 10 times of column volume gradient elutions then, collect main peak.
Embodiment 2:
Step (1) is caught sample with the chromatography column of metal chelate chromatography medium
Filler Chelating is available from GE company, and oneself loads chromatography column, diameter 5cm, and packed height 15cm washes out with purified water and to preserve liquid (20% ethanol)
Cross post with the 0.2M copper-bath of 0.5 times of column volume then, wash out the cupric ion that is not adsorbed with purified water then.
Use balance liquid A:20mmol/L phosphoric acid salt, 600mmol/L sodium-chlor, pH7.5 balance chromatography column, then the sample that contains target protein is adjusted to the damping fluid condition identical with A, i.e. 20mmol/L phosphoric acid salt, 600mmol/L sodium-chlor, pH7.5.Use AKTA then
TMSample on the Purify chromatographic system, flow velocity 50ml/min.
Behind the last sample, with balance liquid A washing, reach basic point to absorption value, effluent liquid discards.
Use buffer B then: the damping fluid of 20mmol/L phosphoric acid salt, 600mmol/L sodium-chlor, 0.3M imidazoles, PH7.5 comes the wash-out target protein, collects the elution peak of buffered soln B.
Step (2) utilizes hydrophobic chromatography filler (HIC) to carry out purifying
Purifying with the B buffered soln elution peak of collecting among the embodiment (1) illustrates and utilizes hydrophobic medium to come purification of target albumen.With phenyl (Phenyl Sepharose
TM6Fast Flow (high sub) GE company) chromatography media loads the chromatography column of diameter 5cm, and the high 15cm of post is used for hydrophobicity and separates.
Other hydrophobic medium also has identical separating effect, but the hydrophobicity of hydrophobicity and phenyl is different, the trier can according to the hydrophobicity of isolating target protein select.
Medium before use need be with damping fluid C:50mmol/L phosphate buffered saline buffer, 0.5M sodium-chlor, the pH6.5 balance of 3 times of column volumes, the solution that contains target protein that obtains in embodiment (1) step (1) is added the concentration that deionized water is diluted to 0.5M NaCl, with phosphoric acid adjust pH to 6.5.
Use AKTA
TMSample on the Purify chromatographic system, flow velocity 50ml/min.Behind the end of the sample, continue wash-out with damping fluid-C:50mmol/L phosphate buffered saline buffer, 0.5M sodium-chlor, the PH6.5 of 2 times of column volumes.Collect that stream is worn the peak and with the elution peak of damping fluid C.With the deionized water wash-out of hydrophobic chromatography post bound fraction with 2 times of column volumes, effluent liquid discards.
The hydrophobic chromatography post is collected liquid, and adding final concentration is sodium tetraborate and the calcium chloride of 0.1M, regulates pH to 9.0, handles 0.5-24 hour, and the centrifugal 20min of 10000rpm gets supernatant liquor then, with the 10K ultra-filtration membrane desalination of Millipore company.
Step (3): use the refining sample of Ion Exchange Medium
Dress Q Sepharose
TMHigh Preformance medium is in the chromatography pillar (80 milliliters of packing volumes) of a high 15cm of diameter 2.6cm.With the deionized water wash of 2 times of column volumes, use damping fluid E (50mM PB, pH7.0) balance of 5 times of column volumes then.Use the damping fluid E washing of 2 column volumes behind the last sample.Use 0-0.5MNaCl through 10 times of column volume gradient elutions then, collect main peak.The Q medium is except having the function of removing foreign protein, also has the function of removing polymkeric substance and concentrating sample in the fusion protein sample.
Claims (12)
1. the method for a purification of Recombinant human serum albumin, this method comprises following chromatographic step:
(1) will contain the fermented supernatant fluid of recombinant human serum albumin, carry out immobilization metal chelating affinity chromatography,
(2) product with step (1) passes through hydrophobic chromatography,
(3) product with step (2) passes through anion-exchange chromatography (hydrophobic type anion-exchange chromatography).
2. method according to claim 1, wherein the matrix of immobilization metal chelating affinity chromatography filler is selected from following one or more: the derivative of agarose, Mierocrystalline cellulose, dextran, polymeric amide, polycarbonate, poly-hydroxyethyl methacrylate, polysulfones, polyvinyl alcohol, polyacrylic acid oxirane, resin or above-mentioned substance and multipolymer or macroporous silica gel, sintered glass, phosphatic rock.
3. method according to claim 2, described substrate material is an agarose.
4. method according to claim 1, the aglucon that contains on the matrix of immobilization metal chelating affinity chromatography filler is selected from: iminodiethanoic acid, trishydroxymethyl quadrol and complexon I.
5. method according to claim 4, wherein aglucon is selected from trishydroxymethyl quadrol or iminodiethanoic acid.
6. method according to claim 1 is selected from Cu with immobilization metal chelating affinity chromatography filler chelated metal ion
++, Ni
++, Zn
++Or Co
++
7. method according to claim 6, described metal ion are Cu
++
8. according to the described method of the arbitrary claim in front,, under the condition that stablizer and reductive agent exist, fermented supernatant fluid is heat-treated wherein in that to enter step (1) preceding.
9. according to the described method of the arbitrary claim in front, wherein in step (2) before, at stablizer, reductive agent and metal chelator exist down, and the product that step (1) obtains is heat-treated.
10. according to Claim 8 or 9 described methods, wherein stablizer is a Sodium octoate, and reductive agent is a halfcystine, and metal chelator is EDTA.
11. according to the described method of the arbitrary claim in front, wherein step (2) hydrophobic chromatoghaphy medium that uses is to contain phenyl, aliphatics and/or the heterocycle hydrophobic chromatoghaphy medium as aglucon.
12. according to the described method of the arbitrary claim in front, wherein in step (3) before, the product that step (2) is obtained passes through the ultra-filtration membrane desalination.
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| CN106046149A (en) * | 2016-06-28 | 2016-10-26 | 中国科学院福建物质结构研究所 | Method for removing impurities in serum albumin and fusion protein thereof |
| CN106076294A (en) * | 2016-07-22 | 2016-11-09 | 杨龙 | A kind of metal chelation resin for albumen affinity purification and preparation method thereof |
| CN107823915A (en) * | 2017-10-31 | 2018-03-23 | 苏州博进生物技术有限公司 | A kind of alkali resistance affinity chromatography medium |
| CN111662944A (en) * | 2019-03-05 | 2020-09-15 | 上海医药工业研究院 | Preparation method and purification method of human serum albumin |
| CN112210002A (en) * | 2020-10-15 | 2021-01-12 | 湖南科众源创科技有限公司 | Purification method of recombinant human serum albumin |
| WO2022120547A1 (en) * | 2020-12-08 | 2022-06-16 | 通化安睿特生物制药股份有限公司 | Method for purifying recombinant protein |
| CN114907469A (en) * | 2021-02-08 | 2022-08-16 | 首都医科大学附属北京朝阳医院 | Method for separating and purifying albumin in human serum |
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| CN106046149B (en) * | 2016-06-28 | 2019-12-24 | 中国科学院福建物质结构研究所 | Method for removing impurities in serum albumin and fusion protein thereof |
| CN106046149A (en) * | 2016-06-28 | 2016-10-26 | 中国科学院福建物质结构研究所 | Method for removing impurities in serum albumin and fusion protein thereof |
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| CN111662944A (en) * | 2019-03-05 | 2020-09-15 | 上海医药工业研究院 | Preparation method and purification method of human serum albumin |
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| WO2022120547A1 (en) * | 2020-12-08 | 2022-06-16 | 通化安睿特生物制药股份有限公司 | Method for purifying recombinant protein |
| CN114885608A (en) * | 2020-12-08 | 2022-08-09 | 通化安睿特生物制药股份有限公司 | Method for purifying recombinant proteins |
| CN114907469A (en) * | 2021-02-08 | 2022-08-16 | 首都医科大学附属北京朝阳医院 | Method for separating and purifying albumin in human serum |
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