CN102234332B - 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
- Publication number
- CN102234332B CN102234332B CN201010154987.4A CN201010154987A CN102234332B CN 102234332 B CN102234332 B CN 102234332B CN 201010154987 A CN201010154987 A CN 201010154987A CN 102234332 B CN102234332 B CN 102234332B
- Authority
- CN
- China
- Prior art keywords
- chromatography
- serum albumin
- human serum
- chromatography column
- recombinant human
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
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 protein separation technical field, particularly to the purifying of the human serum albumin of restructuring.
Background technology
Human serum albumin (HSA) is the protein that in blood plasma, content is maximum, accounts for the 40%-60% of Total plasma protein, it is carrier very important in blood plasma, in the environment of body fluid pH7.4, albumin is negative ion, and per molecule can with more than 200 negative charges.The material of many poorly water-solubles can by being transported with albuminous combination.These materials comprise bilirubin, longer chain fatty acid (per molecule can in conjunction with 4-6 molecule), bile salt, prostaglandin(PG), steroid hormone, metal ion (as Cu
2+, Ni
2+, Ca
2+) medicine (as acetylsalicylic acid, penicillin etc.).Another act as maintenance osmotic pressure.
Albuminous molecular structure was illustrated in 1975, and be the globular proteins containing 585 amino-acid residues, molecular weight is 66458Da, containing 17 disulfide linkage in molecule, and the component not containing sugar.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 expressed after RNA processing and splicing can encode one and have 585 amino acid whose protein.
Human serum albumin has very large application and clinical value.Such as: can be used for treating because the lose blood albumin content that causes of serum albumin dyssynthesis and wound declines the hypoproteinemia caused as medicine; Can as the interpolation composition of cell culture medium, can as the assistant agent of medicine, excipient; The activated hormone of tool or medicine, when with albumin bound, can not show that it is active, and are considered as its storage form, due to this combination reversibility and be in running balance, therefore in the metabolism regulating these hormones and medicine, significant.
The present mankind are separation and purification human serum albumin from human blood.But there is huge problem in this method, such as: the shortage in blood source, be subject to the pollution of the pathogenic micro-organism such as hepatitis B virus, AIDS virus, the multiple method therefore based on the technological method Restruction human serum albumin (rHA) of recombinant DNA obtains research and development.Utilize the scale operation of the recombinant expressed human serum albumin of microbe and purification process feasible.This method obtains exploitation recently.Recombination method quite a lot can use, but the rHA that purifies from fermented liquid is still step that is crucial and that have much room for improvement at any time.
Separation and purification human serum albumin from human blood, antigenicity substance is few, relatively low to the technical requirements of purifying.By comparison, utilize the albumin that genetically engineered recombinant technology is expressed, exogenous material is more, and due to human serum albumin injected dose larger, the antigenicity substance of trace all may cause allergic reaction, even endanger the life security of patient, therefore higher to the purity requirement of genetically engineered recombinant human serum albumin, purification technique difficulty is larger.
EP0612761 discloses a kind of method of producing high purity recombinant human serum albumin, does not contain nonantigenic impurity freely in its product.This method has used hydrophobic chromatography chromatogram (HIC) in certain circumstances, also has other step as ion-exchange chromatography, boric acid or borate process, ultrafiltration and heat treated.But this method is too complicated because step is many, thus is difficult to the requirement meeting large-scale commercial production.
EP0570916 discloses the method for producing rHA with gene manipulation techniques, the method comprises: ultrafiltration fermented supernatant fluid, heat treated, acid treatment and ultrafiltration again, a series of purification step such as use cationic exchange, hydrophobic chromatography and anionresin subsequently and saltout.But because there is 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.
Disclosed in EP0699687, rHA purification process is: heating cells nutrient solution is with inactivated proteases, then through the fluidized bed processing of cationic exchange particulate, elutriant obtains pure rHA through ultrafiltration, HIC and anion-exchange chromatography again.But fluidized-bed is different with the tomography devices of conventional filling to the requirement of equipment, thus, more economical effective means purifying rHA from fermented liquid is still needed.
So, develop a kind of stable, efficiently, extremely important for the separation purification method of recombinant human serum albumin and fusion rotein thereof.
Summary of the invention
An object of the present invention is to provide a kind of method being different from present conventional human serum albumin of purifying from human plasma, and the present invention is a kind of method of separating-purifying recombinant human serum albumin from fermented liquid or Mammals nutrient solution of applicable large-scale operation.
The present invention can be reached by following chromatographic step:
(1) by being fixed of the fermented supernatant fluid metal chelate affinity chromatography containing recombinant human serum albumin,
(2) by the product of step (1) through hydrophobic chromatography,
(3) by the product of step (2) through anion-exchange chromatography (hydrophobic type anion-exchange chromatography).
1975, Poroth proposes first " immobilization metal chelating affinity chromatography (Immobilized Metal-ChelatedAffinity Chromatography) " concept, first successfully coupling iminodiethanoic acid (IDA) sodium on agarose.IDA sodium salt and metal ion are as Cu
++after chelating, can with biomolecules as protein bound, different protein is different from metal ion bonding force, thus by protein separation.Research afterwards shows not only to be combined with chelated forms with matrix through fixing metal ion, can also ionic linkage, covalent linkage form combine, or is dispersed in solid substrate and exists with colloidal.Nineteen eighty-three Poroth is defined as every metal (no matter whether existing with ionic condition) be fixed in matrix " immobilized metal affinity chromatography (IMAC) " with the affinity interaction of solute.At present, immobilization metal chelating affinity chromatography technology becomes protein, particularly one of gene recombinant protein and the most effective instrument of peptide separation purifying.
The ultimate principle of immobilization metal chelating affinity chromatography is: transition metal ion can be combined with coordinate bond with atoms such as electron donor nitrogen, sulphur, oxygen, on metal ion, remaining unoccupied orbital is the hapto of electron donor, is occupied in the solution by water molecules or negatively charged ion.When protein surface amino-acid residue and the bonding force of metal ion are stronger than them, then protein replaces they and metal ion and forms mixture, thus biomolecules is adsorbed.With the solute wash-out affinity matrix of certain and adsorbed proteins competition binding, specifically the various protein of absorption are eluted respectively, as: imidazoles.
The matrix of IMAC filler can be selected from: the derivative of agarose, Mierocrystalline cellulose, dextran, polymeric amide, polycarbonate, poly-hydroxyethyl methacrylate, polysulfones, polyvinyl alcohol, polyacrylic acid oxirane, resin and above-mentioned substance and multipolymer, also has the inorganic materials such as macroporous silica gel, sintered glass, phosphatic rock in addition.
Containing aglucon in the matrix of IMAC filler, conventional aglucon has iminodiethanoic acid (IDA), trishydroxymethyl quadrol (TED), also has complexon I (NTA), carboxymethyl to replace aspartic acid (TEPA) and CMASP, CMDASA.
The IMAC filler having added aglucon has commercially available, wherein aglucon is the filler trade name of trishydroxymethyl quadrol (TED) is IMAC Sepharose 6Fast Flow, and aglucon is the filler trade name of iminodiethanoic acid (IDA) is Chelating Sepharose Fast Flow.
Cu is had with the metal ion of IMAC filler chelating
2+, Ni
2+, Zn
2+, Co
2+deng, with Cu
2+for best.
Step of the present invention (1) operates according to following technique:
We buy IMAC filler (IMAC Sepharose 6Fast Flow 1 liter) from GE company, load XK50/30 chromatography column, then, and chelated metal ions on IMAC filler.Method is: the 0.2M metal ion solution using 0.5 times of chromatography column volume, and be preferably copper ion solution, flow through chromatography column, cupric ion can be combined with aglucon, then washes away the metal ion be not combined with aglucon by purified water.
Then with the buffer A balance chromatography column containing 10-200mmol/L phosphoric acid salt, 10-1500mmol/L sodium-chlor, pH6-8, preferably containing the buffer A of 20mmol/L phosphoric acid salt, 600mmol/L sodium-chlor, pH7.5, until flow into the electric conductivity value of damping fluid of damping fluid with outflow chromatography column of chromatography column, pH value is identical, can be considered and balance.
Then the sample containing recombinant human serum albumin is adjusted to the condition identical with buffer A, preferably containing 10-100mmol/L phosphoric acid salt, 10-1500mmol/L sodium-chlor, pH6-8.
Then by adjust containing the liquid of recombinant human serum albumin sample by chromatography column, wash away not by material that chromatography column adsorbs with buffered soln A.
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 carrys out 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%.
Entering, step (1) is front, under stablizer and reductive agent existent condition, can heat-treat fermented supernatant fluid.Wherein stablizer is Sodium octoate, and reductive agent is halfcystine.
Step of the present invention (1) employs IMAC column chromatography, is applicable to the purifying recombinant proteins under various salt concn, and allows containing the direct loading of fermented supernatant fluid of higher salt concentrations, and the sample even up to 1.5M salt concn also can direct loading.This advantage effectively can reduce sample solution cumulative volume or saves the step of ultrafiltration desalination and reduce costs.Recombinant albumin can also be adsorbed under the condition of pH6-8, avoid (pH3-5) recombinant albumin under low pH value condition and be easily degraded by proteases.IMAC column chromatography is also applicable to effective abstraction and purification work of recombinant human serum albumin of laboratory, pilot scale and suitability for industrialized production.
Another object of the present invention is protein degradation product in removing recombinant human serum albumin and reduces pigment content, further to improve the purity of finished product.Target protein step (1) obtained, further across 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 outside, make it interact with the hydrophobic grouping being connected to chromatography matrix.Such as in patent EP0699687, hydrophobic chromatography is early proposed to be used in the purifying of recombinant human serum albumin.
The hydrophobic chromatography of step of the present invention (2) can remove the degraded product of the recombinant human serum albumin in recombinant human serum albumin, and these degraded product molecular weight are usually at 10-50Kda.The degraded product of using hydrophobic chromatographic adsorption recombinant human serum albumin, and the recombinant human serum albumin of total length at non-bound fraction by wash-out.Hydrophobic interaction intensity between the degraded product of recombinant human serum albumin and medium hydrophobic grouping can by improving the ionic strength of used damping fluid and being strengthened by a small margin.
Current many commercially available hydrophobic mediums are all operable, there are AM General company (GE company) and Japanese Dong Caoda company (TOSOH company) etc. in manufacturer, and the present invention does not limit the aglucon in any specific matrix and/or matrix to hydrophobic medium.The example of hydrophobic medium: the Phenyl Sepharose 6FF being such as but not limited to AM General company
tMhigh sub, Phenyl Sepharose 6FF
tMlow sub, ButylSepharose FF
tM; Butyl-650M, Phenyl-650M, Ether-650S of TOSOH company of Japan etc.
Matrix used can be organic materials or inorganic materials.Organic materials such as natural polymer is as agarose, dextran, Mierocrystalline cellulose, starch etc., or synthetic polymer is as divinylbenzene, vinylbenzene.Inorganic materials has macroporous silica gel, sintered glass etc., and wherein silica gel is well-known widely used one.
Aglucon in matrix can be phenyl, butyl, octyl group, ether, sec.-propyl etc.
One or more action site can be had between the hydrophobic medium of step (2) and recombinant human serum albumin.Be preferably the medium of matrix with agarose, preferably contain phenyl, butyl if normal-butyl, octyl group are as the aglucon of n-octyl etc.Also preferably be matrix with divinylbenzene, be aglucon with ether, sec.-propyl or phenyl medium, the such as Source of GE company
tM.
Step (2) optimum is the medium that application Sepharose links phenyl aglucon, and trade name is the Phenyl Sepharose 6FF that AM General company produces
tMhigh sub.
The hydrophobic chromatography of step (2) can at pH4-8, and preferred pH6.5-7, carries out between salt concn 0.1-0.5M.Process is: first balance hydrophobic chromatography post with the damping fluid C containing 10-100mmol/L phosphoric acid salt, 10-500mmol/L sodium-chlor, pH6-8, preferably containing the damping fluid C of 50mmol/L phosphoric acid salt, 0.5M sodium-chlor, pH6.5; Then the sample that step (1) obtains is adjusted to the condition of damping fluid C; Then sample is flow through hydrophobic chromatography post, and by the part that damping fluid C wash-out does not adsorb with chromatography column; Finally, the part that stream is worn and do not adsorbed with chromatography column is collected.
Before step (2), at stablizer, under reductive agent and metal chelator exist, the product that step (1) obtains can be heat-treated.Wherein stablizer is Sodium octoate, and reductive agent is halfcystine, and metal chelator is EDTA.
Compared with the reverse-phase chromatography that hydrophobic interaction separation principle is similar, the ligand concentra that hydrophobic chromatography uses is much lower.This feature improves its selectivity, and gentle elution requirement can be used to help the biological activity keeping target protein.
Another object of the present invention effectively removes rHA aggressiveness, removes intracellular toxin simultaneously.Target protein step (2) obtained, further across the anion-exchange chromatography of step (3), can achieve the above object.
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 the filler such as the Q CeramicHyperD 20 of PALL company, Q Ceramic HyperF, DEAE Ceramic HyperD F.Q medium removes the function of foreign protein and pigment except having, also have the function removing polymkeric substance and concentrating sample in fusion protein sample.
Compound filler Capto adhere has the double properties of anionresin and hydrophobic interaction, and it at least contains and two of object effect sites, and one provides coulombic interaction, and one provides based on hydrogen bond and/or hydrophobic interaction.This medium can remove nucleic acid, host protein, dimer, polymer, virus effectively.
Before step (3), the salt in solution, through the ultra-filtration membrane desalination of 10K, is taken off by the product that step (2) can be obtained.
In another embodiment of the present invention, before step (3) anion-exchange chromatography, use borate and calcium chloride, the chromatographic solution 0.5-24 hour that treatment step (2) obtains under pH9.0 condition, effectively can reduce the residual quantity of sugar and host protein, the aggressiveness of recombinant human serum albumin can be impelled to recombinant human serum albumin conversion of monomer simultaneously.
The invention provides a kind of method of separating-purifying recombinant human serum albumin and fusion rotein thereof from fermented liquid or Mammals nutrient solution.In this application, fermented supernatant fluid is that the pichia spp built containing human serum albumin base is tired by clone technology is expressed, obtain fermented liquid, then use Hitachi high-capacity and high-speed refrigerated centrifuge (model C R21G) under 10000g condition, centrifugal 20 minutes, obtain after getting supernatant fluid Sterile Filtration.Compared 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, is therefore suitable for laboratory, pilot scale and suitability for industrialized production and amplifies.Particularly the IMAC column chromatography that uses of step (1), is applicable to the purifying recombinant proteins under various salt concn, allows the direct loading of fermented supernatant fluid containing higher salt concentrations.Effectively reduce sample solution cumulative volume, save the cost of ultrafiltration desalination.Recombinant albumin can also be adsorbed under the condition of pH6-8, avoid (pH3-5) recombinant albumin under low pH value condition and be easily degraded by proteases.
Accompanying drawing explanation
Fermentation broth sample before accompanying drawing 1, purifying analyzes collection of illustrative plates
Fermentation broth sample is analyzed through TSK-SW3000, display purity 12%
Equipment: Agilent 1200 liquid chromatograph, liquid-phase chromatographic column TSK-GEL G3000SW
xL7.8 × 300mm chromatographic condition:
Moving phase: the 0.2mol/L phosphate buffered saline buffer containing the pH7.0 of 1% Virahol (gets 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, determined wavelength: 280nm, sample size: 20 μ l, working time: degree of grade runs 30 minutes, column temperature: room temperature
The sample that accompanying drawing 2, sample analysis collection of illustrative plates after this patent technique purifying obtain through embodiment 1 method
After purifying, sample is analyzed through TSK-SW3000, display purity more than 99%
Accompanying drawing 3, non-reduced electrophoresis SDS-PAGE collection of illustrative plates wherein resolving gel concentration (12%), applied sample amount (8ug)
From left to right
1Maker
The human serum albumin sample of 2 references
3 fermented liquids
4 metal chelate chromatography target proteins collect liquid
5 hydrophobic chromatography samples
Sample after 6 anion chromatographies
Sample after 7Capto adhere chromatography
embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are common to the present invention is described and is not restricted to scope of the present invention.
Embodiment 1:
Step (1) immobilization metal chelating affinity chromatography (IMAC) catches sample
Filler IMAC Sepharose 6Fast Flow 1 liter is purchased from GE company, and oneself loads chromatography column, XK50/30 pillar, column diameter 5cm, packed height 15cm, washes out conserving liquid (20% ethanol) by purified water.
Cross post with the 0.2M copper-bath of 0.5 times of column volume, wash out not by the cupric ion adsorbed by purified water.
Then buffer A is used: 20mmol/L phosphoric acid salt, 600mmol/L sodium-chlor, pH7.5, balance chromatography column.
Adding phosphoric acid salt, sodium-chlor, reaching of making it containing the fermented supernatant fluid of human serum albumin: 20mmol/L phosphoric acid salt, 600mmol/L sodium-chlor, pH7.5.
Then AKTA is used
tMpurify chromatographic system loading, flow velocity 50ml/min.After loading, with buffer A washing, reach basic point to absorption value.
By buffer B: 20mmol/L phosphoric acid salt, 600mmol/L sodium-chlor, 0.3M imidazoles, PH7.5 carry out wash-out target protein, collect the elution peak of buffered soln B.
Step (2) hydrophobic chromatography (HIC) carries out purifying
Hydrophobic chromatography purifying is carried out to the B buffered soln elution peak that step (1) is collected.
With phenyl hydrophobic chromatography media Phenyl Sepharose
tM6Fast Flow (high sub) (GE company) loads the chromatography column of diameter 5cm, post height 15cm.
Utilize other hydrophobic medium also to have identical separating effect, but the hydrophobicity of its hydrophobicity and phenyl medium is different, trier can select according to the hydrophobicity of be separated target protein.
Medium needs damping fluid C:50mmol/L phosphoric acid salt, 0.5M sodium-chlor, PH6.5 balance with 3 times of column volumes before use.
Obtain to step (1) adding deionized water in the elutriant of the B buffered soln containing target protein, be diluted to the concentration of 0.5MNaCl, with phosphoric acid adjust pH to 6.5.
Use AKTA
tMpurify chromatographic system loading, flow velocity 50ml/min, after end of the sample, continues wash-out with the damping fluid C:50mmol/L phosphoric acid salt of 2 times of column volumes, 0.5M sodium-chlor, PH6.5.Collect stream and wear peak and the elution peak with damping fluid C.With the part that hydrophobic chromatography post is combined, with the deionized water wash-out of 2 times of column volumes, effluent liquid abandons.
Hydrophobic chromatography post is collected liquid, adds sodium tetraborate and calcium chloride solution that final concentration is 0.1M, regulate pH to 9.0, process 0.5-24 hour, then the centrifugal 20min of 10000rpm, gets supernatant liquor, with the 10K ultra-filtration membrane desalination of MIPPORE company.
Step (3): use anion-exchange chromatography to refine sample
Dress Q Sepharose
tMhigh Preformance filler in the chromatography pillar of a diameter 2.6cm, high 15cm, packing volume 80 milliliters.With the deionized water wash of 2 times of column volumes, the damping fluid E (5OmM PB, pH7.0) of 5 times of column volumes is then used to balance.The damping fluid E of 2 column volumes is used to wash after loading.Then use 0-0.5M NaCl through 10 times of column volume gradient elutions, collect main peak.
Embodiment 2:
Step (1) catches sample with the chromatography column of metal chelate chromatography medium
Filler Chelating is purchased from GE company, and oneself loads chromatography column, diameter 5cm, packed height 15cm, washes out conserving liquid (20% ethanol) by purified water
Then cross post with the 0.2M copper-bath of 0.5 times of column volume, then wash out not by the cupric ion adsorbed by purified water.
Balance liquid A:20mmol/L phosphoric acid salt, 600mmol/L sodium-chlor, pH7.5 balance chromatography column, then the sample containing target protein are adjusted to the damping fluid condition identical with A, i.e. 20mmol/L phosphoric acid salt, 600mmol/L sodium-chlor, pH7.5.Then AKTA is used
tMpurify chromatographic system loading, flow velocity 50ml/min.
After loading, wash with balance liquid A, reach basic point to absorption value, effluent liquid discards.
Then use buffer B: the damping fluid of 20mmol/L phosphoric acid salt, 600mmol/L sodium-chlor, 0.3M imidazoles, PH7.5 carrys out wash-out target protein, collect the elution peak of buffered soln B.
Step (2) utilizes hydrophobic chromatography filler (HIC) to carry out purifying
With the purifying of the B buffered soln elution peak collected in embodiment (1), illustrate and utilize hydrophobic medium to carry out protein of interest.With phenyl (Phenyl Sepharose
tM6Fast Flow (high sub) GE company) chromatography media loads the chromatography column of diameter 5cm, and post height 15cm, is separated for hydrophobicity.
Other hydrophobic medium also has identical separating effect, but the hydrophobicity of hydrophobicity and phenyl is different, and trier can select according to the hydrophobicity of be separated target protein.
Medium needs damping fluid C:50mmol/L phosphate buffered saline buffer, 0.5M sodium-chlor, pH6.5 balance with 3 times of column volumes before use, the solution containing target protein obtained 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
tMpurify chromatographic system loading, flow velocity 50ml/min.After end of the sample, continue wash-out with the damping fluid-C:50mmol/L phosphate buffered saline buffer of 2 times of column volumes, 0.5M sodium-chlor, PH6.5.Collect stream and wear peak and the elution peak with damping fluid C.With the deionized water wash-out of hydrophobic chromatography post bound fraction with 2 times of column volumes, effluent liquid discards.
Hydrophobic chromatography post is collected liquid, adds sodium tetraborate and calcium chloride that final concentration is 0.1M, regulate pH to 9.0, process 0.5-24 hour, then the centrifugal 20min of 10000rpm, gets supernatant liquor, with the 10K ultra-filtration membrane desalination of Millipore company.
Step (3): use Ion Exchange Medium to refine sample
Dress Q Sepharose
tMhigh Preformance medium is in the chromatography pillar (packing volume 80 milliliters) of a diameter 2.6cm height 15cm.With the deionized water wash of 2 times of column volumes, the damping fluid E (50mM PB, pH7.0) of 5 times of column volumes is then used to balance.The damping fluid E of 2 column volumes is used to wash after loading.Then use 0-0.5MNaCl through 10 times of column volume gradient elutions, collect main peak.Q medium removes the function of foreign protein except having, also have the function removing polymkeric substance and concentrating sample in fusion protein sample.
Claims (8)
1. a method for purification of Recombinant human serum albumin, the method comprises following chromatographic step:
(1) by the fermented supernatant fluid containing recombinant human serum albumin, being fixed metal chelate affinity chromatography, first fill XK50/30 chromatography column with the IMAC filler bought from GE company, wherein said IMAC filler is IMAC Sepharose 6Fast Flow 1 liter;
Then, chelated metal ions on IMAC filler, use the 0.2M metal ion solution of 0.5 times of chromatography column volume, wherein metal ion is Cu
2+, flow through chromatography column, cupric ion can be combined with aglucon, then washes away the metal ion be not combined with aglucon by purified water;
Again with the buffer A balance chromatography column containing 10-200mmol/L phosphoric acid salt, 10-1500mmol/L sodium-chlor, pH6-8, until flow into the electric conductivity value of damping fluid of damping fluid with outflow chromatography column of chromatography column, pH value is identical;
Subsequently the sample containing recombinant human serum albumin is adjusted to the condition identical with buffer A;
By adjust containing the liquid of recombinant human serum albumin sample by chromatography column, wash away not by material that chromatography column adsorbs by buffer A;
Finally with containing 10-200mmol/L phosphoric acid salt, 10-1500mmol/L sodium-chlor, 0.1-1.0M imidazoles, the buffer B of pH6-8 carrys out wash-out target protein, collects elution peak;
(2) by the product of step (1) through hydrophobic chromatography,
(3) by the product of step (2) through anion-exchange chromatography.
2. method according to claim 1 is wherein hydrophobic type anion-exchange chromatography in the anion-exchange chromatography of step (3).
3. method according to claim 1, wherein to enter step (1) front, under stablizer and reductive agent existent condition, heat-treats fermented supernatant fluid.
4. method according to claim 1 and 2, wherein before step (2), at stablizer, under reductive agent and metal chelator exist, heat-treats the product that step (1) obtains.
5. method according to claim 4, wherein stablizer is Sodium octoate, and reductive agent is halfcystine.
6. method according to claim 4, wherein stablizer is Sodium octoate, and reductive agent is halfcystine, and metal chelator is EDTA.
7. method according to claim 1, wherein step (2) use hydrophobic chromatoghaphy medium to be the hydrophobic chromatoghaphy medium that is aglucon containing phenyl, aliphatics and/or heterocycle.
8. method according to claim 1, wherein before step (3), product step (2) obtained is through ultra-filtration membrane desalination.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010154987.4A CN102234332B (en) | 2010-04-26 | 2010-04-26 | Process for separating and purifying recombinant human serum albumin and fusion protein thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010154987.4A CN102234332B (en) | 2010-04-26 | 2010-04-26 | Process for separating and purifying recombinant human serum albumin and fusion protein thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102234332A CN102234332A (en) | 2011-11-09 |
| CN102234332B true CN102234332B (en) | 2014-12-17 |
Family
ID=44885501
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201010154987.4A Active CN102234332B (en) | 2010-04-26 | 2010-04-26 | Process for separating and purifying recombinant human serum albumin and fusion protein thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102234332B (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103694343B (en) * | 2013-12-26 | 2016-05-25 | 扬州艾迪生物科技有限公司 | A kind of method of preparing human serum albumin from urine |
| 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 |
| CN106076294A (en) * | 2016-07-22 | 2016-11-09 | 杨龙 | A kind of metal chelation resin for albumen affinity purification and preparation method thereof |
| CN107823915B (en) * | 2017-10-31 | 2020-03-06 | 苏州博进生物技术有限公司 | Alkali-resistant 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 |
| 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 |
| 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 |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1130353A (en) * | 1993-07-09 | 1996-09-04 | 史密丝克莱恩比彻姆公司 | Protein purification |
| CN1138863A (en) * | 1993-09-24 | 1996-12-25 | 赛托医学有限公司 | Chimeric proteins which block complement activation |
| CN1465702A (en) * | 2002-06-21 | 2004-01-07 | 曾位森 | Purifying and use for human vascular endothelial growth factor and granzyme B fusion protein |
| CN1738835A (en) * | 2002-11-15 | 2006-02-22 | 阿雷斯贸易股份有限公司 | Process for the purification of TNF-binding proteins using IMAC |
| CN1854155A (en) * | 2005-04-29 | 2006-11-01 | 华北制药集团新药研究开发有限责任公司 | Purification of rHSA |
| CN101094864A (en) * | 2003-06-25 | 2007-12-26 | 法麦克萨有限公司 | Purification of HER-2 variants |
| WO2009149199A2 (en) * | 2008-06-04 | 2009-12-10 | Talecris Biotherapeutics, Inc. | Composition, method and kit for preparing plasmin |
-
2010
- 2010-04-26 CN CN201010154987.4A patent/CN102234332B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1130353A (en) * | 1993-07-09 | 1996-09-04 | 史密丝克莱恩比彻姆公司 | Protein purification |
| CN1138863A (en) * | 1993-09-24 | 1996-12-25 | 赛托医学有限公司 | Chimeric proteins which block complement activation |
| CN1465702A (en) * | 2002-06-21 | 2004-01-07 | 曾位森 | Purifying and use for human vascular endothelial growth factor and granzyme B fusion protein |
| CN1738835A (en) * | 2002-11-15 | 2006-02-22 | 阿雷斯贸易股份有限公司 | Process for the purification of TNF-binding proteins using IMAC |
| CN101094864A (en) * | 2003-06-25 | 2007-12-26 | 法麦克萨有限公司 | Purification of HER-2 variants |
| CN1854155A (en) * | 2005-04-29 | 2006-11-01 | 华北制药集团新药研究开发有限责任公司 | Purification of rHSA |
| WO2009149199A2 (en) * | 2008-06-04 | 2009-12-10 | Talecris Biotherapeutics, Inc. | Composition, method and kit for preparing plasmin |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102234332A (en) | 2011-11-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102234332B (en) | Process for separating and purifying recombinant human serum albumin and fusion protein thereof | |
| JP3676817B2 (en) | Novel factor IX purification method | |
| US11590486B2 (en) | Solid phase for mixed-mode chromatographic purification of proteins | |
| US7351801B2 (en) | Method, use and kit for separating albumin from contaminants in a liquid | |
| RU2007132851A (en) | METHOD OF SOFT DISTRIBUTION CHROMATOGRAPHY | |
| KR101831300B1 (en) | Method of purifying human granulocyte-colony stimulating factor from recombinant e. coli | |
| CN101921320A (en) | A kind of separation purification method of recombinant protein A | |
| WO2009157415A1 (en) | Separating agent for purification of protein, and protein purification method | |
| Cramer et al. | Preparative chromatography in biotechnology | |
| JP2007238479A (en) | Method of eluting organic matter adsorbed to hydroxyapatite and method for purifying organic matter using the elution method | |
| CA2569821A1 (en) | Process for protein isolation | |
| WO1995029187A1 (en) | Chromatographic process for the copurification of chondroitinase i and ii proteins | |
| JP3476242B2 (en) | Purification method of influenza HA protein | |
| AU759379B2 (en) | Novel factor IX purification methods | |
| CN106749623B (en) | Method for separating human albumin by expanded bed adsorption based on mixed mode | |
| JP2001139600A (en) | Method for purifying fused protein of il-6r and il-6 | |
| KR830001822B1 (en) | Production method of euro kinase | |
| Zhao et al. | Purification of Glutathione by Immobilized Metal Ion Affinity Chromatography with Thiourea as the Ligand | |
| CN117362442A (en) | Elution method of asymmetric bispecific antibody anion exchange chromatography | |
| Bailon et al. | Practical aspects of receptor affinity chromatography | |
| JPS6169799A (en) | Purification of interferon | |
| JPH03277967A (en) | Adsorbent for affinity chromatography | |
| JPS6317898A (en) | Purification of lymphotoxin compound | |
| TH33047B (en) | Methods for the production of rDSPA? 1 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |