CN102382190A - Method for separating and removing oligomer in TNFR-Fc fusion protein - Google Patents
Method for separating and removing oligomer in TNFR-Fc fusion protein Download PDFInfo
- Publication number
- CN102382190A CN102382190A CN2010102684036A CN201010268403A CN102382190A CN 102382190 A CN102382190 A CN 102382190A CN 2010102684036 A CN2010102684036 A CN 2010102684036A CN 201010268403 A CN201010268403 A CN 201010268403A CN 102382190 A CN102382190 A CN 102382190A
- Authority
- CN
- China
- Prior art keywords
- concentration
- tnfr
- fusion rotein
- chromatography
- tris
- 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.)
- Granted
Links
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a method for purifying TNFR-Fc fusion protein. The TNFR-Fc fusion protein can be purified via immunoaffinity chromatography and hydrophobic interaction chromatography, that is, the content of oligomers in the protein can be reduced via hydrophobic interaction chromatography after affinity chromatography so as to meet quality requirements of SFDA (State Food and Drug Administration) for biological products. The oligomers in the TNFR-Fc fusion protein can be effectively separated and removed.
Description
Technical field
The present invention relates to a kind of protein separation and purifying, particularly a kind of method of separating and removing oligomer in the TNFR-Fc fusion rotein.
Background technology
TNFR-Fc fusion rotein (recombinant human tumor necrosis factor's acceptor-Fc fusion rotein) is a kind of protein polypeptide that utilizes genetic engineering technique recombinant human Tumor Necrosis Factor Receptors and immunoglobulin Fc section to form, and promptly is Etanercept, and Chinese benefit match by name is general.It can stop joint deformity, in the treatment, the severe rheumatoid arthritis, also can treat ankylosing spondylitis and severe psoriatic simultaneously.Through combining with the intravital tumour necrosis factor of people (TNF) specifically, the activity of TNF is played down regulation, thereby suppress the inflammatory reaction in the human body osteoarthrosis.One Chinese patent application Publication Specification CN1417334A discloses the recombination that makes up with the preparation soluble part in tumor necrosis factor acceptor; And the technical scheme of antigen-4 fusion protein gene and product; Only mention in the preparation purge process and select for use albumin A-Sepharose to carry out affinity chromatography, can obtain the fusion rotein of purity more than 90%.
Yet a major issue of purifying TNFR-Fc fusion rotein is how to separate and remove the oligomer in the TNFR-Fc fusion rotein.The reason part that oligomer forms is in cell cultures secreting, expressing product, to form automatically, and a part is to cause protein to form aggregate because salt or pH inductive protein denaturation make solvent to expose near the hydrophobic region on surface.The existence of these oligomer makes patient after using medicine, supersensitivity can take place, and causes the side reaction of human body intensive, so in purge process, must remove.The method that tradition is removed oligomer is sieve chromatography (exclusion chromatography SEC); But sieve chromatography is the bottleneck of purifying; A lot of shortcomings are arranged: the one, the sieve chromatography separating effect is not thorough, is not king-sized albumen for molecular weight difference, and separating effect is not fine; The 2nd, sieve chromatography is compared with other chromatographic techniques; The requirement height concentrates, applied sample amount can only column volume 0.5%～4% between, pillar is wanted thin and the long separating effect that just can obtain; Need a large amount of treatment times; Need very big column volume, material expensive and applied sample amount are low, are unfavorable for amplifying producing.
One Chinese patent application Publication Specification CN1898266A discloses and has used hydroxylapatite to remove the technical scheme of HMW oligomer; This scheme is a kind of method from the antibody preparation purification of at least one antibody that contains the HMW oligomer, and it comprises: a) make said antibody preparation contact hydroxylapatite resin and with at least a elution buffer that comprises 1 to 20mM sodium phosphate and 0.2 to 2.5M sodium-chlor from the resin elution antibody purified; Or b) make said hydroxylapatite resin contact be dissolved in the antibody preparation in the sample loading buffer that comprises 1 to 20mM sodium phosphate and 0.2 to 2.5M sodium-chlor and allow antibody purified to flow through said post.This scheme also comprised before hydroxyapatite makes antibody preparation stand A albumen affinity chromatography and two steps of ion exchange chromatography, thereby removes most of aggregate, reaches the purpose of purifying protein.One Chinese patent application Publication Specification CN1703421B discloses the technical scheme that reduces the proteic aggregate levels of PEGization, may further comprise the steps: said PEGization growth hormone antagonist or isoform a) are provided; Said PEGization growth hormone antagonist or its isoform that b) will comprise any impurity and any aggregate are to anionite-exchange resin; Wherein go up appearance and be less than or equal to 10mS/cm in specific conductivity; PH is 5-10, and protein concentration is to be less than or equal under the condition of 10g/L packed bed volume to carry out; And c) separates said PEGization growth hormone antagonist or its isoform through anion-exchange chromatography; It further comprises combining step d) merge the said PEGization growth hormone antagonist isoform of discrete amount, with through being selected from the PEGization growth hormone antagonist that following technology generates merging: capillary electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Ion exchange chromatography, hydrophobic interaction chromatography, anion-exchange chromatography; Cation-exchange chromatography, reverse phase HPLC, size exclusion high pressure liquid chromatography (HPLC); Affinity chromatography and combination thereof, thus the oligomer level that forms reduced.Hydrophobic interaction chromatography reaches as buffering salt and through the anti-phase salt gradient with Trisodium Citrate, sodium acetate removes some free PEG, not PEGization albumen and aggregate.But affinity interaction chromatography and hydrophobic interaction chromatography owing to the change of required buffer salinity or pH in protein concentration increase or the elution process, can induce oligomer to form aspect the removal oligomer.
Summary of the invention
Supersensitivity can take place after making patient use medicine in the existence of oligomer, causes the side reaction of human body intensive, so in purge process, must remove.The contriver is through TE for this reason; Overcome above technical difficulty; Use hydrophobic chromatography after a kind of use affinity chromatography is provided, select suitable chromatographic separation condition for use, will contain the extensive method of removing of oligomer in the animal cell culture supernatant of TNFR-Fc fusion rotein.
For realizing above-mentioned purpose, scheme of the present invention may further comprise the steps:
The first, from the animal cell culture supernatant, reclaim the TNFR-Fc fusion rotein, collect the TNFR-Fc fusion rotein through the albumin A affinity chromatography;
The second, the TNFR-Fc fusion rotein of collecting is separated through hydrophobic interaction chromatography and the removal oligomer.
Purification schemes of the present invention specifically may further comprise the steps:
The first, albumin A affinity chromatography
At first, using concentration is 0.01～0.1mol/LTris damping fluid (containing concentration is 0.1～0.5mol/L sodium-chlor, and pH is 6.0～8.0) balance albumin A affinity column, and the damping fluid consumption is 2～10 column volumes, until effluent lead with the balance liquid electricity and the pH value consistent;
Secondly, will contain the supernatant of TNFR-Fc fusion rotein, and use concentration to regulate pH and identical with the pH of above-mentioned Tris damping fluid as 3mol/LTris solution; With appearance on the flow velocity of 20ml/min; All flow through affinity column until supernatant, it is that (contain concentration is 0.1～0.5mol/L sodium-chlor to 0.01～0.1mol/LTris washings, and also containing concentration simultaneously is 0.5～3.0mol/L urea that concentration is used in the back; PH is 6.0～8.0) the flushing chromatography column; The washings consumption is 2～10 column volumes, and to use concentration then be 0.1～0.2mol/L Hydrocerol A elutriant (containing concentration is 0.5～2.0mol/L urea, and pH is 3.2～3.8) is adsorbed on the TNFR-Fc fusion rotein on the chromatography column with the flow velocity wash-out of 10～50ml/min; According to albumen absorption value under the 280nm wavelength, collect TNFR-Fc fusion rotein elutriant;
Once more, with the TNFR-Fc fusion rotein elutriant of collecting, using concentration to be neutralized to pH as 3mol/L Tris solution is 6.0～8.0, and uses concentration to clean chromatography column as 0.1mol/L citrate buffer solution (pH is 2.0～3.0), and the damping fluid consumption is 2～10 column volumes;
The second, hydrophobic interaction chromatography
At first, using concentration is 0.01～0.05mol/LTris-HCl damping fluid (containing concentration is 0.8～1.5mol/L ammonium sulfate, and pH is 6.0～8.0) balance hydrophobic interaction chromatography post, and the damping fluid consumption is 2～10 column volumes;
Then; With the TNFR-Fc fusion rotein that the albumin A affinity chromatography is collected, uses concentration identical with the pH of above-mentioned Tris-HCl damping fluid as 3mol/LTris solution adjusting pH, and with concentration be that (contain concentration is 1.6～3.0mol/L ammonium sulfate to 0.01～0.05mol/LTris-HCl damping fluid; PH is 6.0～8.0) be to go up an appearance hydrophobic chromatography post behind 1: 1 the ratio mixing with volume ratio; Flow rate control is 10～100ml/min, and using concentration then is 0.01～0.05mol/LTris-HCl washings (containing concentration is 0.7～0.9mol/L ammonium sulfate, and pH is 6.0～8.0) washing; The washings consumption is 2.5～3 column volumes; Using concentration again is that 0.01～0.05mol/LTris-HCl elutriant (containing concentration is 0.1～0.5mol/L ammonium sulfate, and pH is 6.0～8.0) wash-out is adsorbed on the TNFR-Fc fusion rotein on the hydrophobic interaction chromatography post, according to albumen absorption value under the 280nm wavelength; Collect TNFR-Fc fusion rotein elutriant, obtain the TNFR-Fc fusion rotein of low oligomer content.
The present invention has carried out preferably the Tris damping fluid of balance affinity column, and preferably, the used Tris buffer concentration of above-mentioned balance affinity column is 0.02mol/L (containing concentration is 0.2mol/L sodium-chlor, and pH is 7.0).
The present invention has also carried out preferably the washings of affinity chromatography, and preferably, the washings of above-mentioned affinity chromatography is that concentration is 0.02mol/LTris damping fluid (containing concentration is 0.2mol/L sodium-chlor, and also containing concentration simultaneously is 1.5mol/L urea, and pH is 7.0).
The present invention has also carried out preferably the elutriant of affinity chromatography, and preferably, above-mentioned affinity chromatography elutriant is that concentration is 0.1mol/L Hydrocerol A (containing concentration is 1.5mol/L urea, and pH is 3.5).
The present invention has also carried out preferably the flow velocity of affinity chromatography washings, and preferably, the used concentration of above-mentioned affinity chromatography is that the flow velocity of 0.1mol/L Hydrocerol A (containing concentration is 1.5mol/L urea, and pH is 3.5) is 30ml/min.
The pH regulator of the TNFR-Fc fusion rotein elutriant that the present invention also collects affinity chromatography has carried out preferably, and preferably, it is 7.5 as 3mol/L Tris solution adjusting pH that the TNFR-Fc fusion rotein elutriant that above-mentioned affinity chromatography is collected uses concentration.
The present invention has also carried out preferably the damping fluid that cleans affinity column, and preferably, the used concentration of above-mentioned cleaning affinity column is that the pH of 0.1mol/L citrate buffer solution is 2.8.
The present invention has carried out preferably the medium of hydrophobic interaction chromatography, and preferably, the used medium of above-mentioned hydrophobic interaction chromatography is Phenyl Sepharose 6 fast flow or Butyl Sepharose 4 fast flow.
The present invention has also carried out preferably the damping fluid of balance hydrophobic interaction chromatography post, and preferably, the used Tris-HCl buffer concentration of above-mentioned balance hydrophobic interaction chromatography post is 0.02mol/L (containing concentration is 1.0mol/L ammonium sulfate, and pH is 7.5).
The present invention is also to having carried out preferably with TNFR-Fc fusion rotein equal-volume blended Tris-HCl damping fluid; Preferably; Above-mentioned and TNFR-Fc fusion rotein equal-volume blended Tris-HCl buffer concentration is 0.02mol/L (containing concentration is 2.0mol/L ammonium sulfate, and pH is 7.5).
The present invention has also carried out preferably the appearance flow velocity of going up of hydrophobic interaction chromatography, and preferably, the upward appearance flow velocity of above-mentioned hydrophobic interaction chromatography is 50ml/min.
The present invention also carried out preferably the washings of hydrophobic interaction chromatography, and preferably, the washings of above-mentioned hydrophobic interaction chromatography is that concentration is 0.02mol/LTris-HCl washings (containing concentration is 0.8mol/L ammonium sulfate, and pH is 7.5).
The present invention has also carried out preferably the hydrophobic interaction chromatography elutriant, and preferably, the Tris-HCl elutriant concentration of above-mentioned hydrophobic interaction chromatography is 0.02mol/L (containing concentration is 0.4mol/L ammonium sulfate, and pH is 7.5).
The present invention compared with prior art has following outstanding advantage:
Adopt affinity chromatography, choose rProtein A affinity gel, this affinity gel can combine with the Fc section protein-specific in the TNFR-Fc fusion rotein; Reduced purification step greatly; The purity of the TNFR-Fc fusion rotein of this step purifying gained is reached more than 90%, can remove most of impurity simultaneously, for follow-up purifying work brings convenience; Through in washings and elutriant, adding urea, reduced the possibility of protein aggregation formation oligomer.And, select the elution process of ammonium sulfate as elutriant through further hydrophobic interaction chromatography, the low salt concn wash-out well separates oligomer with target protein according to the power of hydrophobic interaction, and the oligomer content of target protein is reduced to below 1.0%.The present invention through after the affinity chromatography with boiled water effect chromatography; The purity of target protein is improved greatly; Reduced the content of oligomer; Purification procedures is saved, is practiced thrift cost, production technology is simple, performance good, has solved shortcoming of the prior art, is applicable to separate the oligomer of removing the TNFR-Fc fusion rotein in laboratory scale, pilot scale and the industrialized production; Through the finished product that this inventive method obtains reached SFDA to biological products in the requirement of oligomer content, have higher utility.
Embodiment
Below further describe the present invention through specific embodiment, but the present invention is not limited only to following examples.
Embodiment 1
Separate and remove the method for oligomer in the TNFR-Fc fusion rotein
The first, albumin A affinity chromatography
With rProtein A Sepharose FF filler, by specification filling LC50/600 synthetic glass chromatography post, colloid amasss 500ml, and loading capacity is in 25mg/ml.Use the distilled water wash chromatography column, flow velocity is 30ml/min, and the zero(ppm) water consumption is 3 column volumes; Use concentration to be 0.1mol/L Tris-HCl damping fluid (pH is 7.0) pre-equilibration, flow velocity is 30ml/min, and the damping fluid consumption is 3 column volumes.
At first, the albumin A affinity column uses concentration to be 0.02mol/LTris damping fluid (contain concentration and be 0.2mol/L sodium-chlor, pH is 7.0) balance chromatography column, and the damping fluid consumption is 2 column volumes;
Secondly, will contain TNFR-Fc fusion rotein and content and be 75.0% supernatant, using concentration is 7.0 as 3mol/LTris solution adjusting pH; With appearance on the flow velocity of 20ml/min; All flow through affinity column until supernatant, use concentration to be 0.02mol/LTris washings (contain concentration and be 1.5mol/L urea as 0.2mol/L sodium-chlor, concentration, pH is 7.0) washing chromatography column subsequently; The washings consumption is 2 column volumes; Use concentration to be adsorbed on the TNFR-Fc fusion rotein on the chromatography column as 0.1mol/L Hydrocerol A elutriant (contain concentration and be 1.5mol/L urea, pH is 3.5) wash-out then, eluent flow rate is controlled to be 30ml/min; According to albumen absorption value under the 280nm wavelength, collect TNFR-Fc fusion rotein elutriant;
Once more, will collect TNFR-Fc fusion rotein elutriant, using concentration to regulate pH as 3.0mol/LTris solution immediately is 7.5, and uses concentration to clean chromatography column as 0.1mol/L citrate buffer solution (pH is 3.0), and the damping fluid consumption is 2 column volumes.
Contrast effect test: go up the appearance step with the above; Use concentration (to contain concentration and be 0.2mol/L sodium-chlor the albumin A affinity column of going up appearance TNFR-Fc fusion rotein as the 0.02mol/LTris washings; PH is 7.0) the washing chromatography column; The washings consumption is 2 column volumes, uses 0.1mol/L Hydrocerol A elutriant (pH is 3.5) wash-out to be adsorbed on the TNFR-Fc fusion rotein on the chromatography column then, and eluent flow rate is controlled to be 30ml/min; According to albumen absorption value under the 280nm wavelength, collect TNFR-Fc fusion rotein elutriant; To collect the TNFR-Fc fusion rotein, to use concentration to regulate pH as 3.0mol/L Tris solution immediately be 7.5, and to use concentration be 0.1mol/L citrate buffer solution (pH is 2.8) cleaning chromatography column, and the damping fluid consumption is 2 column volumes.
Table 1 provides the purity result of the collected TNFR-Fc fusion roteins in albumin A affinity chromatography front and back to compare.Through non-reduced electrophoresis SDS～PAGE, RP～HPLC check and analysis; The preceding purity of appearance is 75.0% on this TNFR-Fc fusion rotein; Oligomer content is 23.1%, and the TNFR-Fc fusion rotein purity of gained reaches 92.3% after selection use washings of the present invention and the elutriant, and oligomer content is 7.1%; The result of simultaneous test is 88.0%, and oligomer content is 11.2%.
Table 1 fusion rotein purity result relatively
| Target protein purity | Oligomer content | |
| Add urea in washings of the present invention and the elutriant | 92.3％ | 7.1％ |
| Do not add urea in simultaneous test washings and the elutriant | 88.0％ | 11.2％ |
| Before the last appearance | 75.0％ | 23.1％ |
The second, be the hydrophobic interaction chromatography of hydrophobic chromatoghaphy medium with Phenyl Sepharose 6 fast flow
As hydrophobic interaction chromatography column packing purifying target protein, by specification loads the BPG100/500 chromatography column with Phenyl Sepharose 6 fast flow hydrophobic chromatoghaphy mediums.
At first, the hydrophobic interaction chromatography post uses concentration to be 0.02mol/LTris-HCl damping fluid (contain concentration and be the 1.0mol/L sulphuric acid soln, pH is 7.5) balance chromatography column, and the damping fluid consumption is 2 column volumes;
Then, with the TNFR-Fc fusion rotein that albumin A affinity chromatography of the present invention is collected, using concentration to regulate pH as 3mol/L Tris solution is 7.5; And with concentration be that (contain concentration is 2.0mol/L ammonium sulfate to the 0.02mol/LTris-HCl damping fluid; PH is 7.5) to go up appearance hydrophobic chromatography post behind 1: 1 the ratio mixing of volume ratio, flow velocity is 50ml/min, last appearance back uses concentration (to contain concentration and be 0.8mol/L ammonium sulfate as the 0.02mol/LTris-HCl washings; PH is 7.5) the washing chromatography column; The washings consumption is 2.5 column volumes, uses concentration to carry out wash-out as 0.02mol/LTris-HCl elutriant (contain concentration and be 0.4mol/L ammonium sulfate, pH is 7.5) again; According to albumen absorption value under the 280nm wavelength, collect TNFR-Fc fusion rotein elutriant.
Through non-reduced electrophoresis SDS～PAGE, RP～HPLC check and analysis; On this TNFR-Fc fusion rotein before the appearance purity be 90.5% and the content of oligomer be 9.0%; Use that the content of oligomer is reduced to 0.6% after the hydrophobic chromatography of the present invention, the purity of fusion rotein is 99.3%.Table 2 provides the hydrophobic interaction chromatography front and back to remove the summary of oligomer situation.
Table 2 oligomer content relatively
| Fusion rotein | Target protein purity | The content of oligomer |
| Before the last appearance | 90.5％ | 9.0％ |
| Result of the present invention | 99.3％ | 0.6％ |
Embodiment 2
Separate and remove the method for oligomer in the TNFR-Fc fusion rotein
The first, albumin A affinity chromatography
This step is with the first step of embodiment 1.
The second, be the hydrophobic interaction chromatography of hydrophobic chromatoghaphy medium with Butyl Sepharose 4fast flow
Come the purifying target protein with Butyl Sepharose 4 fast flow hydrophobic chromatoghaphy mediums as the hydrophobic interaction chromatography filler, by specification filling BPG100/500 chromatography column.
At first, the hydrophobic interaction chromatography post uses concentration to be 0.02mol/LTris-HCl damping fluid (contain concentration and be 1.0mol/L sulfuric acid, pH is 7.5) balance chromatography column, and the damping fluid consumption is 2 column volumes;
Then, with the TNFR-Fc fusion rotein that albumin A affinity chromatography of the present invention is collected, using concentration to regulate pH as 3mol/L Tris solution is 7.5; And with concentration be 0.02mol/LTris-HCl damping fluid (containing concentration is 2.0mol/L ammonium sulfate, and pH is 7.5) to go up an appearance hydrophobic chromatography post behind 1: 1 the ratio mixing of volume ratio, flow velocity is 50ml/min; Last appearance back uses concentration (to contain concentration and be 0.8mol/L ammonium sulfate as the 0.02mol/LTris-HCl washings; PH is 7.5) the washing chromatography column, the washings consumption is 2.5 column volumes, uses concentration (to contain concentration and be 0.4mol/L ammonium sulfate as the 0.02mol/LTris-HCl elutriant again; PH is 7.5) carry out wash-out; According to albumen absorption value under the 280nm wavelength, collect TNFR-Fc fusion rotein elutriant, through non-reduced electrophoresis SDS～PAGE, RP～HPLC testing goal albumen.
Through non-reduced electrophoresis SDS～PAGE, RP～HPLC check and analysis; On this TNFR-Fc fusion rotein before the appearance purity be 91.5% and the content of oligomer be 7.7%; Use that the content of oligomer is reduced to 0.3% after the hydrophobic chromatography of the present invention, the purity of fusion rotein is 99.5%.Table 3 provides the hydrophobic interaction chromatography front and back to remove the summary of oligomer situation.
Table 3 oligomer content relatively
| Fusion rotein | Target protein purity | The content of oligomer |
| Before the last appearance | 91.5％ | 7.7％ |
| Result of the present invention | 99.5％ | 0.3％ |
Embodiment 3
Separate and remove the method for oligomer in the TNFR-Fc fusion rotein
The first, albumin A affinity chromatography
The dress post of affinity column, pre equilibrium process such as embodiment 1 are said.
At first, the albumin A affinity column uses concentration to be 0.1mol/LTris damping fluid (contain 0.5mol/L sodium-chlor, pH is 8.0) balance chromatography column, and the damping fluid consumption is 10 column volumes;
Secondly; To contain TNFR-Fc fusion rotein and content and be 75.4% supernatant, and use concentration to regulate pH for being 8.0, with on the flow velocity of 20ml/min kind as 3mol/LTris solution; All flow through affinity column until supernatant; Use concentration to be 0.1mol/LTris washings (contain concentration and be 3.0mol/L urea as 0.5mol/L sodium-chlor, concentration, pH is 8.0) flushing chromatography column subsequently, the damping fluid consumption is 10 column volumes; Use concentration (to contain concentration and be 2.0mol/L urea then as 0.2mol/L Hydrocerol A elutriant; PH is 3.8) be adsorbed on the TNFR-Fc fusion rotein on the chromatography column with the flow velocity wash-out of 50ml/min, according to albumen absorption value under the 280nm wavelength, collect TNFR-Fc fusion rotein elutriant;
Once more, with the TNFR-Fc fusion rotein elutriant of collecting, using concentration to regulate pH as 3mol/L Tris solution immediately is 8.0, and uses concentration to clean chromatography column as 0.1mol/L citrate buffer solution (pH is 3.0), and the damping fluid consumption is 10 column volumes.
Contrast effect test: go up the appearance step with the above; Use concentration (to contain concentration and be 0.5mol/L sodium-chlor the albumin A affinity column of going up appearance TNFR-Fc fusion rotein as the 0.1mol/LTris washings; PH is 8.0) the flushing chromatography column; The damping fluid consumption is 10 column volumes; Use then to contain concentration and be adsorbed on the TNFR-Fc fusion rotein on the chromatography column with the flow velocity wash-out of 50ml/min,, collect TNFR-Fc fusion rotein elutriant according to albumen absorption value under the 280nm wavelength as 0.2mol/L Hydrocerol A elutriant (pH is 3.8); With the TNFR-Fc fusion rotein elutriant of collecting, using concentration to regulate pH as 3mol/L Tris solution immediately is 8.0, and uses concentration to clean chromatography column as 0.1mol/L citrate buffer solution (pH is 3.0), and the damping fluid consumption is 10 column volumes.
Table 4 provides the purity result of the collected TNFR-Fc fusion roteins in albumin A affinity chromatography front and back to compare.Through non-reduced electrophoresis SDS～PAGE, RP～HPLC check and analysis; The preceding purity of appearance is 75.4% on this TNFR-Fc fusion rotein; Oligomer content is 24.0%, and the TNFR-Fc fusion rotein purity of gained reaches 91.5% after selection use washings of the present invention and the elutriant, and oligomer content is 7.9%; The result of simultaneous test is 86.0%, and oligomer content is 12.9%.
Table 4 fusion rotein purity result relatively
| Target protein purity | Oligomer content | |
| Add urea in washings of the present invention and the elutriant | 91.5％ | 7.9％ |
| Do not add urea in simultaneous test washings and the elutriant | 86.0％ | 12.9％ |
| Before the last appearance | 75.4％ | 24.0％ |
The second, be the hydrophobic interaction chromatography of hydrophobic chromatoghaphy medium with Phenyl Sepharose 6 fast flow
As the hydrophobic interaction chromatography column packing, adorn post process such as embodiment 1 with Phenyl Sepharose 6fast flow hydrophobic chromatoghaphy medium.
At first, use concentration to be 0.05mol/LTris-HCl damping fluid (contain concentration and be 1.5mol/L ammonium sulfate, pH is 8.0) balance chromatography column, the damping fluid consumption is 10 column volumes;
Then; With the TNFR-Fc fusion rotein that albumin A affinity chromatography of the present invention is collected, using concentration is 8.0 as 3mol/L Tris solution accent pH, and with concentration be that (contain concentration is 3.0mol/L ammonium sulfate to the 0.05mol/LTris-HCl damping fluid; PH is 8.0) be to go up an appearance hydrophobic chromatography post behind 1: 1 the ratio mixing with volume ratio; Flow rate control is 100ml/min, uses concentration to wash as 0.05mol/LTris-HCl damping fluid (contain concentration and be 0.9mol/L ammonium sulfate, pH is 8.0) then; The damping fluid consumption is 3 column volumes; Use concentration to be adsorbed on the TNFR-Fc fusion rotein on the hydrophobic interaction chromatography post again, according to albumen absorption value under the 280nm wavelength as 0.05mol/LTris-HCl elutriant (contain concentration and be 0.5mol/L ammonium sulfate, pH is 8.0) wash-out; Collect TNFR-Fc fusion rotein elutriant, through non-reduced electrophoresis SDS～PAGE, RP～HPLC testing goal albumen.
Through non-reduced electrophoresis SDS～PAGE, RP～HPLC check and analysis; On this TNFR-Fc fusion rotein before the appearance purity be 91.0% and the content of oligomer be 8.4%; Use that the content of oligomer is reduced to 0.6% after the hydrophobic chromatography of the present invention, the purity of fusion rotein is 99.2%.Table 5 provides the hydrophobic interaction chromatography front and back to remove the summary of oligomer situation.
Table 5 oligomer content relatively
| Fusion rotein | Target protein purity | The content of oligomer |
| Before the last appearance | 91.0％ | 8.4％ |
| Result of the present invention | 99.2％ | 0.6％ |
Embodiment 4
Separate and remove the method for oligomer in the TNFR-Fc fusion rotein
The first, albumin A affinity chromatography
This step is with the first step of embodiment 3.
The second, be the hydrophobic chromatoghaphy medium hydrophobic interaction chromatography with Butyl Sepharose 4 fast flow
As the hydrophobic interaction chromatography filler, dress post process is as embodiment 2 with Butyl Sepharose 4 fast flow hydrophobic chromatoghaphy mediums.
At first, use concentration to be 0.05mol/LTris-HCl damping fluid (contain concentration and be 1.5mol/L ammonium sulfate, pH is 8.0) balance chromatography column, the damping fluid consumption is 10 column volumes;
Then; With the TNFR-Fc fusion rotein that albumin A affinity chromatography of the present invention is collected, using concentration to transfer pH to save as 3mol/L Tris solution is 8.0, and with concentration be that (contain concentration is 3.0mol/L ammonium sulfate to the 0.05mol/LTris-HCl damping fluid; PH is 8.0) be to go up an appearance hydrophobic chromatography post behind 1: 1 the ratio mixing with volume ratio; Flow rate control is 100ml/min, uses concentration to wash as 0.05mol/LTris-HCl damping fluid (contain concentration and be 0.9mol/L ammonium sulfate, pH is 8.0) then; The damping fluid consumption is 3 column volumes; Use concentration to be adsorbed on the TNFR-Fc fusion rotein on the hydrophobic interaction chromatography post again, according to albumen absorption value under the 280nm wavelength as 0.05mol/L Tris-HCl elutriant (contain concentration and be 0.5mol/L ammonium sulfate, pH is 8.0) wash-out; Collect TNFR-Fc fusion rotein elutriant, through non-reduced electrophoresis SDS～PAGE, RP～HPLC testing goal albumen.
Through non-reduced electrophoresis SDS～PAGE, RP～HPLC check and analysis; On this TNFR-Fc fusion rotein before the appearance purity be 91.1% and the content of oligomer be 8.1%; Use that the content of oligomer is reduced to 0.4% after the hydrophobic chromatography of the present invention, the purity of fusion rotein is 99.4%.Table 6 provides the hydrophobic interaction chromatography front and back to remove the summary of oligomer situation.
Table 6 oligomer content relatively
| Fusion rotein | Target protein purity | The content of oligomer |
| Before the last appearance | 91.1％ | 8.1％ |
| Result of the present invention | 99.4％ | 0.4％ |
Embodiment 5
Separate and remove the method for oligomer in the TNFR-Fc fusion rotein
The first, albumin A affinity chromatography
The dress post of affinity column, pre equilibrium process such as embodiment 1 are said.
At first, the albumin A affinity column uses concentration to be 0.01mol/LTris damping fluid (contain concentration and be 0.1mol/L sodium-chlor, pH is 6.0) balance chromatography column, and the damping fluid consumption is 6 column volumes;
Secondly; To contain TNFR-Fc fusion rotein and content and be 76.1% supernatant, using concentration to transfer pH as 3mol/L Tris solution is 6.0, with appearance on the flow velocity of 20ml/min; All flow through affinity column until supernatant; Use concentration to be 0.01mol/LTris washings (contain concentration and be 0.5mol/L urea as 0.1mol/L sodium-chlor, concentration, pH is 6.0) flushing chromatography column subsequently, the washings consumption is 5 column volumes; Use concentration (to contain concentration and be 0.5mol/L urea then as 0.1mol/L Hydrocerol A elutriant; PH is 3.2) be adsorbed on the TNFR-Fc fusion rotein on the chromatography column with the flow velocity wash-out of 10ml/min, according to albumen absorption value under the 280nm wavelength, collect TNFR-Fc fusion rotein elutriant;
Once more, with the TNFR-Fc fusion rotein elutriant of collecting, using concentration to regulate pH as 3mol/L Tris solution immediately is 6.0, and uses concentration to clean chromatography column as 0.1mol/L citrate buffer solution (pH is 2.0), and the damping fluid consumption is 5 column volumes.
Contrast effect test: go up the appearance step with the above; Use concentration (to contain concentration and be 0.1mol/L sodium-chlor the albumin A affinity column of going up appearance TNFR-Fc fusion rotein as the 0.01mol/LTris washings; PH is 6.0) the flushing chromatography column; The washings consumption is 5 column volumes; Use concentration to be adsorbed on the TNFR-Fc fusion rotein on the chromatography column with the flow velocity wash-out of 10ml/min then,, collect TNFR-Fc fusion rotein elutriant according to albumen absorption value under the 280nm wavelength as 0.1mol/L Hydrocerol A elutriant (pH is 3.2); With the TNFR-Fc fusion rotein elutriant of collecting, using concentration to regulate pH as 3mol/L Tris solution immediately is 6.0, and uses concentration to clean chromatography column as 0.1mol/L citrate buffer solution (pH is 2.0), and the damping fluid consumption is 5 column volumes.
Table 7 provides the purity result of the collected TNFR-Fc fusion roteins in albumin A affinity chromatography front and back to compare.Through non-reduced electrophoresis SDS～PAGE, RP～HPLC check and analysis; The preceding purity of appearance is 76.1% on this TNFR-Fc fusion rotein; Oligomer content is 22.6%, and the TNFR-Fc fusion rotein purity of gained reaches 92.0% after selection use washings of the present invention and the elutriant, and oligomer content is 7.4%; The result of simultaneous test is 87.1%, and oligomer content is 11.8%.
Table 7 fusion rotein purity result relatively
| Target protein purity | Oligomer content | |
| Add urea in washings of the present invention and the elutriant | 92.0％ | 7.4％ |
| Do not add urea in simultaneous test washings and the elutriant | 87.1％ | 11.8％ |
| Before the last appearance | 76.1％ | 22.6％ |
The second, be the hydrophobic interaction chromatography of hydrophobic chromatoghaphy medium with Phenyl Sepharose 6 fast flow
As the hydrophobic interaction chromatography column packing, adorn post process such as embodiment 1 with Phenyl Sepharose 6 fast flow hydrophobic chromatoghaphy mediums.
At first, use concentration to be 0.01mol/L Tris-HCl damping fluid (contain concentration and be 0.8mol/L ammonium sulfate, pH is 6.0) balance chromatography column, the damping fluid consumption is 5 column volumes;
Then; With the TNFR-Fc fusion rotein that albumin A affinity chromatography of the present invention is collected, using concentration is 6.0 as 3mol/L Tris solution accent pH, and with concentration be that (contain concentration is 1.6mol/L ammonium sulfate to the 0.01mol/LTris-HCl damping fluid; PH is 6.0) be to go up an appearance hydrophobic chromatography post behind 1: 1 the ratio mixing with volume ratio; Flow rate control is 10ml/min, uses concentration to wash as 0.01mol/LTris-HCl damping fluid (contain concentration and be 0.7mol/L ammonium sulfate, pH is 6.0) then; The damping fluid consumption is 2.5 column volumes; Use concentration to be adsorbed on the TNFR-Fc fusion rotein on the hydrophobic interaction chromatography post again, according to albumen absorption value under the 280nm wavelength as 0.01mol/LTris-HCl elutriant (contain concentration and be 0.1mol/L ammonium sulfate, and pH is 6.0) wash-out; Collect TNFR-Fc fusion rotein elutriant, through non-reduced electrophoresis SDS～PAGE, RP～HPLC testing goal albumen.
Through non-reduced electrophoresis SDS～PAGE, RP～HPLC check and analysis; On this TNFR-Fc fusion rotein before the appearance purity be 91.7% and the content of oligomer be 7.6%; Use that the content of oligomer is reduced to 0.8% after the hydrophobic chromatography of the present invention, the purity of fusion rotein is 99.1%.Table 8 provides the hydrophobic interaction chromatography front and back to remove the summary of oligomer situation.
Table 8 oligomer content relatively
| Fusion rotein | Target protein purity | The content of oligomer |
| Before the last appearance | 91.7％ | 7.6％ |
| Result of the present invention | 99.1％ | 0.8％ |
Embodiment 6
Separate and remove the method for oligomer in the TNFR-Fc fusion rotein
The first, albumin A affinity chromatography
This step is with the first step of embodiment 5.
The second, be the hydrophobic interaction chromatography of hydrophobic chromatoghaphy medium with Butyl Sepharose 4 fast flow
As the hydrophobic interaction chromatography filler, dress post process is as embodiment 2 with Butyl Sepharose 4 fast flow hydrophobic chromatoghaphy mediums.
At first, use concentration to be 0.01mol/LTris-HCl damping fluid (contain concentration and be 0.8mol/L ammonium sulfate, pH is 6.0) balance chromatography column, the damping fluid consumption is 4 column volumes;
Then; With the TNFR-Fc fusion rotein that albumin A affinity chromatography of the present invention is collected, using concentration is 6.0 as 3mol/L Tris solution accent pH, and with concentration be that (contain concentration is 1.6mol/L ammonium sulfate to the 0.01mol/LTris-HCl damping fluid; PH is 6.0) be to go up an appearance hydrophobic chromatography post behind 1: 1 the ratio mixing with volume ratio; Flow rate control is 10ml/min, uses concentration to wash as 0.01mol/LTris-HCl damping fluid (contain concentration and be 0.7mol/L ammonium sulfate, pH is 6.0) then; The damping fluid consumption is 3 column volumes; Use concentration to be adsorbed on the TNFR-Fc fusion rotein on the hydrophobic interaction chromatography post again, according to albumen absorption value under the 280nm wavelength as 0.01mol/LTris-HCl elutriant (contain concentration and be 0.1mol/L ammonium sulfate, pH is 6.0) wash-out; Collect TNFR-Fc fusion rotein elutriant, through non-reduced electrophoresis SDS～PAGE, RP～HPLC testing goal albumen.
Through non-reduced electrophoresis SDS～PAGE, RP～HPLC check and analysis; On this TNFR-Fc fusion rotein before the appearance purity be 91.5% and the content of oligomer be 7.8%; Use that the content of oligomer is reduced to 0.5% after the hydrophobic chromatography of the present invention, the purity of fusion rotein is 99.3%.Table 9 provides the hydrophobic interaction chromatography front and back to remove the summary of oligomer situation.
Table 9 oligomer content relatively
| Fusion rotein | Target protein purity | The content of oligomer |
| Before the last appearance | 91.5％ | 7.8％ |
| Result of the present invention | 99.3％ | 0.5％ |
Claims (13)
1. method of separating and removing oligomer in the TNFR-Fc fusion rotein comprises following two steps:
The first, from the animal cell culture supernatant, reclaim the TNFR-Fc fusion rotein, collect the TNFR-Fc fusion rotein through the albumin A affinity chromatography;
The second, the TNFR-Fc fusion rotein of collecting is separated through hydrophobic interaction chromatography and the removal oligomer, it is characterized in that the method for described albumin A affinity chromatography is:
At first, use concentration be 0.01～0.1mol/L to contain concentration be 0.1～0.5mol/L sodium-chlor, and pH is 6.0～8.0 Tris damping fluid balance albumin A affinity column, the damping fluid consumption is 2～10 column volumes, until effluent lead with the balance liquid electricity and pH consistent;
Secondly, will contain the supernatant of TNFR-Fc fusion rotein, it is identical with the pH of above-mentioned Tris damping fluid to use concentration to regulate pH as 3mol/LTris solution; With appearance on the flow velocity of 20ml/min; All flow through affinity column until supernatant, the back use concentration be 0.01～0.1mol/L to contain concentration be 0.1～0.5mol/L sodium-chlor, also containing concentration simultaneously is 0.5～3.0mol/L urea; And pH is 6.0～8.0 Tris washings washing chromatography column; The washings consumption is 2～10 column volumes, use then concentration be 0.1～0.2mol/L to contain concentration be 0.5～2.0mol/L urea, and pH to be 3.2～3.8 Hydrocerol A elutriant be adsorbed on the TNFR-Fc fusion rotein on the chromatography column with the flow velocity wash-out of 10～50ml/min; According to albumen absorption value under the 280nm wavelength, collect TNFR-Fc fusion rotein elutriant;
Once more; With the TNFR-Fc fusion rotein elutriant of collecting, using concentration to regulate pH as 3mol/L Tris solution immediately is 6.0～8.0, and uses concentration to be the 0.1mol/L Hydrocerol A; And pH is 2.0～3.0 buffer solution for cleaning chromatography column, and the damping fluid consumption is 2～10 column volumes;
The method of described hydrophobic interaction chromatography is:
At first, use concentration be 0.01～0.05mol/L to contain concentration be 0.8～1.5mol/L ammonium sulfate, and pH is 6.0～8.0 Tris-HCl damping fluid balance hydrophobic interaction chromatography post, the damping fluid consumption is 2～10 column volumes;
Then; With the TNFR-Fc fusion rotein that the albumin A affinity chromatography is collected, uses concentration identical with the pH of above-mentioned Tris-HCl damping fluid as 3mol/LTris solution adjusting pH, and with concentration be that to contain concentration be 1.6～3.0mol/L ammonium sulfate to 0.01～0.05mol/L; And pH is that 6.0～8.0 Tris-HCl damping fluid is to go up an appearance hydrophobic chromatography post behind 1: 1 the ratio mixing with volume ratio; Flow rate control is 10～100ml/min, use then concentration be 0.01～0.05mol/L to contain concentration be 0.7～0.9mol/L ammonium sulfate, and pH is 6.0～8.0 Tris-HCl washings washing; The washings consumption is 2.5～3 column volumes; Use again concentration be 0.01～0.05mol/L to contain concentration be 0.1～0.5mol/L ammonium sulfate, and pH is that 6.0～8.0 Tris-HCl elutriant wash-out is adsorbed on the TNFR-Fc fusion rotein on the hydrophobic interaction chromatography post, according to albumen absorption value under the 280nm wavelength; Collect TNFR-Fc fusion rotein elutriant, obtain the TNFR-Fc fusion rotein of low oligomer content.
2. method according to claim 1, it is characterized in that the used Tris buffer concentration of described balance affinity column be 0.02mol/L to contain concentration be 0.2mol/L sodium-chlor, and pH is 7.0.
3. method according to claim 1, the washings that it is characterized in that described affinity chromatography be concentration be 0.02mol/L to contain concentration be 0.2mol/L sodium-chlor, also containing concentration simultaneously is 1.5mol/L urea, and pH is 7.0 Tris damping fluid.
4. method according to claim 1 it is characterized in that described affinity chromatography elutriant is that concentration is 1.5mol/L urea for the 0.1mol/L Hydrocerol A contains concentration, and pH is 3.5.
5. method according to claim 4 is characterized in that the used concentration of described affinity chromatography is 1.5mol/L urea for the 0.1mol/L Hydrocerol A contains concentration, and pH is that the flow velocity of 3.5 elutriant is 30ml/min.
6. method according to claim 1 is characterized in that it is 7.5 as 3mol/L Tris solution adjusting pH that TNFR-Fc fusion rotein elutriant that described albumin A affinity chromatography is collected uses concentration.
7. method according to claim 1 is characterized in that the used concentration of described cleaning affinity column is that the pH of 0.1mol/L citrate buffer solution is 2.8.
8. method according to claim 1 is characterized in that the used medium of described hydrophobic interaction chromatography is PhenylSepharose 6 fast flow or Butyl Sepharose 4 fast flow.
9. method according to claim 1, it is characterized in that the used Tris-HCl buffer concentration of described balance hydrophobic interaction chromatography post be 0.02mol/L to contain concentration be 1.0mol/L ammonium sulfate, and pH is 7.5.
10. method according to claim 1, it is characterized in that described and TNFR-Fc fusion rotein equal-volume blended Tris-HCl buffer concentration be 0.02mol/L to contain concentration be 2.0mol/L ammonium sulfate, and pH is 7.5.
11. method according to claim 1 is characterized in that the upward appearance flow velocity of described hydrophobic interaction chromatography is 50ml/min.
12. method according to claim 1, the washings that it is characterized in that described hydrophobic interaction chromatography are concentration be 0.02mol/L to contain concentration be 0.8mol/L ammonium sulfate, and pH is 7.5 Tris-HCl washings.
To contain concentration be 0.4mol/L ammonium sulfate 13. method according to claim 1, the Tris-HCl elutriant concentration that it is characterized in that described hydrophobic chromatography are 0.02mol/L, and pH is 7.5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010268403.6A CN102382190B (en) | 2010-09-01 | 2010-09-01 | Method for separating and removing oligomer in TNFR-Fc fusion protein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010268403.6A CN102382190B (en) | 2010-09-01 | 2010-09-01 | Method for separating and removing oligomer in TNFR-Fc fusion protein |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102382190A true CN102382190A (en) | 2012-03-21 |
| CN102382190B CN102382190B (en) | 2014-04-09 |
Family
ID=45822074
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201010268403.6A Active CN102382190B (en) | 2010-09-01 | 2010-09-01 | Method for separating and removing oligomer in TNFR-Fc fusion protein |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102382190B (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012176158A1 (en) * | 2011-06-24 | 2012-12-27 | Dr. Reddy's Laboratories Limited | Purification of chimeric protein |
| CN106967166A (en) * | 2017-05-11 | 2017-07-21 | 合肥知恩生物技术有限公司 | A kind of new purification process of people TNF α albumen |
| JP2018503630A (en) * | 2014-12-31 | 2018-02-08 | エルジー・ケム・リミテッド | Method for producing TNFR-Fc fusion protein containing impurities in desired content |
| CN109563125A (en) * | 2016-07-22 | 2019-04-02 | 美国安进公司 | The purification process of albumen containing Fc |
| EP2975050B1 (en) | 2014-07-18 | 2019-05-29 | Sandoz Ag | Quantification of misfolded TNFR2:Fc |
| CN111220676A (en) * | 2019-11-13 | 2020-06-02 | 上海药明生物技术有限公司 | Method for detecting purity of protein sample containing polyethylene glycol by using capillary electrophoresis technology |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1277206A (en) * | 2000-04-04 | 2000-12-20 | 中国预防医学科学院病毒学研究所 | Purification technology for hepatitis B surface gene fusion antigen containing S-main protein and pre-S1-macroprotein |
| WO2009043191A2 (en) * | 2007-10-05 | 2009-04-09 | Eldgenössische Technische Hochschule Zürich | Method for producing macro-porous materials |
| WO2009111347A1 (en) * | 2008-02-29 | 2009-09-11 | Biogen Idec Ma Inc. | Purified immunoglobulin fusion proteins and methods of their purification |
-
2010
- 2010-09-01 CN CN201010268403.6A patent/CN102382190B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1277206A (en) * | 2000-04-04 | 2000-12-20 | 中国预防医学科学院病毒学研究所 | Purification technology for hepatitis B surface gene fusion antigen containing S-main protein and pre-S1-macroprotein |
| WO2009043191A2 (en) * | 2007-10-05 | 2009-04-09 | Eldgenössische Technische Hochschule Zürich | Method for producing macro-porous materials |
| WO2009111347A1 (en) * | 2008-02-29 | 2009-09-11 | Biogen Idec Ma Inc. | Purified immunoglobulin fusion proteins and methods of their purification |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012176158A1 (en) * | 2011-06-24 | 2012-12-27 | Dr. Reddy's Laboratories Limited | Purification of chimeric protein |
| EP2975050B1 (en) | 2014-07-18 | 2019-05-29 | Sandoz Ag | Quantification of misfolded TNFR2:Fc |
| JP2018503630A (en) * | 2014-12-31 | 2018-02-08 | エルジー・ケム・リミテッド | Method for producing TNFR-Fc fusion protein containing impurities in desired content |
| US10988527B2 (en) | 2014-12-31 | 2021-04-27 | Lg Chem, Ltd. | Method for preparing TNFR-Fc fusion protein containing target content of impurities |
| CN109563125A (en) * | 2016-07-22 | 2019-04-02 | 美国安进公司 | The purification process of albumen containing Fc |
| US11312745B2 (en) | 2016-07-22 | 2022-04-26 | Amgen Inc. | Methods of purifying Fc-containing proteins |
| CN106967166A (en) * | 2017-05-11 | 2017-07-21 | 合肥知恩生物技术有限公司 | A kind of new purification process of people TNF α albumen |
| CN111220676A (en) * | 2019-11-13 | 2020-06-02 | 上海药明生物技术有限公司 | Method for detecting purity of protein sample containing polyethylene glycol by using capillary electrophoresis technology |
| CN111220676B (en) * | 2019-11-13 | 2022-11-29 | 上海药明生物技术有限公司 | Method for detecting purity of protein sample containing polyethylene glycol by using capillary electrophoresis technology |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102382190B (en) | 2014-04-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5948343B2 (en) | Method for purifying human serum albumin from seeds of transgenic rice | |
| US11590486B2 (en) | Solid phase for mixed-mode chromatographic purification of proteins | |
| US10954295B2 (en) | Correctly folded etanercept in high purity and excellent yield | |
| CN102382190B (en) | Method for separating and removing oligomer in TNFR-Fc fusion protein | |
| US10556942B2 (en) | Process for the purification of TNFR:Fc fusion protein | |
| US6627737B1 (en) | Factor IX purification methods | |
| KR20130069515A (en) | A method of antibody purification | |
| EP0467992A1 (en) | Process for purifying a protein | |
| CN105777904A (en) | Cation exchange chromatographic purification method anti-TNF alpha-type monoclonal antibody | |
| CN102911250B (en) | Method for purifying acidic recombinant protein medicament | |
| CN101260145B (en) | Technique for separating and purifying recombination human serum albumin and fusion protein thereof | |
| CN105624251A (en) | Peony seed blood sugar decreasing peptide and purification method and application thereof | |
| CN108623677A (en) | A kind of method of purification of intravenous human immunoglobulin(HIg) | |
| CN103497248B (en) | A kind of method of isolated and purified antibody from cells and supernatant | |
| CN113355309B (en) | Process for preparing recombined truncated human fibrinolysin | |
| JPH0545600B2 (en) | ||
| CN106867984A (en) | A kind of purification process of recombinant human urokinase zymogen | |
| JPS59231097A (en) | Manufacture of homogeneous human immunological interferon subtype 26k and 21k | |
| CN112898387B (en) | Purification method of recombinant staphylococcus aureus vaccine antigen protein | |
| CN102443055B (en) | A kind of purifying process of recombinant soluble human TRAIL | |
| KR20210083173A (en) | Method for Purifying Follicle stimulating hormone | |
| CN101111511A (en) | Process for protein isolation | |
| CN103102417B (en) | Purification method of recombinant human interferon alpha 2b-CTP fusion protein | |
| Katoh et al. | Purification of α-amylase by specific elution from anti-peptide antibodies | |
| CN102952836A (en) | Recombinant human long-acting interleukin-1 receptor antagonist fusion protein purification method |
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 | ||
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20161230 Address after: 276005 Hongqi Road, Shandong, Linyi, No. 209 Patentee after: Lunan Pharmaceutical Group Co., Ltd. Address before: 276005 Shandong province Feixian County North Ring Road No. 1 Patentee before: Shangdong New Time Pharmaceutical Co., Ltd. |