CN102382190B - 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
- CN102382190B CN102382190B CN201010268403.6A CN201010268403A CN102382190B CN 102382190 B CN102382190 B CN 102382190B CN 201010268403 A CN201010268403 A CN 201010268403A CN 102382190 B CN102382190 B CN 102382190B
- Authority
- CN
- China
- Prior art keywords
- concentration
- tris
- tnfr
- fusion rotein
- damping fluid
- 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
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 oligomer in separation and removal TNFR-Fc fusion rotein.
Background technology
TNFR-Fc fusion rotein (recombinant human tumor necrosis factor 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, be Etanercept, Chinese benefit match by name is general.It can stop joint deformity, in treatment, severe rheumatoid arthritis, also can treat ankylosing spondylitis and severe psoriatic simultaneously.By specifically with human body in tumour necrosis factor (TNF) combination, the activity of TNF is played to down regulation, thereby suppresses the IA inflammatory reaction of human bone.Chinese patent application Publication Specification CN1417334A discloses and has built and the recombination of preparing soluble part in tumor necrosis factor acceptor, and the technical scheme of antigen-4 fusion protein gene and product, only mention in preparation purge process and select albumin A-Sepharose to carry out affinity chromatography, can obtain more than 90% fusion rotein of purity.
Yet how separated and remove the oligomer in TNFR-Fc fusion rotein a major issue of purifying TNFR-Fc fusion rotein be.The reason part that oligomer forms is automatically to form in cell cultures secreting, expressing product, and a part is that the protein denaturation due to salt or pH induction makes solvent can approach surperficial hydrophobic region to expose and cause Protein formation aggregate.The existence of these oligomer makes patient, after using medicine, supersensitivity can occur, and causes the side reaction that human body is strong, so must remove in purge process.The method that tradition is removed oligomer is sieve chromatography (exclusion chromatography SEC), but sieve chromatography is the bottleneck of purifying, there are a lot of shortcomings: the one, sieve chromatography separating effect is not thorough, for molecular weight difference, is not king-sized albumen, and separating effect is not fine; The 2nd, sieve chromatography is compared with other chromatographic techniques, require sample height concentrated, applied sample amount can only column volume 0.5%～4% between, pillar is wanted the thin and long separating effect that just can obtain, need a large amount for the treatment of times, need very large column volume, material expensive and applied sample amount are low, are unfavorable for amplifying producing.
Chinese patent application Publication Specification CN1898266A discloses and has used hydroxylapatite to remove the technical scheme of high molecular oligomer, this scheme is a kind of method from the antibody preparation purification of at least one antibody that contains high molecular oligomer, and it comprises: the antibody that a) makes described antibody preparation contact hydroxylapatite resin and purify from resin elution with at least one elution buffer that comprises 1 to 20mM sodium phosphate and 0.2 to 2.5M sodium-chlor; Or b) make described 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 the antibody of purifying to flow through described 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 object of purifying protein.Chinese patent application Publication Specification CN1703421B discloses the technical scheme that reduces the aggregate levels of PEGization albumen, comprises the following steps: described PEGization growth hormone antagonist or isoform a) are provided; B) will comprise that the described PEGization growth hormone antagonist of any impurity and any aggregate or its isoform are to anionite-exchange resin, wherein loading is 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) by the separated described PEGization growth hormone antagonist of anion-exchange chromatography or its isoform; It further comprises combining step d) merge the described PEGization growth hormone antagonist isoform of discrete amount, to generate by being selected from following technology the PEGization growth hormone antagonist 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 forming reduced.Trisodium Citrate for hydrophobic interaction chromatography, sodium acetate reach and remove some PEGylation, not PEGization albumen and aggregate as buffering salt and by anti-phase salt gradient.But affinity interaction chromatography and hydrophobic interaction chromatography, aspect removal oligomer, due to the change of required buffer salinity or pH in protein concentration increase or elution process, can induce oligomer to form.
Summary of the invention
Can there is supersensitivity after making patient use medicine in the existence of oligomer, cause the side reaction that human body is strong, so must remove in purge process.Contriver is through repetition test for this reason, overcome above technical difficulty, after a kind of use affinity chromatography is provided, use hydrophobic chromatography, select applicable chromatographic separation condition, by the extensive method of removing of the oligomer in the animal cell culture supernatant liquor that contains TNFR-Fc fusion rotein.
For achieving the above object, the solution of the present invention comprises the following steps:
The first, from animal cell culture supernatant, by protein A affinity chromatography, reclaim TNFR-Fc fusion rotein, collect TNFR-Fc fusion rotein;
The second, the TNFR-Fc fusion rotein of collecting passed through to hydrophobic interaction chromatography separation and remove oligomer.
Purification schemes of the present invention specifically comprises the following steps:
The first, protein A affinity chromatography
First, by concentration, be that 0.01～0.1mol/LTris damping fluid (is 0.1～0.5mol/L sodium-chlor containing concentration, pH is 6.0～8.0) balance protein A affinity chromatography post, damping fluid consumption is 2～10 column volumes, until effluent liquid is led with balance liquid electricity and pH value is consistent;
Secondly, the supernatant liquor of TNFR-Fc fusion rotein will be contained, by concentration, be that 3mol/LTris solution regulates pH and identical with the pH of above-mentioned Tris damping fluid, flow velocity loading with 20ml/min, until supernatant liquor all flows through affinity column, by concentration, be that 0.01～0.1mol/LTris washings (is 0.1～0.5mol/L sodium-chlor containing concentration afterwards, also containing concentration, be 0.5～3.0mol/L urea simultaneously, pH is 6.0～8.0) flushing chromatography column, washings consumption is 2～10 column volumes, then by concentration, be that (contain concentration is 0.5～2.0mol/L urea to 0.1～0.2mol/L citric acid elutriant, pH is 3.2～3.8) with the flow velocity wash-out of 10～50ml/min, be adsorbed on the TNFR-Fc fusion rotein on chromatography column, according to albumen absorption value under 280nm wavelength, collect TNFR-Fc fusion rotein elutriant,
Again, by the TNFR-Fc fusion rotein elutriant of collecting, with concentration be 3mol/L Tris solution to be neutralized to pH be 6.0～8.0, and be that 0.1mol/L citrate buffer solution (pH is 2.0～3.0) cleans chromatography column by concentration, damping fluid consumption is 2～10 column volumes;
The second, hydrophobic interaction chromatography
First, by concentration, be 0.01～0.05mol/LTris-HCl damping fluid (be 0.8～1.5mol/L ammonium sulfate containing concentration, pH is 6.0～8.0) balance hydrophobic interaction chromatography post, damping fluid consumption is 2～10 column volumes;
Then, the TNFR-Fc fusion rotein that protein A affinity chromatography is collected, by concentration, be that 3mol/LTris solution regulates pH identical with the pH of above-mentioned Tris-HCl damping fluid, and with concentration be 0.01～0.05mol/LTris-HCl damping fluid (containing concentration be 1.6～3.0mol/L ammonium sulfate, pH is 6.0～8.0) take the ratio that volume ratio is 1: 1 and mix rear loading hydrophobic chromatography post, flow rate control is 10～100ml/min, then by concentration, be that 0.01～0.05mol/LTris-HCl washings (is 0.7～0.9mol/L ammonium sulfate containing concentration, pH is 6.0～8.0) washing, washings consumption is 2.5～3 column volumes, by concentration, be that 0.01～0.05mol/LTris-HCl elutriant (is 0.1～0.5mol/L ammonium sulfate containing concentration again, pH is 6.0～8.0) wash-out is adsorbed on the TNFR-Fc fusion rotein on hydrophobic interaction chromatography post, according to albumen absorption value under 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, above-mentioned balance affinity column Tris buffer concentration used is 0.02mol/L (be 0.2mol/L sodium-chlor containing concentration, 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 (be 0.2mol/L sodium-chlor containing concentration, be also 1.5mol/L urea containing concentration simultaneously, 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 citric acid (be 1.5mol/L urea containing concentration, pH is 3.5).
The present invention has also carried out preferably the flow velocity of affinity chromatography washings, and preferably, above-mentioned affinity chromatography concentration used is that the flow velocity of 0.1mol/L citric acid (be 1.5mol/L urea containing concentration, 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, the TNFR-Fc fusion rotein elutriant concentration that above-mentioned affinity chromatography is collected is that 3mol/L Tris solution adjusting pH is 7.5.
The present invention has also carried out preferably cleaning the damping fluid of affinity column, and preferably, above-mentioned cleaning affinity column concentration used 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, above-mentioned hydrophobic interaction chromatography medium used 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, above-mentioned balance hydrophobic interaction chromatography post Tris-HCl buffer concentration used is 0.02mol/L (be 1.0mol/L ammonium sulfate containing concentration, pH is 7.5).
The present invention has also carried out preferably the Tris-HCl damping fluid mixing with TNFR-Fc fusion rotein equal-volume, preferably, the above-mentioned Tris-HCl buffer concentration mixing with TNFR-Fc fusion rotein equal-volume is 0.02mol/L (be 2.0mol/L ammonium sulfate containing concentration, pH is 7.5).
The present invention has also carried out preferably the loading flow velocity of hydrophobic interaction chromatography, and preferably, the loading flow velocity of above-mentioned hydrophobic interaction chromatography is 50ml/min.
The present invention has 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 (be 0.8mol/L ammonium sulfate containing concentration, pH is 7.5).
The present invention has also carried out preferably hydrophobic interaction chromatography elutriant, and preferably, the Tris-HCl elutriant concentration of above-mentioned hydrophobic interaction chromatography is 0.02mol/L (be 0.4mol/L ammonium sulfate containing concentration, pH is 7.5).
The present invention compared with prior art has advantages of following outstanding:
Adopt affinity chromatography, choose rProtein A affinity gel, this affinity gel can be combined by the Fc section protein-specific in TNFR-Fc fusion rotein, reduced greatly purification step, can make the purity of the TNFR-Fc fusion rotein of this single step purification gained reach more than 90%, can remove most of impurity, for follow-up purifying work brings convenience simultaneously, by add urea in washings and elutriant, reduced the possibility of protein aggregation formation oligomer.And by further hydrophobic interaction chromatography, select ammonium sulfate as the elution process of elutriant, and low salt concn wash-out, according to the power of hydrophobic interaction, oligomer is well separated with target protein, the oligomer content of target protein is down to below 1.0%.The present invention after affinity chromatography with boiled water effect chromatography, the purity of target protein is improved greatly, reduced the content of oligomer, purification procedures is saved, cost-saving, production technology is simple, performance is good, solved shortcoming of the prior art, be applicable to the oligomer of the separated TNFR-Fc of removal fusion rotein in laboratory scale, pilot scale and industrialized production, the finished product obtaining through this inventive method reached SFDA to biological products in the requirement of oligomer content, there is higher practical value.
Embodiment
By specific embodiment, further describe the present invention below, but the present invention is not limited only to following examples.
Embodiment 1
The separated method with removing oligomer in TNFR-Fc fusion rotein
The first, protein 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 distilled water wash chromatography column, flow velocity is 30ml/min, and distilled water consumption is 3 column volumes; By concentration, be 0.1mol/L Tris-HCl damping fluid (pH is 7.0) pre-equilibration, flow velocity is 30ml/min, and damping fluid consumption is 3 column volumes.
First, protein A affinity chromatography post concentration is 0.02mol/LTris damping fluid (be 0.2mol/L sodium-chlor containing concentration, pH is 7.0) balance chromatography column, and damping fluid consumption is 2 column volumes;
Secondly, by containing TNFR-Fc fusion rotein and content, it is 75.0% supernatant liquor, by concentration, be that 3mol/LTris solution adjusting pH is 7.0, flow velocity loading with 20ml/min, until supernatant liquor all flows through affinity column, by concentration, be that 0.02mol/LTris washings (is 0.2mol/L sodium-chlor containing concentration subsequently, concentration is 1.5mol/L urea, pH is 7.0) washing chromatography column, washings consumption is 2 column volumes, then by concentration, be that 0.1mol/L citric acid elutriant (is 1.5mol/L urea containing concentration, pH is 3.5) wash-out is adsorbed on the TNFR-Fc fusion rotein on chromatography column, eluent flow rate is controlled as 30ml/min, according to albumen absorption value under 280nm wavelength, collect TNFR-Fc fusion rotein elutriant,
Again, will collect TNFR-Fc fusion rotein elutriant, be that 3.0mol/LTris solution adjusting pH is 7.5 by concentration, and be that 0.1mol/L citrate buffer solution (pH is 3.0) cleans chromatography column by concentration immediately, and damping fluid consumption is 2 column volumes.
Contrast effect test: loading step is same as above, by the protein A affinity chromatography post concentration of loading TNFR-Fc fusion rotein, be that 0.02mol/LTris washings (is 0.2mol/L sodium-chlor containing concentration, pH is 7.0) washing chromatography column, washings consumption is 2 column volumes, then use 0.1mol/L citric acid elutriant (pH is 3.5) wash-out to be adsorbed on the TNFR-Fc fusion rotein on chromatography column, eluent flow rate is controlled as 30ml/min, according to albumen absorption value under 280nm wavelength, collect TNFR-Fc fusion rotein elutriant; To collect TNFR-Fc fusion rotein is that 3.0mol/L Tris solution adjusting pH is 7.5 by concentration immediately, and is that 0.1mol/L citrate buffer solution (pH is 2.8) cleans chromatography column by concentration, and damping fluid consumption is 2 column volumes.
Table 1 provides the purity result comparison of the collected TNFR-Fc fusion roteins in protein A affinity chromatography front and back.By non-reduced electrophoresis SDS～PAGE, RP～HPLC, detect analysis, before this TNFR-Fc fusion rotein loading, purity is 75.0%, oligomer content is 23.1%, after choice for use washings of the present invention and elutriant, the TNFR-Fc fusion rotein purity of gained reaches 92.3%, oligomer content is 7.1%, the result of simultaneous test is 88.0%, and oligomer content is 11.2%.
The comparison of table 1 fusion rotein purity result
| Target protein purity | Oligomer content | |
| In washings of the present invention and elutriant, add urea | 92.3％ | 7.1％ |
| In simultaneous test washings and elutriant, do not add urea | 88.0％ | 11.2％ |
| Before loading | 75.0％ | 23.1％ |
The second, take the hydrophobic interaction chromatography that Phenyl Sepharose 6 fast flow are hydrophobic chromatoghaphy medium
With Phenyl Sepharose 6 fast flow hydrophobic chromatoghaphy mediums, as hydrophobic interaction chromatography column packing, purify target protein, by specification filling BPG100/500 chromatography column.
First, hydrophobic interaction chromatography post concentration is 0.02mol/LTris-HCl damping fluid (be 1.0mol/L sulphuric acid soln containing concentration, pH is 7.5) balance chromatography column, and damping fluid consumption is 2 column volumes;
Then, the TNFR-Fc fusion rotein that protein A affinity chromatography of the present invention is collected, by concentration, be that 3mol/L Tris solution adjusting pH is 7.5, and with concentration be 0.02mol/LTris-HCl damping fluid (containing concentration be 2.0mol/L ammonium sulfate, pH is 7.5) with the volume ratio ratio of 1: 1, mix rear loading hydrophobic chromatography post, flow velocity is 50ml/min, after loading, by concentration, be that 0.02mol/LTris-HCl washings (is 0.8mol/L ammonium sulfate containing concentration, pH is 7.5) washing chromatography column, washings consumption is 2.5 column volumes, by concentration, be that 0.02mol/LTris-HCl elutriant (is 0.4mol/L ammonium sulfate containing concentration again, pH is 7.5) carry out wash-out, according to albumen absorption value under 280nm wavelength, collect TNFR-Fc fusion rotein elutriant.
By non-reduced electrophoresis SDS～PAGE, RP～HPLC, detect analysis, before this TNFR-Fc fusion rotein loading purity be 90.5% and the content of oligomer be 9.0%, use the content of oligomer after hydrophobic chromatography of the present invention to be reduced to 0.6%, the purity of fusion rotein is 99.3%.Table 2 provides hydrophobic interaction chromatography front and back to remove the summary of oligomer situation.
The comparison of table 2 oligomer content
| Fusion rotein | Target protein purity | The content of oligomer |
| Before loading | 90.5％ | 9.0％ |
| Result of the present invention | 99.3％ | 0.6％ |
Embodiment 2
The separated method with removing oligomer in TNFR-Fc fusion rotein
The first, protein A affinity chromatography
This step is with the first step of embodiment 1.
The second, take the hydrophobic interaction chromatography that Butyl Sepharose 4fast flow is hydrophobic chromatoghaphy medium
With Butyl Sepharose 4 fast flow hydrophobic chromatoghaphy mediums, as hydrophobic interaction chromatography filler, carry out purifying target protein, by specification filling BPG100/500 chromatography column.
First, hydrophobic interaction chromatography post concentration is 0.02mol/LTris-HCl damping fluid (be 1.0mol/L sulfuric acid containing concentration, pH is 7.5) balance chromatography column, and damping fluid consumption is 2 column volumes;
Then, the TNFR-Fc fusion rotein that protein A affinity chromatography of the present invention is collected, by concentration, be that 3mol/L Tris solution adjusting pH is 7.5, and with concentration be 0.02mol/LTris-HCl damping fluid (containing concentration be 2.0mol/L ammonium sulfate, pH is 7.5) with the volume ratio ratio of 1: 1, mix rear loading hydrophobic chromatography post, flow velocity is 50ml/min, after loading, by concentration, be that 0.02mol/LTris-HCl washings (is 0.8mol/L ammonium sulfate containing concentration, pH is 7.5) washing chromatography column, washings consumption is 2.5 column volumes, by concentration, be that 0.02mol/LTris-HCl elutriant (is 0.4mol/L ammonium sulfate containing concentration again, pH is 7.5) carry out wash-out, according to albumen absorption value under 280nm wavelength, collect TNFR-Fc fusion rotein elutriant, by non-reduced electrophoresis SDS～PAGE, RP～HPLC testing goal albumen.
By non-reduced electrophoresis SDS～PAGE, RP～HPLC, detect analysis, before this TNFR-Fc fusion rotein loading purity be 91.5% and the content of oligomer be 7.7%, use the content of oligomer after hydrophobic chromatography of the present invention to be reduced to 0.3%, the purity of fusion rotein is 99.5%.Table 3 provides hydrophobic interaction chromatography front and back to remove the summary of oligomer situation.
The comparison of table 3 oligomer content
| Fusion rotein | Target protein purity | The content of oligomer |
| Before loading | 91.5％ | 7.7％ |
| Result of the present invention | 99.5％ | 0.3％ |
Embodiment 3
The separated method with removing oligomer in TNFR-Fc fusion rotein
The first, protein A affinity chromatography
The dress post of affinity column, pre equilibrium process are as described in Example 1.
First, protein A affinity chromatography post concentration is 0.1mol/LTris damping fluid (containing 0.5mol/L sodium-chlor, pH is 8.0) balance chromatography column, and damping fluid consumption is 10 column volumes;
Secondly, by containing TNFR-Fc fusion rotein and content, it is 75.4% supernatant liquor, by concentration, be that 3mol/LTris solution regulates pH for being 8.0, flow velocity loading with 20ml/min, until supernatant liquor all flows through affinity column, by concentration, be that 0.1mol/LTris washings (is 0.5mol/L sodium-chlor containing concentration subsequently, concentration is 3.0mol/L urea, pH is 8.0) flushing chromatography column, damping fluid consumption is 10 column volumes, then with there being concentration, be that 0.2mol/L citric acid elutriant (is 2.0mol/L urea containing concentration, pH is 3.8) with the flow velocity wash-out of 50ml/min, be adsorbed on the TNFR-Fc fusion rotein on chromatography column, according to albumen absorption value under 280nm wavelength, collect TNFR-Fc fusion rotein elutriant,
Again, by the TNFR-Fc fusion rotein elutriant of collecting, be 3mol/L Tris solution adjusting pH be 8.0 by concentration immediately, and be that 0.1mol/L citrate buffer solution (pH is 3.0) cleans chromatography column by concentration, damping fluid consumption is 10 column volumes.
Contrast effect test: loading step is same as above, by the protein A affinity chromatography post concentration of loading TNFR-Fc fusion rotein, be that 0.1mol/LTris washings (is 0.5mol/L sodium-chlor containing concentration, pH is 8.0) flushing chromatography column, damping fluid consumption is 10 column volumes, then with being 0.2mol/L citric acid elutriant (pH is 3.8) containing concentration, with the flow velocity wash-out of 50ml/min, be adsorbed on the TNFR-Fc fusion rotein on chromatography column, according to albumen absorption value under 280nm wavelength, collect TNFR-Fc fusion rotein elutriant; By the TNFR-Fc fusion rotein elutriant of collecting, be 3mol/L Tris solution adjusting pH be 8.0 by concentration immediately, and be that 0.1mol/L citrate buffer solution (pH is 3.0) cleans chromatography column by concentration, damping fluid consumption is 10 column volumes.
Table 4 provides the purity result comparison of the collected TNFR-Fc fusion roteins in protein A affinity chromatography front and back.By non-reduced electrophoresis SDS～PAGE, RP～HPLC, detect analysis, before this TNFR-Fc fusion rotein loading, purity is 75.4%, oligomer content is 24.0%, after choice for use washings of the present invention and elutriant, the TNFR-Fc fusion rotein purity of gained reaches 91.5%, oligomer content is 7.9%, the result of simultaneous test is 86.0%, and oligomer content is 12.9%.
The comparison of table 4 fusion rotein purity result
| Target protein purity | Oligomer content | |
| In washings of the present invention and elutriant, add urea | 91.5％ | 7.9％ |
| In simultaneous test washings and elutriant, do not add urea | 86.0％ | 12.9％ |
| Before loading | 75.4％ | 24.0％ |
The second, take the hydrophobic interaction chromatography that Phenyl Sepharose 6 fast flow are hydrophobic chromatoghaphy medium
With Phenyl Sepharose 6fast flow hydrophobic chromatoghaphy medium, as hydrophobic interaction chromatography column packing, dress post process is as embodiment 1.
First, by concentration, be 0.05mol/LTris-HCl damping fluid (be 1.5mol/L ammonium sulfate containing concentration, pH is 8.0) balance chromatography column, damping fluid consumption is 10 column volumes;
Then, the TNFR-Fc fusion rotein that protein A affinity chromatography of the present invention is collected, by concentration, be that 3mol/L Tris solution tune pH is 8.0, and with concentration be 0.05mol/LTris-HCl damping fluid (containing concentration be 3.0mol/L ammonium sulfate, pH is 8.0) take the ratio that volume ratio is 1: 1 and mix rear loading hydrophobic chromatography post, flow rate control is 100ml/min, then by concentration, be that 0.05mol/LTris-HCl damping fluid (is 0.9mol/L ammonium sulfate containing concentration, pH is 8.0) washing, damping fluid consumption is 3 column volumes, by concentration, be that 0.05mol/LTris-HCl elutriant (is 0.5mol/L ammonium sulfate containing concentration again, pH is 8.0) wash-out is adsorbed on the TNFR-Fc fusion rotein on hydrophobic interaction chromatography post, according to albumen absorption value under 280nm wavelength, collect TNFR-Fc fusion rotein elutriant, by non-reduced electrophoresis SDS～PAGE, RP～HPLC testing goal albumen.
By non-reduced electrophoresis SDS～PAGE, RP～HPLC, detect analysis, before this TNFR-Fc fusion rotein loading purity be 91.0% and the content of oligomer be 8.4%, use the content of oligomer after hydrophobic chromatography of the present invention to be reduced to 0.6%, the purity of fusion rotein is 99.2%.Table 5 provides hydrophobic interaction chromatography front and back to remove the summary of oligomer situation.
The comparison of table 5 oligomer content
| Fusion rotein | Target protein purity | The content of oligomer |
| Before loading | 91.0％ | 8.4％ |
| Result of the present invention | 99.2％ | 0.6％ |
Embodiment 4
The separated method with removing oligomer in TNFR-Fc fusion rotein
The first, protein A affinity chromatography
This step is with the first step of embodiment 3.
The second, take Butyl Sepharose 4 fast flow is hydrophobic chromatoghaphy medium hydrophobic interaction chromatography
With Butyl Sepharose 4 fast flow hydrophobic chromatoghaphy mediums, as hydrophobic interaction chromatography filler, dress post process is as embodiment 2.
First, by concentration, be 0.05mol/LTris-HCl damping fluid (be 1.5mol/L ammonium sulfate containing concentration, pH is 8.0) balance chromatography column, damping fluid consumption is 10 column volumes;
Then, the TNFR-Fc fusion rotein that protein A affinity chromatography of the present invention is collected, by concentration, be that 3mol/L Tris solution tune pH joint is 8.0, and with concentration be 0.05mol/LTris-HCl damping fluid (containing concentration be 3.0mol/L ammonium sulfate, pH is 8.0) take the ratio that volume ratio is 1: 1 and mix rear loading hydrophobic chromatography post, flow rate control is 100ml/min, then by concentration, be that 0.05mol/LTris-HCl damping fluid (is 0.9mol/L ammonium sulfate containing concentration, pH is 8.0) washing, damping fluid consumption is 3 column volumes, by concentration, be that 0.05mol/L Tris-HCl elutriant (is 0.5mol/L ammonium sulfate containing concentration again, pH is 8.0) wash-out is adsorbed on the TNFR-Fc fusion rotein on hydrophobic interaction chromatography post, according to albumen absorption value under 280nm wavelength, collect TNFR-Fc fusion rotein elutriant, by non-reduced electrophoresis SDS～PAGE, RP～HPLC testing goal albumen.
By non-reduced electrophoresis SDS～PAGE, RP～HPLC, detect analysis, before this TNFR-Fc fusion rotein loading purity be 91.1% and the content of oligomer be 8.1%, use the content of oligomer after hydrophobic chromatography of the present invention to be reduced to 0.4%, the purity of fusion rotein is 99.4%.Table 6 provides hydrophobic interaction chromatography front and back to remove the summary of oligomer situation.
The comparison of table 6 oligomer content
| Fusion rotein | Target protein purity | The content of oligomer |
| Before loading | 91.1％ | 8.1％ |
| Result of the present invention | 99.4％ | 0.4％ |
Embodiment 5
The separated method with removing oligomer in TNFR-Fc fusion rotein
The first, protein A affinity chromatography
The dress post of affinity column, pre equilibrium process are as described in Example 1.
First, protein A affinity chromatography post concentration is 0.01mol/LTris damping fluid (be 0.1mol/L sodium-chlor containing concentration, pH is 6.0) balance chromatography column, and damping fluid consumption is 6 column volumes;
Secondly, by containing TNFR-Fc fusion rotein and content, it is 76.1% supernatant liquor, by concentration, be that 3mol/L Tris solution tune pH is 6.0, flow velocity loading with 20ml/min, until supernatant liquor all flows through affinity column, by concentration, be that 0.01mol/LTris washings (is 0.1mol/L sodium-chlor containing concentration subsequently, concentration is 0.5mol/L urea, pH is 6.0) flushing chromatography column, washings consumption is 5 column volumes, then by concentration, be that 0.1mol/L citric acid elutriant (is 0.5mol/L urea containing concentration, pH is 3.2) with the flow velocity wash-out of 10ml/min, be adsorbed on the TNFR-Fc fusion rotein on chromatography column, according to albumen absorption value under 280nm wavelength, collect TNFR-Fc fusion rotein elutriant,
Again, by the TNFR-Fc fusion rotein elutriant of collecting, be 3mol/L Tris solution adjusting pH be 6.0 by concentration immediately, and be that 0.1mol/L citrate buffer solution (pH is 2.0) cleans chromatography column by concentration, damping fluid consumption is 5 column volumes.
Contrast effect test: loading step is same as above, by the protein A affinity chromatography post concentration of loading TNFR-Fc fusion rotein, be that 0.01mol/LTris washings (is 0.1mol/L sodium-chlor containing concentration, pH is 6.0) flushing chromatography column, washings consumption is 5 column volumes, then by concentration, being 0.1mol/L citric acid elutriant (pH is 3.2) is adsorbed on the TNFR-Fc fusion rotein on chromatography column with the flow velocity wash-out of 10ml/min, according to albumen absorption value under 280nm wavelength, collect TNFR-Fc fusion rotein elutriant; By the TNFR-Fc fusion rotein elutriant of collecting, be 3mol/L Tris solution adjusting pH be 6.0 by concentration immediately, and be that 0.1mol/L citrate buffer solution (pH is 2.0) cleans chromatography column by concentration, damping fluid consumption is 5 column volumes.
Table 7 provides the purity result comparison of the collected TNFR-Fc fusion roteins in protein A affinity chromatography front and back.By non-reduced electrophoresis SDS～PAGE, RP～HPLC, detect analysis, before this TNFR-Fc fusion rotein loading, purity is 76.1%, oligomer content is 22.6%, after choice for use washings of the present invention and elutriant, the TNFR-Fc fusion rotein purity of gained reaches 92.0%, oligomer content is 7.4%, the result of simultaneous test is 87.1%, and oligomer content is 11.8%.
The comparison of table 7 fusion rotein purity result
| Target protein purity | Oligomer content | |
| In washings of the present invention and elutriant, add urea | 92.0％ | 7.4％ |
| In simultaneous test washings and elutriant, do not add urea | 87.1％ | 11.8％ |
| Before loading | 76.1％ | 22.6％ |
The second, take the hydrophobic interaction chromatography that Phenyl Sepharose 6 fast flow are hydrophobic chromatoghaphy medium
With Phenyl Sepharose 6 fast flow hydrophobic chromatoghaphy mediums, as hydrophobic interaction chromatography column packing, dress post process is as embodiment 1.
First, by concentration, be 0.01mol/L Tris-HCl damping fluid (be 0.8mol/L ammonium sulfate containing concentration, pH is 6.0) balance chromatography column, damping fluid consumption is 5 column volumes;
Then, the TNFR-Fc fusion rotein that protein A affinity chromatography of the present invention is collected, by concentration, be that 3mol/L Tris solution tune pH is 6.0, and with concentration be 0.01mol/LTris-HCl damping fluid (containing concentration be 1.6mol/L ammonium sulfate, pH is 6.0) take the ratio that volume ratio is 1: 1 and mix rear loading hydrophobic chromatography post, flow rate control is 10ml/min, then by concentration, be that 0.01mol/LTris-HCl damping fluid (is 0.7mol/L ammonium sulfate containing concentration, pH is 6.0) washing, damping fluid consumption is 2.5 column volumes, by concentration, be that 0.01mol/LTris-HCl elutriant (is 0.1mol/L ammonium sulfate containing concentration again, and pH is 6.0) wash-out is adsorbed on the TNFR-Fc fusion rotein on hydrophobic interaction chromatography post, according to albumen absorption value under 280nm wavelength, collect TNFR-Fc fusion rotein elutriant, by non-reduced electrophoresis SDS～PAGE, RP～HPLC testing goal albumen.
By non-reduced electrophoresis SDS～PAGE, RP～HPLC, detect analysis, before this TNFR-Fc fusion rotein loading purity be 91.7% and the content of oligomer be 7.6%, use the content of oligomer after hydrophobic chromatography of the present invention to be reduced to 0.8%, the purity of fusion rotein is 99.1%.Table 8 provides hydrophobic interaction chromatography front and back to remove the summary of oligomer situation.
The comparison of table 8 oligomer content
| Fusion rotein | Target protein purity | The content of oligomer |
| Before loading | 91.7％ | 7.6％ |
| Result of the present invention | 99.1％ | 0.8％ |
Embodiment 6
The separated method with removing oligomer in TNFR-Fc fusion rotein
The first, protein A affinity chromatography
This step is with the first step of embodiment 5.
The second, take the hydrophobic interaction chromatography that Butyl Sepharose 4 fast flow are hydrophobic chromatoghaphy medium
With Butyl Sepharose 4 fast flow hydrophobic chromatoghaphy mediums, as hydrophobic interaction chromatography filler, dress post process is as embodiment 2.
First, by concentration, be 0.01mol/LTris-HCl damping fluid (be 0.8mol/L ammonium sulfate containing concentration, pH is 6.0) balance chromatography column, damping fluid consumption is 4 column volumes;
Then, the TNFR-Fc fusion rotein that protein A affinity chromatography of the present invention is collected, by concentration, be that 3mol/L Tris solution tune pH is 6.0, and with concentration be 0.01mol/LTris-HCl damping fluid (containing concentration be 1.6mol/L ammonium sulfate, pH is 6.0) take the ratio that volume ratio is 1: 1 and mix rear loading hydrophobic chromatography post, flow rate control is 10ml/min, then by concentration, be that 0.01mol/LTris-HCl damping fluid (is 0.7mol/L ammonium sulfate containing concentration, pH is 6.0) washing, damping fluid consumption is 3 column volumes, by concentration, be that 0.01mol/LTris-HCl elutriant (is 0.1mol/L ammonium sulfate containing concentration again, pH is 6.0) wash-out is adsorbed on the TNFR-Fc fusion rotein on hydrophobic interaction chromatography post, according to albumen absorption value under 280nm wavelength, collect TNFR-Fc fusion rotein elutriant, by non-reduced electrophoresis SDS～PAGE, RP～HPLC testing goal albumen.
By non-reduced electrophoresis SDS～PAGE, RP～HPLC, detect analysis, before this TNFR-Fc fusion rotein loading purity be 91.5% and the content of oligomer be 7.8%, use the content of oligomer after hydrophobic chromatography of the present invention to be reduced to 0.5%, the purity of fusion rotein is 99.3%.Table 9 provides hydrophobic interaction chromatography front and back to remove the summary of oligomer situation.
The comparison of table 9 oligomer content
| Fusion rotein | Target protein purity | The content of oligomer |
| Before loading | 91.5％ | 7.8％ |
| Result of the present invention | 99.3％ | 0.5％ |
Claims (11)
1. separation and a method of removing oligomer in TNFR-Fc fusion rotein, comprises following two steps:
The first, from animal cell culture supernatant, by protein A affinity chromatography, reclaim TNFR-Fc fusion rotein, collect TNFR-Fc fusion rotein;
The second, the TNFR-Fc fusion rotein of collecting passed through to hydrophobic interaction chromatography separation and remove oligomer,
It is characterized in that,
Step 1, protein A affinity chromatography:
First, by concentration, it is the Tris damping fluid balance protein A affinity chromatography post that 0.01 ~ 0.1mol/L and pH are 6.0 ~ 8.0, in described Tris damping fluid, containing concentration, be 0.1 ~ 0.5mol/L sodium-chlor, damping fluid consumption is 2 ~ 10 column volumes, until effluent liquid is led with balance liquid electricity and pH value is consistent;
Secondly, the supernatant liquor of TNFR-Fc fusion rotein will be contained, by concentration, be that 3mol/L Tris solution regulates pH identical with the pH of above-mentioned Tris damping fluid, flow velocity loading with 20ml/min, until supernatant liquor all flows through affinity column, by concentration, be the Tris washings washing chromatography column that 0.01 ~ 0.1mol/L and pH are 6.0 ~ 8.0 afterwards, in described Tris washings, containing concentration, be 0.1 ~ 0.5 mol/L sodium-chlor, also containing concentration, be 0.5 ~ 3.0mol/L urea simultaneously, washings consumption is 2 ~ 10 column volumes, then by concentration, being 0.1 ~ 0.2mol/L and the pH citric acid elutriant that is 3.2 ~ 3.8 is adsorbed on the TNFR-Fc fusion rotein on chromatography column with the flow velocity wash-out of 10 ~ 50ml/min, in described citric acid elutriant, containing concentration, be 0.5 ~ 2.0mol/L urea, according to albumen absorption value under 280nm wavelength, collect TNFR-Fc fusion rotein elutriant,
Again, by the TNFR-Fc fusion rotein elutriant of collecting, be 3mol/L Tris solution adjusting pH be 6.0 ~ 8.0 by concentration immediately, and be 0.1mol/L citric acid by concentration, and the buffer solution for cleaning chromatography column that pH is 2.0 ~ 3.0, damping fluid consumption is 2 ~ 10 column volumes;
Step 2, hydrophobic interaction chromatography:
First, by concentration, it is the Tris-HCl damping fluid balance hydrophobic interaction chromatography post that 0.01 ~ 0.05mol/L and pH are 6.0 ~ 8.0, in described Tris-HCl damping fluid, containing concentration, be 0.8 ~ 1.5mol/L ammonium sulfate, damping fluid consumption is 2 ~ 10 column volumes, and described hydrophobic interaction chromatography medium used is Phenyl Sepharose 6 fast flow or Butyl Sepharose 4 fast flow;
Then, the TNFR-Fc fusion rotein that protein A affinity chromatography is collected, by concentration, be that 3mol/L Tris solution regulates pH identical with the pH of above-mentioned Tris-HCl damping fluid, and with concentration be that Tris-HCl damping fluid that 0.01 ~ 0.05mol/L and pH are 6.0 ~ 8.0 be take the ratio that volume ratio is 1:1 and mixed rear loading hydrophobic chromatography post, in described Tris-HCl damping fluid, containing concentration, be 1.6 ~ 3.0mol/L ammonium sulfate, flow rate control is 10 ~ 100ml/min, then by concentration, be the Tris-HCl washings washing that 0.01 ~ 0.05mol/L and pH are 6.0 ~ 8.0, in described Tris-HCl washings, containing concentration, be 0.7 ~ 0.9mol/L ammonium sulfate, washings consumption is 2.5 ~ 3 column volumes, by concentration, be that the Tris-HCl elutriant wash-out that 0.01 ~ 0.05mol/L and pH are 6.0 ~ 8.0 is adsorbed on the TNFR-Fc fusion rotein on hydrophobic interaction chromatography post again, in described Tris-HCl elutriant, containing concentration, be 0.1 ~ 0.5mol/L ammonium sulfate, according to albumen absorption value under 280nm wavelength, collect TNFR-Fc fusion rotein elutriant, obtain the TNFR-Fc fusion rotein of low oligomer content.
2. method according to claim 1, is characterized in that the Tris damping fluid described in step 1 is that concentration is that 0.02mol/L, pH are 7.0, and containing concentration, is 0.2mol/L sodium-chlor in this Tris damping fluid.
3. method according to claim 1, it is characterized in that the Tris washings described in step 1 is that concentration is the Tris damping fluid that 0.02mol/L and pH are 7.0, in this Tris washings, containing concentration, be 0.2 mol/L sodium-chlor, be also 1.5mol/L urea containing concentration simultaneously.
4. method according to claim 1, is characterized in that the citric acid elutriant described in step 1 is that concentration is that 0.1 mol/L, pH are 3.5, and containing concentration, is 1.5mol/L urea in this citric acid elutriant.
5. method according to claim 1, is characterized in that the flow velocity of the citric acid elutriant described in step 1 is 30ml/min.
6. method according to claim 1, is characterized in that the TNFR-Fc fusion rotein elutriant concentration of collecting described in step 1 is that to regulate pH be 7.5 to 3mol/L Tris solution.
7. method according to claim 1, is characterized in that described in step 2 that balance hydrophobic interaction chromatography post Tris-HCl buffer concentration used is that 0.02mol/L, pH are 7.5, and containing concentration, is 1.0mol/L ammonium sulfate in this Tris-HCl damping fluid.
8. method according to claim 1, it is characterized in that the Tris-HCl buffer concentration mixing with TNFR-Fc fusion rotein equal-volume described in step 2 is that 0.02mol/L, pH are 7.5, and containing concentration, be 2.0mol/L ammonium sulfate in this Tris-HCl damping fluid.
9. method according to claim 1, is characterized in that the loading flow velocity of the hydrophobic interaction chromatography described in step 2 is 50ml/min.
10. method according to claim 1, is characterized in that the Tris-HCl washings described in step 2 is that concentration is that 0.02mol/L, pH are 7.5, and containing concentration, is 0.8mol/L ammonium sulfate in this Tris-HCl washings.
11. methods according to claim 1, is characterized in that the Tris-HCl elutriant described in step 2 is that concentration is that 0.02mol/L, pH are 7.5, and containing concentration, are 0.4mol/L ammonium sulfate in this Tris-HCl elutriant.
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 CN102382190A (en) | 2012-03-21 |
| CN102382190B true 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) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140128577A1 (en) * | 2011-06-24 | 2014-05-08 | Dr. Reddy's Laboratories Limited | Purification of chimeric protein |
| EP2975050B1 (en) | 2014-07-18 | 2019-05-29 | Sandoz Ag | Quantification of misfolded TNFR2:Fc |
| WO2016108569A1 (en) | 2014-12-31 | 2016-07-07 | 주식회사 엘지생명과학 | Method for producing tnfr-fc fusion protein containing target content of impurities |
| AU2017298984B2 (en) | 2016-07-22 | 2023-08-31 | 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 |
| 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 |
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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102382190A (en) | 2012-03-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102382190B (en) | Method for separating and removing oligomer in TNFR-Fc fusion protein | |
| JP5948343B2 (en) | Method for purifying human serum albumin from seeds of transgenic rice | |
| US5714583A (en) | Factor IX purification methods | |
| US20170152298A1 (en) | Process for the purification of tnfr:fc fusion protein | |
| KR20130069515A (en) | A method of antibody purification | |
| EP2768843B1 (en) | Solid phase for mixed-mode chromatographic purification of proteins | |
| CN106146660B (en) | Method for separating and purifying monoclonal antibody | |
| CN102911250B (en) | Method for purifying acidic recombinant protein medicament | |
| MX2012011066A (en) | A process for purifying vitamin k dependent proteins such as coagulation factor ix. | |
| CN105624251A (en) | Peony seed blood sugar decreasing peptide and purification method and application thereof | |
| CN103497248B (en) | A kind of method of isolated and purified antibody from cells and supernatant | |
| CN101260145B (en) | Technique for separating and purifying recombination human serum albumin and fusion protein thereof | |
| CN106167522A (en) | A kind of method of extensive isolated and purified teriparatide (Teriparatide) | |
| CN113355309A (en) | Process for preparing recombined truncated human fibrinolysin | |
| CN109929027B (en) | Method for purifying recombinant fusion protein by linear elution step | |
| WO2016009049A1 (en) | Methods for purifying tnfr:fc | |
| CN109336970A (en) | The method of cation exchange chromatography antibody purification | |
| CN113121637B (en) | Separation and purification method of recombinant protein | |
| CN111849945A (en) | Method for purifying human blood coagulation factor VIIa | |
| CN106867984A (en) | A kind of purification process of recombinant human urokinase zymogen | |
| US20230079633A1 (en) | Optimized method for bevacizumab purification | |
| CN105777903B (en) | A kind of cation exchange chromatography method of adalimumab | |
| CN115925890A (en) | Method for purifying anti-new coronavirus neutralizing antibody | |
| CN106349384A (en) | Method for purifying recombinant interleukin 12 | |
| KR20210083174A (en) | Method for Purifying Follicle stimulating hormone |
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. |