CN105273042A - Separation and purification method capable of separating anti-Fc antibody from anti-P75 antibody and hinge region antibodies in rabbit anti-human polyclonal antibody - Google Patents
Separation and purification method capable of separating anti-Fc antibody from anti-P75 antibody and hinge region antibodies in rabbit anti-human polyclonal antibody Download PDFInfo
- Publication number
- CN105273042A CN105273042A CN201410322503.0A CN201410322503A CN105273042A CN 105273042 A CN105273042 A CN 105273042A CN 201410322503 A CN201410322503 A CN 201410322503A CN 105273042 A CN105273042 A CN 105273042A
- Authority
- CN
- China
- Prior art keywords
- antibody
- rabbit
- human
- target protein
- protein
- 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.)
- Pending
Links
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a separation and purification method capable of separating an anti-Fc antibody from an anti-P75 antibody and hinge region antibodies in a rabbit anti-human polyclonal antibody. Specifically, the invention discloses a method for separating and purifying target protein from a mixture of target protein and non-target protein. The method is characterized in that the mixture undergoes affinity chromatography by an affinity chromatographic column filled with a filling material coupled with ligand related to the non-target protein. With the method, the anti-Fc antibody can be effectively separated from the anti-P75 antibody and the hinge region antibodies in the rabbit anti-human polyclonal antibody.
Description
Technical field
The invention belongs to separation and purification of protein field, more specifically, the present invention relates to the separation purification method of specific region polyclonal antibody.
Background technology
Affinity chromatography is a kind of adsorption chromatography, there is specific binding in antigen (or antibody) and corresponding antibody (or antigen), and this combination is reversible under certain condition, so by after antigen (or antibody) immobilization, the corresponding antibodies (or antigen) deposited in the liquid phase just can be made optionally to be combined on solid phase carrier, so as to separating with other protein in liquid phase, reach the object of separating-purifying.
Sepharose is that wetting ability is strong as its advantage of mounting medium of a kind of affinity chromatography, stable in physicochemical property, is not subject to the effect of bacterium and enzyme, has loose reticulated structure, when damping fluid ionic concn is greater than 0.05Mol/L, almost non-specific adsorption is not had to protein.
Current, preparing in recombinant human tumor necrosis factor-Fc fusion protein process, the antibody protein that the immune serum of the rabbit of recombinant human II type Tumor Necrosis Factor Receptors immunity obtains after affinity chromatography comprises the mixture of the fragment gene (as anti-P75, hinge area antibody) of recombinant human tumor necrosis factor-Fc fusion protein (hereinafter referred to as anti-FC antibody) and anti-FC antibody etc. often, how effective from many anti-isolate target antibody and be still current urgent problem.
Summary of the invention
The present invention by prepare with epoxy activation method can be separated rabbit anti-human many anti-in anti-FC antibody and anti-P75, hinge area antibody affinity chromatography filler, with the method for affinity chromatography be separated rabbit anti-human many anti-in anti-FC antibody and anti-P75, hinge area antibody, thus complete the present invention.Particularly,
A kind of method containing separation and purification target protein in the mixture of target protein and non-target protein of disclosure of the invention, it is characterized in that, described mixture carries out affinity chromatography through the affinity column of filling with non-target protein related ligand coupling filler.
Above-mentioned non-target protein related ligand includes but not limited to: antibody, antibody fusion protein, IgG, ScFv, Fab etc., preferred monoclonal antibody, is most preferably recombinate anti-HER 2 humanized monoclonal antibody for aglucon.
Above-mentioned purpose albumen includes but not limited to: antibody or antibody fusion protein, preferred antibody fusion rotein, more preferably recombinant human tumor necrosis factor-Fc fusion protein.
Preferably, above-mentioned purpose albumen obtains or recombinate to obtain from immune serum.
Invention also discloses a kind of separation purification method being separated anti-Fc antibody and anti-P75, hinge area antibody from the anti-human how anti-mixture of rabbit, it is characterized in that, the affinity column that the anti-human how anti-process of described rabbit is filled with the anti-human many anti-uncorrelated ligand cou fillers of rabbit carries out affinity chromatography, uses the citric acid wash-out anti-FC antibody main peak of pH acidity and reclaims.Preferably, the anti-human many anti-uncorrelated aglucons of described rabbit are anti-HER 2 humanized monoclonal antibodies of recombinating.More preferably, the anti-human many anti-uncorrelated ligand cou fillers of described rabbit are prepared by the following method: drain after ethanol is removed in blank glue water cleaning, continue after adding NaOH room temperature treatment 10min to add DMSO and glycerine fan room temperature epoxy activation 6h, after cleaning with straight alcohol and water the glue activated respectively, glue and the anti-human many anti-uncorrelated ligand cou 21h of rabbit are closed with thanomin again and spend the night, be kept in the ethanol of 20% after the glue finally closed with water and 20% ethanol purge.
In addition, a kind of rabbit of disclosure of the invention anti-human many anti-in anti-FC antibody consummate method, described method comprises step:
The preparation of A, filler: drain after ethanol is removed in blank glue water cleaning, continue after adding NaOH room temperature treatment 10min to add DMSO and glycerine fan room temperature epoxy activation 6h, the anti-human many anti-uncorrelated ligand cou 21h of anti-human many decorrelations aglucon, rabbit close with thanomin and spend the night with rabbit respectively after cleaning with straight alcohol and water the glue activated respectively, glue to be divided into 2 parts, are kept in the ethanol of 20% after the glue finally closed with water and 20% ethanol purge.
The separation of B, non-antibody protein: the rabbit anteserum after the immunity of recombinant human II type Tumor Necrosis Factor Receptors crosses rProteinA affinity chromatography, uses the citric acid of pH acidity carry out antibody elution albumen main peak and reclaim.
The separation of non-anti-object antibody in C, rabbit anteserum: affinity chromatography column packing is rabbit anti-human many decorrelations ligand cou filler, loading sample is the antibody protein that in above-mentioned B, wash-out reclaims, and uses the citric acid of pH acidity carry out antibody elution albumen main peak and reclaim.
Anti-FC domain antibodies and anti-P75 in D, rabbit anteserum, being separated of hinge area antibody: affinity chromatography column packing is the anti-human many anti-uncorrelated ligand cou fillers of rabbit, loading sample is the antibody protein that in above-mentioned C, wash-out reclaims, use the citric acid of pH acidity carry out antibody elution albumen main peak and reclaim, obtain anti-FC antibody.
The anti-human many decorrelations aglucon of described rabbit take recombinant human tumor necrosis factor-Fc fusion protein as aglucon, and the anti-human many anti-uncorrelated aglucons of rabbit recombinate anti-HER 2 humanized monoclonal antibody for aglucon.
By aforesaid method, can effectively target protein (as antibody fusion protein) be separated from containing the mixture of other non-target protein, especially from containing the rabbit of anti-Fc antibody and anti-P75, hinge area antibody anti-human many anti-the anti-Fc antibody of separating-purifying.
Accompanying drawing explanation
Fig. 1. the affinity chromatography collection of illustrative plates of the separation of non-antibody protein;
Fig. 2. the affinity chromatography collection of illustrative plates of the separation of non-anti-object antibody in rabbit anteserum;
Fig. 3. the affinity chromatography collection of illustrative plates be separated of anti-FC domain antibodies and anti-P75, hinge area antibody in rabbit anteserum;
Fig. 4. the electrophoretogram of embodiment is (in figure, what left figure showed is the non-reduced electrophoretogram of 8%SDS-PAGE, what right figure showed is 12%SDS-PAGE reduction electrophoretogram, M:Proteinmarker200kDa150kDa120kDa100kDa85kDa70kDa60kDa50 kDa40kDa30kDa, the rabbit anteserum of 1: sample recombinant human II type Tumor Necrosis Factor Receptors fusion protein immunization, 2: chromatography column 1 loading flows out sample, 3: chromatography column 1 main elution peak sample (chromatography column 2 loading), 4: chromatography column 2 loading flows out sample, 5: chromatography column 2 main elution peak sample (chromatography column 3 loading), 6: chromatography column 3 loading flows out sample (anti-P75 and hinge area antibody), 7: chromatography column 3 main elution peak sample (anti-FC domain antibodies), 8: commercially available recombinant human II type Tumor Necrosis Factor Receptors fusion rotein),
Fig. 5. and the elisa assay collection of illustrative plates of embodiment (in figure, RS: rabbit anti-recombinant human II type Tumor Necrosis Factor Receptors fusion rotein resists more; Sample1: anti-P75 and hinge area antibody; Sample2: anti-FC domain antibodies; Sample3: negative control).
Embodiment
Following examples are only further described the present invention, should not be construed as any limitation of the invention.
Rabbit anteserum, the recombinant human II type Tumor Necrosis Factor Receptors-antibody fusion protein of recombinant human II type Tumor Necrosis Factor Receptors immunity in example and recombinate anti-HER 2 humanized monoclonal antibody protein all from upper marine Xin Guojian Bioteknologisk Institut.
Embodiment: recombinant human II type Tumor Necrosis Factor Receptors-antibody fusion protein (hereinafter referred to as, anti-FC antibody) rabbit anti-human many anti-in anti-FC antibody and anti-P75, hinge area antibody separation and purification.
Prepared by filler: blank glue (purchased from GE company) is drained after removing ethanol with water cleaning, continue after adding NaOH room temperature treatment 10min to add dimethyl sulfoxide (DMSO) (DMSO) and glycerine fan room temperature epoxy activation 6h, after cleaning with straight alcohol and water the glue activated respectively, glue is divided into 2 parts respectively with recombinant human II type Tumor Necrosis Factor Receptors-antibody fusion protein (from upper marine Xin Guojian Bioteknologisk Institut, as follows), recombinate anti-HER 2 humanized monoclonal antibody protein (from upper marine Xin Guojian Bioteknologisk Institut, as follows) coupling 21h again with thanomin close spend the night, be kept in the ethanol of 20% after the glue finally closed with water and 20% ethanol purge.
Chromatography column 1:XK16/20, rProteinA, 1CV=20ml, H=10cm, flow=2ml/min
Chromatography column 2:XK16/20, recombinant human II type Tumor Necrosis Factor Receptors-antibody fusion protein is ligand cou, 1CV=7ml, H=3.5cm, flow=1.4ml/min
Chromatography column 3:XK16/20, anti-HER 2 humanized monoclonal antibody protein of recombinating is ligand cou, 1CV=7ml, H=3.5cm, flow=1.4ml/min
Chromatographic system: AKTA-PURIFIER
Operating system: UNICORN5.20 system
Sample: the rabbit anteserum (from upper marine Xin Guojian Bioteknologisk Institut, as follows) of recombinant human II type Tumor Necrosis Factor Receptors immunity.
1, the separation of non-antibody protein: filler is rProteinA
Solution:
A balances buffer20mMPB+150mMNacLpH7.0 ice bath
B wash-out buffer25mm citrate buffer solution pH:3 ice bath
Operating process:
Get chromatography column 1, loading is immunize rabbit serum: the mixed solution of balance buffer=1:5 mixing, pH7.0 ± 0.1.
Balance (3CV)-loading-balance (3CV)-wash-out (3CV)-balance (3CV)-purified water (3CV)-20% ethanol (3CV) CV=post bed
Collection elution peak: protein content, by the protein concentration of spectrophotometer sweep measuring component, records when receiving peak and receives peak volume, according to protein concentration and receipts peak volume computing protein content.
Elution peak: 26.6ml*6mg/ml=159.6mg
Fig. 1 is shown in by affinity chromatography collection of illustrative plates
2, the separation of non-anti-object antibody in rabbit anteserum: affinity chromatography column packing group people II type Tumor Necrosis Factor Receptors-antibody fusion protein of attaching most importance to is ligand cou.
Solution:
A balances buffer20mMPB+150mMNacLpH7.0 ice bath
B wash-out buffer25mm citrate buffer solution pH:3 ice bath
Operating process:
Get chromatography column 2, loading is the elution peak of above-mentioned steps: the mixed solution of balance buffer=1:5 mixing, pH7.0 ± 0.1.
Balance (3CV)-loading-balance (3CV)-wash-out (3CV)-balance (3CV)-purified water (3CV)-20% ethanol (3CV) CV=post bed
Collection elution peak: protein content, by the protein concentration of spectrophotometer sweep measuring component, records when receiving peak and receives peak volume, according to protein concentration and receipts peak volume computing protein content.
Elution peak: 12.5ml*0.2mg/ml=2.5mg
Fig. 2 is shown in by affinity chromatography collection of illustrative plates.
3, recombinant human II type Tumor Necrosis Factor Receptors-antibody fusion protein rabbit anti-human many anti-in anti-FC antibody and anti-P75, being separated of hinge area antibody: affinity chromatography column packing is attached most importance to, and to organize anti-HER 2 humanized monoclonal antibody protein be ligand cou.
Solution:
A balances buffer20mMPB+150mMNacLpH7.0 ice bath
B wash-out buffer25mm citrate buffer solution pH:3 ice bath
Operating process:
Get chromatography column 3, loading is that the elution peak evaporating pipe of above-mentioned steps concentrates and uses balance buffer to replace loading sample system pH7.0 ± 0.1.
Balance (3CV)-loading-balance (3CV)-wash-out (3CV)-balance (3CV)-purified water (3CV)-20% ethanol (3CV) CV=post bed
Collect outflow, elution peak: protein content, by the protein concentration of spectrophotometer sweep measuring component, flow out and record efflux volume, record receipts peak volume during receipts peak, according to protein concentration and receipts peak volume computing protein content.
Flow out: 3.2ml*0.028mg/ml=0.09mg
Elution peak: 3ml*0.165mg/ml=0.495mg
Fig. 3 is shown in by affinity chromatography collection of illustrative plates.
The experimental result of embodiment is in table 1, and its electrophoretogram is shown in Fig. 4.
Table 1. experimental result table
The ELISA of experimental result detects and analyzes:
1 reagent and instrument
1.1 reagent, to wrap by plate sample and recombinant human II type Tumor Necrosis Factor Receptors-antibody fusion protein (from upper marine Xin Guojian Bioteknologisk Institut) of having marked.
1.2 microplate reader: U.S.'s molecule instrument company SpectraMaxM5 type or other identical type microplate reader
2 experimental procedures
2.1 bags are spent the night by elisa plate 4 DEG C.
2.2 add 1%BSA closes 2h.
2.3 resist with elisa plate in conjunction with 1.5h with 1%BSA with containing the antibody through chromatography column 3 purifying in 1% human serum (HuSe) buffer dilution above-mentioned steps four as 1 respectively.
2.4 the recombinant human II type Tumor Necrosis Factor Receptors-antibody fusion protein of labelling resists with elisa plate in conjunction with 1h as 2.
2.5 add HRP-TMB colour developing.
2.6 stop reading.
3 experimental results are shown in Fig. 5, and result shows:
Reference material contrast serum-free reference material relative reactivity in 3.1 serum: 22.5%, namely rabbit anti-recombinant human II type Tumor Necrosis Factor Receptors-antibody fusion protein many anti-in containing a large amount of anti-Fc antibody (~ 80%), this part antibody capable is neutralized by IgG in human serum.
3.2 chromatography column 3 flows out Sample serum dilution contrast serum-free dilution relative reactivity: 89.7%, namely most anti-Fc antibody is eliminated, and in sample, remaining antibody about 90% is anti-P75, hinge area antibody.
3.3 chromatography column 3 elution samples serum-dilution contrast serum-frees dilution relative reactivities: 8.56%, namely about have 9% for anti-P75, hinge area antibody after chromatography column 3 purifying in sample, other about 91% is anti-FC antibody.
3.4 negative control results become negative.
Claims (10)
1. from a method for separation and purification target protein the mixture containing target protein and non-target protein, it is characterized in that, described mixture carries out affinity chromatography through the affinity column of filling with non-target protein related ligand coupling filler.
2. method according to claim 1, it is characterized in that, described non-target protein related ligand includes but not limited to: antibody, antibody fusion protein, IgG, ScFv, Fab etc.
3. method according to claim 1, it is characterized in that, described target protein includes but not limited to: antibody, antibody fusion protein.
4. method according to claim 1, it is characterized in that, described target protein is recombinant human II type Tumor Necrosis Factor Receptors-antibody fusion protein, and described non-target protein is anti-P75, hinge area antibody.
5. method according to claim 1, it is characterized in that, described non-target protein related ligand recombinates anti-HER 2 humanized monoclonal antibody for aglucon.
6. method according to claim 1, described target protein obtains or recombinate to obtain from immune serum.
7. one kind is separated the separation purification method of anti-Fc antibody and anti-P75, hinge area antibody from the anti-human how anti-mixture of rabbit, it is characterized in that, the affinity column that the anti-human how anti-process of described rabbit is filled with the anti-human many anti-uncorrelated ligand cou fillers of rabbit carries out affinity chromatography, uses the citric acid wash-out anti-FC antibody main peak of pH acidity and reclaims.
8. method according to claim 7, is characterized in that, the anti-human many anti-uncorrelated aglucons of described rabbit recombinate anti-HER 2 humanized monoclonal antibody for aglucon.
9. method according to claim 8, it is characterized in that, describedly to prepare by the following method for ligand cou filler with anti-HER 2 humanized monoclonal antibody of recombinating: drain after ethanol is removed in blank glue water cleaning, continue after adding NaOH room temperature treatment 10min to add DMSO and glycerine fan room temperature epoxy activation 6h, after cleaning with straight alcohol and water the glue activated respectively, glue and the anti-HER 2 humanized monoclonal antibody ligand cou 21h of restructuring are closed with thanomin again and spend the night, be kept in the ethanol of 20% after the glue finally closed with water and 20% ethanol purge.
10. rabbit anti-human many anti-in the consummate method of anti-FC antibody, described method comprises step:
A) preparation of filler: drain after ethanol is removed in blank glue water cleaning, continue after adding NaOH room temperature treatment 10min to add DMSO and glycerine fan room temperature epoxy activation 6h, the anti-human many anti-uncorrelated ligand cou 21h of anti-human many decorrelations aglucon, rabbit close with thanomin and spend the night with rabbit respectively after cleaning with straight alcohol and water the glue activated respectively, glue to be divided into 2 parts, are kept in the ethanol of 20% after the glue finally closed with water and 20% ethanol purge.
B) separation of non-antibody protein: the rabbit anteserum after the immunity of recombinant human II type Tumor Necrosis Factor Receptors crosses rProteinA affinity chromatography, uses the citric acid of PH acidity carry out antibody elution albumen main peak and reclaim.
C) separation of non-anti-object antibody in rabbit anteserum: affinity chromatography column packing is rabbit anti-human many decorrelations ligand cou filler, loading sample is the antibody protein that in above-mentioned B, wash-out reclaims, and uses the citric acid of pH acidity carry out antibody elution albumen main peak and reclaim.
D) anti-FC domain antibodies and anti-P75 in rabbit anteserum, being separated of hinge area antibody: affinity chromatography column packing is the anti-human many anti-uncorrelated ligand cou fillers of rabbit, loading sample is the antibody protein that in above-mentioned C, wash-out reclaims, use the citric acid of pH acidity carry out antibody elution albumen main peak and reclaim, obtain anti-FC antibody.
Described rabbit anti-human many decorrelations aglucon is with anti-FC antibody for aglucon, and the anti-human many anti-uncorrelated aglucons of described rabbit recombinate anti-HER 2 humanized monoclonal antibody for aglucon.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410322503.0A CN105273042A (en) | 2014-07-08 | 2014-07-08 | Separation and purification method capable of separating anti-Fc antibody from anti-P75 antibody and hinge region antibodies in rabbit anti-human polyclonal antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410322503.0A CN105273042A (en) | 2014-07-08 | 2014-07-08 | Separation and purification method capable of separating anti-Fc antibody from anti-P75 antibody and hinge region antibodies in rabbit anti-human polyclonal antibody |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105273042A true CN105273042A (en) | 2016-01-27 |
Family
ID=55142935
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410322503.0A Pending CN105273042A (en) | 2014-07-08 | 2014-07-08 | Separation and purification method capable of separating anti-Fc antibody from anti-P75 antibody and hinge region antibodies in rabbit anti-human polyclonal antibody |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105273042A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4472508A (en) * | 1982-12-30 | 1984-09-18 | The Beth Israel Hospital Association | Test for detecting and measuring the graves' disease-specific immunoglobulins |
| CN102675465A (en) * | 2011-03-09 | 2012-09-19 | 上海赛金生物医药有限公司 | Purification method for recombined human II-type tumor necrosis factor receptor and antibody fusion protein |
-
2014
- 2014-07-08 CN CN201410322503.0A patent/CN105273042A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4472508A (en) * | 1982-12-30 | 1984-09-18 | The Beth Israel Hospital Association | Test for detecting and measuring the graves' disease-specific immunoglobulins |
| CN102675465A (en) * | 2011-03-09 | 2012-09-19 | 上海赛金生物医药有限公司 | Purification method for recombined human II-type tumor necrosis factor receptor and antibody fusion protein |
Non-Patent Citations (3)
| Title |
|---|
| 户义等: "抗Ig 融合蛋白Fc 段单克隆抗体的制备、鉴定与应用研究", 《细胞与分子免疫学杂志》 * |
| 李湛勇等: "环氧活化法制备免疫吸附剂及其对乙型肝炎表面抗原的吸附", 《离子交换与吸附》 * |
| 黄祯祥主编: "《医学病毒学基础及实验技术》", 28 February 1990 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Roque et al. | Affinity-based methodologies and ligands for antibody purification: advances and perspectives | |
| Li et al. | Affinity monolith chromatography: A review of general principles and applications | |
| US20180215786A1 (en) | Removal of protein aggregates from biopharmaceutical preparations in a flow-through mode | |
| Delaunay et al. | Immunoaffinity solid-phase extraction for the trace-analysis of low-molecular-mass analytes in complex sample matrices | |
| EP1974215B1 (en) | Affinity chromatography matrices and methods of making and using the same | |
| Josić et al. | Application of membranes and compact, porous units for the separation of biopolymers | |
| Grodzki et al. | Antibody purification: affinity chromatography–protein A and protein G Sepharose | |
| Vergara-Barberan et al. | Current trends in affinity-based monoliths in microextraction approaches: A review | |
| KR101838039B1 (en) | Method of increasing protein purity using protein a based chromatography | |
| AU2005317279B2 (en) | Purification of immunoglobulins | |
| Fitzgerald et al. | Immunoaffinity chromatography: concepts and applications | |
| US20170369527A1 (en) | Method for affinity purification | |
| JP6457479B2 (en) | Removal of leaked affinity purified ligand | |
| Kovács et al. | Medicinal chemistry meets proteomics: fractionation of the human plasma proteome | |
| JP2015502959A (en) | Separation of IgG2 disulfide isoform | |
| Gomes et al. | Magnetic hydrophobic‐charge induction adsorbents for the recovery of immunoglobulins from antiserum feedstocks by high‐gradient magnetic fishing | |
| Hermans et al. | Purification of antibodies and antibody fragments using CaptureSelect™ affinity resins | |
| Matlschweiger et al. | Secretory immunoglobulin purification from whey by chromatographic techniques | |
| Fitzgerald et al. | Immunoaffinity chromatography | |
| Inui et al. | A scFv antibody-based immunoaffinity chromatography column for clean-up of bisphenol A-contaminated water samples | |
| CN105273042A (en) | Separation and purification method capable of separating anti-Fc antibody from anti-P75 antibody and hinge region antibodies in rabbit anti-human polyclonal antibody | |
| Pavan et al. | Phospho‐l‐tyrosine‐agarose chromatography: Adsorption of human IgG and its proteolytic fragments | |
| Pichon | Immunoaffinity extraction | |
| EP3812400A1 (en) | Affinity carrier using mutant vhh antibody | |
| Pichon et al. | Immunosorbents in sample preparation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| CB02 | Change of applicant information |
Address after: Shanghai city 201203 libing road Pudong New Area Zhangjiang hi tech Park No. 399 Applicant after: Three country Kin Pharmaceutical (Shanghai) Limited by Share Ltd Address before: Shanghai city 201203 libing road Pudong New Area Zhangjiang hi tech Park No. 399 Applicant before: Shanghai CP Guojian Pharmaceutical Co., Ltd. |
|
| COR | Change of bibliographic data | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160127 |