CN101892279A - Method for preparing medicinal recombinant human interleukin-11 - Google Patents
Method for preparing medicinal recombinant human interleukin-11 Download PDFInfo
- Publication number
- CN101892279A CN101892279A CN 201010201215 CN201010201215A CN101892279A CN 101892279 A CN101892279 A CN 101892279A CN 201010201215 CN201010201215 CN 201010201215 CN 201010201215 A CN201010201215 A CN 201010201215A CN 101892279 A CN101892279 A CN 101892279A
- Authority
- CN
- China
- Prior art keywords
- recombinant human
- human interleukin
- preparation
- medicinal recombinant
- medicinal
- 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
Images
Landscapes
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a method for preparing medicinal recombinant human interleukin-11, which adopts the following preparation steps of: 1) initial purification of a fusion protein in the interleukin-11, which comprises steps of crushing thalli and separating a chelate column; 2) digesting the fusion protein containing the interleukin-11 by enterokinase; and 3) fine purification of the interleukin-11, wherein Macro Cap SP column chromatography and Superdex-75 column chromatography are carried out to obtain the medicinal recombinant human interleukin-11. The method has the characteristics of simple process, high expression amount, high stability, uniform product, low production cost, environmental protection and high activity, improves the safety, controllability and validity of medicaments and is suitable for industrial production.
Description
Technical field
The present invention relates to a kind of preparation method of medicinal recombinant human interleukin element-11.Belong to biological technical field.
Background technology
Human interleukin-11 (IL-11) is multiple tissues such as a kind of human bone marrow of being present in, nerve, respiratory tract and digestive tube epithelium, has the cytokine of extensive physiological function, and particularly it is to the effect of hemopoietic system and make it have important clinic value.At present, recombinant human IL-11 (
Genetics Institute) ratified official listing in November, 1997 by FDA, its indication is the thrombopenia that chemotherapy causes, becomes the unique choice drug that is used for the treatment of the thrombocytopenia that chemotherapy causes at present.
IL-11 is just found in late 1980s, and it finds that the nutrient solution of derived from bone marrow stroma cell strain PU-34 can stimulate the growth of excessive IL-6 antibody neutral T1165 cell strain.Nineteen ninety, Paul SR etc. is cloned into complete people IL-11cDNA, and after this its structure and function are disclosed gradually.The gene of people IL-11 is positioned at karyomit(e) q13.3-13.4 district No. 19, and total length is 7Kb, contains 5 exons and 4 introns.People IL-11cDNA 199 the amino acid whose polypeptide of encoding, 21 aa of N end form signal peptides, and maturation protein is made up of 178 aa residues.Do not have cysteine residues in the maturation protein, thereby do not have the structure of intramolecularly and intermolecular disulfide bond, and also do not have the potential glycosylation site, simultaneously its pI value is up to 11.7, the homology from cytokine 6 no dna levels relevant with function.
IL-11 is mainly produced by the adherent cell (as marrow stromal cell, matrix inoblast, embryo lung fibroblast etc.) in mesenchyme source.IL-11 is the growth factor family member, and described family comprises tethelin, G-CSF and other somatomedins.IL-11 also is the cytokine family member, and described family comprises IL-6, leukaemia inhibitory factor (LIF), oncostatin M (OSM) and ciliary neurotrophic factor (CNTF), and they all transmit signal by coreceptor subunit gp130.Along with going deep into of research, it is found that IL-11 is a kind of multi-functional cytokine, has biologic activity widely.The main biological function of IL-11 is respectively to be the stimulation and the promoter action of cell proliferation to hemopoietic system.For example, IL-11 and cytokine (comprising IL-3, IL-4, SCF, the GM-CSF etc.) synergy that some successively play a role in the cell proliferation and differentiation different steps, the propagation of stimulation pluripotential hemopoietic stem cell, multipotency committed stem cell and monoenergetic committed stem cell; IL-11 uses separately or is collaborative with other cytokine, and all there is hormesis in erythropoietic a plurality of stages; The differentiation and maturation of medullary system progenitor cell and the growth of bone-marrow-derived lymphocyte also there is promoter action; IL-11 all has hormesis to megalokaryocyte and thrombopoietic different steps, megakaryocyte colony number and size is increased, and can improve the hematoblastic quantity of peripheral blood.In addition, in the different animals model, also observe IL-11 some non-hemopoietic tissues are also had certain biological effect, for example: in brain, spinal nerves unit, enteron aisle and testis tissue, can detect the expression of IL-11; Regulate the growth of intestinal epithelial cells; Suppressing lipid forms; Induce the synthetic of acute phase reactive protein; Stimulate the growth of osteoclast and the propagation of neural progenitor cell etc.
Thrombocyte is extremely important for the effect in the formation of keeping hemostasis and initial blood clot in the injury.In the patient of thrombocytopenia, can't form blood clot is the most direct consequence, and serious thrombocytopenia causes the hemorrhage of typical module, and severe gi tract and central nervous system are hemorrhage may to be fatal.Caused to form distribute unusual, thrombocyte of thrombocyte, thrombocyte if a certain step in the thrombocyte formation process disturbs and damage and increase and/or loss increases, will show as thrombocytopenia.The factor that causes thrombocytopenia can be divided into geneogenous and acquired, for example: congenital no hyperplasia megakaryocytic; Cytotoxic chemotherapy etc.Thrombocytopenia is the potential mortality complication of tumor chemoradiotherapy method
In the biologic activity of IL-11, the most outstanding and hematoblastic quantity of dose-dependently rising peripheral blood has importantly been pointed out the important clinic value of IL-11, promptly may be a kind of medicine for the treatment of thrombocytopenia.After IL-11 is found, U.S. Genetics Institute has just begun the R﹠D work of recombinant human IL-11 (rhIL-11), through the clinical preceding and clinical study more than 6 years, obtained FDA approval listing in 1997, commodity are called Neumega, be used for the treatment of the thrombocytopenia that tumor chemoradiotherapy causes, Blood substitute platelet infusion.
As one of ordinary skill understood, many both at home and abroad at present employing intestinal bacteria (as Chinese patent 99112322.0,99125600.X) and yeast (as Chinese patent 200710162970.1) expression system are produced the IL-11 product.With regard to expression system itself, intestinal bacteria and yeast expression system respectively have its relative merits.Yet, with regard to the people IL-11 of no intramolecularly/intermolecular disulfide bond, no potential glycosylation site, glycosylation can appear during with yeast expression, degree of glycosylation is wayward, simultaneously, and with respect to escherichia expression system, the fermentation period of yeast expression system is long, technology difficulty is big, the cost height, and technology stability is poor; Use escherichia coli expression, the expression amount height is beneficial to purifying.Escherichia expression system is because of its expression amount height, and technology is simple, and is with low cost, so adopt escherichia expression system expressing human IL-11 at present both at home and abroad mostly.
Summary of the invention
In order to address the above problem, the object of the present invention is to provide a kind of preparation method of medicinal recombinant human interleukin element-11, it is simple that the present invention has technology, expression amount height, good stability, the product homogeneous, production cost is low, environmental protection, active high characteristics, and improve security, controllability and the validity of medicine, be applicable to suitability for industrialized production.
For reaching above-mentioned purpose, the present invention adopts following technical scheme:
A kind of preparation method of medicinal recombinant human interleukin element-11, adopt following preparation process:
1) to the preliminary purification of fusion rotein in the interleukin 11 (rhIL-11), comprises bacterial cell disruption and huge legendary turtle zygostyle separating step;
2) enteropeptidase (EK enzyme) enzyme that contains the fusion rotein of interleukin 11 (rhIL-11) is cut;
3) polishing purification of interleukin 11 (rhIL-11) comprises MacroCap SP column chromatography and S-75 column chromatography, obtains medicinal recombinant human interleukin element-11.
The elution requirement of huge legendary turtle zygostyle separating step adopts 50mMTris-Cl, the imidazoles of 285-325mM, 1% tween 80 gradient elution in the described step 1).
Described step 2) the enteropeptidase prescription is as follows in: 50mMTris-Cl, pH8.0+500mMNaCl, volume percent content are 50% glycerine.
Described step 2) enzyme is cut the buffer system of the pH value of employing for 6-10 in, contains ethanol or DMSO organic solvent in the system, and contains metal ion as activator.
Described pH value is preferably 7-9.
Described pH value more preferably 8.0.
Described metal ion adopts K+ or Ca+.
Described K+ concentration is: 5-8mM; Ca+ concentration is: 1-2mM.
Described alcoholic acid volume percent content is 5-20%, and the volume percent content of DMSO is 1%-15%.
Described ethanol content is preferably 5-8%, and DMSO content is preferably 1-5%.
The invention has the beneficial effects as follows: the present invention has adopted advanced huge legendary turtle zygostyle elution requirement, and the single step purification after the fragmentation of fusion rotein high pressure behind the mistake metal chelating column just can obtain the purity about 90%.Also have the present invention to adopt homemade highly purified recombinant enterokinase, enteropeptidase is quality controllable, no thermal source and animal source protein contamination, good reproducibility between batch.And the present invention has found the enzyme tangent condition of optimizing, and when pH8.0, contains K2+ at 5-50mM or Ca2+1-2mM, and under ethanol 5-8% or the DMSO 1-5% condition IL-11 fusion protease being cut yield can reach more than 80%.So N end and the C end of the IL-11 that produces by route of the present invention are complete, the product homogeneous is active high, and environmental protection is for the security and the validity of the use of product provides safeguard.
Description of drawings
Fig. 1 is that recombinant human IL-11 metal ion of the present invention is cut fusion protease and influenced the SDS-PAGE collection of illustrative plates;
Fig. 2 is that recombinant human IL-11 organic solvent of the present invention is cut fusion protease and influenced the SDS-PAGE collection of illustrative plates;
Fig. 3 is the non-reduced SDS-PAGE collection of illustrative plates of recombinant human IL-11 stoste of the present invention;
Fig. 4 is a recombinant human IL-11 stoste reduction SDS-PAGE collection of illustrative plates of the present invention;
Fig. 5 is that not the present invention recombinant human IL-11 product purity detects (SEC-HPLC) collection of illustrative plates.
Embodiment
Below by specific embodiment, the invention will be further described.
1) preliminary purification of rhIL-11
A. bacterial cell disruption: thalline is used 50mM Tris+500mMNaCl in 1: 10 (w/v) ratio, and the pH8.0 damping fluid is resuspended.Can adopt ultrasonic disruption when a small amount of, adopt the high pressure fragmentation when a large amount of.Adopt 100-900Psi pressure the thalline that is suspended in broken liquid to be carried out the comparison of centrifuged supernatant.The result shows: 200Psi has higher yield and higher purity.
B. the huge legendary turtle zygostyle separates: according to the characteristics that have the MB residue in the fusion rotein, selected the filler of Chelating Sepharose FF effect preliminary purification for use.Adopt the imidazoles flush away foreign protein of 100mM, the target protein elution requirement adopts 50mMTris-Cl, the imidazoles of 285-325mM, 1% tween 80 gradient elution, purification result shows, this step purifying single step purification just can make the proteic purity of Target Fusion up to more than 90%, has reached to can be used for the purpose requirement that enzyme is cut.Simultaneously, a large amount of inhibitions have also been removed to the EK enzyme.
2) the enteropeptidase enzyme that contains the fusion rotein of rhIL-11 is cut
EK enzyme enzyme is cut: adopt highly purified reorganization EK enzyme, the EK enzyme following 50mMTris-Cl that fills a prescription, pH8.0+500mMNaCl, 50% (V/V) glycerine.The enzyme tangent condition is studied, adopted 20 ℃ temperature to prevent that fusion rotein from precipitating.
A. grope by the pH gradient condition that the EK enzyme is cut, under 20 ℃ of conditions, adopt the Tris-Cl of 50mM, the enzyme system of cutting of pH5-9 is carried out enzyme and is cut test, and the result shows, it is more satisfactory to cut effect at the scope endoenzyme of pH7-9, and it is 8.0 system that enzyme is cut optimal pH.
B. investigated the influence test that each metal ion species is cut enzyme under this buffer system, wherein K2+, Ca2+ have promoter action to enzymic activity, and Zn2+ is inhibited.And investigated K2+ in the 5-200mM scope, Ca2+ in the 1-30mM scope concentration gradient to the influence of enzymic activity, K2+ is at 5-50mM, the Ca2+1-2mM enzymic activity is best, then K2+, Ca2+ is cut influence to fusion protease and is SDS-PAGE, as shown in Figure 1, Fig. 1 is that present embodiment recombinant human IL-11 metal ion is cut fusion protease and influenced the SDS-PAGE collection of illustrative plates, wherein Lane1-5:K+ concentration is 5mM, 50mM, 100mM, 150mM, 200mM; Lane6-10Ca2+ concentration is 1mM, 2mM, 10mM, 20mM, 30mM; Lane11 control (enzyme is not cut contrast)
C. investigated the influence that organic solvent is cut the EK enzyme, to methyl alcohol, ethanol, DMSO, n-propyl alcohol, acetonitrile, acetone is to the influence of enzymic activity.Wherein ethanol and DMSO have beyond thought promoter action to enzyme activity, and methyl alcohol is to the not influence of the enzyme activity of BEK, and n-propyl alcohol, acetonitrile, acetone have certain restraining effect to BEK.Then ethanol and DMSO are cut influence to fusion protease and be SDS-PAGE, as shown in Figure 2, Fig. 2 is that present embodiment recombinant human IL-11 organic solvent is cut fusion protease and influenced the SDS-PAGE collection of illustrative plates, Lane1 control (enzyme is not cut contrast) wherein, the Lane2-6 alcohol concn is 5%, 8%, 10%, 15%, 20%Lane7-11DMSO concentration is 1%, 5%, 8%, 20%, 15%, as seen from the figure, ethanol is in the 5-20% scope, interior enzyme is cut of DMSO1%-15% scope all has promoter action, and wherein with ethanol 5-8%, DMSO has beyond thought effect at 1-5%.
Present embodiment adopts under 20 ℃ of conditions, and the Tris-Cl of 50mM carries out enzyme and cuts step under the condition of pH8, and activator adopts 5-8mM K2+ or 1-2mM Ca2+, and organic solvent adopts 5-8% ethanol, 1-5%DMSO.
3): the polishing purification of rhIL-11 (MacroCap SP and S-75 column chromatography)
Utilize rhIL-11 that characteristics than higher isoelectric point are arranged.Descended MacroCap SP post in the PH8.0 condition, adopt 50mM PBNa, pH8.0 conditional equilibrium MacroCap SP post, last sample, elution requirement adopts 450mM PBNa, the high salt condition wash-out target protein of pH8.0, this step has been removed most foreign proteins, and make one step of rh I L-11 obtain efficiently purifying, adopt 10mM PBNa, sample and wash-out collection target protein can make the purity of purified product-medicinal recombinant human interleukin element-11 reach 99% on the pH8.0 equilibrated Superdex-75 post.As Fig. 3,4, shown in 5, Fig. 3 is the proteic non-reduced type SDS-PAGE electrophorogram of the rhIL-11 of purifying, last sample 20ug albumen, compare with 100ng (0.5%) with last sample control band 400ng (2), 200ng (1%), no sightless impurity greater than 200ng (1%) illustrates that electrophoresis purity is greater than 99%; Fig. 4 is the reduced form SDS-PAGE electrophorogram of purifying protein, and last sample 20ug albumen is compared with 100ng (0.5%) with last sample control band 400ng (2), 200ng (1%), and no sightless impurity greater than 200ng (1%) illustrates that electrophoresis purity is greater than 99%; Fig. 5 is the proteic high performance liquid phase purity of the rhIL-11 of a purifying collection of illustrative plates, the integration statistics sees Table 1, in the table 1 statistical conditions at 4 peaks of high performance liquid phase purity collection of illustrative plates appearance, wherein RT is the retention time that the peak, Area is an integral area, %Area is the per-cent that accounts for the total mark area at each peak, Height is the height at each peak, as can be seen from Table 1, table 1 is the integration statistical conditions of HPLC collection of illustrative plates, retention time is that the rhIL-11 purpose peak that 13.17min occurs accounts for 99.33% of total liquid phase integral area, and the HPLC purity that rhIL-11 is described is greater than 99%.
Table 1
| RT | Area | ％ | Height | ||
| 1 | 10.915 | 25250 | 0.13 | 757 | |
| 2 | 12.233 | 60501 | 0.31 | 3447 | |
| 3 | 13.170 | 194533409 | 99.33 | 594591 | |
| 4 | 14.250 | 46293 | 0.24 | 3517 |
Claims (10)
1. the preparation method of a medicinal recombinant human interleukin element-11 is characterized in that: adopt following preparation process:
1), comprises bacterial cell disruption and huge legendary turtle zygostyle separating step to the preliminary purification of fusion rotein in the interleukin 11;
2) the enteropeptidase enzyme that contains the fusion rotein of interleukin 11 is cut;
3) polishing purification of interleukin 11 comprises MacroCap SP column chromatography and S-75 column chromatography, obtains medicinal recombinant human interleukin element-11.
2. the preparation method of a kind of medicinal recombinant human interleukin element-11 as claimed in claim 1 is characterized in that: the elution requirement of huge legendary turtle zygostyle separating step adopts 50mMTris-Cl, the imidazoles of 285-325mM, 1% tween 80 gradient elution described step 2).
3. the preparation method of a kind of medicinal recombinant human interleukin element-11 as claimed in claim 1 is characterized in that: the enteropeptidase prescription is as follows in the described step 3): 50mMTris-Cl, pH8.0+500mMNaCl, the glycerine of volume percent content 50%.
4. the preparation method of a kind of medicinal recombinant human interleukin element-11 as claimed in claim 1, it is characterized in that: enzyme is cut and is adopted the buffer system of pH value for 6-10 in the described step 3), contain ethanol or DMSO organic solvent in the system, and contain metal ion as activator.
5. the preparation method of a kind of medicinal recombinant human interleukin element-11 as claimed in claim 4 is characterized in that: described pH value is 7-9.
6. the preparation method of a kind of medicinal recombinant human interleukin element-11 as claimed in claim 5 is characterized in that: described pH value is 8.0.
7. the preparation method of a kind of medicinal recombinant human interleukin element-11 as claimed in claim 4 is characterized in that: described metal ion adopts K+ or Ca+.
8. the preparation method of a kind of medicinal recombinant human interleukin element-11 as claimed in claim 7 is characterized in that: described K+ concentration is: 5-8mM; Ca+ concentration is: 1-2mM.
9. the preparation method of a kind of medicinal recombinant human interleukin element-11 as claimed in claim 4 is characterized in that: described alcoholic acid volume percent content is 5-20%, and the volume percent content of DMSO is 1%-15%.
10. the preparation method of a kind of medicinal recombinant human interleukin element-11 as claimed in claim 9 is characterized in that: described alcoholic acid volume percent content is 5-8%, and the volume percent content of DMSO is 1-5%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010201215 CN101892279A (en) | 2010-06-10 | 2010-06-10 | Method for preparing medicinal recombinant human interleukin-11 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010201215 CN101892279A (en) | 2010-06-10 | 2010-06-10 | Method for preparing medicinal recombinant human interleukin-11 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101892279A true CN101892279A (en) | 2010-11-24 |
Family
ID=43101624
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201010201215 Pending CN101892279A (en) | 2010-06-10 | 2010-06-10 | Method for preparing medicinal recombinant human interleukin-11 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101892279A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102644983A (en) * | 2012-05-18 | 2012-08-22 | 苏州市时代工程咨询设计管理有限公司 | Ground-source heat pump system |
| WO2024092879A1 (en) * | 2022-10-31 | 2024-05-10 | 杭州先为达生物科技股份有限公司 | High-specificity enterokinase digestion method and use of ethanol in improving enterokinase digestion specificity |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1670034A (en) * | 2004-11-25 | 2005-09-21 | 杭州九源基因工程有限公司 | Method for producing Pichia anomala expression recombination human interleukin 11 |
-
2010
- 2010-06-10 CN CN 201010201215 patent/CN101892279A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1670034A (en) * | 2004-11-25 | 2005-09-21 | 杭州九源基因工程有限公司 | Method for producing Pichia anomala expression recombination human interleukin 11 |
Non-Patent Citations (2)
| Title |
|---|
| 《中国实验血液学杂志》 20081231 赵阳等 利用肠激酶加工融合蛋白的方法制备rhIL-11 903-909 2-10 第16卷, 第4期 2 * |
| 《中国生物工程杂志》 20091231 李智华等 人白细胞介素11的定点聚乙二醇修饰 20-24 1-10 第29卷, 第6期 2 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102644983A (en) * | 2012-05-18 | 2012-08-22 | 苏州市时代工程咨询设计管理有限公司 | Ground-source heat pump system |
| WO2024092879A1 (en) * | 2022-10-31 | 2024-05-10 | 杭州先为达生物科技股份有限公司 | High-specificity enterokinase digestion method and use of ethanol in improving enterokinase digestion specificity |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2022100741A1 (en) | Fusion protein of human serum albumin and interleukin-2, and use thereof | |
| CN114671941B (en) | Nerve growth factor mutant | |
| JP2002514887A (en) | Method of secreting thrombopoietin polypeptide | |
| CN108314722B (en) | Antibacterial peptide and application thereof | |
| CN116535486A (en) | Keratin YK93-1, preparation method, pharmaceutical composition and application thereof | |
| CN107286233B (en) | Low-pain nerve growth factor mutant | |
| CN110128544B (en) | Hybrid peptide with immunoregulation and anti-inflammatory functions and preparation method and application thereof | |
| US20150337025A1 (en) | Use of fusion protein | |
| CN101892279A (en) | Method for preparing medicinal recombinant human interleukin-11 | |
| US5437988A (en) | Expression and secretion of mature human beta interleukin-1 in Bacillus subtilis and means and methods for its achievement | |
| KR20120069221A (en) | Bee venom composition | |
| US4879374A (en) | Bovine interleukin-1β DNA sequence | |
| CN102020710B (en) | Novel mutant EN-46 of human epidermal growth factor | |
| WO2004020576A2 (en) | Glycosylated human granulocyte colony-stimulating factor (g-csf) isoform | |
| EP4026555A1 (en) | Human recombinant hyposialylated erythropoietin, methods of purification and therapeutic uses thereof | |
| CN1170927C (en) | Method for producing medicine using silkworm expressed numan epidermal growth factor | |
| KR20240047459A (en) | Persistent recombinant human interleukin-2 fusion protein and method and application thereof | |
| KR101626291B1 (en) | Composition comprising hydrolysate of whey protein using alkaline protease for preventing or treating inflammatory diseases | |
| KR20180046021A (en) | Composition including neoagarooligosaccharide for preventing, improving or treating inflammatory disease | |
| CN110577587B (en) | Isolated plant defensin polypeptide and preparation method and application thereof | |
| CA2410702A1 (en) | Production of human granulocyte macrophage-colony stimulating factor (gm-csf) in the seeds of transgenic rice plants | |
| WO2024074111A1 (en) | Keratin yk93-9, preparation method therefor, pharmaceutical composition thereof, and use thereof | |
| EP0500589B1 (en) | Purified protein and bioactive pharmaceutical | |
| JP2018527025A (en) | Enzyme fraction with anti-inflammatory activity | |
| CN114891122A (en) | Cow antibacterial polypeptide synthesized by primary cow mammary epithelial cell bioreactor and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20101124 |