BRPI1010035A2 - Células deficientes em fucosilação - Google Patents
Células deficientes em fucosilaçãoInfo
- Publication number
- BRPI1010035A2 BRPI1010035A2 BRPI1010035-0A BRPI1010035A BRPI1010035A2 BR PI1010035 A2 BRPI1010035 A2 BR PI1010035A2 BR PI1010035 A BRPI1010035 A BR PI1010035A BR PI1010035 A2 BRPI1010035 A2 BR PI1010035A2
- Authority
- BR
- Brazil
- Prior art keywords
- fucosylation
- deficient cells
- deficient
- cells
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/005—Glycopeptides, glycoproteins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2863—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2866—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01271—GDP-L-fucose synthase (1.1.1.271)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
- C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
- C07K2317/41—Glycosylation, sialylation, or fucosylation
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Enzymes And Modification Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US18340009P | 2009-06-02 | 2009-06-02 | |
| US34885810P | 2010-05-27 | 2010-05-27 | |
| PCT/US2010/036914 WO2010141478A1 (en) | 2009-06-02 | 2010-06-01 | Fucosylation-deficient cells |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| BRPI1010035A2 true BRPI1010035A2 (pt) | 2015-08-25 |
Family
ID=42338231
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| BRPI1010035-0A BRPI1010035A2 (pt) | 2009-06-02 | 2010-06-01 | Células deficientes em fucosilação |
Country Status (16)
| Country | Link |
|---|---|
| US (8) | US8409838B2 (enExample) |
| EP (3) | EP2438171B1 (enExample) |
| JP (4) | JP5744856B2 (enExample) |
| KR (2) | KR101441437B1 (enExample) |
| CN (1) | CN102459603B (enExample) |
| AU (1) | AU2010256753B2 (enExample) |
| BR (1) | BRPI1010035A2 (enExample) |
| CA (1) | CA2764370C (enExample) |
| DK (3) | DK3279326T3 (enExample) |
| ES (2) | ES2527173T3 (enExample) |
| HU (1) | HUE038596T2 (enExample) |
| IL (1) | IL216624A (enExample) |
| PL (3) | PL2438171T3 (enExample) |
| SG (1) | SG176251A1 (enExample) |
| TW (1) | TWI513818B (enExample) |
| WO (1) | WO2010141478A1 (enExample) |
Families Citing this family (33)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140161782A1 (en) | 2008-06-09 | 2014-06-12 | Targazyme, Inc. | Augmentation of cell therapy efficacy including treatment with alpha 1-3 fucoslytransferase |
| BRPI1010035A2 (pt) | 2009-06-02 | 2015-08-25 | Regeneron Pharma | Células deficientes em fucosilação |
| WO2012120500A2 (en) | 2011-03-06 | 2012-09-13 | Merck Serono S.A. | Low fucose cell lines and uses thereof |
| WO2013013013A2 (en) * | 2011-07-21 | 2013-01-24 | Alnylam Pharmaceuticals, Inc. | Compositions and methods for producing modified glycoproteins |
| TWI641687B (zh) | 2012-05-29 | 2018-11-21 | 美商再生元醫藥公司 | 生產細胞株增強子 |
| EP2722673B1 (en) | 2012-10-17 | 2017-07-12 | Hexal AG | Improved method of mapping glycans of glycoproteins |
| AR095196A1 (es) | 2013-03-15 | 2015-09-30 | Regeneron Pharma | Medio de cultivo celular libre de suero |
| WO2015033343A1 (en) | 2013-09-03 | 2015-03-12 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. | Compositions and methods for expressing recombinant polypeptides |
| WO2015057393A1 (en) * | 2013-10-01 | 2015-04-23 | University Of Wyoming | Compositions and methods for reducing fucosylation of glycoproteins in insect cells and methods of use thereof for production recombinant glycoproteins |
| RS59077B1 (sr) | 2014-03-11 | 2019-09-30 | Regeneron Pharma | Anti-egfrviii antitela i njihova primena |
| EP3149034B1 (en) * | 2014-05-30 | 2022-07-13 | New England Biolabs, Inc. | Deglycosylation reagents and methods |
| CN106687581A (zh) * | 2014-07-07 | 2017-05-17 | 塔尔加泽梅股份有限公司 | 用于治疗用途的岩藻糖基化细胞的制备和冷冻干燥 |
| CN105671109B (zh) * | 2014-11-20 | 2019-08-23 | 中国人民解放军军事医学科学院生物工程研究所 | 用糖基工程酵母制备具有动物细胞糖基化修饰流感血凝素糖蛋白的方法 |
| TWI710573B (zh) | 2015-01-26 | 2020-11-21 | 美商再生元醫藥公司 | 抗伊波拉病毒醣蛋白之人類抗體 |
| TWI899515B (zh) | 2015-08-04 | 2025-10-01 | 美商再生元醫藥公司 | 補充牛磺酸之細胞培養基及用法 |
| CN108431026B (zh) * | 2015-11-02 | 2023-02-17 | 豪夫迈·罗氏有限公司 | 制备岩藻糖基化和去岩藻糖基化形式的蛋白质的方法 |
| EP3400241B1 (en) | 2016-01-06 | 2020-07-22 | Outlook Therapeutics, Inc. | Modulation of afucosylated species in a monoclonal antibody composition |
| EP3411401A1 (en) | 2016-02-03 | 2018-12-12 | Oncobiologics, Inc. | Buffer formulations for enhanced antibody stability |
| SG11201807881VA (en) | 2016-04-20 | 2018-10-30 | Regeneron Pharma | Compositions and methods for making antibodies based on use of an expression-enhancing locus |
| KR102906454B1 (ko) * | 2016-04-20 | 2026-01-02 | 리제너론 파마슈티칼스 인코포레이티드 | 발현 강화 유전자좌의 사용에 기초하여 항체를 만들기 위한 조성물 및 방법 |
| EP3339444A1 (en) * | 2016-12-20 | 2018-06-27 | Mabxience Research, S.L. | Method for obtaining a glycoprotein with an increased percentage of afucosylated glycans |
| KR20200135781A (ko) | 2018-03-26 | 2020-12-03 | 암젠 인크 | 세포 배양에서 생산된 항체의 총 비푸코실화 당형태 |
| KR102596303B1 (ko) * | 2018-08-29 | 2023-10-30 | 유나이티드 바이오파마, 인크. | 비푸코실화된 항체 및 그의 제조법 |
| CA3118073A1 (en) | 2018-10-29 | 2020-05-07 | Immuno-Biological Laboratories Co., Ltd. | Anti-hiv antibody and method for producing same |
| US12297451B1 (en) | 2019-10-25 | 2025-05-13 | Regeneron Pharmaceuticals, Inc. | Cell culture medium |
| WO2021242815A1 (en) | 2020-05-26 | 2021-12-02 | Regeneron Pharmaceuticals, Inc. | Anti-sars-cov-2-spike glycoprotein antibodies and antigen-binding fragments |
| JP2023528441A (ja) | 2020-06-03 | 2023-07-04 | リジェネロン・ファーマシューティカルズ・インコーポレイテッド | 抗ＳＡＲＳ－ＣｏＶ－２スパイク糖タンパク質抗体を用いてＳＡＲＳ－ＣｏＶ－２感染及びＣＯＶＩＤ－１９を治療又は予防するための方法 |
| CN112843234B (zh) * | 2021-01-20 | 2022-09-06 | 四川大学华西第二医院 | 一种调控生物机体内gdp-甘露糖的浓度的装置 |
| AU2022289365A1 (en) | 2021-06-07 | 2023-12-14 | Amgen Inc. | Using fucosidase to control afucosylation level of glycosylated proteins |
| CA3212850A1 (en) | 2021-06-22 | 2022-12-29 | Frank DELFINO | Anti-egfrviii antibody drug conjugates and uses thereof |
| CA3225575A1 (en) | 2021-07-14 | 2023-01-19 | Regeneron Pharmaceuticals, Inc. | Anti-sars-cov-2-spike glycoprotein antibodies and antigen-binding fragments |
| WO2023081434A2 (en) | 2021-11-07 | 2023-05-11 | Regeneron Pharmaceuticals, Inc. | Methods for treating or preventing sars-cov-2 infections and covid-19 with anti-sars-cov-2 spike glycoprotein antibodies |
| AU2023228815A1 (en) | 2022-03-02 | 2024-09-12 | Regeneron Pharmaceuticals, Inc. | Manufacturing process for high titer antibody |
Family Cites Families (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US1006070A (en) | 1909-05-11 | 1911-10-17 | George Walter Durbrow | Method of and means for extracting micro-organisms and solids from water. |
| EP1176195B1 (en) | 1999-04-09 | 2013-05-22 | Kyowa Hakko Kirin Co., Ltd. | Method for controlling the activity of immunologically functional molecule |
| JP2001145488A (ja) | 1999-11-19 | 2001-05-29 | Natl Inst Of Advanced Industrial Science & Technology Meti | シロイヌナズナ由来のｇｄｐ−４−ケト−６−デオキシ−ｄ−マンノース−３，５−エピメラーゼ−４−レダクターゼ遺伝子 |
| FR2807767B1 (fr) | 2000-04-12 | 2005-01-14 | Lab Francais Du Fractionnement | Anticorps monoclonaux anti-d |
| US6946292B2 (en) | 2000-10-06 | 2005-09-20 | Kyowa Hakko Kogyo Co., Ltd. | Cells producing antibody compositions with increased antibody dependent cytotoxic activity |
| CN102311986B (zh) | 2000-10-06 | 2015-08-19 | 协和发酵麒麟株式会社 | 产生抗体组合物的细胞 |
| US20020150968A1 (en) | 2001-01-10 | 2002-10-17 | Wang Peng G. | Glycoconjugate and sugar nucleotide synthesis using solid supports |
| WO2002057423A2 (en) | 2001-01-16 | 2002-07-25 | Regeneron Pharmaceuticals, Inc. | Isolating cells expressing secreted proteins |
| US20030157108A1 (en) | 2001-10-25 | 2003-08-21 | Genentech, Inc. | Glycoprotein compositions |
| KR100585140B1 (ko) | 2004-04-26 | 2006-05-30 | 삼성전자주식회사 | 와이어 본딩 장치 및 이를 이용한 반도체 패키지의 와이어본딩 방법 |
| US20060223147A1 (en) * | 2004-08-05 | 2006-10-05 | Kyowa Hakko Kogyo Co., Ltd., | Process for producing glycoprotein composition |
| EP1888638A2 (en) | 2005-06-03 | 2008-02-20 | Genentech, Inc. | Method of producing antibodies with modified fucosylation level |
| JP5825756B2 (ja) | 2006-08-14 | 2015-12-02 | ゼンコー・インコーポレイテッドＸｅｎｃｏｒ、 Ｉｎｃ． | Ｃｄ１９を標的とする最適化抗体 |
| RU2479629C2 (ru) | 2007-03-07 | 2013-04-20 | Гликофи, Инк. | Продукция гликопротеинов с модифицированным фукозилированием |
| ES2522615T3 (es) | 2007-06-04 | 2014-11-17 | Regeneron Pharmaceuticals, Inc. | Regiones de expresión y estabilidad potenciadas |
| EP2311864A4 (en) | 2008-08-13 | 2013-07-31 | Kyowa Hakko Kirin Co Ltd | RECOMBINANT PROTEIN-S COMPOSITION |
| BRPI1010035A2 (pt) | 2009-06-02 | 2015-08-25 | Regeneron Pharma | Células deficientes em fucosilação |
| WO2012120500A2 (en) | 2011-03-06 | 2012-09-13 | Merck Serono S.A. | Low fucose cell lines and uses thereof |
-
2010
- 2010-06-01 BR BRPI1010035-0A patent/BRPI1010035A2/pt not_active Application Discontinuation
- 2010-06-01 DK DK17192430.1T patent/DK3279326T3/da active
- 2010-06-01 KR KR1020117031320A patent/KR101441437B1/ko not_active Expired - Fee Related
- 2010-06-01 AU AU2010256753A patent/AU2010256753B2/en not_active Ceased
- 2010-06-01 JP JP2012514045A patent/JP5744856B2/ja not_active Expired - Fee Related
- 2010-06-01 CA CA2764370A patent/CA2764370C/en active Active
- 2010-06-01 HU HUE14179885A patent/HUE038596T2/hu unknown
- 2010-06-01 US US12/791,637 patent/US8409838B2/en active Active
- 2010-06-01 EP EP10721096.5A patent/EP2438171B1/en active Active
- 2010-06-01 KR KR1020147007025A patent/KR101641526B1/ko not_active Expired - Fee Related
- 2010-06-01 PL PL10721096T patent/PL2438171T3/pl unknown
- 2010-06-01 EP EP17192430.1A patent/EP3279326B1/en active Active
- 2010-06-01 DK DK14179885.0T patent/DK2808393T3/en active
- 2010-06-01 CN CN2010800294912A patent/CN102459603B/zh not_active Expired - Fee Related
- 2010-06-01 ES ES10721096.5T patent/ES2527173T3/es active Active
- 2010-06-01 PL PL17192430T patent/PL3279326T3/pl unknown
- 2010-06-01 EP EP14179885.0A patent/EP2808393B1/en active Active
- 2010-06-01 ES ES14179885.0T patent/ES2661074T3/es active Active
- 2010-06-01 WO PCT/US2010/036914 patent/WO2010141478A1/en not_active Ceased
- 2010-06-01 TW TW099117652A patent/TWI513818B/zh not_active IP Right Cessation
- 2010-06-01 PL PL14179885T patent/PL2808393T3/pl unknown
- 2010-06-01 DK DK10721096.5T patent/DK2438171T3/en active
- 2010-06-01 SG SG2011087517A patent/SG176251A1/en unknown
-
2011
- 2011-11-27 IL IL216624A patent/IL216624A/en active IP Right Grant
-
2013
- 2013-02-28 US US13/779,952 patent/US9206455B2/en active Active
-
2014
- 2014-12-12 JP JP2014251541A patent/JP2015051021A/ja not_active Withdrawn
-
2015
- 2015-12-08 US US14/963,178 patent/US9550823B2/en active Active
-
2016
- 2016-03-07 JP JP2016043151A patent/JP6211642B2/ja not_active Expired - Fee Related
- 2016-10-14 JP JP2016202634A patent/JP2017006147A/ja active Pending
- 2016-12-14 US US15/378,670 patent/US10006070B2/en active Active
-
2018
- 2018-05-23 US US15/987,286 patent/US20190024129A1/en not_active Abandoned
-
2019
- 2019-08-30 US US16/556,334 patent/US20200080124A1/en not_active Abandoned
-
2021
- 2021-05-04 US US17/307,255 patent/US11560550B2/en active Active
-
2022
- 2022-12-15 US US18/066,303 patent/US12054751B2/en active Active
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| B06F | Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette] | ||
| B07A | Application suspended after technical examination (opinion) [chapter 7.1 patent gazette] | ||
| B09B | Patent application refused [chapter 9.2 patent gazette] | ||
| B12B | Appeal against refusal [chapter 12.2 patent gazette] | ||
| B11D | Dismissal acc. art. 38, par 2 of ipl - failure to pay fee after grant in time |