AU2021222032A1 - Protein-based purification matrices and methods of using the same - Google Patents
Protein-based purification matrices and methods of using the same Download PDFInfo
- Publication number
- AU2021222032A1 AU2021222032A1 AU2021222032A AU2021222032A AU2021222032A1 AU 2021222032 A1 AU2021222032 A1 AU 2021222032A1 AU 2021222032 A AU2021222032 A AU 2021222032A AU 2021222032 A AU2021222032 A AU 2021222032A AU 2021222032 A1 AU2021222032 A1 AU 2021222032A1
- Authority
- AU
- Australia
- Prior art keywords
- biologic
- seq
- purification matrix
- amino acid
- gvpgv
- 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
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
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 and B01D15/30 - B01D15/36, e.g. affinity, ligand exchange or chiral chromatography
- B01D15/3804—Affinity chromatography
- B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G or L chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
- C07K14/08—RNA viruses
- C07K14/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus human T-cell leukaemia-lymphoma virus
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
- C07K14/08—RNA viruses
- C07K14/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus human T-cell leukaemia-lymphoma virus
- C07K14/155—Lentiviridae, e.g. human immunodeficiency virus [HIV], visna-maedi virus or equine infectious anaemia virus
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
- C07K14/08—RNA viruses
- C07K14/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus human T-cell leukaemia-lymphoma virus
- C07K14/155—Lentiviridae, e.g. human immunodeficiency virus [HIV], visna-maedi virus or equine infectious anaemia virus
- C07K14/16—HIV-1 ; HIV-2
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- 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
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
- C12N7/02—Recovery or purification
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
- C12N2710/10351—Methods of production or purification of viral material
-
- 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
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
-
- 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
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15051—Methods of production or purification of viral material
-
- 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
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14151—Methods of production or purification of viral material
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Virology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Hematology (AREA)
- Analytical Chemistry (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Urology & Nephrology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Cell Biology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Physics & Mathematics (AREA)
- Oncology (AREA)
- Food Science & Technology (AREA)
- Toxicology (AREA)
- General Engineering & Computer Science (AREA)
- Communicable Diseases (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Water Supply & Treatment (AREA)
- Tropical Medicine & Parasitology (AREA)
- AIDS & HIV (AREA)
- Peptides Or Proteins (AREA)
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US202062978615P | 2020-02-19 | 2020-02-19 | |
| US62/978,615 | 2020-02-19 | ||
| PCT/US2021/018805 WO2021168270A1 (en) | 2020-02-19 | 2021-02-19 | Protein-based purification matrices and methods of using the same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| AU2021222032A1 true AU2021222032A1 (en) | 2022-09-15 |
| AU2021222032A9 AU2021222032A9 (en) | 2023-06-29 |
Family
ID=77391793
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU2021222032A Pending AU2021222032A1 (en) | 2020-02-19 | 2021-02-19 | Protein-based purification matrices and methods of using the same |
Country Status (5)
| Country | Link |
|---|---|
| US (2) | US20230099707A1 (https=) |
| EP (1) | EP4107167A4 (https=) |
| JP (1) | JP2023514611A (https=) |
| AU (1) | AU2021222032A1 (https=) |
| WO (1) | WO2021168270A1 (https=) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US12269847B2 (en) | 2018-08-16 | 2025-04-08 | Donaldson Company, Inc. | Genetically encoded polypeptide for affinity capture and purification of biologics |
| US20230099707A1 (en) * | 2020-02-19 | 2023-03-30 | Isolere Bio, Inc. | Protein-based purification matrices and methods of using the same |
| EP4554958A2 (en) * | 2022-07-12 | 2025-05-21 | Donaldson Company, Inc. | Compositions and methods for improving viral transduction |
| EP4622990A1 (en) | 2022-11-21 | 2025-10-01 | CHRETO ApS | Method for purification of a target product using affinity purification technology |
| EP4630444A1 (en) * | 2022-12-08 | 2025-10-15 | CSL Behring LLC | Dissociable envelope binding proteins and uses thereof |
| WO2024169990A1 (zh) * | 2023-02-13 | 2024-08-22 | 浙江大学绍兴研究院 | 双特异性抗体及其应用 |
| EP4467563A1 (en) * | 2023-05-23 | 2024-11-27 | JLP Health GmbH | Ldlr derived polypeptides for anti-viral uses |
| EP4688822A1 (en) * | 2023-05-23 | 2026-02-11 | JLP Health GmbH | Ldlr derived polypeptides for anti-viral uses |
| CN121794390A (zh) | 2023-07-18 | 2026-04-03 | 维伟生物技术有限公司 | 纯化用树脂及其使用方法 |
Family Cites Families (24)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5496926A (en) * | 1992-01-19 | 1996-03-05 | Yeda Research And Development Co. Ltd. | Process of preparing a soluble LDL receptor |
| AU3867400A (en) | 1999-03-19 | 2000-10-09 | Duke University | Methods of using bioelastomers |
| US20050255554A1 (en) * | 2000-03-20 | 2005-11-17 | Ashutosh Chilkoti | Fusion peptides isolatable by phase transition |
| WO2006110292A2 (en) * | 2005-03-25 | 2006-10-19 | The Regents Of The University Of California | Temperature-triggered immobilization and purification of antibodies |
| CA2663047A1 (en) * | 2006-09-06 | 2008-03-13 | Phase Bioscience, Inc. | Therapeutic elastin-like polypeptide (elp) fusion proteins |
| US8609621B2 (en) | 2010-11-15 | 2013-12-17 | E I Du Pont De Nemours And Company | Acid-cleavable linkers exhibiting altered rates of acid hydrolysis |
| EP2673297A2 (en) * | 2011-02-11 | 2013-12-18 | Zyngenia, Inc. | Monovalent and multivalent multispecific complexes and uses thereof |
| EP2668962B1 (en) * | 2012-05-29 | 2016-10-26 | Albert-Ludwigs-Universität Freiburg | Protein assembler |
| WO2014026054A2 (en) * | 2012-08-10 | 2014-02-13 | University Of Southern California | CD20 scFv-ELPs METHODS AND THERAPEUTICS |
| US9616138B1 (en) * | 2013-09-26 | 2017-04-11 | University Of South Florida | Cargo carrying nanoparticles |
| EP3052521A1 (en) * | 2013-10-03 | 2016-08-10 | Moderna Therapeutics, Inc. | Polynucleotides encoding low density lipoprotein receptor |
| CN104725515B (zh) * | 2015-03-20 | 2017-11-14 | 扬州大学 | 一种类弹性蛋白多肽与柯萨奇腺病毒受体融合蛋白及其制备方法和应用 |
| US10385115B2 (en) * | 2015-03-26 | 2019-08-20 | Duke University | Fibronectin type III domain-based fusion proteins |
| CN105175554B (zh) | 2015-10-26 | 2018-09-25 | 扬州大学 | 一种类弹性蛋白多肽与热应激蛋白90α融合蛋白及其制备方法和应用 |
| US10633662B2 (en) | 2015-11-10 | 2020-04-28 | The Board Of Trustees Of The Leland Stanford Junior University | Methods and compositions for modulating AAV infection |
| US11149060B2 (en) * | 2016-02-19 | 2021-10-19 | University Of Delaware | Functionalized nanoparticles for enhanced affinity precipitation of proteins |
| JP2020500150A (ja) * | 2016-09-23 | 2020-01-09 | デューク ユニバーシティ | 下限臨界溶液温度挙動を有する非反復かつ非構造的ポリペプチド |
| WO2018132732A1 (en) | 2017-01-12 | 2018-07-19 | Duke University | Genetically encoded lipid-polypeptide hybrid biomaterials that exhibit temperature triggered hierarchical self-assembly |
| US11464867B2 (en) | 2018-02-13 | 2022-10-11 | University Of Southern California | Multimeric elastin-like polypeptides |
| CA3098565A1 (en) * | 2018-04-29 | 2019-11-07 | Claire G. ZHANG | Scalable clarification process for recombinant aav production |
| US12269847B2 (en) | 2018-08-16 | 2025-04-08 | Donaldson Company, Inc. | Genetically encoded polypeptide for affinity capture and purification of biologics |
| CN114746552B (zh) | 2019-11-25 | 2024-11-08 | 东曹株式会社 | 改良腺相关病毒结合性蛋白、该蛋白的制造方法以及使用了该蛋白的腺相关病毒吸附剂 |
| US20230340426A1 (en) * | 2020-02-19 | 2023-10-26 | Isolere Bio, Inc. | Protein-based purification matrices and methods of using the same |
| US20230099707A1 (en) * | 2020-02-19 | 2023-03-30 | Isolere Bio, Inc. | Protein-based purification matrices and methods of using the same |
-
2021
- 2021-02-19 US US17/904,535 patent/US20230099707A1/en not_active Abandoned
- 2021-02-19 EP EP21757068.8A patent/EP4107167A4/en active Pending
- 2021-02-19 AU AU2021222032A patent/AU2021222032A1/en active Pending
- 2021-02-19 WO PCT/US2021/018805 patent/WO2021168270A1/en not_active Ceased
- 2021-02-19 JP JP2022549696A patent/JP2023514611A/ja active Pending
- 2021-10-05 US US17/493,938 patent/US11591576B2/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| US20220010288A1 (en) | 2022-01-13 |
| US20230099707A1 (en) | 2023-03-30 |
| EP4107167A1 (en) | 2022-12-28 |
| JP2023514611A (ja) | 2023-04-06 |
| EP4107167A4 (en) | 2024-04-03 |
| US11591576B2 (en) | 2023-02-28 |
| WO2021168270A1 (en) | 2021-08-26 |
| AU2021222032A9 (en) | 2023-06-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11591576B2 (en) | Protein-based purification matrices and methods of using the same | |
| JP2023515469A (ja) | Aav結合ポリペプチドを含む精製マトリックス及びその使用方法 | |
| CN112048005B (zh) | 新型冠状病毒s蛋白片段多倍体及其制备方法、检测试剂盒、疫苗及药物 | |
| de las Mercedes Segura et al. | A novel purification strategy for retrovirus gene therapy vectors using heparin affinity chromatography | |
| US20230340426A1 (en) | Protein-based purification matrices and methods of using the same | |
| ES2354715T3 (es) | Procedimiento para mejorar el aislamiento de proteínas producidas de manera recombinante. | |
| CN108473964B (zh) | 用于稳定慢病毒制剂的缓冲液 | |
| US11739126B2 (en) | Genetically encoded polypeptide for affinity capture and purification of biologics | |
| JP2023514611A5 (https=) | ||
| US20240390517A1 (en) | Aav8 capsid variants with enhanced liver targeting | |
| CA3183557A1 (en) | Methods for the removal of free factor viii from preparations of lentiviral vectors modified to express said protein | |
| Olgun et al. | High-grade purification of third-generation HIV-based lentiviral vectors by anion exchange chromatography for experimental gene and stem cell therapy applications | |
| EP3248984A2 (en) | Method for purifying protein, fusion protein containing peptide tag, and production method thereof | |
| Gashti et al. | Purification of recombinant vesicular stomatitis virus-based HIV vaccine candidate | |
| US20250066759A1 (en) | Fusion proteins for purifying nucleic acids | |
| US20250382336A1 (en) | Functionalized ubx protein materials for enhanced purification of antibodies | |
| Gunnels et al. | CELLISA-a cell-cell binding assay for evaluation of nanovesicle targeting proteins | |
| RU2821947C2 (ru) | Способ получения мембранного белка | |
| TW202417469A (zh) | 純化供用於疫苗組成物之具有疏水性膜結構域的病毒蛋白的下游程序 | |
| KR20230147593A (ko) | 생체분자 정제용 중합체 | |
| WO2023180527A1 (en) | Adenoviral mediated targeting of activated immune cells | |
| CN103421093A (zh) | 介导zmiz1靶向敲除的转录激活子样效应因子核酸酶及其制法和应用 | |
| Labisch et al. | Lab-Scale Tools for Advanced LV Downstream Processing |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| SREP | Specification republished | ||
| PC1 | Assignment before grant (sect. 113) |
Owner name: DONALDSON COMPANY, INC. Free format text: FORMER APPLICANT(S): ISOLERE BIO, INC. |