WO2019066485A1 - Procédé d'analyse de pureté pour vésicules extracellulaires, faisant appel à une chromatographie d'exclusion stérique - Google Patents

Procédé d'analyse de pureté pour vésicules extracellulaires, faisant appel à une chromatographie d'exclusion stérique Download PDF

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Publication number
WO2019066485A1
WO2019066485A1 PCT/KR2018/011415 KR2018011415W WO2019066485A1 WO 2019066485 A1 WO2019066485 A1 WO 2019066485A1 KR 2018011415 W KR2018011415 W KR 2018011415W WO 2019066485 A1 WO2019066485 A1 WO 2019066485A1
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WIPO (PCT)
Prior art keywords
endoplasmic reticulum
extracellular endoplasmic
absorbance
extracellular
peak
Prior art date
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Ceased
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PCT/KR2018/011415
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English (en)
Korean (ko)
Inventor
고용송
이창진
박현택
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POSTECH Academy Industry Foundation
Rosetta Exosome Co Ltd
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POSTECH Academy Industry Foundation
Rosetta Exosome Co Ltd
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Publication date
Application filed by POSTECH Academy Industry Foundation, Rosetta Exosome Co Ltd filed Critical POSTECH Academy Industry Foundation
Priority claimed from KR1020180114980A external-priority patent/KR102109921B1/ko
Publication of WO2019066485A1 publication Critical patent/WO2019066485A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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
    • B01D15/34—Size-selective separation, e.g. size-exclusion chromatography; Gel filtration; Permeation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86

Definitions

  • SEC size exclusion chromatography
  • a porous stationary phase such as a gel, a matrix, or a bead
  • large molecules that can not pass through the hole in the stationary phase can not enter the hole,
  • the small molecules exit the column quickly, while the small molecules move relatively slowly through the hole in the column to exit the column.
  • This method is generally used for desalting for buffer exchange, separation for purification, or molecular weight measurement according to solute size.
  • extracellular < / RTI > endoplasmic reticulum " of the present invention collectively refers to a living body nanoparticle derived from cells of Archaea, Prokarya or Eukarya and includes extracellular endoplasmic reticulum (exosome), argosomes , Dexosomes, ectosomes, exovesicles, oncosomes, prominosomes, prostasomes, tolerosomes, microparticles (e. G.
  • the purity analysis method of the extracellular endoplasmic reticulum of the present invention includes a step (a) of injecting a sample containing extracellular endoplasmic reticulum into a size exclusion chromatography column and developing the same.
  • a stationary phase with holes sized to separate nanoparticles, extracellular endoplasmic reticulum, from proteins of various sizes.
  • Gels most commonly used in size exclusion chromatography is Sepharose (Sepharose; GE Healthcare), Super Rose (Superose; GE Healthcare), Sephadex (Sephadex; Pharmacia), Bio- Gel P (Bio-Rad) and TSKgel ® ( silica-based; Sigma).
  • a Sephacryl S500 stationary phase having pores having a size capable of separating the extracellular endoplasmic reticulum, which is a nanoparticle, from proteins of various sizes, But is not limited thereto.
  • sample includes a biological sample containing cell extracellular medium, a cell culture fluid, a tissue sample, and the like, and specifically includes mammalian cell culture medium, bacterial cell culture medium, yeast culture medium, , Serum, plasma, saliva, tears, sweat, urine, feces, CSF, ascites, amniotic fluid, semen, milk, dust, fresh water, seawater, soil and fermentation One or more of which may be selected from the group consisting of foods.
  • the specific wavelength may be selected from one or more values selected from the range of 200 nm to 800 nm. In the present invention, the specific wavelength may be selected from four or more wavelengths including at least one of wavelengths in the range of 330 to 450 nm, 230 nm, 260 nm, and 280 nm, but is not limited thereto.
  • the extinction band of the extracellular endoplasmic reticulum is an extinction band satisfying all of the following conditions:
  • the extinction band of the impurity is an extinction band which does not satisfy at least one of the following conditions:
  • the absorbance band area (A CONT ) of the impurity of the present invention can be obtained by calculating the corresponding peak area of the 230 nm chromatogram as an impurity absorption band satisfying the above conditions.
  • the purity analysis method of the extracellular endoplasmic reticulum of the present invention includes the step of calculating the purity of the extracellular endoplasmic reticulum from the value of the extinction band of the extracellular endoplasmic reticulum and the value of the extinction band of the impurity [step (e)].
  • the method for analyzing an extracellular endoplasmic reticulum of the present invention includes a step (a) of injecting a sample containing an extracellular endoplasmic reticulum into a size exclusion chromatography column and developing the same.
  • the method for analyzing an extracellular endoplasmic reticulum of the present invention comprises the steps of: (a) detecting an absorbance chromatogram for a first wavelength of the developed sample; (b) detecting a light intensity for a second wavelength different from the first wavelength of the developed sample And detecting the chromatogram (step (c)).
  • the first and second wavelengths of the present invention may be selected from the range of 200 nm to 800 nm, preferably 230 nm, 260 nm, 280 nm and 450 nm, but are not limited thereto Do not.
  • extracellular endoplasmic reticulum and albumin exhibit completely different absorbance characteristics at wavelengths of 260 nm, 280 nm and 450 nm, respectively, according to one embodiment of the present invention.
  • the method for analyzing an extracellular endoplasmic reticulum of the present invention includes a step (d) of calculating an extinction band area of an extracellular vesicle or an extinction band area of an impurity from each of the above absorbance chromatograms.
  • the method for quantitatively analyzing an extracellular vesicle of the present invention comprises the steps of injecting a sample containing an extracellular endoplasmic reticulum into a size exclusion chromatography column (step (a)), detecting the absorbance chromatogram for a specific wavelength of the developed sample (Step (b)), calculating the extinction band area of the extracellular endoplasmic reticulum from the absorbance chromatogram (step (c)), and calculating the total amount of extracellular endoplasmic reticulum from the extinction band area of the extracellular endoplasmic reticulum [(d) step].
  • the method of analyzing the extracellular endoplasmic reticulum of the present invention can quickly and effectively analyze the total amount of the extracellular endoplasmic reticulum or impurities contained in the sample, and the purity of the extracellular endoplasmic reticulum without limiting the amount of the sample.
  • it can be used to measure the yield and efficiency of the extracellular ER separation method by comparing and analyzing purity before and after extracellular ER separation and purification.
  • the colon cancer cell line SW480 culture was centrifuged at 500 xg for 10 minutes and at 2,000 xg for 20 minutes to remove the precipitate.
  • a polyethylene glycol solution (final 8.4% Polyethylene Glycol 6000, 250 mM NaCl, 20 mM HEPES, pH 7.4) was added to the supernatant for the first purification of the extracellular endoplasmic reticulum and incubated at 4 ° C for 16 hours, xg for 30 minutes to dissolve the precipitated extracellular endoplasmic reticulum in HEPES buffered saline (20 mM HEPES, 150 mM NaCl, pH 7.4).
  • the second purified sample was injected into a column packed with Sephacryl S500 connected to a high performance liquid chromatography (HPLC) system (10 x 100 mm)
  • HPLC high performance liquid chromatography
  • Example 4 Absorption chromatogram analysis of extracellular ER and albumin using size exclusion chromatography at 230 nm and its application

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  • Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

La présente invention concerne un procédé d'analyse de pureté pour vésicules extracellulaires, faisant appel à une chromatographie d'exclusion stérique, et plus particulièrement, la présente invention concerne un procédé d'utilisation de la capacité de séparation en fonction de la taille d'une chromatographie d'exclusion stérique et d'une spectroscopie, qui est unique ou combinant au moins deux types, afin de quantifier, dans divers échantillons, le volume de vésicules extracellulaires et divers contaminants, simultanément, et ainsi d'analyser la pureté, la quantité totale et le rendement de vésicules extracellulaires. Le procédé d'analyse de pureté pour vésicules extracellulaires, selon la présente invention, a l'avantage de pouvoir réaliser une analyse efficace tout en conservant la forme ou les propriétés des vésicules extracellulaires. De plus, le procédé d'analyse de pureté pour vésicules extracellulaires, selon la présente invention, permet de déterminer rapidement, simultanément, la pureté, la quantité totale et le rendement de vésicules extracellulaires qui ont été séparées à l'aide d'un procédé classique, et peut être utilisé, par exemple, dans le diagnostic de maladies, le traitement de maladies, la recherche multiomique et la recherche des propriétés de vésicules extracellulaires qui utilisent des vésicules extracellulaires séparées. En outre, le procédé d'analyse de pureté selon la présente invention peut également être appliqué au développement d'un nouveau procédé de séparation.
PCT/KR2018/011415 2017-09-27 2018-09-27 Procédé d'analyse de pureté pour vésicules extracellulaires, faisant appel à une chromatographie d'exclusion stérique Ceased WO2019066485A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR10-2017-0124848 2017-09-27
KR20170124848 2017-09-27
KR1020180114980A KR102109921B1 (ko) 2017-09-27 2018-09-27 크기 배제 크로마토그래피를 이용한 세포밖 소포체의 순도 분석 방법
KR10-2018-0114980 2018-09-27

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113189067A (zh) * 2021-04-27 2021-07-30 浙江大学 一种细胞外囊泡的富集和检测方法及装置
US20220305405A1 (en) * 2019-05-01 2022-09-29 The Research Foundation For The State University Of New York System and method for isolation of intact extracellular vesicles with near-single-vesicle resolution coupled with on-line characterization
CN117384741A (zh) * 2023-12-11 2024-01-12 上海晟燃生物科技有限公司 细胞外囊泡分离设备
US12269003B2 (en) 2019-12-30 2025-04-08 Industrial Technology Research Institute Extracellular vesicle separation method, colloidal particle and preparation method thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6002986A (en) * 1990-11-16 1999-12-14 Shimadzu Corporation Fraction purity measuring apparatus for chromatogram peak
KR20140139122A (ko) * 2012-03-30 2014-12-04 샹하이 테크웰 바이오파마슈티컬 컴퍼니, 리미티드 고순도 고리형 펩티드 화합물 및 그 제조방법과 용도

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6002986A (en) * 1990-11-16 1999-12-14 Shimadzu Corporation Fraction purity measuring apparatus for chromatogram peak
KR20140139122A (ko) * 2012-03-30 2014-12-04 샹하이 테크웰 바이오파마슈티컬 컴퍼니, 리미티드 고순도 고리형 펩티드 화합물 및 그 제조방법과 용도

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Title
BOND, M. D.: "Evaluation of a dual-wavelength size exclusion HPLC method with improved sensitivity to detect protein aggregates and its use to better characterize degradation pathways of an IgG1 monoclonal antibody", JOURNAL *
CASTLEDINE, J. B. ET AL.: "A multiwavelength approach to the selection of absorbance ratios for the assessment of chromatographic peak purity", JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS, 8 September 1991 (1991-09-08), pages 619 - 624, XP055586021 *
CASTLEDINE, J. B.: "Assessment of chromatographic peak purity by means of multi-wavelength detection and correlation-based algorithms", JOURNAL OF - CHROMATOGRAPHY, vol. 592, 1992, pages 27 - 36, XP026503508, DOI: doi:10.1016/0021-9673(92)85069-6 *
KOTMAKCI, M.: "Exosome isolation: Is there an optimal method with regard to diagnosis or treatment?", NOVEL IMPLICATIONS OF EXOSOMES IN DIAGNOSIS AND TREATMENT OF CANCER AND INFECTIOUS DISEASES, 12 July 2017 (2017-07-12), pages 163 - 182, XP055586020 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220305405A1 (en) * 2019-05-01 2022-09-29 The Research Foundation For The State University Of New York System and method for isolation of intact extracellular vesicles with near-single-vesicle resolution coupled with on-line characterization
US12269003B2 (en) 2019-12-30 2025-04-08 Industrial Technology Research Institute Extracellular vesicle separation method, colloidal particle and preparation method thereof
CN113189067A (zh) * 2021-04-27 2021-07-30 浙江大学 一种细胞外囊泡的富集和检测方法及装置
CN117384741A (zh) * 2023-12-11 2024-01-12 上海晟燃生物科技有限公司 细胞外囊泡分离设备
CN117384741B (zh) * 2023-12-11 2024-03-15 上海晟燃生物科技有限公司 细胞外囊泡分离设备

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