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WO2024144117A1 - Procédé permettant d'analyser la teneur d'une forme mono-glycosylée de l'hormone de stimulation folliculaire (fsh) - Google Patents

Procédé permettant d'analyser la teneur d'une forme mono-glycosylée de l'hormone de stimulation folliculaire (fsh) Download PDF

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Publication number
WO2024144117A1
WO2024144117A1 PCT/KR2023/021322 KR2023021322W WO2024144117A1 WO 2024144117 A1 WO2024144117 A1 WO 2024144117A1 KR 2023021322 W KR2023021322 W KR 2023021322W WO 2024144117 A1 WO2024144117 A1 WO 2024144117A1
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WO
WIPO (PCT)
Prior art keywords
uplc
mobile phase
fsh
glycosylated form
column
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.)
Ceased
Application number
PCT/KR2023/021322
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English (en)
Korean (ko)
Inventor
김효주
김선영
정영란
이은정
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
LG Chem Ltd
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LG Chem Ltd
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Filing date
Publication date
Application filed by LG Chem Ltd filed Critical LG Chem Ltd
Priority to CN202380087995.7A priority Critical patent/CN120418651A/zh
Publication of WO2024144117A1 publication Critical patent/WO2024144117A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • 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
    • 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
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/30—Control of physical parameters of the fluid carrier of temperature
    • 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
    • G01N2030/022—Column chromatography characterised by the kind of separation mechanism
    • G01N2030/027—Liquid chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G01N2440/38—Post-translational modifications [PTMs] in chemical analysis of biological material addition of carbohydrates, e.g. glycosylation, glycation

Definitions

  • the present invention relates to a novel method for detecting the mono-glycosylated form of FSH (Follicle-stimulating hormone). More specifically, it relates to a novel method for accurately analyzing the content of a single glycosylated form of FSH by comparatively analyzing the single glycosylated forms of the beta and alpha subunits of human FSH.
  • FSH Follicle-stimulating hormone
  • FSH Ficle-stimulating hormone
  • alpha and beta subunits form a non-covalent bond with each other.
  • N-glycan sites There are two N-glycan sites in each of the alpha and beta subunits; the alpha subunit has N-glycans at Asn52 and Asn78, and the beta subunit has N-glycans at Asn7 and Asn24.
  • N-glycans are known to play an important role in the interaction between alpha and beta subunits, clearance in vivo, or receptor binding affinity.
  • carbohydrate bound to the alpha-subunit of FSH is important for dimer assembly, integrity, secretion, and signaling, while the beta-subunit carbohydrate is responsible for dimer assembly, secretion, and removal of the heterodimer from circulation. It is important for
  • the purpose of the present invention is to provide a method for analyzing the mono-glycosylated form of a novel follicle-stimulating hormone (FSH).
  • FSH novel follicle-stimulating hormone
  • One aspect of the present invention aims to provide a method for analyzing the novel mono-glycosylated form of FSH (Follicle-stimulating hormone).
  • the single glycosylated form of FSH which has conventionally been difficult to analyze quantitatively, can be quantitatively evaluated with accuracy and reproducibility without removing the sugar.
  • Figure 1 is a graph showing the UV chromatogram results of FSH analyzed using a HILIC column.
  • Figure 3 is a graph showing the UV chromatogram results of FSH analyzed using a C3 column.
  • Figure 5 shows the results of a UV chromatogram of FSH analyzed by UPLC-Q-TOF using a C3 column.
  • Figure 6 is a result showing the deconvoluted mass spectrum of the beta-subunit peak. Green indicates the normal form, and red indicates the N-terminal (Asn-Ser) cleaved form.
  • Figure 7a is the result showing the deconvoluted mass spectrum of the alpha subunit and the single glycosylated alpha subunit peak
  • Figure 7b is the deconvoluted mass spectrum of the oxidized alpha subunit peak.
  • Figure 8 is a graph showing the gradient test results of the C3 column.
  • Figure 9 is a graph showing a content analysis chromatogram according to column temperature conditions.
  • Figure 10 is a graph showing the content analysis chromatogram of the single glycosylated form of rhFSH.
  • Figure 14 is a graph confirming the intra-laboratory precision (Intermediate Precision) (by date, Agilent UPLC) for rhFSH of the content analysis method according to the present invention.
  • Figure 15 is a graph confirming the intra-laboratory precision (Intermediate Precision, Waters UPLC) for rhFSH of the content analysis method according to the present invention.
  • Figure 16 is a graph confirming the intra-laboratory precision (Intermediate Precision, Agilent UPLC) for rhFSH of the content analysis method according to the present invention.
  • rhFSH used in the present invention refers to Recombinant Human Follicle Stimulating Hormone.
  • the term “mono-glycosylated form” or “mono-glycosylated form” or “Mono-glycosylated form” used in the present invention refers to the presence of two N-glycosylation sites in each of the alpha and beta subunits of rhFSH, Among these, the structure in which N-glycan is glycosylated at only one site is called the mono-glycosylated form.
  • the term “glycosylation” refers to the attachment of a polysaccharide to FSH.
  • the polysaccharide consists of 2 to 12 simple sugars linked to each other through glycosidic bonds.
  • N-glycosylation means that a polysaccharide is attached to an asparagine residue of an amino acid chain.
  • the present invention provides a method for analyzing the content of a single glycosylated form of FSH.
  • the method includes isolating the analyte from the single glycosylated form of FSH using ultra-performance liquid chromatography (UPLC).
  • UPLC ultra-performance liquid chromatography
  • the analyte is a substance that may include a mono-glycosylate form of FSH.
  • FSH mono-glycosylate form of FSH.
  • it may be a pharmaceutical composition comprising FSH, which may also include a mono-glycosylate form.
  • the UPLC may be RP-UPLC (Reversed-phase Ultra performance liquid chromatography), but any UPLC analysis method known in the art can be used without limitation.
  • any device for UPLC analysis can be used without limitation.
  • UPLC systems from Agilent or Waters were used, and it was confirmed that both can precisely separate the mono-glycosylated form of FSH.
  • the column used in the UPLC may be a C8 or C3 reversed-phase (RP) column, and preferably, the C3 column is more advantageous in terms of resolution because the single glycosylated form of FSH is intended to be separated into protein units. It can show an effect.
  • RP reversed-phase
  • the method according to the present invention is characterized in that it is performed without any pretreatment process.
  • Weight values of beta subunit peaks Peak No. N-glycan Structure Theoretical Mw (Da) Delta (Da) 1) -NS 2) +NS 3) -NS +NS One A2G2FS2+A2G2FS2 16976.07 17177.25 -0.40 -0.07 2 A2G2FS2+A3G3FS2 17341.39 17542.57 0.11 0.06 3 A2G2FS2+A3G3FS3 17632.65 17833.83 -0.04 -0.04 4 A3G3FS2+A3G3FS3 17997.97 18199.15 -0.44 -0.14 5 A3G3FS3+A3G3FS3 18289.23 18490.41 -0.19 -0.04 6 A3G3FS3+A4G4FS3 18654.63 18855.73 -0.12 0.02 7 A3G3FS3+A4G4FS4 18945.89 19146.99 -0.85 0.
  • A antenna structure (antennary); G, galactose; F, fucose; S, sialic acid
  • A antenna structure (antennary); G, galactose; F, fucose; S, sialic acid
  • A antenna structure (antennary); G, galactose; F, fucose; S, sialic acid
  • A antenna structure (antennary); G, galactose; F, fucose; S, sialic acid
  • A2G2S2+A3G3S2 14988.87 -0.64 1.32 A2G2S2+A3G3S3 15280.12 0.89 1.89 10 A3G3S2+A3G3S2 15354.19 0.72 1.36 11 A3G3S2+A3G3S3 15645.44 1.46 N.D. 12 A3G3S3+A3G3S3 15936.70 N.D. N.D.
  • a column temperature test was conducted to find the conditions that best separate the single glycosylated form using a C3 column.
  • the maximum operating temperature of the column indicated by the manufacturer is 80°C. Accordingly, a total of four temperature conditions of 60°C, 65°C, 70°C, and 80°C were tested, and the UV chromatogram results analyzed under each condition are shown in Figure 9.
  • the alpha subunit (Alpha) and the single glycosylated alpha subunit (Alpha-mono) were separated well, but the resolution of the beta subunit (Beta) was poor at 60°C. Therefore, the single glycosylated beta subunit (Beta-Mono) peak was not clear.
  • the UV chromatogram of test substance 2 analyzed by the final analysis method is shown in Figure 10, and the relative contents of each single glycosylated form of the beta and alpha subunits were calculated and summarized in Table 12.
  • the content of the beta single glycosylated form of test substance 2 was 31.0%, and the content of the alpha single glycosylated form was 2.5%.
  • beta and alpha subunit peaks and single glycosylated beta/alpha subunit peaks appeared, respectively, and in the UV chromatogram in which only purified water was injected, no peaks that interfered with the rhFSH sample peak were detected.
  • rhFSH test substance 2 samples were each diluted three times and analyzed.
  • the UV chromatogram is summarized in Figure 12, and the retention time and relative content for each peptide peak are summarized in Tables 13 and 14, respectively.
  • the overall peak profile was similar, and the retention times of major peaks were consistent within 1 minute.
  • the contents of monoglycosylated forms of beta and alpha were 30.8% and 2.5%, respectively, and the %RSD was 1.4% and 0.0%.

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

Abstract

La présente invention se rapporte à un procédé permettant d'analyser une forme mono-glycosylée de l'hormone folliculostimulante (FSH) à l'aide d'une chromatographie liquide à ultra-haute performance (UPLC). Selon la présente invention, une chromatographie liquide à ultra-haute performance est effectuée à l'aide d'une colonne spécifique et d'une phase mobile et, ainsi, le procédé est utile pour détecter et/ou quantifier une forme mono-glycosylée de l'hormone FSH sans prétraitement séparé.
PCT/KR2023/021322 2022-12-29 2023-12-21 Procédé permettant d'analyser la teneur d'une forme mono-glycosylée de l'hormone de stimulation folliculaire (fsh) Ceased WO2024144117A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202380087995.7A CN120418651A (zh) 2022-12-29 2023-12-21 分析促卵泡激素（fsh）的单糖基化形式的含量的方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR20220189348 2022-12-29
KR10-2022-0189348 2022-12-29

Publications (1)

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WO2024144117A1 true WO2024144117A1 (fr) 2024-07-04

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PCT/KR2023/021322 Ceased WO2024144117A1 (fr) 2022-12-29 2023-12-21 Procédé permettant d'analyser la teneur d'une forme mono-glycosylée de l'hormone de stimulation folliculaire (fsh)

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KR (1) KR20240106989A (fr)
CN (1) CN120418651A (fr)
TW (1) TW202436871A (fr)
WO (1) WO2024144117A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20060135656A (ko) * 2003-12-22 2006-12-29 아레스 트레이딩 에스.에이. Ｆｓｈ를 정제하는 방법
JP2013153770A (ja) * 2013-05-22 2013-08-15 Jcr Pharmaceuticals Co Ltd 組換え体ヒトｆｓｈの製造方法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20060135656A (ko) * 2003-12-22 2006-12-29 아레스 트레이딩 에스.에이. Ｆｓｈ를 정제하는 방법
JP2013153770A (ja) * 2013-05-22 2013-08-15 Jcr Pharmaceuticals Co Ltd 組換え体ヒトｆｓｈの製造方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BOUSFIELD GEORGE R.; BUTNEV VLADIMIR Y.; BUTNEV VIKTOR Y.; HIROMASA YASUAKI; HARVEY DAVID J.; MAY JEFFREY V.: "Hypo-glycosylated human follicle-stimulating hormone (hFSH21/18) is much more activein vitrothan fully-glycosylated hFSH (hFSH24)", MOLECULAR AND CELLULAR ENDOCRINOLOGY, ELSEVIER IRELAND LTD, IE, vol. 382, no. 2, 1 December 2013 (2013-12-01), IE , pages 989 - 997, XP028671325, ISSN: 0303-7207, DOI: 10.1016/j.mce.2013.11.008 *
BUTNEV VIKTOR Y., MAY JEFFREY V., BROWN ALAN R., SHARMA TARAK, BUTNEV VLADIMIR Y., WHITE WILLIAM K., HARVEY DAVID J., BOUSFIELD GE: "Human FSH Glycoform α-Subunit Asparagine52 Glycans: Major Glycan Structural Consistency, Minor Glycan Variation in Abundance", FRONTIERS IN ENDOCRINOLOGY, FRONTIERS RESEARCH FOUNDATION, CH, vol. 13, 18 October 2022 (2022-10-18), CH , XP093188007, ISSN: 1664-2392, DOI: 10.3389/fendo.2022.767661 *
LOUREIRO, R.F. ; DE OLIVEIRA, J.E. ; TORJESEN, P.A. ; BARTOLINI, P. ; RIBELA, M.T.C.P.: "Analysis of intact human follicle-stimulating hormone preparations by reversed-phase high-performance liquid chromatography", JOURNAL OF CHROMATOGRAPHY A, ELSEVIER, AMSTERDAM, NL, vol. 1136, no. 1, 8 December 2006 (2006-12-08), AMSTERDAM, NL, pages 10 - 18, XP024967136, ISSN: 0021-9673, DOI: 10.1016/j.chroma.2006.09.037 *

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CN120418651A (zh) 2025-08-01
KR20240106989A (ko) 2024-07-08
TW202436871A (zh) 2024-09-16

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