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WO2025063917A1 - Procédé pour déterminer des concentrations d'acide hyaluronique de poids moléculaire élevé et de faible poids moléculaire dans des produits - Google Patents

Procédé pour déterminer des concentrations d'acide hyaluronique de poids moléculaire élevé et de faible poids moléculaire dans des produits Download PDF

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Publication number
WO2025063917A1
WO2025063917A1 PCT/TR2024/050533 TR2024050533W WO2025063917A1 WO 2025063917 A1 WO2025063917 A1 WO 2025063917A1 TR 2024050533 W TR2024050533 W TR 2024050533W WO 2025063917 A1 WO2025063917 A1 WO 2025063917A1
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WO
WIPO (PCT)
Prior art keywords
hyaluronic acid
molecular weight
products
analysis
hplc
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
Application number
PCT/TR2024/050533
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English (en)
Inventor
Mesut Celil ONCEYIZ
Murat GUMUS
Safak CELIK
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.)
VSY Biyoteknoloji ve Ilac Sanayi AS
Original Assignee
VSY Biyoteknoloji ve Ilac Sanayi AS
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Filing date
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Priority claimed from TR2023/011840 external-priority patent/TR2023011840A1/tr
Application filed by VSY Biyoteknoloji ve Ilac Sanayi AS filed Critical VSY Biyoteknoloji ve Ilac Sanayi AS
Publication of WO2025063917A1 publication Critical patent/WO2025063917A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • 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
    • G01N30/50—Conditioning of the sorbent material or stationary liquid
    • G01N30/52—Physical parameters
    • G01N30/54—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
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • 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
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • 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
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G01N2030/8836—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving saccharides
    • 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
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds
    • 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
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/74—Optical detectors
    • 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
    • G01N30/86—Signal analysis
    • G01N30/8624—Detection of slopes or peaks; baseline correction

Definitions

  • the invention relates to a method for determining the amount of high molecular weight and/or low molecular weight hyaluronic acid in commercial products or raw materials.
  • Hyaluronic acid (or hyaluronan or hyaluronic acid) is a type of natural polysaccharide and a component found in our body. It is especially found in the skin, joint fluid and eyes. Hyaluronic acid is widely used in skin care products and aesthetic applications. It can increase the skin's moisture levels, strengthen the skin barrier, and help reduce wrinkles. It is also used to support joint health and can be administered in the form of injections in the treatment of joint conditions such as osteoarthritis.
  • the molecular weight of hyaluronic acid is important to understand how effective this substance can be in various applications and for what purposes it can be used.
  • the molecular weight of hyaluronic acid is divided into two as low molecular weight hyaluronic acid and high molecular weight hyaluronic acid.
  • Determining the concentration of hyaluronic acid in commercial products is important to evaluate the efficacy and safety of the product, to ensure that users obtain accurate results, and to comply with regulatory requirements.
  • the lower and upper values of the target hyaluronic acid concentration for the effectiveness of the final products may affect the effectiveness of the product.
  • UV-Vis spectroscopy is used to determine the concentration of hyaluronic acid in products in the relevant technical field.
  • hyaluronic acid is broken down in the presence of strong acids such as H2SO4 at high temperature and converted into D-glucuronic acid, and then carbazole is added to color the hyaluronic acid solution to be analyzed. Analysis of the obtained colored solution is carried out.
  • the disadvantage of this method is the difficulties in the preparation of the solution during the sample preparation phase, the inefficient disintegration during the conversion of the hyaluronic acid to be analyzed in the presence of strong acid to D- glucuronic acid during the preparation of the solution, the insufficient temperature required for the disintegration, the lack of discoloration of the solution in the last step while preparing the solution, the low reproducibility of the analysis results and the high number of factors affecting the measurement uncertainty.
  • other substances in formulations comprising hyaluronic acid interfere at the same wavelength.
  • HPLC high-pressure liquid chromatography
  • the invention application No. US 2004/0175769 A1 relates to a method for the quantitative analysis of hyaluronic acid.
  • a substance with affinity for hyaluronic acid (HA) binding protein modified with a labeled substance was prepared and treated with anti-HA binding protein antibody.
  • This reagent (Fab') was cross-linked to horseradish peroxidase (POD) with the help of a cross-linking agent (SMPB).
  • SMPB cross-linking agent
  • Fluorescence measurements of HA with cross-linking reagent (Fab'-POD) were taken at 450 nm as a result of various reactions using LiBASys (Liquid Phase Bonding Auto-Immunoassay system) and HA concentration was calculated.
  • LiBASys Liquid Phase Bonding Auto-Immunoassay system
  • the invention with publication No. US 8367818 B2 relates to the analysis of low molecular weight HA (average molecular weight 5-50 kDa) used in cosmetic, pharmaceutical and food formulations in an HPLC device.
  • high molecular weight hyaluronic acids obtained from cockscomb and by the fermentation method thereof were converted into low molecular weight form by various processes and the low molecular weight hyaluronic acids obtained were analyzed by measuring the absorbance at 660 nm.
  • the resulting HAs were used to create a cosmetic formulation absorbable on the body surface and applied to mice. After the application, the amount of HA remaining unabsorbed on the skin surface of the mice was determined by HPLC method.
  • the HPLC method is as follows:
  • Detector Fluorescent detector, excitation wavelength at 365 nm; fluorescence wavelength at 447 nm
  • the invention with publication No. US 8163498 B2 relates to the ability to measure the molecular weight of HA in a short time by the GPC method.
  • the concentrations of HAs, whose molecular weight is determined by the HPLC method are measured.
  • HA samples were converted into unsaturated disaccharides with the help of enzymes and their concentrations were determined by HPLC device.
  • the method for HPLC device concentration analysis is as follows:
  • Detector Fluorescent detector, excitation wavelength at 346 nm; fluorescence wavelength at 410 nm
  • Mobile phase 0-200 nM sodium sulfate (gradient system).
  • the invention with publication No. EP 2216412 B1 relates to the production of good quality HA that can be used in the biomedical field by a process that does not contain surfactants or detergents.
  • HA was produced by fermentation method, its molecular weight was determined by HPLC device and purified by appropriate methods. The following parameters were used:
  • Hyaluronic acid is an important ingredient that is frequently used in the fields of pharmacology and cosmetic technique. It is especially used as an ingredient in the elimination of skin-related diseases or defects, in the regeneration of the skin and in the treatment of bone diseases.
  • concentrations in the final products and raw materials such as the ingredients used in each pharmacology and cosmetic technical field in order to inform the user about the efficacy, safety and quality.
  • research and development activities continue to obtain a test analysis method in which hyaluronic acid concentrations in products or raw materials are fast, suitable for procedures, have low costs, no measurement uncertainty, and low reproducibility.
  • HPLC analysis test method has become a test method in which measurement uncertainty is eliminated, precision is high, analysis costs are not high, reproducibility is not low, there is no need to use harmful chemicals in order to determine the hyaluronic acid concentration in products and raw materials, and it provides advanced technical teachings for the relevant technical field.
  • the subject of the invention relates to a method for determining the concentration of hyaluronic acid in products and is explained only with examples that will not create any limiting effect for a better understanding of the subject.
  • products refers to all compositions, mixtures, solutions and substances in which hyaluronic acid is included in certain amounts as an ingredient. These products can be in a final product, or they can also be a raw material.
  • the protection scope of the invention is independent of the product in which the concentration of hyaluronic acid is determined.
  • HPLC test-analysis method is used as the method.
  • This method has been tried as an analysis-test method in previous studies in the relevant technical field, but it has been determined that it is not a suitable method for determining the concentration of hyaluronic acid in products in authorized guidelines and by the experts in the technical field.
  • the present inventors have carried out research and development activities for the use of the HPLC method as an analysis-test method in determining the amount of hyaluronic acid in products.
  • Quantity analysis for hyaluronic acid is reported in the EP guideline, and it is known that the measurement uncertainty is high for personnel in the method specified in the guideline, its reproducibility is low, the precision when determining the quantity for the product is low, chemicals that are very harmful to both human and nature are used, and the analysis cost is quite high. In particular, it is known that when performing raw material analysis, the results depend on the person.
  • the peak performance data should be as follows:
  • %RSD should be ⁇ 2.0
  • hyaluronic acid has a long chain structure, and therefore, when analyze-test is performed with HPLC, it is possible to obtain peaks in chromatograms where the ratio of height to width is very low.
  • an HPLC test setup was prepared in which two columns were connected in series to each other. In this way, the column length of the HPLC test setup was extended and the targeted column type feature was provided.
  • the preferred column height here is between 600 mm and 1200 mm.
  • Shodex Ohpak SB-806M HQ 300 mm-8 mm-13 pm and Shodex Ohpak SB-807 HQ 300 mmx8 mm-13 pm columns are used.
  • the protection scope of the invention is not limited to this column type. When it comes to determining the appropriate column type in the invention, it is meant that the column types of appropriate heights are used.
  • sodium chloride has a value of 0.1 M. It was chosen as the mobile phase due to the high solubility of hyaluronic acid in solutions containing NaCI and the presence of an ion balance with hyaluronic acid. In addition, it was considered that NaCI is suitable because it is easily available and does not harm the nature.
  • the concentration (mg/L, ppm) of the product containing hyaluronic acid in standard solution (product equivalent to hyaluronic acid mg)/0.1 M sodium chloride solution (L) is between 10-1000 ppm.
  • the concentration range should be in the range of 100-800 ppm. More preferably, the concentration range should be in the range of 200- 500 ppm.
  • the concentrations indicated here are determined so that the signal-to-noise ratio in the device is above 10.0.
  • Predetermined amounts of sodium hyaluronate are added to a volumetric flask and sodium chloride solution is added to dissolve it. It is then vortexed for at least 1 minute. Magnetic fish is placed in the volumetric flask and mixing is carried out for at least 1 hour with the help of a magnetic stirrer at room temperature. As a result of the mixing process, the mixture is passed through filters and vialized and a standard solution is prepared for use.
  • UV detectors provide an important function in the formation of peaks with high length and narrow width compared to RID detectors, thanks to their high ability to detect analytes at low concentrations.
  • the flow rate of 1 mL/min of the samples to the HPLC test setup is the most appropriate flow rate.
  • the appropriate flow rate parameter is mostly found by trial and error method.
  • the present inventors studied in the range of 1.0-1.5 mL/min and determined the appropriate flow rate by analyzing the peak performance data obtained.
  • the tailing factor will also increase as the width of the peak increases, that is, the peak becomes wider.
  • the peak will occur earlier and there will be a possibility of coinciding with the solvent peak, so a flow of 1 mL has been determined as the best flow in terms of both the form and location of the peak.
  • Tray temperature is the waiting temperature of the prepared standard and sample solutions in a chamber in the device for analysis.
  • the waiting temperature affects the time the solution prepared for the analysis remains without decomposition.
  • Some materials need to be stored at 5 °C, while some materials are stored at 40 °C. This is related to the stability of the solution.
  • the solutions referred to in the present invention are suitable for the stability of standard tray temperature and sample solutions in the temperature ranges of 15-30 °C. Since this temperature is also room temperature, it is the ideal temperature for the operation of a column oven.
  • Table 2 values provided below were obtained for the HPLC method used to determine the concentration of hyaluronic acid.
  • the HPLC method is an applicable analysis method to determine the hyaluronic acid concentrations in the products as discussed in the present invention. But the important thing here is to use the appropriate parameters and elements.
  • the present inventors have addressed the drawbacks known in the art and carried out research and development studies on the HPLC method, whose technical advantages are known for the relevant technical field, and have shown that it is suitable for use in determining the concentration of hyaluronic acid in products.
  • Samples were prepared with high molecular weight and low molecular weight standards and the most appropriate parameters for the method of the present invention were determined.

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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)

Abstract

L'invention concerne un procédé pour déterminer la quantité d'acide hyaluronique de poids moléculaire élevé et/ou de faible poids moléculaire dans des produits commerciaux ou des matières premières.
PCT/TR2024/050533 2023-09-22 2024-05-23 Procédé pour déterminer des concentrations d'acide hyaluronique de poids moléculaire élevé et de faible poids moléculaire dans des produits Pending WO2025063917A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
TR2023011840 2023-09-22
TR2023/011840 TR2023011840A1 (tr) 2023-09-22 Ürünlerdeki̇ yüksek molekül ağirlikli ve düşük molekül ağirlikli hyaluroni̇k asi̇t konsantrasyonlarinin beli̇rlenmesi̇ i̇çi̇n bi̇r yöntem

Publications (1)

Publication Number Publication Date
WO2025063917A1 true WO2025063917A1 (fr) 2025-03-27

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PCT/TR2024/050533 Pending WO2025063917A1 (fr) 2023-09-22 2024-05-23 Procédé pour déterminer des concentrations d'acide hyaluronique de poids moléculaire élevé et de faible poids moléculaire dans des produits

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WO (1) WO2025063917A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2058659A1 (fr) * 2006-08-08 2009-05-13 Seikagaku Corporation Procédé de détermination du poids moléculaire d'un acide hyaluronique
CN112255345A (zh) * 2020-10-20 2021-01-22 山东华熙海御生物医药有限公司 一种测定高分子量透明质酸分子量的方法
CN115774066A (zh) * 2022-11-16 2023-03-10 南京乐韬生物科技有限公司 一种分析超低分子量透明质酸组分比例的方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2058659A1 (fr) * 2006-08-08 2009-05-13 Seikagaku Corporation Procédé de détermination du poids moléculaire d'un acide hyaluronique
CN112255345A (zh) * 2020-10-20 2021-01-22 山东华熙海御生物医药有限公司 一种测定高分子量透明质酸分子量的方法
CN115774066A (zh) * 2022-11-16 2023-03-10 南京乐韬生物科技有限公司 一种分析超低分子量透明质酸组分比例的方法

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