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WO2004096863A1 - Procede de purification d'acide hyaluronique a poids moleculaire eleve - Google Patents

Procede de purification d'acide hyaluronique a poids moleculaire eleve Download PDF

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Publication number
WO2004096863A1
WO2004096863A1 PCT/KR2003/001092 KR0301092W WO2004096863A1 WO 2004096863 A1 WO2004096863 A1 WO 2004096863A1 KR 0301092 W KR0301092 W KR 0301092W WO 2004096863 A1 WO2004096863 A1 WO 2004096863A1
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WIPO (PCT)
Prior art keywords
hyaluronic acid
molecular hyaluronic
high molecular
low molecular
density
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Ceased
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PCT/KR2003/001092
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English (en)
Inventor
Hyun Ik Yang
Alla Aleksandrovna Kornilova
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Individual
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Priority claimed from KR1020030028331A external-priority patent/KR20030090501A/ko
Application filed by Individual filed Critical Individual
Priority to AU2003232660A priority Critical patent/AU2003232660A1/en
Publication of WO2004096863A1 publication Critical patent/WO2004096863A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates

Definitions

  • the present invention relates to a method for purifying a high molecular hyaluronic acid.
  • Hyaluronic acid is a kind of glycosaminoglycans
  • GAGs GAGs
  • hyaluronic acid exists in human connective tissues, humor of human eyes, human umbilical cords and hens' crests. It exists also as proteoglycans by binding with proteins of metabolic products from streptococos .
  • hyaluronic acid plays important roles in retention and lubrication of tissue structures, immunity against bacteria and cushioning against shocks. Therefore, it can be implemented for treatments for vascular diseases, ulcers, burns, joint pains, rheumatism, and cataract. Also, hyaluronic acid is capable of retaining about 200 grams of water per 1 gram of hyaluronic acid, and this excellent capability of holding water leads hyaluronic acid to be popularly used in moisturizing cosmetics and plastic surgical purposes.
  • hyaluronic acid exists as proteoglycans in human body, which acts as an antigen when injected into a foreign human body.
  • an egg-white protein is an allergen in a foreign human body. Therefore, it is necessary to remove proteins binding with hyaluronic acid.
  • the most effective hyaluronic acid is hyaluronic acid with heavy molecular weights formed in a dynamic secondary structure with a strong bonding force.
  • the hyaluronic acid with heavy molecular weights is referred to as high molecular hyaluronic acid.
  • Hyaluronic acid with light molecular weights is not suitable for the medical and cosmetic purposes since it is easily dissolved into water due to its weak bonding force and has a short effective half-life time in the body.
  • the hyaluronic acid with light molecular weights is referred to as low molecular hyaluronic acid.
  • a high molecular hyaluronic acid is needed to be separated from hyaluronic acid with diverse molecular weights. Furthermore, it is also necessary to enhance yields of high molecular hyaluronic acid from low molecular hyaluronic acid.
  • the high molecular hyaluronic acid has a molecular weight of above about 1.5 x 10 6 Dalton.
  • There are many known methods for purifying hyaluronic acid For instance, the Russian Patent No.1616926, C 08 B 37/08, EMNO.48, 1990/1616926, C 08 B introduces a purification method with increased yields of hyaluronic acid.
  • the purification and extraction of the filtrate with use of the activated carbon causes a higher loss of hyaluronic acid. Furthermore, although it is possible to obtain hyaluronic acid with diverse molecular weights, high molecular hyaluronic acid that is useful in an actual implementation may not be easily obtained from low molecular hyaluronic acid in an actual practice.
  • this fine filtration may cause depolymerization of hyaluronic acid so that the high molecular hyaluronic acid becomes low molecular hyaluronic acid, further resulting in losses of high molecular hyaluronic acid. It may not be also possible to exclude a chance that low molecular hyaluronic acid and some hydrolyzed proteins may still remain on the filter membrane. As a result, it is difficult to obtain highly purified hyaluronic acid.
  • an object of the present invention to provide a method for purifying high molecular hyaluronic acid with increased yields by effectively removing proteins from hyaluronic acid, separating high molecular hyaluronic acid from low molecular hyaluronic acid and facilitating high polymerization of low molecular hyaluronic acid.
  • a method for purifying high molecular hyaluronic acid from a natural hyaluronic acid compound containing proteins and hyaluronic acid including the steps of: hydrolyzing the proteins contained in the natural hyaluronic acid compound and then performing a first highspeed centrifugation to separate the hydrolyzed proteins from the hyaluronic acid; and performing a second highspeed centrifugation to separate high molecular hyaluronic acid and low molecular hyaluronic acid each from the centrifuged hyaluronic acid.
  • Fig. 1 is a diagram schematically showing a method for purifying high molecular hyaluronic acid in accordance with the present invention.
  • a first step is to separate proteins from hyaluronic acid through a high-speed centrifugation after proteins contained in the natural hyaluronic acid compound are hydrolyzed.
  • a second step is to separate high molecular hyaluronic acid and low molecular hyaluronic acid from each other through another high-speed centrifugation.
  • hyaluronic acid is classified into two groups based on a specific molecular weight suitable for the use in commercializing hyaluronic acid.
  • the known molecular weight of the hyaluronic acid for such purpose is about 1.5 X 10 6 Dolton. That is, in this preferred embodiment, the low molecular hyaluronic acid is defined to have a molecular weight below about 1.5 X 10 6 Dolton, while the high molecular hyaluronic acid is defined to have a molecular weight above 1.5 X 10 6 Dolton.
  • papain solution is used to enzymatically hydrolyze proteins.
  • the high-speed centrifugation for separating the hydrolyzed proteins from the hyaluronic acid is carried out in a water/salt solution 1 of which density pi is higher than the density p L of the high molecular hyaluronic acid and the density p s of low molecular hyaluronic acid but less than the density p P of the hydrolyzed protein.
  • the high-speed centrifugation for separating the high molecular hyaluronic acid and the low molecular hyaluronic acid from each other can be also carried out in a water/salt solution 2 of which density p 2 is higher than the density of the high molecular hyaluronic acid p L but less than the density of the low molecular hyaluronic acid p s .
  • a is an acceleration
  • ⁇ is a high speed angular velocity
  • R is a radius of the high-speed centrifuge.
  • the separation of hydrolyzed proteins from the high molecular hyaluronic acid is based on a structural characteristic of hyaluronic acid in a water/salt solution.
  • Hyaluronic acid has a left-wise helical structure wherein there are four disaccharides for every turn in a solid state without water.
  • the unit of four disaccharides forms a basic structure in a rhombic shape.
  • Each of the basic structures binds with at least one water molecule so that they exist in irregular clusters having a volume that is about 1000 to about 10000 times higher than the volume of the high molecular hyaluronic acid itself.
  • the molecule of hyaluronic acid has 8 units of CH, forming a hydrophobic dehydration region.
  • This structural characteristic leads hyaluronic acid to have different densities based on molecular weights of hyaluronic acid. That is, high molecular hyaluronic acid has a heavy molecular weight since it contains lots of disaccharides but its volume increase extensively due to formation of dehydration regions in a water soluble medium.
  • the density p L of the high molecular hyaluronic acid is less than the density p s of most of the low molecular hyaluronic acid which barely form a secondary structure in a water soluble medium.
  • the enzymatically hydrolyzed proteins are in small fractions which do not nearly have the volume, and thus, the density of the enzymatically hydrolyzed proteins p L is higher than the density of the low molecular hyaluronic acid p s .
  • This order of density i.e., p L ⁇ Ps ⁇ pp, makes it possible to purify high molecular hyaluronic acid through the highspeed centrifugation in the water/salt solution.
  • compositions of a water/salt solution mixed compound having a low density are positioned in a top portion, while other compositions of the compound having a higher density are positioned in a bottom portion.
  • density difference between the compositions of the compound is small, the separation process based merely on the density difference is progressed very slowly. Therefore, it is necessary to exert the same force of gravity to accelerate the separation rate of the compositions of the compound. This object can be achieved by high-speed centrifugation.
  • the density pi or p 2 of the water/salt solution 1 or 2 can be controlled by an amount of salt added to the solution.
  • the high molecular hyaluronic acid can be separated from the low molecular hyaluronic acid based on the density difference between the hyaluronic acid molecules and the water/salt solution. That is, in case that the density pi of the water/salt solution 1 is higher than that p s or p L of the hyaluronic acid but less than that p P of the hydrolyzed proteins, the high-speed centrifugation separates the hyaluronic acid and the hydrolyzed proteins from each other.
  • the high-speed centrifugation separates the high molecular hyaluronic acid from the low molecular hyaluronic acid.
  • a ⁇ R
  • compositions with high density are moved rapidly along the radius of the high-speed centrifuge from a central region of the highspeed centrifuge towards an edge region of the high-speed centrifuge.
  • compositions with low density are moved towards the central region of the highspeed centrifuge.
  • the hydrolyzed proteins with high density are moved towards the edge region, while the hyaluronic acid with low density is moved towards the central region.
  • the second step of carrying out the high-speed centrifugation for separating the high molecular hyaluronic acid from the low molecular hyaluronic acid the low molecular hyaluronic acid with a high density is moved towards the edge region, while the high molecular hyaluronic acid with a lower density is moved towards the central region.
  • the compositions moved towards the central region of the high-speed centrifuge can be easily collected by using a pump.
  • the low molecular hyaluronic acid is moved towards the central region due to high polymerization caused by the spontaneous self-binding reaction at the point that the density p s of the low molecular hyaluronic acid is the same as that p 2 of the water/salt solution 2.
  • the yield of the high molecular hyaluronic acid is further increased.
  • a continuous high-speed centrifugation leads the remaining low molecular hyaluronic acid to become high molecular hyaluronic acid in more extents in proportion to a time elapsed, and this fact results in further yields of the high molecular hyaluronic acid.
  • Ultrasound waves can be also used in the high-speed centrifuge in order to accelerate the separation of hyaluronic acid with different molecular weight fractions both having a small density difference.
  • the separation of the high molecular hyaluronic acid from the low molecular hyaluronic acid is accelerated when a wavelength of the ultrasound wave is greater than an average linear length of the low molecular hyaluronic acid but less than an average linear length of the high molecular hyaluronic acid.
  • the ultrasound wave is an effective tool for mixing and getting the low molecular hyaluronic acid collided so that the self-binding reaction of the low molecular hyaluronic acid is accelerated.
  • a rate that the low molecular hyaluronic acid becomes the high molecular hyaluronic acid increases.
  • Fig. 1 is a diagram describing directions of movements of compositions during the high-speed centrifugation. With reference to Fig. 1, more detailed descriptions on the high-speed centrifugation will be provided in accordance with a preferred embodiment of the present invention.
  • First step Separation of hydrolyzed proteins from hyaluronic acid
  • proteins are initially hydrolyzed by adding an enzyme to a mixture of protein, high molecular hyaluronic acid and low molecular hyaluronic acid all obtained from a natural compound. Then, a first water/salt solution 1 is added to the mixture containing enzymatically hydrolyzed proteins and hyaluronic acid.
  • the density p x of the first water/salt solution 1 is set to be greater than the density p L , P S or an arbitrary median density of any type of the hyaluronic acid but less than the density p P of the hydrolyzed proteins.
  • the hydrolyzed proteins are moved towards the edge region along a radius of the high-speed centrifuge so as to be accumulated on a surface of the high-speed centrifuge.
  • the hyaluronic acid is moved towards a central region of the high-speed centrifuge.
  • the hyaluronic acid moved towards the central region is collected through a pumping.
  • Second Step Separation of high molecular hyaluronic acid from low molecular hyaluronic acid
  • the collected hyaluronic acid is mixed with a second water/salt solution 2.
  • the density p 2 of the second water/salt solution 2 is set to be greater than the density p L of high molecular hyaluronic acid but less than the density p s of low molecular hyaluronic acid.
  • the high molecular hyaluronic acid is moved towards the central region of the high-speed centrifuge.
  • the low molecular hyaluronic acid is more actively collided and an accumulation rate on the surface of the high-speed centrifuge is enhanced.
  • the high molecular hyaluronic acid moved towards the central region is collected through a pumping.
  • a third high-speed centrifugation is additionally carried out.
  • the low molecular hyaluronic acid accumulated on the surface of the high-speed centrifuge gets highly polymerized and are moved towards the central region of the high-speed centrifuge.
  • Vibrations of the additionally used ultrasound wave enhance the movement of the high molecular hyaluronic acid from the surface of the high-speed centrifuge towards the central region of the high-speed centrifuge.
  • high molecular hyaluronic acid can be purified cost-effectively by using a high-speed centrifuge consuming less energy. Also, it is possible to minimize the loss of hyaluronic acid as well as to obtain highly purified high molecular hyaluronic acid with increased yields by removing proteins and inducing high polymerization of the low molecular hyaluronic acid.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Materials Engineering (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
  • Polymers & Plastics (AREA)
  • Organic Chemistry (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)

Abstract

L'invention concerne un procédé de purification d'un acide hyaluronique à poids moléculaire élevé à partir d'un composé d'acide hyaluronique naturel contenant des protéines et un acide hyaluronique. Ce procédé comprend les étapes consistant: à hydrolyser les protéines contenues dans le composé d'acide hyaluronique naturel et à mettre en oeuvre une première centrifugation à haute vitesse afin de séparer les protéines hydrolysées et l'acide hyaluronique, puis une seconde centrifugation haute vitesse afin de séparer un acide hyaluronique à poids moléculaire élevé et un acide hyaluronique à poids moléculaire faible de l'acide hyaluronique centrifugé. Selon cette invention, l'acide hyaluronique et les protéines peuvent être séparés de façon économique par utilisation d'une centrifugation haute vitesse consommant peu d'énergie. De même, il est possible d'obtenir séparément de l'acide hyaluronique à poids moléculaire élevé hautement purifié et de l'acide hyaluronique à poids moléculaire faible. Enfin, l'acide hyaluronique à poids moléculaire faible peut être induit de manière à devenir de l'acide hyaluronique à poids moléculaire élevé en vue d'augmenter les rendements de l'acide hyaluronique à poids moléculaire élevé.
PCT/KR2003/001092 2003-05-02 2003-06-03 Procede de purification d'acide hyaluronique a poids moleculaire eleve Ceased WO2004096863A1 (fr)

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Application Number Priority Date Filing Date Title
AU2003232660A AU2003232660A1 (en) 2003-05-02 2003-06-03 Method for purification of high molecular hyaluronic acid

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KR10-2003-0028331 2003-05-02
KR1020030028331A KR20030090501A (ko) 2002-05-20 2003-05-02 고분자 히알루론산의 정제 방법

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021108790A1 (fr) * 2019-11-30 2021-06-03 Lacerta Life Sciences, Llc Purification d'acide hyaluronique reptilien et son utilisation pour la réparation et la régénération de tissus mous et durs

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4141973A (en) * 1975-10-17 1979-02-27 Biotrics, Inc. Ultrapure hyaluronic acid and the use thereof
JP2001158801A (ja) * 1999-12-02 2001-06-12 Medicaraise Corp 複合ムコ多糖及びその抽出方法
US20020120132A1 (en) * 2000-12-15 2002-08-29 Al Prescott Method for purifying high molecular weight hyaluronic acid
KR20020071390A (ko) * 2001-03-06 2002-09-12 이종하 미생물을 이용한 히알루론산의 제조방법

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4141973A (en) * 1975-10-17 1979-02-27 Biotrics, Inc. Ultrapure hyaluronic acid and the use thereof
US4141973B1 (fr) * 1975-10-17 1989-08-08
JP2001158801A (ja) * 1999-12-02 2001-06-12 Medicaraise Corp 複合ムコ多糖及びその抽出方法
US20020120132A1 (en) * 2000-12-15 2002-08-29 Al Prescott Method for purifying high molecular weight hyaluronic acid
KR20020071390A (ko) * 2001-03-06 2002-09-12 이종하 미생물을 이용한 히알루론산의 제조방법

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021108790A1 (fr) * 2019-11-30 2021-06-03 Lacerta Life Sciences, Llc Purification d'acide hyaluronique reptilien et son utilisation pour la réparation et la régénération de tissus mous et durs

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