WO2004096863A1

WO2004096863A1 - Method for purification of high molecular hyaluronic acid

Info

Publication number: WO2004096863A1
Application number: PCT/KR2003/001092
Authority: WO
Inventors: Hyun Ik Yang; Alla Aleksandrovna Kornilova
Original assignee: Hyun Ik Yang; Alla Aleksandrovna Kornilova
Priority date: 2003-05-02
Filing date: 2003-06-03
Publication date: 2004-11-11
Also published as: AU2003232660A1

Abstract

A method for purifying high molecular hyaluronic acid from a natural hyaluronic acid compound containing proteins and hyaluronic acid includes the steps of: hydrolyzing proteins contained in the natural hyaluronic acid compound and performing a first high-speed centrification to separate the hydrolyzed proteins and the hyaluronic acid from each other; and performing a second high-speed centrification to separate a high molecular hyaluronic acid and a low molecular hyaluronic acid from the centrifuged hyaluronic acid. According to the present invention, the hyaluronic acid and proteins can be cost-effectively separated from each other by employing the high-speed centrification consuming a low power. Also, it is possible to obtain separately the highly purified high molecular hyaluronic acid and the low molecular hyaluronic acid. Furthermore, the low molecular hyaluronic acid can be induced to be the high molecular hyaluronic acid to thereby augment yields of the high molecular hyaluronic acid.

Description

METHOD FOR PURIFICATION OF HIGH MOLECULAR HYALURONIC ACID

Technical Field

The present invention relates to a method for purifying a high molecular hyaluronic acid.

Background Art

Hyaluronic acid is a kind of glycosaminoglycans

(GAGs) , and more particularly, it exists as a linear biopolymer composed of repeating disaccharides formed through a β(l -> 3) glycosidal linkage of D-glucuronic acid and N-acetylglucosamine . Generally, 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 .

Medicines containing hyaluronic acid play 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.

However, a highly purified hyaluronic acid is reguired for such medical or cosmetic purposes. As mentioned above, hyaluronic acid exists as proteoglycans in human body, which acts as an antigen when injected into a foreign human body. For instance, an egg-white protein is an allergen in a foreign human body. Therefore, it is necessary to remove proteins binding with hyaluronic acid.

Among various types of synthetic hyaluronic acid synthesized from the same natural source material, the most effective hyaluronic acid is hyaluronic acid with heavy molecular weights formed in a dynamic secondary structure with a strong bonding force. Hereinafter, 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. Hereinafter, the hyaluronic acid with light molecular weights is referred to as low molecular hyaluronic acid. Accordingly, 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. For commercialization, it is preferable that the high molecular hyaluronic acid has a molecular weight of above about 1.5 x 10⁶ 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. Firstly, blood of hens' crests is drained out, and the hens' crests are ground and extracted with water. The extract is subjected to a heat treatment at a temperature from about 80 °C to about 100 °C. After the heat treatment, lipids are separated from the heat treated extract, which is subsequently filtered after the lipid separation. Then, the filtrate is added with activated carbon to be purified and extracted. The extract is again filtered as being stirred. This filtrate is mixed with raw material sediments and is extracted again in two steps in the presence of penicillin. Finally, the extract is dried. However, this method has disadvantages of procedural complexity due to repeated steps of purification and extraction and low yields of hyaluronic acid. Particularly, 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.

In addition, in the Russian Patent NO.2157381, 7C 08 B 37/08, A 61 K 31/728, there is described another known method for purifying hyaluronic acid. Hens' crests are finely ground and extracted in several steps. Then, the extracts are put together and hyaluronic acid is settled thereafter. The sediments are dissolved in water and are treated with sodium hydroxide (NaOH) . Afterwards, enzyme is added thereto to enzymatically hydrolyze proteins. Then, the hydrolyzed proteins are removed through a fine filtration. The resultant is settled by using ethanol, and the sediment is settled again in water. However, there are disadvantages of employing the above fine filtration allowing molecules/particles to pass through a filter membrane under a certain pressure. First, in the filter membrane with micro pores of different diameters, a small quantity of large pores can be formed when nearly allocated micro pores are connected together. Through these large pores, a partial quantity of high molecular hyaluronic acid can be filtered. Second, during the fine filtration under pressure, a partial quantity of hyaluronic acid with different molecular weights, accumulated on the filer membrane, may be destructed due to pressure. Also, 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.

Disclosure of the Invention

It is, therefore, 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.

In accordance with an aspect of the present invention, there is provided 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.

Brief Description of the Drawings

Other objects and aspects of the invention will become apparent from the following description of the embodiments with reference to the accompanying drawings, in which: Fig. 1 is a diagram schematically showing a method for purifying high molecular hyaluronic acid in accordance with the present invention.

Best Mode for Carrying Out the Invention

Hereinafter, a method for purifying hyaluronic acid according to the present invention will be described in detail referring to the accompanying drawings.

In accordance with a preferred embodiment of the Present invention, there are two sequential steps for purifying high molecular hyaluronic acid from a natural hyaluronic acid compound containing hyaluronic acid and proteins. 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.

As a reference, 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 10⁶ Dolton. That is, in this preferred embodiment, the low molecular hyaluronic acid is defined to have a molecular weight below about 1.5 X 10⁶ Dolton, while the high molecular hyaluronic acid is defined to have a molecular weight above 1.5 X 10⁶ Dolton.

In the first step of the present invention, 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 p₂ 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.

The acceleration of the high-speed centrifuge is expressed as the following equations:

a = ΩR Eq. 1

ΩR » g Eq. 2

Herein, in Eq. 1, a is an acceleration; Ω is a high speed angular velocity; and R is a radius of the high-speed centrifuge. Also, g in Eq. 2 is an acceleration of gravity, i.e., g = 9.8 m/sec².

In addition to the above two steps of high-speed centrifugations, it is further possible to centrifuge the remaining low molecular hyaluronic acid in high speed to increase yields of the high molecular hyaluronic acid. It is also possible to use ultrasonic waves during the highspeed centrifugation to catalyze the separation of the high molecular hyaluronic acid from the low molecular hyaluronic acid and to increase yields of the high molecular hyaluronic acid. The ultrasonic wave has a preferable wavelength larger than an average linear length of the low molecular hyaluronic acid but less than an average linear length of the high molecular hyaluronic acid.

In accordance with the present invention related to a method for purifying hyaluronic acid, 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. On the other hand, in a state of retaining 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. Under this structure, 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. As a result, 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. Also, 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.

Under gravity, some 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. However, since the 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. At this time, the density pi or p₂ of the water/salt solution 1 or 2 can be controlled by an amount of salt added to the solution. This fact makes it possible to separate the hyaluronic acid and the hydrolyzed proteins from each other by using the density difference between the hyaluronic acid, hydrolyzed proteins and water/salt solution. Also, 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. In case that the density p₂ of the water/salt solution 2 is higher than that p_L of the high molecular hyaluronic acid but less than that p_s of the low molecular hyaluronic acid, the high-speed centrifugation separates the high molecular hyaluronic acid from the low molecular hyaluronic acid.

The acceleration of the high-speed centrifuge, i.e., a = ΩR, is exactly the same as the acceleration of gravity when the mixture is closely placed into an outer surface of the high-speed centrifuge, i.e., into a region with large radius R. When if the acceleration of gravity g is under the satisfactory condition, i.e., g << ΩR, 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. On the other hand, compositions with low density are moved towards the central region of the highspeed centrifuge. That is, in the first step of carrying out the high-speed centrifugation for separating the hydrolyzed protein from the hyaluronic acid, 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. Also, in 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.

In case that the low molecular hyaluronic acid moved towards the edge region is accumulated on a surface of the high-speed centrifuge, there is a self-binding reaction between chains of the linear hyaluronic acid itself, and this self-binding reaction induces polymerization of the low molecular hyaluronic acid so as to become high molecular hyaluronic acid. Consequently, the yield of the high molecular hyaluronic acid is also increased. In case that a third high-speed centrifugation is additionally carried out after the second high-speed centrifugation for obtaining the high molecular hyaluronic acid through a pumping, 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 p₂ of the water/salt solution 2. As a result, 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. In more detail, 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. Also, 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. As a result of the accelerated spontaneous self-binding reaction, 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.

(Preferred Embodiment)

First step: Separation of hydrolyzed proteins from hyaluronic acid At the first step of high-speed centrifugation for separating 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. Herein, 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. Thereafter, the mixture of the hydrolyzed proteins, the hyaluronic acid with different molecular weights and the water/salt solution 1 is injected to an edge region of the high-speed centrifuge, which is, in turn, rotated rapidly with a predetermined angular velocity Ω. The first section of Fig. 1 shows directional movements of each components of the mixture. As shown, 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. In the mean time, 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

After the first step, the collected hyaluronic acid is mixed with a second water/salt solution 2. Herein, the density p₂ 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. Then, as the same manner of the first high-speed centrifugation, the mixture is centrifuged in high speed. As shown in the second section of Fig. 1, the low molecular hyaluronic acid is moved towards the edge region along the radius of the high-speed centrifuge so as to be accumulated on a surface of the high-speed centrifuge. Meanwhile, the high molecular hyaluronic acid is moved towards the central region of the high-speed centrifuge. As a result of the use of ultrasound wave, 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.

After the collection of the high molecular hyaluronic acid, a third high-speed centrifugation is additionally carried out. As time elapsed, it is observed that 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.

Although the preferred embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Industrial Applicability

In accordance with the present invention, 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.

Claims

What is claimed is

1. A method for purifying high molecular hyaluronic acid from a natural hyaluronic acid compound containing proteins and hyaluronic acid, comprising 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 high-speed centrifugation to separate high molecular hyaluronic acid and low molecular hyaluronic acid each from the centrifuged hyaluronic acid.

2. The method as recited in claim 1, wherein a water/salt solution is used at the step of performing the first high-speed centrifugation for separating the hydrolyzed proteins from the hyaluronic acid and the density of the water/salt solution is greater than the density of the high molecular hyaluronic acid and the density of the low molecular hyaluronic acid but less than the density of the hydrolyzed proteins.

3. The method as recited in claim 1, wherein a water/salt solution is used at the step of performing the second high-speed centrifugation for separating the high molecular hyaluronic acid from the low molecular hyaluronic acid and the density of the water/salt solution is greater than the density of the high molecular hyaluronic acid but less than the density of the low molecular hyaluronic acid.

4. The method as recited in claim 1, wherein, at the step of performing the second high-speed centrifugation for separating the high molecular hyaluronic acid and the low molecular hyaluronic acid from each other, ultrasound waves are used to accelerate the separation of the high molecular hyaluronic acid from the low molecular hyaluronic acid .

5. The method as recited in claim 4, wherein the ultrasound wave has a wave length that is longer than an average linear length of the low molecular hyaluronic acid but shorter than an average linear length of the high molecular hyaluronic acid.

6. The method as recited in claim 1, wherein a solution of papain is used for enzymatically hydrolyzing proteins .

7. The method as recited in claim 1, wherein the acceleration of the high-speed centrifugation is expressed as :

a = ΩR

where a is an acceleration, Ω is an angular velocity and R is a radius of the high-speed centrifuge; and

ΩR » g

where g is an acceleration of gravity, which is about 9.8 m/sec2.

8. The method as recited in claim 1, further comprising the step of performing a third high-speed centrifugation for the remaining low molecular hyaluronic acid to increase yields of high molecular hyaluronic acid.

9. The method as recited in claim 8, wherein ultrasound waves are used during the third high-speed centrifugation to accelerate the separation of the high molecular hyaluronic acid from the low molecular hyaluronic acid and to increase yields of high molecular hyaluronic acid .

10. The method as recited in claim 9, wherein a wavelength of the ultrasound wave is longer than an average linear length of the low molecular hyaluronic acid but shorter than an average linear length of the high molecular hyaluronic acid.