KR102150744B1 - A novel process for crosslinked hyaluronic acid derivatives - Google Patents

Abstract

The present invention is characterized by providing a novel method for effectively preparing a crosslinked hyaluronic acid derivative by washing the crosslinked hyaluronic acid derivative using a washing apparatus equipped with a washing basket.

Description

A novel process for crosslinked hyaluronic acid derivatives {A novel process for crosslinked hyaluronic acid derivatives}

The present invention relates to a method for effectively washing a crosslinked hyaluronic acid derivative in a gel state in a sterile state, while maintaining the shape of a crosslinked hyaluronic acid cut to a certain size, quickly and uniformly washing the crosslinking agent used in the crosslinking reaction and washing the crosslinked It relates to an apparatus and a method for easily recovering water.

Hyaluronic acid is composed of D-type glucuronic acid and N-acetylglucosamine, and the glycosidic bonds of β-1,4 and β-1,3 alternately and repeatedly. As a biopolymer substance that is linearly connected of disaccharides that appear, it is widely distributed throughout the human body in connective tissue, epithelial tissue, and nervous tissue. Since hyaluronic acid is a bio-derived polymer, side effects rarely occur when applied to a patient, and have hydrophilic properties due to the chemical structure of sugar. In addition, it contains a lot of moisture, has a buffering effect of physical shock and a lubricating effect against friction in joints, and is known as a biomaterial with high biocompatibility as well as being involved in skin flexibility. In addition, it has protective properties against bacterial invasion from the outside, and has a hyaluronidase in the body that is biodegradable when inserted into the human body.

Hyaluronic acid is the main component of the skin and is also involved in skin recovery. Due to its excellent biocompatibility and viscoelasticity, hyaluronic acid is a medical and medical device such as an ophthalmic surgical aid, a joint function improving agent, a drug delivery material, an anti-adhesion agent after surgery, an anti-wrinkle agent, a cosmetic aid, a scaffold and an eye drop. It is widely used for cosmetic purposes. However, since it is easily decomposed in vivo or under conditions such as acid and alkali, its use is limited, and thus efforts have been made to develop structurally stable hyaluronic acid derivatives.

Recently, active research has been conducted on a composition of a cross-linked hyaluronic acid derivative for commercial use in order to increase skin such as wrinkle improvement agents and molding aids. Typically, such hyaluronic acid derivative compositions are more resistant to degradation than non-crosslinked hyaluronic acid compositions, so that frequent re-injection is not required, and a crosslinking agent is used to form a polymer hydrogel network that is less water soluble. It is a crosslink that connects.

Crosslinked hyaluronic acid derivatives are generally prepared by stirring and mixing hyaluronic acid and a crosslinking agent in an aqueous solution, and chemically bonding between the hyaluronic acid polymer chains with a crosslinking agent. Here, when such a cross-linked hyaluronic acid derivative is administered in a living body, there is a great concern that the cross-linking agent component is recognized as a foreign substance in the living body and exerts adverse effects such as causing an inflammatory reaction. In fact, according to a paper published in 2003 by cancer research under the U.S. Food and Drug Administration (FDA), it is reported that BDDE, which is most commonly used as a crosslinking agent, is dangerous when the residual amount is 2 ppm or more. It's an important issue. It is not easy to completely remove the crosslinking agent, but the hyaluronic acid crosslinked product induces a crosslinking reaction in a liquid phase by mixing a crosslinking agent and hyaluronic acid in an alkaline aqueous solution, but in general, the reactivity is very low. It is necessary to increase the reaction concentration or add a large amount of crosslinking agent. In this case, it has a problem that it is more difficult to completely remove the crosslinking agent added in a large amount during the purification process.

US Patent Application Publication No. 2003/0094719A1 discloses a method of preparing a crosslinked hyaluronic acid derivative by a simple method. A hyaluronic acid solution is prepared and the pH is adjusted with sodium hydroxide to provide a method of crosslinking with a multifunctional epoxy compound. As a preferred multifunctional epoxy compound used as a crosslinking agent, 1,4-butanediol diglycidyl ether (BDDE), ethylene glycol diglycidyl ether (EGDGE), sorbitol polyglycidyl ether, etc. are illustrated. However, this invention does not suggest an efficient method for removing the residual crosslinking agent after the crosslinking reaction.

On the other hand, U.S. Patent Application Publication No. 2006/0105022A1 discloses that the crosslinked hyaluronic acid derivative gel is decomposed after being used in the human body. Disclosed is a method for preparing a crosslinked hyaluronic acid derivative gel having a binding rate. Examples of crosslinking agents include divinyl sulfone, BDDE, and EGDGE. However, this invention also does not suggest a method for effective removal of the crosslinking agent remaining after the crosslinking reaction.

In Example 2 of U.S. Patent No.8357795, in order to remove the unreacted crosslinking agent and stabilize the pH close to neutral, the crosslinked product is filled in a dialysis membrane, and the bath is usually changed while dialysis with a buffer solution for up to several days. Is disclosed. This method is not suitable for mass production, and it is difficult to remove the crosslinking agent or it takes a long time.

The present invention is to solve the problem that it is difficult to remove the crosslinking agent remaining after the crosslinking reaction of hyaluronic acid by the conventional method, it takes a long time, or is not suitable for mass production, and is prepared by effectively washing the crosslinked hyaluronic acid derivative. I would like to provide a way to do it.

Cross-linking agents used in the cross-linking reaction of hyaluronic acid are recognized as a very important problem because there is a great concern about side effects such as causing an inflammatory reaction in the human body. However, in the process of preparing a crosslinked hyaluronic acid derivative, it is very difficult to sufficiently remove the remaining crosslinking agent after the hyaluronic acid and the crosslinking agent crosslink.

Accordingly, the present invention is to provide a method and apparatus for effectively and in large quantities washing of the crosslinking agent remaining after the crosslinking reaction in a short time in the preparation of a crosslinked hyaluronic acid derivative, and easily recovering the washed crosslinked product.

To this end, the present invention provides a method of washing crosslinked hyaluronic acid derivatives using a washing device equipped with a basket.

The method for efficiently washing the crosslinked hyaluronic acid derivative according to the present invention is

Putting a crosslinked product cut to a certain size in a washing basket in which a mesh is formed,

Fixing and mounting the washing basket inside the washing tank,

Injecting the buffer solution into the washing tank,

Washing the crosslinked product in the washing basket in a buffer solution by rotating the washing tank,

Draining the washed buffer solution,

Injecting the buffer solution, rotating the washing tank, and draining the buffer solution repeatedly until the crosslinking agent used in the reaction is completely removed,

And recovering the washed crosslinked hyaluronic acid derivative.

In order to completely remove the crosslinking agent by washing the crosslinked hyaluronic acid derivative and to make the hyaluronic acid derivative in a gel state, washing through dialysis for a long time was a classic method.

However, the present invention provides a simple and efficient method of removing the crosslinking agent by using a washing apparatus equipped with a washing basket having a mesh formed thereon. After crosslinking reaction by mixing the crosslinking agent and hyaluronic acid, in the present invention, the crosslinked product is cut into a certain size and the crosslinking agent used in the reaction is washed while maintaining the size. At this time, the alkali component (usually sodium hydroxide) used in the crosslinking reaction is also removed at the same time. As the washing buffer solution, a conventional solution, for example, a phosphate buffer solution (PBS) may be used.

If the lumped crosslinked product is not cut or is cut too large, it takes too long to proceed with the washing process, and the washing is not performed evenly to the inside of the crosslinked product, which may cause problems in uniformity. There may be losses. Therefore, it is possible to efficiently produce hyaluronic acid derivatives reproducibly only by removing the crosslinking agent used in the reaction while maintaining the shape and removing the crosslinking agent used in the reaction while maintaining the shape by uniformly cutting the side length of 0.5~1.0cm and easily in a cube shape have.

Through this process, the efficiency and convenience of washing are increased, and the formation of particles of the hyaluronic acid derivative having a uniform size is advantageous.

The method of washing crosslinked hyaluronic acid using the washing apparatus of the present invention can proceed while maintaining the size and shape of the crosslinked hyaluronic acid derivative having a certain size and shape in a sterile state, as well as fast without loss of the crosslinked hyaluronic acid derivative. It can be easily and efficiently recovered within time. The method according to the present invention also has the advantage that the final crosslinked hyaluronic acid product can be repeatedly and reproducibly obtained in large quantities with homogeneous quality after removal of the crosslinking agent. It is also possible to simultaneously remove the alkali component (usually sodium hydroxide) used in the crosslinking reaction.

1 is a front view of a cleaning device according to the present invention.
2 is a plan view of the cleaning apparatus of FIG. 1.
3 is a front view of a washing tank in the washing apparatus of FIG. 1.
4 is a right cross-sectional view of the washing tank of FIG. 3.
5 is a plan view of the washing tank of FIG. 3.
6 is a front view of a washing basket in the washing tank of FIG. 3.
7 is a plan view of a washing basket in the washing tank of FIG. 3.

Hereinafter, a technical problem to be achieved by the present invention will be described in detail with reference to the accompanying drawings. However, the present invention is not limited or limited by the following description.

1 is a front view of the washing apparatus according to the present invention, FIG. 2 is a plan view of the washing apparatus of FIG. 1, FIG. 3 is a front view of a washing tank among the washing apparatus of FIG. 1, and FIG. 4 is a right side of the washing tank of FIG. It is a cross-sectional view, FIG. 5 is a plan view of the washing tank of FIG. 3, FIG. 6 is a front view of a washing basket in the washing tank of FIG. 3, and FIG. 7 is a plan view of a washing basket in the washing tank of FIG. 3.

), 컨트롤 패널(

)을 포함한다.
Referring to FIGS. 1 to 7, the washing apparatus for washing the crosslinked hyaluronic acid derivative includes a washing tank (①), a washing basket (④), and a rotary motor (

), control panel (

).

)로 이루어져 있다. 세척 바스켓(④)의 옆면과 밑면은 전체에 메쉬(mesh)가 형성되어 있어 완충 용액의 출입은 자유롭지만 가교된 히알루론산 유도체는 세척 바스켓에서 빠져나가지 않도록 제공된다. 세척 바스켓은 세척 탱크(①)의 상부와 하부에 장착할 수 있으며, 세척 탱크(①)에 탈부착이 용이하다.
The washing basket (④) is made of a hollow hexahedron or a cylindrical shape, preferably a cylindrical shape, and the upper surface is a washing basket cover (

). The side and bottom of the washing basket (④) are entirely meshed, so that the buffer solution can be freely accessed, but the crosslinked hyaluronic acid derivative is provided to prevent it from escaping from the washing basket. The washing basket can be mounted on the upper and lower portions of the washing tank (①), and it is easy to attach and detach to the washing tank (①).

The mesh of the washing basket (④) is 500㎛ ~ 4,000㎛, preferably 1,500㎛ ~ 2,500㎛ is suitable. If the mesh is too small, it is difficult to enter the buffer solution during the washing process, so the efficiency of washing decreases. If the mesh is too large, the crosslinked material cut into a certain size will exit the washing basket (④) or the washing process The pulverized fragments of the crosslinked hyaluronic acid derivative that may occur in may escape, thereby reducing the yield.

The washing tank (①) can be formed in a hexahedron, a cylindrical shape, or a double cone type.

)를 이용하여 360도 회전이 가능하며, 필요에 따라 회전 각도는 1도~360도로 설정하여 사용할 수 있다. 회전 속도의 설정이 가능하고, 회전 시간도 설정이 가능하다. 완충용액을 채워 일정 시간 회전하여 세척하면 완충용액을 배출하고, 새로운 완충용액을 주입하고 세척하는 작업을 반복하여 세척한다.The washing tank (①) is a rotating motor (

) Can be used to rotate 360 degrees, and the rotation angle can be set from 1 to 360 degrees as needed. The rotation speed can be set and the rotation time can be set. If the buffer solution is filled and rotated for a certain period of time and washed, the buffer solution is discharged, and a new buffer solution is injected and washing is repeated.

In addition, the rotational speed of the washing tank (①) is 1rpm ~ 10rpm, preferably at a slow speed of 2rpm ~ 6rpm so that the shape of the crosslinked hyaluronic acid derivative in the buffer solution is crushed and not damaged. If the rotational speed is high, it is crushed and lost through the mesh of the washing basket (④), so that the yield may decrease. The rotation time is 10 to 90 minutes, preferably 30 to 50 minutes, and the efficiency of washing is increased by repeating the discharge and injection of the buffer solution. It was confirmed that the number of washing repetitions for washing is usually 3 to 5 times, and the crosslinking agent is completely removed. Therefore, it is possible to provide a method suitable for mass production by easily removing the crosslinking agent in a short time.

)을 이용하여 이송하고, 배출은 배출부 패럴(⑫)을 이용하여 진행한다. The injection and discharge of the buffer solution can be easily carried out through a parallel. The buffer solution prepared in a separate container can be used as a parallel (⑬ or ⑭ or ⑮ or

) To transfer, and discharging is carried out by using the discharging parallel (⑫).

,

)를 통해 세척 탱크의 내부 진행 사항을 확인하며 작업할 수 있다.Also sight glass (

,

), you can check the internal progress of the washing tank and work.

As described above, the present invention has been described with reference to specific details for the implementation of the present invention, but those skilled in the art can variously modify and modify the present invention within the scope not departing from the technical concept of the present invention described in the following claims. The present invention is not limited thereto as it can be changed.

주입부 패럴

,

사이트 글라스

컨트롤 패널

회전 모터

안전 커버

세척 바스켓 커버

세척 바스켓 손잡이 ① Washing tank
④ Washing basket
⑦ Wash tank cover
⑫ Discharge part parallel
⑬, ⑭, ⑮,

Injection part parallel

,

Sight glass

Control panel

Rotary motor

Safety cover

Washing basket cover

Washing basket handle

Claims (4)

In the washing device for removing the crosslinking agent used in the reaction after the crosslinking reaction by mixing the crosslinking agent and hyaluronic acid,
Equipped with a rotating motor, the rotating motor is connected to the washing tank,
The washing tank is equipped with a washing basket,
The washing basket is formed with a mesh,
A washing apparatus, characterized in that it has a structure in which the washing buffer solution in the washing tank can flow freely into and out of the washing basket. The cleaning apparatus according to claim 1, wherein the mesh of the cleaning basket is 500 µm to 4,000 µm. The washing apparatus according to claim 1 or 2, wherein a buffer solution injection panel and a discharge panel are disposed in the washing tank. Equipped with a rotating motor, the rotating motor is connected to the washing tank,
The washing tank is equipped with a washing basket,
The washing basket is formed with a mesh,
Using a washing device having a structure in which the washing buffer solution in the washing tank can flow freely into and out of the washing basket,
Adding a crosslinked hyaluronic acid derivative in a washing basket in which a mesh is formed;
Injecting a buffer solution into the washing tank;
Washing the crosslinked product in the washing basket in a buffer solution by rotating the washing tank;
Discharging the washed buffer solution;
Including
A method of washing the crosslinking agent used in the reaction after the crosslinking reaction by mixing the crosslinking agent and hyaluronic acid.

Images