WO2022062011A1 - Method for purifying low-molecular weight fucosylated glycosaminoglycan by means of tangential flow ultrafiltration - Google Patents

Abstract

Provided is a method for purifying low-molecular weight fucosylated glycosaminoglycan by means of tangential flow ultrafiltration. The method comprises the following steps: (1) dissolving a crude product in a certain proportion of water, and filtering same to obtain a mother solution; (2) installing a 30 kd membrane bag ultrafiltration system, and carrying out ultrafiltration on the mother solution, and then collecting the ultrafiltrate; (3) carrying out ultrafiltration by using a 3 kd membrane bag ultrafiltration system to remove small molecule organic and inorganic matter, and sampling trapped fluid; (4) carrying out ultrafiltration by using a 10 kd membrane ultrafiltration system, and collecting a percolate; and (5) finally, carrying out concentration by using the 3 kd membrane ultrafiltration system. The method provided by the invention has the advantages of a high efficiency, rapidness, mild conditions and a high repeatability, and provides a stable ultrafiltration process with a consistent product quality between batches for industrial production.

Description

A method of purifying low molecular weight fucosylated glycosaminoglycans using tangential flow ultrafiltration technical field

The present invention relates to the field of medical technology, more particularly to the purification of low molecular weight fucosylated glycosaminoglycan compounds.

Background technique:

Cardiovascular and cerebrovascular diseases have high morbidity, disability, mortality, recurrence and complications, seriously endangering human health and quality of life. Thrombosis is one of the main causes of cardiovascular and cerebrovascular diseases. Antithrombotic drugs, including anticoagulants, are the first-line clinical drugs for the treatment of cardiovascular and cerebrovascular diseases and occupy an important position in the pharmaceutical market. At present, the research on anticoagulant drugs at home and abroad is still in full swing, and generations of drugs with low side effects and good anticoagulant effects have been developed, such as apixaban, rivaroxaban, dabigatran etexilate, heparin sodium, enol Drugs such as heparin sodium, but the defects of such drugs are mainly the risk of severe bleeding and thrombocytopenia related to the target. Therefore, there is a need for new anticoagulant drugs with superior pharmacological and pharmacodynamic characteristics. The core of the research and development of new anticoagulant drugs is to effectively avoid bleeding tendency, while the research on innovative drugs with low bleeding tendency has not yet made a breakthrough.

Fucosylated glycosaminoglycans (FG) derived from sea cucumbers are a class of glycosaminoglycan derivatives with fucose side chain substitution, with a main and side chain structure. esterification (J. Biol. Chem., 1996, 271: 23973-23984; Mar. Drugs, 2013, 11: 399-417). Naturally derived FG has strong anticoagulant activity (Thromb. Haemost., 2008, 100:420-428; J. Biol. Chem., 1996, 271:23973-23984). However, natural FG still has extensive and contradictory pharmacological effects, including induction of platelet aggregation, bleeding tendency and activation of XII, etc. (Thromb. Haemost., 1988, 59: 432-434; Thromb. Haemost., 1997, 65(4) : 369-373). After proper depolymerization by chemical means, low-molecular-weight fucosylated glycosaminoglycans can be obtained. The separation of macromolecular fucosylated glycosaminoglycans and other substances has not been reported so far, so the present invention provides a method for purifying low molecular weight fucosylated glycosaminoglycans.

Tangential flow filtration is a widely used separation method in the field of biotechnology, usually used for the concentration, desalination or purification of biological products. In the tangential flow filtration process, the liquid flow direction is perpendicular to the filtration direction. Substances smaller than the die pore or nominal molecular weight (NMWL) can pass through the membrane, while substances larger than the die pore are generally retained. The retention performance of ultrafiltration membrane refers to the retention and permeation performance of the membrane, which is affected by the composition, concentration, tangential flow rate, pressure, etc. of the filtrate.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a method for purifying low-molecular-weight fucosylated glycosaminoglycans, using ultrafiltration, a purification method with high efficiency, rapidity, mild conditions and high repeatability, to provide stable, batch-to-batch products for industrial production. Consistent quality ultrafiltration process.

For achieving the above object, the technical scheme adopted in the present invention is as follows:

(1) Dissolving the low molecular weight fucosylated glycosaminoglycan crude product obtained by semi-synthesis in a certain proportion of purified water, and filtering through 1 μm and 0.45 μm filter elements, respectively, to obtain an ultrafiltration mother liquor;

(2) Install the ultrafiltration system: take out the 30kd ultrafiltration membrane package from the protective solution, install it in the middle of the fixture and clamp it, connect the pipeline, first wash it with a small amount of purified water, then circulate it with 0.1M NaOH for 30min, and then wash it with purified water After reaching neutrality and passing the air tightness test, turn on the peristaltic pump and adjust the flow rate to a certain flow rate, adjust the inlet and outlet pressures to make the transmembrane pressure meet the requirements, start ultrafiltration, and replenish water 3-6 times after reaching a certain volume, and combine all the permeation liquid;

(2) Replace the 3kd ultrafiltration membrane package, take out the 3kd ultrafiltration membrane package from the protective solution, install it in the middle of the fixture and clamp it, connect the pipeline, first wash with a small amount of purified water, and then circulate it with 0.1M NaOH for 30min, and then use Wash the purified water to neutrality, test the air tightness, adjust the speed of the peristaltic pump to a certain flow rate after passing the test, adjust the inlet and outlet pressure to make the transmembrane pressure meet the requirements, start ultrafiltration, and add water after ultrafiltration to a certain volume remaining until the salt is detected. After the content is qualified, the ultrafiltration is stopped, and the retentate is collected;

(3) Replace the 10kd ultrafiltration membrane package, take out the 10kd ultrafiltration membrane package from the protective solution, install it in the middle of the fixture and clamp it, connect the pipeline, first wash with a small amount of purified water, and then circulate it with 0.1M NaOH for 30min, then Wash with purified water to neutrality. After passing the air tightness test, adjust the speed of the peristaltic pump to a certain flow rate, adjust the inlet and outlet pressures to make the transmembrane pressure meet the requirements, start ultrafiltration and collect the permeate, and add water after ultrafiltration to a certain volume. 3-6 times, combine all the permeate;

(4) 3kd ultrafiltration and concentration, take out the 10kd ultrafiltration membrane package from the protective solution, install it in the middle of the clamp and clamp it, connect the pipeline, first wash with a small amount of purified water, then circulate with 0.1M NaOH for 30min, and then wash with purified water To neutrality, after passing the air tightness test, adjust the speed of the peristaltic pump to a certain flow rate, adjust the inlet and outlet pressures to make the transmembrane pressure meet the requirements, start ultrafiltration, stop after ultrafiltration to a certain volume remaining, and release the retentate, a small amount Rinse the membrane package and pipeline with water, combine with the retentate, and freeze-dry;

(5) Detect its molecular weight and small molecular impurities such as inorganic salts.

In the method of the present invention, the concentration range of the crude product after dissolving with purified water is 10-60 mg/ml, preferably 20-40 mg/ml;

The ultrafiltration membrane package material is regenerated fiber material or polyethersulfone material, and the flow channel is C-type or V-type, wherein regenerated cellulose material is preferred, and the flow channel is preferably V-type. According to the requirements of the product volume, the membrane package can be separately or superimposed;

During 30kd ultrafiltration, the peristaltic pump flow rate is 1 to 5L/min, preferably 2 to 3.5L/min, and during 10kd ultrafiltration, the peristaltic pump flow rate is 1 to 5L/min, preferably 2 to 3.5L/min, and 3kd ultrafiltration. During filtration, the peristaltic pump flow rate is 3~10L/min, preferably 4~7L/min; the transmembrane pressure is 0.8~1.2bar, the inlet pressure is 1.0~1.5bar, and the outlet pressure is 0.6~0.9bar;

In the process of removing small molecular impurities by 3kd ultrafiltration, water replenishment starts when the volume of the retentate is 1/5 to 1/10 of the total volume, the replenishment volume is 1/5 to 1/10 of the total volume, and the number of replenishment times is 5 to 10 times.

The entire ultrafiltration process must be carried out in the order of 30kd→3kd→10kd→3kd, and the order cannot be reversed.

Due to its own viscosity, and the composition of the low molecular weight fucosylated glycosaminoglycan crude product in the present invention, in addition to the low molecular weight fucosylated glycosaminoglycan of the target product, it also contains small molecular inorganic salts, and other macromolecular saccharides, it is difficult to separate and purify using conventional methods, while the tangential flow ultrafiltration because its liquid flow direction is perpendicular to the filtration direction, the viscous macromolecular saccharides are not easy to aggregate on the surface of the membrane envelope, As ultrafiltration progresses, there is no blockage of the membrane cartridges that causes flux to drop, so efficient operation can be maintained all the time. According to the difference of target molecular weight and the composition of impurities, ultrafiltration membrane packages with different molecular weight cut-off can be used. The interception range of 30kd ultrafiltration membrane package is the substance with molecular weight greater than 30000D, and the interception range of 10kd ultrafiltration membrane package is the substance with molecular weight greater than 10000D , while the cut-off range of the 3kd ultrafiltration membrane package is greater than 3000D molecular weight.

The present invention adopts three kinds of ultrafiltration membrane packages with different molecular weight cut-offs for ultrafiltration, and through the purification method of controlling parameters such as different crude product concentrations, different transmembrane pressures and pump flow rates, the final product low molecular weight fucosylated glycosamine is obtained. The molecular weight of the glycan meets the requirements of 3000-10000 Da, and at the same time, it is efficient, fast, mild in conditions and high in repeatability, and can be used in industrial production.

detailed description

The present invention can be better understood from the following examples. However, those skilled in the art can easily understand that the content described in the embodiments is only used to illustrate the specific embodiments of the present invention, and should not and will not limit the scope of the present invention.

Materials and Reagents

The crude product of low molecular weight fucosylated glycosaminoglycan is prepared with reference to the method disclosed in Chinese Patent 201410007855. The fucosylated glycosaminoglycan extracted from sea cucumber is used as the starting material, and is subjected to carboxyl esterification, beta elimination and depolymerization. , alkaline hydrolysis deesterification, decolorization, end group reduction and other chemical processes to obtain a crude product of low molecular weight fucosylated glycosaminoglycan, which mainly contains the target product, some large molecular weight heteropolysaccharides that do not undergo depolymerization, inorganic salt etc.

30kd, 10kd, 3kd ultrafiltration membrane packs and peristaltic pumps were purchased from Millipore, 0.5 square meters, sodium hydroxide was commercially available analytical grade, purchased from Beijing Chemical Factory; purified water was made by the workshop.

Example 1

The crude low molecular weight fucosylated glycosaminoglycan was purified using the following steps:

(1) Add 15 L of purified water to the crude product, the concentration is about 28 mg/ml, and pass through 1 μm and 0.45 μm filter membranes respectively. The purpose of filtration here is to remove some insoluble particles introduced from outside, so as to prevent damage or blockage of the membrane package, Therefore, the initial concentration before and after filtration did not change. A total of about 23L of filtrate was obtained by washing the filtration system, and ultrafiltration was carried out.

(2) Take a 30kd regenerated cellulose membrane package, install the ultrafiltration equipment, rinse with 10L purified water for 10 minutes, and rinse the membrane package with 0.1M sodium hydroxide solution for 30 minutes, then wash it with water until it is neutral and has good air tightness, and starts ultrafiltration. sample. The outlet pressure is 0.8bar, the inlet pressure is 0.8, 1.0bar, the pump flow rate is 1.2L/min, and the water is replenished after ultrafiltration to about 1L, 2.5L each time, a total of 6 times, and the combined permeate is about 38L. After the ultrafiltration, wash with water, pack with 0.1N sodium hydroxide solution membrane, and end the ultrafiltration.

(3) Take a 3kd regenerated cellulose membrane pack, install the ultrafiltration equipment, rinse with 10L purified water for 10 minutes, and rinse the membrane pack with 10L 0.1M sodium hydroxide solution for 30 minutes, then wash it with water until it is neutral, and the air tightness is good. Start Ultrafiltration sample, the outlet pressure is 1.0bar, the inlet pressure is 1.2bar, the pump flow rate is 3.9L/min, and the water is replenished when the ultrafiltration reaches 5L, 5L each time, and the permeating end is detected by AgNO ₃ until there is no ^Cl- residue, and the ultrafiltration is ended. Dilute the retentate to 18L.

(4) Take a 10kd regenerated cellulose membrane package, install the ultrafiltration equipment, rinse with 10L purified water for 10 minutes, and then rinse the membrane package with 10L 0.1M sodium hydroxide solution for 30 minutes, then wash it with water until it is neutral, and test the air tightness. Ultrafiltration sample, outlet pressure 0.7bar, inlet pressure 1.1bar, pump flow rate 1.2L/min, ultrafiltered to 1.0L, and then replenished with 2.5L of water for a total of 6 times, and combined all the permeate.

(5) Take a 3kd regenerated cellulose membrane pack, install the ultrafiltration equipment, rinse with 10L purified water for 10 minutes, and rinse the membrane pack with 10L 0.1M sodium hydroxide solution for 30 minutes, then wash it with water until it is neutral, and test the air tightness. Ultrafiltration sample, outlet pressure 1.1bar, inlet pressure 1.3bar, pump flow rate 4.0L/min, stop ultrafiltration when ultrafiltration reaches 1.5L, rinse membrane capsule and pipeline with 500ml×3 times of purified water, combine retentate, filter 0.22 μm filter, then lyophilized.

(6) HPLC detects the weight-average molecular weight of the freeze-dried product, and the results are as follows:

The Mw of the sample LGAG-3 obtained in Example 1 is 13709, which has achieved the purpose of separation and purification, but still exceeds the molecular weight requirement range of 3000-10000, and requires further optimization of ultrafiltration conditions. Through the previous research, it was found that the pump flow rate of the 30kd and 10kd ultrafiltration stages has a great influence on the molecular weight, and the small pump flow rate may cause some large molecular weight substances to pass through the membrane package, making the proportion of large molecular weight substances high, and the increase of Mw. Big.

Example 2

The crude low molecular weight fucosylated glycosaminoglycan was purified using the following steps:

(1) Add 22 L of purified water to the crude product, pass through 1 μm and 0.45 μm filter membranes respectively, and rinse the filtration system to obtain a total of about 33 L of filtrate with a concentration of 36 mg/ml.

(2) Take a 30kd regenerated cellulose membrane package, install the ultrafiltration equipment, rinse with 10L purified water for 10 minutes, and rinse the membrane package with 0.1M sodium hydroxide solution for 30 minutes, then wash it with water until it is neutral and has good air tightness, and starts ultrafiltration. Crude product sample. The outlet pressure is 0.8bar, the inlet pressure is 1.2bar, the pump flow rate is 2.3L/min, and the water is replenished after ultrafiltration to about 1.5L, 3.6L each time, a total of 6 times, and the combined permeate is about 54L. After the ultrafiltration, wash with water, pack with 0.1M sodium hydroxide solution membrane, and end the ultrafiltration.

(3) Take two 3kd regenerated cellulose membrane packs, install the ultrafiltration equipment, rinse with 10L of purified water for 10 minutes, and then rinse the membrane packs with 10L of 0.1M sodium hydroxide solution for 30 minutes, then wash with water until neutral, and the air tightness is good. , start the ultrafiltration of the sample, the outlet pressure is 1.0bar, the inlet pressure is 1.2bar, the pump flow rate is 3.5L/min, and the water is replenished when the ultrafiltration reaches 5L, each 5L, and the permeating end is detected by AgNO ₃ until there is no ^Cl- residue, and the ultrafiltration is over. Filter and dilute the retentate to 22L.

(4) Take a 10kd regenerated cellulose membrane package, install the ultrafiltration equipment, rinse with 10L purified water for 10 minutes, and then rinse the membrane package with 10L 0.1M sodium hydroxide solution for 30 minutes, then wash it with water until it is neutral, and test the air tightness. Ultrafiltration sample, outlet pressure 0.8bar, inlet pressure 1.1bar, pump flow rate 2.3L/min, ultrafiltration to 1.5L, and then replenishing 3.6L of water for a total of 6 times, and combining all the permeate.

(5) Take a 3kd regenerated cellulose membrane pack, install the ultrafiltration equipment, rinse with 10L purified water for 10 minutes, and rinse the membrane pack with 10L 0.1M sodium hydroxide solution for 30 minutes, then wash it with water until it is neutral, and test the air tightness. Ultrafiltration sample, outlet pressure 1.0bar, inlet pressure 1.2bar, pump flow rate 3.5L/min, stop ultrafiltration when ultrafiltration reaches 1.5L, rinse the membrane package and pipeline with 500ml×3 times of purified water, combine the retentate, filter 0.22 μm filter, then lyophilized.

(6) HPLC detects the weight-average molecular weight of the purified low-molecular-weight fucosylated glycosaminoglycan, and the results are as follows:

The weight-average molecular weight of the purified low-molecular-weight fucosylated glycosaminoglycan is in the range of 3000-10000 Da, and the result of the method meets the requirements of the relevant production technology.

Example 3

The following steps were used to purify the crude low molecular weight fucosylated glycosaminoglycan

(1) Add 18 L of purified water to the crude product, pass through a 1 μm and 0.45 μm filter membrane, and rinse the filtration system to obtain a total of about 23 L of filtrate with a concentration of 25 mg/ml.

(2) Take a 30kd regenerated cellulose membrane package, install the ultrafiltration equipment, rinse with 10L purified water for 10 minutes, and rinse the membrane package with 0.1M sodium hydroxide solution for 30 minutes, then wash it with water until it is neutral and has good air tightness, and starts ultrafiltration. sample. The outlet pressure is 0.7bar, the inlet pressure is 1.4bar, the pump flow rate is 2.7L/min, and the water is replenished after ultrafiltration to about 1.5L, 3.6L each time, a total of 6 times, and the combined permeate is about 45L. After the ultrafiltration, wash with water, pack with 0.1MN sodium hydroxide solution membrane, and end the ultrafiltration.

(3) Take two 3kd regenerated cellulose membrane packs, install the ultrafiltration equipment, rinse with 10L of purified water for 10 minutes, and then rinse the membrane packs with 10L of 0.1M sodium hydroxide solution for 30 minutes, then wash with water until neutral, and the air tightness is good. , start the ultrafiltration of the sample, the outlet pressure is 1.1bar, the inlet pressure is 1.3bar, the pump flow rate is 4.0L/min, and the water is replenished when the ultrafiltration reaches 5L, 5L each time, and the permeating end is detected by AgNO ₃ until there is no ^Cl- residue, and the ultrafiltration is over. Filter and dilute the retentate to 18L.

(4) Take a 10kd regenerated cellulose membrane package, install the ultrafiltration equipment, rinse with 10L purified water for 10 minutes, and then rinse the membrane package with 10L 0.1M sodium hydroxide solution for 30 minutes, then wash it with water until it is neutral, and test the air tightness. Ultrafiltration sample, outlet pressure 0.6bar, inlet pressure 1.3bar, pump flow rate 2.9L, ultrafiltered to 1.5L, and then replenished with 3.6L of water, a total of 6 times of water, and combined all the permeate.

(5) Take a 3kd regenerated cellulose membrane pack, install the ultrafiltration equipment, rinse with 10L purified water for 10 minutes, and rinse the membrane pack with 10L 0.1M sodium hydroxide solution for 30 minutes, then wash it with water until it is neutral, and test the air tightness. Ultrafiltration sample, outlet pressure 1.1bar, inlet pressure 1.3bar, pump flow rate 4.0L/min, stop ultrafiltration when ultrafiltration reaches 1.5L, rinse membrane capsule and pipeline with 500ml×3 times of purified water, combine retentate, filter 0.22 μm filter, then lyophilized.

(6) HPLC detects its weight-average molecular weight, and the results are as follows:

The weight-average molecular weight of the purified low-molecular-weight fucosylated glycosaminoglycan is in the range of 3000-10000 Da, and the result of the method meets the requirements of the relevant production technology.

Claims (10)

1. A method for purifying low molecular weight fucosylated glycosaminoglycans using tangential flow ultrafiltration, comprising the following steps:

   (1) The crude product of low molecular weight fucosylated glycosaminoglycan was dissolved in purified water, and the mother liquor was obtained by ultrafiltration through 1 μm and 0.45 μm filter elements respectively;

   (2) use the ultrafiltration system installed with the 30kd ultrafiltration membrane package to carry out tangential flow ultrafiltration to the mother liquor obtained in step (1), and collect the permeate;

   (3) use the ultrafiltration system installed with the 3kd ultrafiltration membrane package to carry out tangential flow ultrafiltration to the permeate collected in step (2), and collect the retentate;

   (4) use the ultrafiltration system installed with 10kd ultrafiltration membrane package to carry out tangential flow ultrafiltration to the retentate collected in step (3), and collect permeate;

   (5) Use an ultrafiltration system equipped with a 3kd ultrafiltration membrane package to perform tangential flow ultrafiltration on the permeate collected in step (4), and collect the retentate, which is the purified low molecular weight fucosylated glycosaminoglycan .
2. The method for purifying low-molecular-weight fucosylated glycosaminoglycans using tangential flow ultrafiltration according to claim 1, wherein the concentration range of the low-molecular-weight fucosylated glycosaminoglycan crude product after dissolving in purified water It is 10～60mg/ml.
3. The method for purifying low-molecular-weight fucosylated glycosaminoglycans using tangential flow ultrafiltration according to claim 1, wherein the ultrafiltration membrane package material is regenerated fiber material or polyethersulfone material, and the flow channel Type C or V.
4. The method for purifying low molecular weight fucosylated glycosaminoglycans using tangential flow ultrafiltration according to claim 1, wherein:

   The step (2) is to place the 30kd ultrafiltration membrane package in the middle of the fixture to install an ultrafiltration system, connect the pipeline, and after the air tightness test is qualified, turn on the peristaltic pump and adjust to a certain flow rate, adjust the inlet and outlet pressures, and make the membrane across the membrane. When the pressure meets the requirements, start ultrafiltration, and after ultrafiltration to a certain volume, add water for 3 to 6 times, and collect all the permeate together;

   The step (3) is to replace the ultrafiltration system with a 3kd ultrafiltration membrane package, test the air tightness after installation, adjust the peristaltic pump rotational speed to a certain flow rate after passing the test, and adjust the inlet and outlet pressures to make the transmembrane pressure meet the requirements. , start ultrafiltration, replenish water after ultrafiltration to a certain volume remaining, detect the filtrate through the end until there is no Cl ^- residue, stop ultrafiltration, and collect the retentate;

   The step (4) is to replace the ultrafiltration system with a 10kd ultrafiltration membrane package, test the air tightness after installation, adjust the peristaltic pump rotational speed to a certain flow rate after passing the test, and adjust the inlet and outlet pressures to make the transmembrane pressure meet the requirements. , start ultrafiltration and collect the permeate, add water 3 to 6 times after ultrafiltration to a certain volume, and then combine and collect all permeate;

   The step (5) is to replace the ultrafiltration system with a 3kd ultrafiltration membrane package, test the air tightness after installation, adjust the peristaltic pump rotational speed to a certain flow rate after passing the test, and adjust the inlet and outlet pressures to make the transmembrane pressure meet the requirements. , start ultrafiltration, stop after ultrafiltration to a certain volume remaining, release the retentate, rinse the membrane package and pipeline with a small amount of water, and combine with the retentate to collect for freeze-drying.
5. The method for purifying low-molecular-weight fucosylated glycosaminoglycans using tangential flow ultrafiltration according to claim 4, wherein the flow rate of the peristaltic pump is 1 to 5L/min when using the 30kd membrane package for ultrafiltration, and the 10kd membrane package The flow rate of peristaltic pump is 1-5L/min during ultrafiltration, and the flow rate of peristaltic pump is 3-10L/min during ultrafiltration with 3kd membrane.
6. The method for purifying low-molecular-weight fucosylated glycosaminoglycans using tangential flow ultrafiltration according to claim 5, wherein the peristaltic pump flow rate is 2-3.5L/min when using 30kd membrane-packed ultrafiltration, and the 10kd membrane The flow rate of the peristaltic pump is 2-3.5L/min during the ultrafiltration of the membrane, and the flow rate of the peristaltic pump is 4-7L/min during the ultrafiltration of the 3kd membrane.
7. The method for purifying low-molecular-weight fucosylated glycosaminoglycans using tangential flow ultrafiltration according to claim 5, wherein in the ultrafiltration process, the transmembrane pressure is 0.8-1.2 bar, and the inlet pressure is 1.0-1.5 bar , the outlet pressure is 0.6 ~ 0.9bar.
8. The method for purifying low-molecular-weight fucosylated glycosaminoglycans using tangential flow ultrafiltration according to claim 5, wherein in the 3kd membrane-encapsulated ultrafiltration process, the volume of the retentate is 1/1 of the total volume before ultrafiltration Start to replenish water at 5-1/10, the replenishment volume is 1/5 to 1/10 of the total volume before ultrafiltration, and the number of replenishment is 5 to 10 times.
9. The method for purifying low-molecular-weight fucosylated glycosaminoglycans using tangential flow ultrafiltration according to claim 5, wherein in the step ( ₃ ), the filtrate through the end is detected by AgNO until no ^Cl- remains. Ultrafiltration.
10. The method for purifying low-molecular-weight fucosylated glycosaminoglycans using tangential flow ultrafiltration according to claim 5, wherein the weight-average molecular weight Mw of the freeze-dried product is 3000-10000 Da.