N-deacetylated heparosan-containing composition

Abstract

Disclosed is an N-deacetylated heparosan-containing composition, wherein the electrical conductivity per unit concentration (g / L) of N-deacetylated heparosan is less than 0.9 mS / cm.

Description

N-deacetylated heparosan-containing composition 【0001】 This disclosure relates to N-deacetylated heparosan-containing compositions and methods for producing the same. This disclosure also relates to methods for producing sulfated heparosan-containing compositions. 【0002】 N-acetylheparosan (hereinafter referred to as "heparosan") is a polysaccharide having a repeating disaccharide structure composed of glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc). Heparosan is known as a capsular polysaccharide produced by Escherichia coli K5 strain and Pasteurella multicida, among others. 【0003】 Heparin is a type of sulfated polysaccharide with heparosan as its backbone and is widely used as an anticoagulant. Industrially, heparin extracted and purified from the intestinal mucosa of pigs has been mainly used. However, since a fatal accident occurred in 2008 due to the contamination of porcine-derived heparin with impurities, there is a need for the production of non-animal-derived heparin with controlled manufacturing processes and quality. As a method for producing non-animal-derived heparin, a method has been reported in which heparosan is deacetylated, isomerized, and sulfated by combining chemical and enzymatic methods to obtain heparin (Patent Documents 1-3, Non-Patent Document 1). 【0004】 In the enzymatic method for producing heparin, it is known that the sulfation reaction of the intermediate N-deacetylated heparosan is inhibited by the presence of salt (Non-Patent Literature 2). Therefore, in order to efficiently produce non-animal-derived heparin using N-deacetylated heparosan, there is a need for a desalting method for N-deacetylated heparosan that can be implemented on an industrial scale and is highly efficient, as well as an N-deacetylated heparosan composition with a low salt content. 【0005】For example, Patent Document 1 discloses a method for desalting an N-deacetylated heparosan solution obtained by deacetylating heparosan using a dialysis membrane. However, dialysis is said to be unsuitable for mass production, and it is known that when industrial diffusion dialysis is used, nearly half of the salt remains in the product without being permeated away (Non-Patent Document 3). 【0006】 Japanese Patent Publication No. 5-271305, International Publication No. 2018 / 048973, International Publication No. 2021 / 201282 【0007】 Wang et al., "Response surface optimization of the heparosan N-deacetylation in producing bioengineered heparin", J Biotechnol., 2011, 156(3): 188-196. Jansson et al., "Biosynthesis of heparin. Solubilization and partial characterization of N- and O-sulfotransferases", Biochem J., 1975, 149(1):49-55. Kawakita, "Desalination technology in seasoning production", Journal of the Japan Society for Marine Science, 1998, Vol. 52, No. 6, pp. 336-351. 【0008】 The present inventors, in the same manner as in Patent Document 1, desalted an N-deacetylated heparosan-containing solution by dialysis, but confirmed that the electrical conductivity of the solution did not decrease sufficiently, indicating insufficient desalting. Therefore, the object of this disclosure is to provide an N-deacetylated heparosan-containing composition with a low salt content, and to provide a method for producing such a composition. 【0009】 The inventors of the present invention have discovered that a highly desalted N-deacetylated heparosan-containing composition can be obtained by treating a solution containing N-deacetylated heparosan with an ultrafiltration membrane, and have completed the present invention. 【0010】In other words, this disclosure relates to the following [1] to

[10] : [1] A composition containing N-deacetylated heparosan, wherein the electrical conductivity per N-deacetylated heparosan unit concentration (g / L) is less than 0.9 mS / cm. [2] The composition according to [1], wherein the electrical conductivity per N-deacetylated heparosan unit concentration (g / L) is 0.56 mS / cm or less. [3] The composition according to [1] or [2], wherein the pH at 25°C is 6.5 or higher and the concentration of N-deacetylated heparosan is 4 g / L or higher. [4] The composition according to any one of [1] to [3], wherein the electrical conductivity per N-deacetylated heparosan unit concentration (g / L) in a solution with an N-deacetylated heparosan concentration of 7.4 g / L or higher is 0.56 mS / cm or less. [5] The composition according to [4], wherein the N-deacetylated heparosan concentration is 7.4 g / L or more and the electrical conductivity per unit concentration (g / L) of N-deacetylated heparosan is 0.56 mS / cm or less. [6] A method for producing an N-deacetylated heparosan-containing composition, comprising the step of desalting a mixture containing N-deacetylated heparosan using an ultrafiltration membrane, wherein the N-deacetylated heparosan-containing composition obtained after desalting is (1) an N-deacetylated heparosan-containing composition having an electrical conductivity of less than 0.9 mS / cm per unit concentration (g / L) of N-deacetylated heparosan. A method comprising: (3) an N-deacetylated heparosan-containing composition having an electrical conductivity of less than 0.9 mS / cm, a pH of 6.5 or higher at 25°C, and a concentration of N-deacetylated heparosan of 4 g / L or higher; or (4) an N-deacetylated heparosan-containing composition in a solution with an N-deacetylated heparosan concentration of 7.4 g / L or higher, wherein the electrical conductivity per unit concentration (g / L) of N-deacetylated heparosan is 0.56 mS / cm or less.[7] The N-deacetylated heparosan-containing composition obtained after desalting is (1') an N-deacetylated heparosan-containing composition in which the electrical conductivity per unit concentration (g / L) of N-deacetylated heparosan is 0.56 mS / cm or less, (2') an N-deacetylated heparosan-containing composition in which the electrical conductivity per unit concentration (g / L) of N-deacetylated heparosan is 0.56 mS / cm or less, the pH at 25°C is 6.5 or higher and the concentration of N-deacetylated heparosan is 4 g / L or higher, or (3') an N-deacetylated heparosan concentration of 7.4 g / L or higher and the electrical conductivity per unit concentration (g / L) of N-deacetylated heparosan is 0.56 mS / cm or less, as described in [6]. [8] The molecular weight cutoff of the ultrafiltration membrane is 30 × 10. ３ The method according to [6] or [7], which is as follows: [9] A method for producing a sulfated heparosan-containing composition, comprising the step of sulfated N-deacetylated heparosan using the composition according to any one of [1] to [5].

[10] A method for producing a sulfated heparosan-containing composition, comprising the step of obtaining a desalted N-deacetylated heparosan-containing composition according to any one of [6] to [8], and the step of sulfated N-deacetylated heparosan using the desalted N-deacetylated heparosan-containing composition. 【0011】 This disclosure provides a composition containing highly desalted N-deacetylated heparosan and a method for producing the same. Because such a composition has a low salt content, the sulfation reaction necessary for heparin production is less likely to be inhibited, making it possible to produce heparin with high efficiency. 【0012】 The embodiments of this disclosure will be described in detail below. 【0013】The first embodiment of the present disclosure relates to a composition containing N - deacetylated heparosan. N - deacetylated heparosan is one of the important intermediates in the production method of heparin derived from non - animal sources, especially from microorganisms, and is a compound obtained by deacetylating heparosan. For N - deacetylated heparosan, it is only necessary that part of the acetyl groups of heparosan be deacetylated. The degree of deacetylation may be, for example, 40% or more, 50% or more, 60% or more, 70% or more, or 80% or more, and may also be 100% or less, 95% or less, or 90% or less, and may be 60% or more and 100% or less, 70% or more and 95% or less, or 80% or more and 90% or less. The measurement of the degree of deacetylation follows the method described in Analytical Example 1. The weight - average molecular weight of N - deacetylated heparosan contained in the composition of the present disclosure is 2×10 ３ or more, 5×10 ３ or more, or 10×10 ３ or more, and may be 100×10 ３ or less, 50×10 ３ or less, or 40×10 ３ or less, and may be 2×10 ３ or more and 100×10 ３ or less, 5×10 ３ or more and 50×10 ３ or less, or 10×10 ３ or more and 40×10 ３ or less. In this specification, the weight - average molecular weight means the value determined by gel filtration chromatography (GPC). The above - mentioned gel filtration chromatography is performed under the conditions described later. 【0014】The N-deacetylated heparosan-containing composition of this embodiment has an electrical conductivity of less than 0.9 mS / cm per unit concentration (g / L) of N-deacetylated heparosan. Electrical conductivity is proportional to the salt content, and a lower electrical conductivity indicates a lower salt content. The lower the salt content in the composition, the less the sulfation reaction of N-deacetylated heparosan is inhibited, making it possible to produce heparin with high efficiency. The electrical conductivity per unit concentration (g / L) of N-deacetylated heparosan may be 0.8 mS / cm or less, 0.7 mS / cm or less, 0.6 mS / cm or less, 0.56 mS / cm or less, 0.5 mS / cm or less, 0.4 mS / cm or less, 0.32 mS / cm or less, 0.24 mS / cm or less, or 0.2 mS / cm or less, and may be 0.01 mS / cm or more, 0.02 mS / cm or more, or 0. The electrical conductivity may be 0.3 mS / cm or higher, 0.04 mS / cm or higher, 0.05 mS / cm or higher, or 0.1 mS / cm or higher, and may be 0.1 mS / cm or higher and 0.56 mS / cm or lower, 0.1 mS / cm or higher and 0.5 mS / cm or lower, 0.1 mS / cm or higher and 0.4 mS / cm or lower, 0.1 mS / cm or higher and 0.32 mS / cm or lower, or 0.1 mS / cm or higher and 0.24 mS / cm or lower. The N-deacetylated heparosan-containing composition may be, for example, in the form of an aqueous solution, and the electrical conductivity refers to the value in the aqueous solution state. The aqueous solution may be obtained by dissolving a solid containing N-deacetylated heparosan in a solvent, or it may be a solution obtained in the manufacturing process of N-deacetylated heparosan (for example, a reaction solution, an extract, a concentrated solution or permeate after membrane treatment, or a dilution thereof). Furthermore, the solvent is not limited to water, but may be a buffer solution or a mixed solvent with a water-soluble organic solvent (such as methanol, ethanol, acetone, or dimethyl sulfoxide). The electrical conductivity per unit concentration (g / L) of N-deacetylated heparosan means σ / c, where the electrical conductivity of an aqueous solution with a concentration of c (g / L) of N-deacetylated heparosan is σ (mS / cm). In this specification, electrical conductivity refers to values ​​measured with Horiba, Ltd.'s compact electrical conductivity (EC) meter LAQUA twin, benchtop pH / electrical conductivity meter F-74, benchtop electrical conductivity meter SD-72, etc. 【0015】A composition containing N-deacetylated heparosan may contain, in addition to N-deacetylated heparosan, salts (e.g., sodium chloride, sodium sulfate, sodium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, magnesium chloride, magnesium sulfate, magnesium acetate, magnesium phosphate, ammonium chloride, ammonium sulfate, ammonium phosphate, sodium acetate, ammonium acetate, potassium acetate, potassium chloride, potassium sulfate, potassium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, calcium chloride, calcium sulfate, calcium acetate, sodium citrate, potassium citrate, magnesium citrate, calcium citrate, ammonium citrate), defoaming agents (e.g., polyalkylene glycol, polyethylene glycol, polypropylene glycol, polyvinyl alcohol), and pH adjusters (e.g., sodium hydroxide, potassium hydroxide, aqueous ammonia). Furthermore, if the composition containing N-deacetylated heparosan is an aqueous solution, it naturally contains water and, if necessary, a buffer and a water-soluble organic solvent. When the N-deacetylated heparosan-containing composition is an aqueous solution, the concentration of N-deacetylated heparosan in the aqueous solution can be 4 g / L or more and 400 g / L or less, preferably 5 g / L or more and 200 g / L or less. 【0016】When an N-deacetylated heparosan-containing composition is in the state of an aqueous solution, depending on the concentration and pH of the N-deacetylated heparosan, turbidity may occur in the aqueous solution, which is undesirable from the standpoint of the composition's physical properties. Therefore, from the viewpoint of the stability of the composition, it is preferable that the N-deacetylated heparosan-containing composition has a pH of 6.5 or higher at 25°C and a concentration of N-deacetylated heparosan of 4 g / L or higher, a pH of 6.5 or higher at 25°C and a concentration of N-deacetylated heparosan of 4 g / L to 400 g / L, a pH of 6.5 or higher at 25°C and a concentration of N-deacetylated heparosan of 4 g / L to 300 g / L, and a pH of 6.5 or higher at 25°C and N-deacetylated heparosan. The concentration of N-deacetylated heparosan is 4 g / L or more and 200 g / L or less, the pH at 25°C is 6.5 or higher and the concentration of N-deacetylated heparosan is 4 g / L or more and 100 g / L or less, the pH at 25°C is 6.5 or higher and the concentration of N-deacetylated heparosan is 4 g / L or more and less than 50 g / L, the pH at 25°C is 6.5 or higher and the concentration of N-deacetylated heparosan is 10 g / L or more, the pH at 25°C is 6.5 or higher and the concentration of N-deacetylated heparosan is 10 g / L or more and 40 The concentration is 0 g / L or less, the pH at 25°C is 6.5 or higher and the concentration of N-deacetylated heparosan is 10 g / L or more and 300 g / L or less, the pH at 25°C is 6.5 or higher and the concentration of N-deacetylated heparosan is 10 g / L or more and 200 g / L or less, the pH at 25°C is 6.5 or higher and the concentration of N-deacetylated heparosan is 10 g / L or more and 100 g / L or less, the pH at 25°C is 6.5 or higher and the concentration of N-deacetylated heparosan is 10 g / L or more and less than 50 g / L In some cases, the pH at 25°C is 6.5 or higher and 12 or lower and the concentration of N-deacetylated heparosan is 4 g / L or higher; the pH at 25°C is 6.5 or higher and 12 or lower and the concentration of N-deacetylated heparosan is 4 g / L or higher and 400 g / L or lower; the pH at 25°C is 6.5 or higher and 12 or lower and the concentration of N-deacetylated heparosan is 4 g / L or higher and 300 g / L or lower; the pH at 25°C is 6.5 or higher and 12 or lower and the concentration of N-deacetylated heparosan is 4 g / L or higher and 200 g / L or lower.The pH at 25°C is 6.5 or higher and 12 or lower and the concentration of N-deacetylated heparosan is 4 g / L or higher and 100 g / L or lower; the pH at 25°C is 6.5 or higher and 12 or lower and the concentration of N-deacetylated heparosan is 4 g / L or higher and less than 50 g / L; the pH at 25°C is 6.5 or higher and 12 or lower and the concentration of N-deacetylated heparosan is 10 g / L or higher; the pH at 25°C is 6.5 or higher and 12 or lower and the concentration of N-deacetylated heparosan is 10 g / L or higher and 400 g / L or lower; the pH at 25°C is 6.5 or higher and 12 or lower and the concentration of N-deacetylated heparosan is 10 g / L or higher and 400 g / L or lower The concentration of parosan is 10 g / L or more and 300 g / L or less, the pH at 25°C is 6.5 or more and 12 or less and the concentration of N-deacetylated heparosan is 10 g / L or more and 200 g / L or less, the pH at 25°C is 6.5 or more and 12 or less and the concentration of N-deacetylated heparosan is 10 g / L or more and 100 g / L or less, the pH at 25°C is 6.5 or more and 12 or less and the concentration of N-deacetylated heparosan is 10 g / L or more and less than 50 g / L, the pH at 25°C is 6.5 or more and 10 or less and the concentration of N-deacetylated heparosan is 4 g / L or more, 25°C The pH at 6.5 to 10 and the concentration of N-deacetylated heparosan is 4 g / L to 400 g / L, the pH at 25°C is 6.5 to 10 and the concentration of N-deacetylated heparosan is 4 g / L to 300 g / L, the pH at 25°C is 6.5 to 10 and the concentration of N-deacetylated heparosan is 4 g / L to 200 g / L, the pH at 25°C is 6.5 to 10 and the concentration of N-deacetylated heparosan is 4 g / L to 100 g / L, the pH at 25°C is 6.5 to 10 and the concentration of N-deacetylated heparosan is 4 g / L to 100 g / L The concentration of N-deacetylated heparosan is 4 g / L or more and less than 50 g / L, the pH at 25°C is 6.5 or more and 10 or less and the concentration of N-deacetylated heparosan is 10 g / L or more, the pH at 25°C is 6.5 or more and 10 or less and the concentration of N-deacetylated heparosan is 10 g / L or more and 400 g / L or less, the pH at 25°C is 6.5 or more and 10 or less and the concentration of N-deacetylated heparosan is 10 g / L or more and 300 g / L or less, the pH at 25°C is 6.5 or more and 10 or less and the concentration of N-deacetylated heparosan is 10 g / L or more and 200 g / L or less,The pH at 25°C is 6.5 or higher and 10 or lower and the concentration of N-deacetylated heparosan is 10 g / L or higher and 100 g / L or lower, the pH at 25°C is 6.5 or higher and 10 or lower and the concentration of N-deacetylated heparosan is 10 g / L or higher and less than 50 g / L, the pH at 15°C is 8 or higher and the concentration of N-deacetylated heparosan is 50 g / L or higher, the pH at 15°C is 8 or higher and the concentration of N-deacetylated heparosan The pH is 50 g / L or more and 400 g / L or less, the pH at 15°C is 8 or higher and the concentration of N-deacetylated heparosan is 50 g / L or more and 300 g / L or less, the pH at 15°C is 8 or higher and the concentration of N-deacetylated heparosan is 50 g / L or more and 200 g / L or less, the pH at 15°C is 8 or more and 12 or less and the concentration of N-deacetylated heparosan is 50 g / L or more, the pH at 15°C is 8 or more and 12 or less The following conditions apply: the pH at 15°C is 8 or higher and the concentration of N-deacetylated heparosan is 50 g / L or higher and 400 g / L or lower; the pH at 15°C is 8 or higher and 12 or lower and the concentration of N-deacetylated heparosan is 50 g / L or higher and 300 g / L or lower; the pH at 15°C is 8 or higher and 12 or lower and the concentration of N-deacetylated heparosan is 50 g / L or higher and 200 g / L or lower; the pH at 15°C is 8 or higher and 10 or lower and the concentration of N-deacetylated heparosan is 5 It is more preferable that the concentration is 0 g / L or higher, the pH at 15°C is 8 to 10 and the concentration of N-deacetylated heparosan is 50 g / L to 400 g / L, the pH at 15°C is 8 to 10 and the concentration of N-deacetylated heparosan is 50 g / L to 300 g / L, or the pH at 15°C is 8 to 10 and the concentration of N-deacetylated heparosan is 50 g / L to 200 g / L. 【0017】When the N - deacetylated heparosan - containing composition is an aqueous solution containing an antifoaming agent, the aqueous solution becomes opaque from transparent or translucent when the temperature exceeds a certain value. This temperature is called the cloud point. The cloud point can be measured by visually observing the aqueous solution while heating it. When the aqueous solution contains insoluble turbidity, the influence of the insoluble matter can be excluded by centrifuging the solution in advance. The cloud point depends on factors such as the content of the antifoaming agent and the salt content in the composition, but it is within the range of 2°C to 60°C. When the N - deacetylated heparosan - containing composition is an aqueous solution containing an antifoaming agent, the operation of determining whether there is turbidity in the aqueous solution can exclude the influence of the antifoaming agent when performed under temperature conditions below the cloud point. The temperature conditions below the cloud point mean a temperature lower than the cloud point and higher than 0°C. 【0018】The present inventors have found that when an N-deacetylated heparosan-containing composition is in the state of an aqueous solution, the lower the concentration of N-deacetylated heparosan, the higher the electrical conductivity per unit concentration (g / L) of N-deacetylated heparosan tends to be.Therefore, when the N-deacetylated heparosan-containing composition is in the state of an aqueous solution, it is preferable that the aqueous solution has a high concentration of N-deacetylated heparosan. For example, (A) a solution with an N-deacetylated heparosan concentration of 7.4 g / L or more, in which the electrical conductivity per unit concentration (g / L) of N-deacetylated heparosan is 0.56 mS / cm or less, and (B) a solution with an N-deacetylated heparosan concentration of 12 g / L or more, in which the electrical conductivity per unit concentration (g / L) of N-deacetylated heparosan is 0.24 mS / cm or less. It may be a solution, (C) a solution with an N-deacetylated heparosan concentration of 31.4 g / L or more in which the electrical conductivity per N-deacetylated heparosan unit concentration (g / L) is 0.18 mS / cm or less, (D) a solution with an N-deacetylated heparosan concentration of 32.2 g / L or more in which the electrical conductivity per N-deacetylated heparosan unit concentration (g / L) is 0.15 mS / cm or less, (E) a solution with an N-deacetylated heparosan concentration of 89 g / L or more in which the electrical conductivity per N-deacetylated heparosan unit concentration ( (F) A solution in which the electrical conductivity per g / L is 0.18 mS / cm or less, (G) A solution in which the electrical conductivity per g / L of N-deacetylated heparosan is 0.12 mS / cm or less, in a solution with an N-deacetylated heparosan concentration of 100.9 g / L or more, (H) A solution in which the electrical conductivity per g / L of N-deacetylated heparosan is 0.1 mS / cm or less, in a solution with an N-deacetylated heparosan concentration of 111.7 g / L or more, (H) A solution in which the electrical conductivity per g / L of N-deacetylated heparosan is 0.1 mS / cm or less, in a solution with an N-deacetylated heparosan concentration of 121. (I) A solution in which the electrical conductivity per unit concentration (g / L) of N-deacetylated heparosan is 0.12 mS / cm or less in a solution of 1 g / L or more; (J) A solution in which the electrical conductivity per unit concentration (g / L) of N-deacetylated heparosan is 0.14 mS / cm or less in a solution with an N-deacetylated heparosan concentration of 27 g / L or more; (J) A solution in which the electrical conductivity per unit concentration (g / L) of N-deacetylated heparosan is 0.1 mS / cm or less in a solution with an N-deacetylated heparosan concentration of 110.3 g / L or more. 【0019】Furthermore, when the N-deacetylated heparosan-containing composition is in aqueous solution form, for example, (A) may be a solution in which the N-deacetylated heparosan concentration is 7.4 g / L or more and the electrical conductivity per unit concentration (g / L) of N-deacetylated heparosan is 0.56 mS / cm or less, (B) may be a solution in which the N-deacetylated heparosan concentration is 12 g / L or more and the electrical conductivity per unit concentration (g / L) of N-deacetylated heparosan is 0.24 mS / cm or less, and (C) may be an aqueous solution containing N-deacetylated heparosan. (D) A solution having an N-deacetylated heparosan concentration of 31.4 g / L or more and an electrical conductivity of 0.18 mS / cm or less per N-deacetylated heparosan unit concentration (g / L), (E) A solution having an N-deacetylated heparosan concentration of 32.2 g / L or more and an electrical conductivity of 0.15 mS / cm or less per N-deacetylated heparosan unit concentration (g / L), The solution may be mS / cm or less, (F) a solution having an N-deacetylated heparosan concentration of 100.9 g / L or more and an electrical conductivity of 0.12 mS / cm or less per unit concentration (g / L) of N-deacetylated heparosan, (G) a solution having an N-deacetylated heparosan concentration of 111.7 g / L or more and an electrical conductivity of 0.1 mS / cm or less per unit concentration (g / L) of N-deacetylated heparosan, (H) a solution having an N-deacetylated heparosan concentration of 121.1 g / L or more The solution may be (I) a solution in which the N-deacetylated heparosan concentration is 27 g / L or more and the electrical conductivity per N-deacetylated heparosan unit concentration (g / L) is 0.14 mS / cm or less, or (J) a solution in which the N-deacetylated heparosan concentration is 110.3 g / L or more and the electrical conductivity per N-deacetylated heparosan unit concentration (g / L) is 0.1 mS / cm or less. 【0020】The second embodiment of the present disclosure relates to a method for producing an N - deacetylated heparosan - containing composition. By including a step of desalting a mixture containing N - deacetylated heparosan using an ultrafiltration membrane, the above - mentioned (1) N - deacetylated heparosan - containing composition having an electrical conductivity of less than 0.9 mS / cm per unit concentration (g / L) of N - deacetylated heparosan, (2) N - deacetylated heparosan - containing composition having an electrical conductivity of less than 0.9 mS / cm per unit concentration (g / L) of N - deacetylated heparosan, a pH of 6.5 or more at 25°C, and a concentration of N - deacetylated heparosan of 4 g / L or more, or (3) N - deacetylated heparosan - containing composition having an electrical conductivity of 0.56 mS / cm or less per unit concentration (g / L) of N - deacetylated heparosan in a solution with a concentration of N - deacetylated heparosan of 7.4 g / L or more can be obtained. 【0021】 The mixture containing N - deacetylated heparosan means any mixture containing N - deacetylated heparosan and salts, and examples include a reaction solution obtained by deacetylating heparosan or a mixture obtained by post - treating the reaction solution. The deacetylation of heparosan can be carried out, for example, by adding an aqueous sodium hydroxide solution to heparosan and stirring at a predetermined temperature (for example, under heating or at room temperature). Post - treatment includes, but is not limited to, the addition of a neutralizing agent (such as an acidic carrier like hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, citric acid, strongly acidic cation - exchange resin, etc.) to stop the deacetylation reaction, concentration of the reaction solution under reduced pressure, filtration through a membrane filter, etc. 【0022】 The molecular weight cut - off of the ultrafiltration membrane is 10×10 ３ or less, 20×10 ３ [ or less, or 30×10 ３ or less, and may also be 10 or more, 50 or more, 100 or more, or 150 or more. These upper and lower limits can be arbitrarily combined. 【0023】Examples of materials for ultrafiltration membranes include porous ceramics, cellulose acetate, cellulose nitrate, regenerated cellulose, porous cellulose, polysulfone, polyethersulfone, polyethylene glycol, polyacrylonitrile, polyamide, polyvinylidene fluoride, polytetrafluoroethylene, and polyimide. 【0024】 The ultrafiltration conditions can be optimized within the service life of the ultrafiltration membrane used. For example, when using SUEZ's DK series, GE series, GH series, or GK series membranes, the permeate velocity should be 5 to 40 L / m². ２ A flow rate of 0.0 / h is preferred, and the circulation flow rate is preferably 5 to 20 times the product of the permeate flow rate and the membrane area. The transmembrane pressure difference (TMP) can be set to 0.1 to 3.0 MPa. The temperature conditions can be optimized within the service life range of the ultrafiltration membrane used, but generally, higher temperatures are preferred because the permeate flow rate increases. Examples include 4 to 80°C, 10 to 60°C, 15 to 50°C, and 20 to 40°C. The pH can be optimized within the service life range of the ultrafiltration membrane used and within the range in which N-deacetylated heparosan does not precipitate, but examples include pH 6.5 to 13.5, pH 7.0 to 12.5, pH 7.5 to 11.5, and pH 8.0 to 10.5. 【0025】 Desalting by ultrafiltration efficiently removes the salt contained in the mixture containing N-deacetylated heparosan before desalting, resulting in the N-deacetylated heparosan-containing composition of the first embodiment. When the desalting rate is defined by the following formula (1), the desalting rate is greater than 64.4%, and may be 70% or more, 80% or more, 85% or more, 90% or more, or 90.7% or more, and may be less than 100%, 99% or less, or 98% or less, and may be 85% or more but less than 100%, 90% or more but 99% or less, or 90.7% or more but 98% or less. Desalting rate = [(Electrical conductivity per 1 g / L of N-deacetylated heparosan in the solution before desalting) - (Electrical conductivity per 1 g / L of N-deacetylated heparosan in the desalted solution or desalted concentrate) / (Electrical conductivity per 1 g / L of N-deacetylated heparosan in the solution before desalting) * 100] ... Equation (1) 【0026】 A third embodiment of this disclosure relates to a method for producing a sulfated heparosan-containing composition. The N-deacetylated heparosan-containing composition obtained by the method for producing the N-deacetylated heparosan-containing composition according to the first embodiment and the N-deacetylated heparosan-containing composition according to the second embodiment has a low salt content, which makes it less likely for the sulfation reaction to be inhibited. By using these as raw materials to carry out the sulfation reaction, it is possible to efficiently produce a sulfated heparosan-containing composition. 【0027】 A method for producing a sulfated heparosan-containing composition is, for example, a method of producing sulfated heparosan by subjecting the N-deacetylated heparosan-containing composition of this disclosure to one or more of the following treatments (1) to (5): (1) N-sulfation of glucosamine residues (2) C5-epimery of uronic acid residues by C5 epimerase (3) 2-O-sulfation of uronic acid residues by 2-O-sulfotransferase (4) 6-O-sulfation of glucosamine residues by 6-O-sulfotransferase (5) 3-O-sulfation of glucosamine residues by 3-O-sulfotransferase Since the treatments (3) to (5) above are easily affected by the salt content in the raw materials, it is preferable to produce sulfated heparosan by subjecting the N-deacetylated heparosan-containing composition of this disclosure to one or more of the following treatments (3) to (5). 【0028】The N-sulfation reaction can be carried out by known methods, such as an enzymatic reaction using a sulfotransferase, or a chemical reaction using a sulfur trioxide trimethylamine complex. For example, the enzymatic reaction can be carried out by the method described in Duncan et al., "Characterization of the N-deacetylase domain from the heparan sulfate N-deacetylase / N-sulfotransferase 2", Biochem Biophys Res Commun. 2006 Jan 27;339(4):1232-1237, or Douaisi et al., "Synthesis of bioengineered heparin chemically and biologically similar to porcine-derived products and convertible to low MW heparin", Proc Natl Acad Sci USA.,2024;121(14):e2315586121. For example, the chemical reaction can be carried out by the method described in Patent Document 1 or Patent Document 2. The degree of N-sulfation in the sulfated heparosan of the present invention is not particularly limited, but may be, for example, 30% or more, 40% or more, 50% or more, 60% or more, or 70% or more, and may be 100% or less, 95% or less, or 90% or less, and may be 50% or more and 100% or less, 60% or more and 95% or less, or 70% or more and 90% or less. The degree of N-sulfation can be confirmed, for example, by disaccharide analysis. That is, the degree of N-sulfation can be calculated as the ratio (area percentage by HPLC) of the amount of disaccharide units having N-sulfate groups to the total amount of disaccharide units when the polysaccharide is subjected to disaccharide analysis. 【0029】C5 epimerase, 2-O-sulfotransferase, 6-O-sulfotransferase, and 3-O-sulfotransferase can be prepared according to conventionally known methods (for example, the method described in International Publication No. 2021 / 201282). In one embodiment of this production method, for example, C5 epimerase, 2-O-sulfotransferase, 6-O-sulfotransferase, or 3-O-sulfotransferase may be from microorganisms expressing each protein, or may be extracted and purified from microorganisms. In this embodiment, each protein may be added to the reaction mixture initially, sequentially, or in combination. 【0030】 Instead of using 2-O-sulfotransferase, 6-O-sulfotransferase, and 3-O-sulfotransferase, 2-O-sulfation, 6-O-sulfation, or 3-O-sulfation may be carried out by a chemical reaction using a sulfur trioxide trimethylamine complex or the like. 【0031】 The treatment of N-deacetylated heparosan according to (1) to (5) above can be carried out by methods well known in the art. The above treatments can be carried out in any order. For example, the treatments (3) to (5) above can be carried out in any order, but typically they can be carried out in the order of 2-O-sulfation, 3-O-sulfation and 6-O-sulfation, or in the order of 2-O-sulfation, 6-O-sulfation and 3-O-sulfation. The above treatments can also be carried out in numerical order. Two or more of the above treatments can be carried out simultaneously or separately. 【0032】 The sulfated heparosan produced by the manufacturing method of this disclosure is preferably a compound that is N-sulfated and / or O-sulfated. Examples of N-sulfated and / or O-sulfated sulfated heparosan include heparan sulfate, heparin, dermatan sulfate, acetylated heparosan, methylated heparosan, and silylated heparosan, with heparan sulfate and heparin being preferred, and heparin being more preferred. 【0033】[Analysis Example 1] Measurement of the Deacetylation Degree of Deacetylated Heparosan In the comparative examples and examples described later, unless otherwise specified, the degree of deacetylation of N-deacetylated heparosan was measured after converting N-deacetylated heparosan to N-sulfated heparosan using the method shown below. 【0034】 (1-1. Pretreatment 1) First, the N-deacetylated heparosan-containing solution was diluted with deionized water to a concentration of 8-10 g / L of N-deacetylated heparosan. Next, 15 mg each of sodium carbonate and sulfur trioxide trimethylamine complex were added to 1 mL of this diluted solution and stirred. Then, the solution was left to stand at 49°C for 24 hours to carry out the sulfation reaction and obtain an N-sulfated heparosan-containing solution. 【0035】 (1-2. Pretreatment 2) 500 μL of the obtained N-sulfated heparosan-containing solution is placed in Amicon (registered trademark) Ultra-0.5 3 × 10 ３ In addition to using a Merck centrifugation system, the solution was centrifuged (13,400 rpm, room temperature, 20 minutes). 400 μL of ultrapure water was added to the resulting concentrate, and this centrifugal separation (13,400 rpm, room temperature, 20 minutes) was repeated twice. Finally, ultrapure water was added to the resulting concentrate to a total volume of 500 μL, and the solution was recovered as N-sulfated heparosan desalinated solution. 【0036】 Two μL of the desalted N-sulfated heparosan solution, six μL of heparinase (New England Biolabs or equivalent), fourteen μL of decomposition buffer [ammonium acetate 0.143 mol / L, calcium chloride 5.7 mmol / L], and eighteen μL of ultrapure water were thoroughly mixed. The mixture was then reacted at 35°C for two hours to cleave the sugar chains and produce disaccharides. Subsequently, the mixture was heat-treated at 95°C for 15 minutes to inactivate the enzyme. After the reaction was complete, the mixture was centrifuged, and the resulting supernatant was used as a sample for disaccharide analysis. 【0037】(1-3. Disaccharide Analysis Using HPLC) Using the disaccharide analysis samples obtained above, disaccharide analysis was performed using HPLC under the following conditions. <HPLC Conditions> Column: Spheresorb® SAX chromatography column 4.0 × 250 mm, 5 μm (Waters) Column temperature: 40°C Mobile phase: (Mobile phase A) 1.80 mmol / L sodium dihydrogen phosphate aqueous solution (pH adjusted to 3.0 with phosphoric acid) (Mobile phase B) 1 mol / L sodium perchlorate (dissolved in 1.80 mmol / L sodium dihydrogen phosphate aqueous solution) Mixing ratio of mobile phase A and mobile phase B: (0-9 min) Slope from 97:3 to 65:35 (9-18 min) Slope from 65:35 to 20:80 (18-20 min) Slope from 20:80 to 0:100 (20-26 min) Slope from 0:100 to 97:3 (26-35 min) 97:3 Flow rate: 1.0 mL / min Detector: UV, 232 nm, 40°C (Hitachi, Ltd.) Measurement time: 35 minutes Injection volume: 20 μL 【0038】 (1-4. Measurement of Deacetylation Degree) Using a heparin preparation, the peaks of nine major disaccharides (FA, 0S, NS, 6S, 2S, NS6S, NS2S, 2S6S, TriS, in order of shortest retention time) contained in the disaccharide analysis sample were identified. The heparin preparation used was prepared by enzymatic reaction treatment using heparinase as described in 1-1 above. The sum of the area values ​​of the nine identified peaks was set to 100%, and the peak area value of each disaccharide was calculated as the percentage (CR) [%]. Of these, the sum of the percentages of FA, NS, NS6S, NS2S, and TriS was defined as the degree of deacetylation (DA%). 【0039】 [Analysis Example 2] Measurement of Heparosan Concentration and Weight-Average Molecular Weight In the comparative examples and examples described later, unless otherwise specified, the concentration and weight-average molecular weight of heparosan were measured using gel filtration chromatography (GPC) by the method shown below. 【0040】 (2-1. Pretreatment) When analyzing a solution containing heparosan, the solution was filtered through a membrane filter with a pore size of 0.45 μm before being subjected to GPC analysis. 【0041】(2-2. Acquisition of Chromatograms by GPC) Separation and detection of heparosan-containing solutions by GPC were performed under the following GPC analysis conditions. GPC chromatograms were recorded using Labsolutions software (Shimadzu Corporation, version 6.117) and analyzed using the GPC reanalysis function of the same software. <GPC Analysis Conditions> Guard column: TSKgel guard column PWH (7.5 mm inner diameter x 75 mm length, Tosoh Corporation) Column: TSKgel G6000PW (7.5 mm inner diameter x 300 mm length, Tosoh Corporation) Column temperature: 30°C Detector: Differential refractive index detector (RID) Cell temperature: 30°C Sample injection volume: 20 μL Mobile phase: 0.1 mol / L ammonium acetate aqueous solution Flow rate: 0.6 mL / min Analysis time: 35 minutes 【0042】 (2-3. Measurement of Heparosan Concentration) The heparosan concentration in the heparosan-containing solution was calculated by converting it to the concentration of heparin sodium using the following method. A calibration curve was created using heparin sodium reagent (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) as a standard. When creating the calibration curve, the water content of the heparin sodium reagent was determined to be 15.35% by mass using the method described in 2-4 below. Therefore, the calibration curve was created after converting the heparin sodium concentration of the standard solution, based on the powder weighing value of the heparin sodium reagent, to dry weight by multiplying it by 0.8465. Using the obtained calibration curve, the heparin sodium concentration was calculated from the area value of the main peak of the chromatogram obtained in 2-2 above. The calculated heparin sodium concentration was taken as the heparosan concentration. The retention times for the main peaks were estimated to be between 17 and 22 minutes for heparin sodium in the standard solution and between 14 and 22 minutes for heparosan in the sample solution. Specifically, the area of ​​the peak appearing around 17-22 minutes was treated as the peak area for heparin sodium, and the area of ​​the peak appearing around 14-22 minutes was treated as the peak area for heparosan. 【0043】(2-4. Measurement of the water content of the heparin sodium reagent) The water content of the heparin sodium reagent was measured using an automated water content analyzer AQV-2260S (manufactured by HIRANUMA Corporation) equipped with an AUTO SOLID EVAPORATOR EV-2010. The conditions for setting the EV file, blank measurement, and sample measurement were as follows, and were carried out according to the instruction manual. <EV File> Step 1 Temperature 200°C, Time 0 minutes Number of BG stabilization waits: 60 Pre-purge mode: OFF Pre-purge wait time: 10 seconds Blank volume correction: OFF Sample density: 1.00000 g / cm³ ３ Gas flow rate: 105-205 mL / min <Blank measurement> Calculation formula: Automatic input of blank measurement value Waiting time: 30 seconds Electrolytic current: MEDIUM S. Timer: 0 minutes Blank automatic input: ON BG automatic correction: ON Minimum electrolytic amount: 5 μg Electrolytic cell: Two chambers <Sample measurement> Waiting time: 30 seconds Electrolytic current: MEDIUM S. Timer: 0 minutes Moisture content unit: Auto BG automatic correction: ON Auto interval: 0.0000 mg Minimum electrolytic amount: 5 μg Electrolytic cell: Two chambers 【0044】 (2-5. Measurement of the weight-average molecular weight (Mw) of heparosan) The weight-average molecular weight of heparosan was measured by the following method. First, six types of standards (P-20, P-50, P-100, P-200, P-400, and P-800) contained in STANDARD P-82 (standard sample for size exclusion chromatography; manufactured by Shodex) were dissolved in ultrapure water to a concentration of 10 mg / mL without water correction, and these were used as molecular weight standard solutions to create a calibration curve. Using the calibration curve, the molecular weight of the major peaks in the chromatogram obtained in 2-2 above was calculated. 【0045】[Analysis Example 3] Measurement of the concentration and weight-average molecular weight of deacetylated heparosan and N-sulfated heparosan In the comparative examples and examples described later, unless otherwise specified, the concentration and weight-average molecular weight of deacetylated heparosan were measured using gel filtration chromatography (GPC) after converting N-deacetylated heparosan to N-sulfated heparosan by the method described below. Also, in the reference examples described later, unless otherwise specified, the concentration and weight-average molecular weight of N-sulfated heparosan were measured using gel filtration chromatography (GPC) by the method described below. 【0046】 (3-1. Pretreatment) When measuring a solution containing deacetylated heparosan, a desalted N-sulfated heparosan solution was prepared using the solution according to the methods described in (1-1. Pretreatment 1) and (1-2. Pretreatment 2) of Analysis Example 1. The obtained desalted solution was filtered through a membrane filter with a pore size of 0.45 μm and used as a sample for GPC analysis. When measuring a solution containing N-sulfated heparosan, a desalted N-sulfated heparosan solution was prepared using the solution according to the method described in (1-2. Pretreatment 2) of Analysis Example 1. The obtained desalted solution was filtered through a membrane filter with a pore size of 0.45 μm and used as a sample for GPC analysis. 【0047】 (3-2. Acquisition of chromatograms by GPC) The separation and detection of solutions containing deacetylated heparosan or N-sulfated heparosan by GPC were performed under the following GPC analysis conditions. The GPC chromatograms were recorded using LabSolutions software (Shimadzu Corporation, version 6.117) and analyzed using the GPC reanalysis function of the same software. <GPC Analysis Conditions> Guard column: TSKgel guard column SWXL (6.0 mm inner diameter x 40 mm length, manufactured by Tosoh Corporation) Columns: TSKgel G4000 SWXL and TSKgel G3000 SWXL (both 7.8 mm inner diameter x 300 mm length, manufactured by Tosoh Corporation) from the pump side Column temperature: 30°C Detector: Differential refractive index detector (RID) Cell temperature: 30°C Autosampler rack temperature: 10°C Sample injection volume: 20 μL Mobile phase: 0.1 mol / L ammonium acetate aqueous solution Flow rate: 0.6 mL / min Analysis time: 60 minutes 【0048】 (3-3. Measurement of the concentrations of deacetylated heparosan and N-sulfated heparosan) The concentration of deacetylated heparosan in the deacetylated heparosan-containing solution, or the concentration of N-sulfated heparosan in the N-sulfated heparosan-containing solution, was calculated by converting it to the concentration of heparin sodium using the following method. A calibration curve was created using heparin sodium reagent (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) as a standard. When creating the calibration curve, the heparin sodium concentration of the standard solution based on the powder weighing value of the heparin sodium reagent was multiplied by 0.8465 to convert it to dry weight, as in 2-3 above. Using the obtained calibration curve, the heparin sodium concentration was calculated from the area value of the main peak of the chromatogram obtained in 3-2 above. The calculated heparin sodium concentration was taken as the concentration of deacetylated heparosan or N-sulfated heparosan. The retention time for the main peak was estimated to be between 20 and 40 minutes for heparin sodium in the standard solution, and between 20 and 40 minutes for deacetylated heparosan and N-sulfated heparosan in the sample solution. 【0049】 (3-4. Measurement of the weight-average molecular weight (Mw) of deacetylated heparosan and N-sulfated heparosan) The weight-average molecular weight of deacetylated heparosan and N-sulfated heparosan was measured by the following method. First, 10 mg of Heparin Sodium Molecular Weight Calibrant (manufactured by the American Pharmacopeia, molecular weight range 6000-40000) was weighed without water correction and dissolved in 2 mL of 0.1 mol / L ammonium acetate aqueous solution. This was used as the molecular weight standard solution, and a calibration curve was created. Using the calibration curve, the molecular weight of the main peaks that eluted between 20 and 30 minutes of retention time, as contained in the chromatogram obtained in 3-2 above, was calculated. The peak that appeared around 38 minutes of retention time was excluded by tail-cutting. 【0050】 [Analysis Example 4] Electrical Conductivity Measurement A compact electrical conductivity meter (model number: AS-EC-33, manufactured by Horiba, Ltd.) was used to measure the electrical conductivity of the solution. 【0051】Comparative Example 1: Desalting treatment of N-deacetylated heparosan-containing solution by dialysis 1 (1) Preparation of desalted heparosan solution First, a culture solution containing heparosan was obtained according to the method described in Japanese Patent Application Publication No. 2024-060562. Specifically, an Escherichia coli strain was created into which the heparosan synthesis gene group kfiA, kfiC, and kfiD, as well as the glmS gene involved in heparosan precursor supply, were introduced, and the Escherichia coli strain was cultured in the medium and culture conditions described in Japanese Patent Application Publication No. 2024-060562. Next, 2 L of the obtained culture solution was centrifuged (9000 rpm, 15 min, 25°C) to remove the bacterial cells, and the obtained supernatant was filtered through a membrane filter with a pore size of 0.22 μm. 3600 mL of 95% (v / v) ethanol was added to 900 mL of the obtained filtrate and mixed, and then allowed to stand at 4°C for 8 hours or more to form a precipitate. The supernatant was removed by suction and centrifugation, and the resulting precipitate was dried under reduced pressure at 40°C overnight to obtain a solid containing heparosan. The heparosan content in the obtained solid was 28.2%. 13.1 g of the obtained solid (equivalent to 3.1 g of heparosan) was dissolved in 70 mL of deionized water. The solution was then stored in a visking tube (cellulose, 28.6 mm in diameter, molecular weight cutoff approximately 3.5 × 10⁶). ３ The sample was placed in a pore size of 1.25 nm and dialyzed with six times the volume (420 mL) of deionized water for 24 hours to obtain a desalinated heparosan solution. 【0052】 (2) Preparation of N-deacetylated heparosan-containing solution Based on Patent Document 1, N-deacetylated heparosan was prepared by the following procedure. 1.6 mL of 48% sodium hydroxide aqueous solution was added to 28.4 mL of the desalted heparosan solution obtained, so that the sodium hydroxide concentration was 1N. The mixture was then stirred at 50°C for 8 hours to carry out the N-deacetylation reaction of heparosan. After that, the pH of the solution was adjusted to 8.0 using 36% hydrochloric acid to stop the reaction. The solution after the reaction was obtained as an N-deacetylated heparosan-containing solution. The concentration of N-deacetylated heparosan in the solution was 18.4 g / L. 【0053】(3) Desalting treatment by dialysis Based on Patent Document 1, the N-deacetylated heparosan-containing solution was desalted. 28 mL of the N-deacetylated heparosan-containing solution prepared in (1) was desalted by dialysis with 6 times the volume (168 mL) of deionized water for 24 hours using a visking tube, as in (1). The concentration of N-deacetylated heparosan in the desalted solution, the electrical conductivity of the desalted solution, and the degree of N-deacetylation of the N-deacetylated heparosan contained in the desalted solution were measured according to the method described in the analytical example. As a result, the concentration of N-deacetylated heparosan in the desalted solution obtained by the method described in Patent Document 1 was 14.9 g / L, the electrical conductivity was 13.4 mS / cm, and the electrical conductivity per 1 g / L of N-deacetylated heparosan was 0.90 mS / cm. Furthermore, the degree of N-deacetylation of N-deacetylated heparosan contained in the desalting solution was 42%, which reproduced the experimental results of Patent Document 1. 【0054】 Comparative Example 2: Desalting treatment of N-deacetylated heparosan-containing solution by dialysis 2 (1) Preparation of heparosan desalting solution 1100 L of heparosan culture solution obtained by the same method as in Comparative Example 1 (1) was mixed with 35% by mass sulfuric acid to adjust the pH to 3.0 at room temperature. Then, 400 mL of 20% (v / v) P-2000 (polyglycol, manufactured by Dow Chemical Japan) was added as an antifoaming agent, and the mixture was heated at 70°C for 24 hours while stirring. After heating, the mixture was cooled to below 30°C, and a portion of the solution was centrifuged to collect the supernatant. A 48% by mass aqueous sodium hydroxide solution was added to the obtained supernatant to adjust the pH to 10.0 at room temperature. The entire volume was filtered through filter paper (quantitative filter paper No. 5C, manufactured by ADVANTEC), and then filtered at room temperature using a microfiltration membrane (ULP-143, hollow fiber membrane: polyvinylidene fluoride, nominal pore size: 0.45 μm, manufactured by Asahi Kasei). The obtained filtrate was filtered through an ultrafiltration membrane (SEP-3013, hollow fiber membrane: polysulfone, nominal molecular weight cutoff: 3 × 10⁻⁶). ３ The solution was passed through a (manufactured by Asahi Kasei Corporation) and desalted. Desalting was performed by adding 3.26 times the volume of deionized water to the filtrate and ultrafiltration. The heparosan concentration of the obtained desalted solution was 11.0 g / L, and the weight-average molecular weight of the heparosan contained in the solution was 129 × 10⁻¹⁶. ３ That was the case. 【0055】 (2) Preparation of N-deacetylated heparosan-containing solution 58.68 mL of deionized water was added to 232 mL of the desalting solution obtained in (1) above so that the heparosan concentration was 8.5 g / L. The resulting solution was heated to 51.9°C, and then 9.62 mL of 48% sodium hydroxide aqueous solution was added to the total volume of the solution. The mixture was stirred at 51.9°C for 49.4 hours to carry out the N-deacetylation reaction of heparosan. After that, the mixture was cooled to below 35°C, and the pH of the solution was adjusted to pH 8.45 using 35% hydrochloric acid to stop the reaction. The solution after the reaction was obtained as an N-deacetylated heparosan-containing solution. The concentration of N-deacetylated heparosan in the solution was 8.0 g / L, and the degree of N-deacetylation of the N-deacetylated heparosan contained in the solution was 83.5%. 【0056】 (3) Desalination treatment by dialysis 15 mL of the N-deacetylated heparosan-containing solution prepared in (2) is placed in a visking tube (cellulose, diameter 28.6 mm, fractional molecular weight approximately 3.5 × 10 ３ The solution was placed in a pore size of 1.25 nm and desalted by dialysis overnight in 6 times the volume (90 mL) of ultrapure water. The concentration of N-deacetylated heparosan, electrical conductivity, and degree of N-deacetylation of the contained N-deacetylated heparosan were examined in the obtained desalted solution. As a result, the concentration of N-deacetylated heparosan in the desalted solution obtained by the above method was 6.1 g / L, the electrical conductivity was 7.4 mS / cm, and the electrical conductivity per 1 g / L of N-deacetylated heparosan was 1.21 mS / cm. Furthermore, the degree of N-deacetylation of the N-deacetylated heparosan contained in the desalted solution was 83.7%. 【0057】Example 1: Desalting treatment of N-deacetylated heparosan-containing solution using ultrafiltration (UF membrane) 1 (1) Preparation of heparosan desalting solution 35% by mass sulfuric acid was added to 1100 L of heparosan culture solution, the same as in Comparative Example 2 (1), to adjust the pH to 3.0 at room temperature. Then, 400 mL of 20% (v / v) P-2000 (polyglycol, manufactured by Dow Chemical Japan) was added as an antifoaming agent, and a portion of the solution was withdrawn and heated at 70°C for 7 hours while stirring. After heating, it was cooled to below 30°C, and the supernatant was collected by centrifugation. The obtained supernatant was filtered at room temperature using a microfiltration membrane (ULP-143, hollow fiber membrane: polyvinylidene fluoride, nominal pore size: 0.45 μm, manufactured by Asahi Kasei Corporation). The obtained filtrate is processed using an ultrafiltration membrane (RC70PP, membrane material: regenerated cellulose, molecular weight cutoff: 10 × 10). ３ , 0.6m ２ Using Alfa Laval, the solution was concentrated 1.4 times by volume, and then desalted by ultrafiltration after adding 4.6 times the volume of deionized water to the concentrated solution to obtain a desalted solution. The heparosan concentration of the obtained desalted solution was 9.8 g / L, and the weight-average molecular weight of the heparosan contained in the solution was 219 × 10⁻¹⁴. ３ That was the case. 【0058】 (2) Preparation of N-deacetylated heparosan-containing solution 20.5 L of the heparosan desalting solution prepared in (1) was mixed with 0.94 L of deionized water and heated to 52.9°C. Then, 2.38 L of 10 mol / L sodium hydroxide aqueous solution was added to the total volume of the solution so that the final concentration of sodium hydroxide was 1.0 mol / L. The solution after the addition of the sodium hydroxide aqueous solution (heparosan concentration 8.5 g / L) was stirred at 52.9°C for 18.5 hours to carry out the N-deacetylation reaction of heparosan. Then, the solution was cooled to below 47°C, and the pH of the solution was adjusted to 8.51 using 35% hydrochloric acid to stop the reaction. The solution after the reaction was obtained as an N-deacetylated heparosan-containing solution. The concentration of N-deacetylated heparosan in this solution was 8.0 g / L. 【0059】(3) Desalting treatment using a UF membrane The N-deacetylated heparosan-containing solution prepared in (2) was filtered through a membrane filter (0.45 μm, Merck). 13.2 L of the filtered solution was then filtered through one UF module (SUEZ membrane (DK2540C50), 2.3 m ２ The solution was passed through a fractionation filter (SUEZ, molecular weight cutoff 150-300) for concentration and desalting. The flow conditions were a supply flow rate of 320 L / h and a TMP of 0.8 MPa. Concentration was carried out up to 1.83 times the flow rate (7.2 L). Desalting was performed by gradually adding 6.39 times the volume (46 L) of deionized water to the concentrated solution while maintaining a liquid volume of approximately 7.2 L. The concentration of N-deacetylated heparosan, electrical conductivity, and degree of deacetylation of the contained N-deacetylated heparosan were checked in the obtained desalted solution. As a result, the concentration of N-deacetylated heparosan in the desalted solution obtained by the above method was 9.9 g / L, the electrical conductivity was 3.2 mS / cm, and the electrical conductivity per 1 g / L of N-deacetylated heparosan was 0.32 mS / cm. Furthermore, the molecular weight (Mw) of N-deacetylated heparosan contained in the desalting solution is 40.6 × 10⁻⁶. ３ The degree of deacetylation was 86.8%. There was no permeation loss to the filtrate during the desalting process, and the yield of N-deacetylated heparosan was 89.4%. The desalted solution obtained in the above desalting process was centrifuged at 4000 G for 10 minutes, and the cloud point was measured by visually observing the occurrence of turbidity while gradually increasing the temperature of the supernatant. No cloud point was observed in the range up to 47.3°C. 【0060】 (4) Concentration of the desalted solution The desalted solution obtained in (3) was concentrated under reduced pressure at 40°C using an evaporator to obtain a solution with an N-deacetylated heparosan concentration of 48.0 g / L and a pH (at room temperature) of 8.73. The obtained solution was further concentrated under reduced pressure in the same manner as above to obtain a desalted concentrate. The concentration of N-deacetylated heparosan in the obtained desalted concentrate was 89.0 g / L, the electrical conductivity was 15.6 mS / cm, and the electrical conductivity per 1 g / L of N-deacetylated heparosan was 0.18 mS / cm. 【0061】Example 2: Desalting treatment of N-deacetylated heparosan-containing solution using a UF membrane 2 (1) Preparation of heparosan desalting solution 1010 L of heparosan culture solution obtained by the same method as in Comparative Example 1 (1) was mixed with 35% by mass sulfuric acid to adjust the pH to 3.0 at room temperature. Then, 20% (v / v) P-2000 (polyglycol, manufactured by Dow Chemical Japan) was added as an antifoaming agent and the mixture was heated at 70°C for 10 hours while stirring. After heating, the mixture was cooled to below 30°C and then centrifuged to collect the supernatant. The obtained supernatant was microfiltered using a microfiltration membrane (ULW-348, hollow fiber membrane: polyvinylidene fluoride, nominal pore size: 0.45 μm, manufactured by Asahi Kasei Corporation) to obtain a filtrate. The filtrate obtained by this microfiltration was mixed with an ultrafiltration membrane (RC10PE-3838 / 48, membrane material: regenerated cellulose, molecular weight cutoff: 10 × 10) ３ , 4.8m ２ Desalination was performed by diafiltration using two Alfa Laval diafiltration systems. The operating conditions for the diafiltration system were 12-17°C and the intermembrane pressure differential was 0.4 MPa. First, the filtrate obtained by microfiltration was concentrated to 3.7 times its volume. Next, desalination was performed by gradually adding 5 times its volume of deionized water to the obtained concentrate, and the solution was further heated at 70°C for 17 hours to obtain the desalination solution of heparosan. The obtained desalination solution of heparosan had a heparosan content of 15.5 g / L and a weight-average molecular weight of 149 × 10⁻¹⁶. ３ That was the case. 【0062】 (2) Preparation of N-deacetylated heparosan-containing solution 78.15 L of deionized water was added to 97.27 L of the desalting solution prepared in (1), and the temperature was raised to 57.0°C. Then, 4.90 L of 48% sodium hydroxide aqueous solution was added so that the final concentration of sodium hydroxide was 0.50 mol / L. The solution after the addition of the sodium hydroxide aqueous solution (heparosan concentration 8.5 g / L) was stirred at 57.0°C for 45 hours to carry out the deacetylation reaction of heparosan. Then, the pH of the solution (at room temperature) was adjusted to 8.54 using 35% hydrochloric acid, and the reaction was stopped by cooling to 20.3°C. The solution after the reaction was obtained as an N-deacetylated heparosan-containing solution. The concentration of N-deacetylated heparosan in this solution was 7.9 g / L. 【0063】 (3) Desalting treatment using a UF membrane The N-deacetylated heparosan-containing solution prepared in (2) was filtered using a membrane filter (TCF-G100-T1MS, 1.0 μm, manufactured by ADVANTEC). 202 L of the filtrate was filtered using two UF modules connected in series (SUEZ membrane (GK2540F30), 2.6 m ２ , fractional molecular weight 3.5 × 10 ３ The solution was passed through polyethylene glycol (manufactured by SUEZ) for concentration and desalting. The flow conditions were a supply flow rate of 14.17 L / min and a TMP of 1.0 MPa. Concentration was carried out up to 2.13 times the flow volume (95 L). Desalting was performed by gradually adding 3.90 times the volume (370 L) of deionized water to the concentrated solution while maintaining a liquid volume of approximately 95 L. Desalting was terminated after confirming that the electrical conductivity of the solution reached 3.0 mS / cm. After desalting, the solution was concentrated again to approximately 36 g / L using the above UF module, and then the concentrated solution in the apparatus was recovered by passing 5.8 L of water through it. The obtained solution was used as the desalted solution, and the concentration of N-deacetylated heparosan and its electrical conductivity were confirmed. As a result, the concentration of N-deacetylated heparosan in the desalted solution obtained by the above method was 32.2 g / L, and the electrical conductivity was 4.7 mS / cm. The electrical conductivity per 1 g / L of N-deacetylated heparosan was 0.15 mS / cm. There was no permeation loss to the filtrate during the desalting process, and the yield of N-deacetylated heparosan was 100.1%. The cloud point was measured by gradually increasing the temperature of the desalted solution obtained in the above desalting process and visually observing the occurrence of turbidity. The cloud point was found to be 22-23°C. 【0064】 (4) Concentration of desalted solution The desalted solution obtained in (3) was concentrated under reduced pressure at 45°C using an evaporator to obtain a desalted concentrate. The N-deacetylated heparosan concentration of the obtained desalted concentrate was 111.7 g / L, the electrical conductivity was 11.5 mS / cm, the electrical conductivity per 1 g / L of N-deacetylated heparosan was 0.10 mS / cm, and the pH (at room temperature) was 8.45. The molecular weight (Mw) of N-deacetylated heparosan contained in the desalted concentrate was 219 × 10⁻⁶. ３The degree of deacetylation was 84.3%. The used UF module was regenerated using the following method. First, warm water at 30-50°C was circulated for 30 minutes, and then a sodium hydroxide aqueous solution adjusted to pH 10.5 was circulated for another 30 minutes. Next, water was flowed until the pH of the solution that permeated the module became neutral. When the amount of permeate water was measured after regeneration, it was 2.5 L / min after regeneration compared to 2.7 L / min before use, confirming that it had recovered to more than 80% of the amount of permeate water before use. 【0065】 Example 3: Desalting treatment of N-deacetylated heparosan-containing solution using a UF membrane 3 (1) Preparation of heparosan desalting solution 35% by mass sulfuric acid was added to 1030 L of heparosan culture solution obtained by the same method as in Comparative Example 1 (1) to adjust the pH to 3.0 at room temperature. Then, 20% (v / v) P-2000 (polyglycol, manufactured by Dow Chemical Japan) was added as an antifoaming agent, and the mixture was heated at 70°C for 10 hours while stirring. After heating, the mixture was cooled to below 30°C, and the supernatant was collected by centrifugation. The obtained supernatant was microfiltered at below 10°C using a microfiltration membrane (ULW-348, hollow fiber membrane: polyvinylidene fluoride, nominal pore size: 0.45 μm, manufactured by Asahi Kasei Corporation) to obtain a filtrate. The filtrate obtained by this microfiltration is then subjected to an ultrafiltration membrane (RC10PE-3838 / 48, membrane material: regenerated cellulose, molecular weight cutoff: 10 × 10). ３ , 4.8m ２ Desalination was performed by diafiltration using one Alfa Laval (ALFA) filter. The operating conditions for the diafiltration were 10-15°C and the intermembrane pressure differential was 0.4 MPa. First, the filtrate obtained by the above microfiltration was concentrated to 3.3 times its volume. Next, desalination was performed by gradually adding 5 times its volume of deionized water to the obtained concentrate to obtain a desalination solution of heparosan. The obtained desalination solution of heparosan had a heparosan content of 16.2 g / L and a weight-average molecular weight of 197 × 10⁻¹⁶. ３ That was the case. 【0066】(2) Preparation of N-deacetylated heparosan-containing solution 77.39 L of deionized water was added to 88.73 L of the desalting solution prepared in (1), and the temperature was raised to 62.0°C. Then, 3.42 L of 48% sodium hydroxide aqueous solution was added so that the final concentration of sodium hydroxide was 0.375 mol / L. The solution after the addition of the sodium hydroxide aqueous solution (heparosan concentration 8.5 g / L) was stirred at 62.0°C for 49 hours to carry out the deacetylation reaction of heparosan. Then, the pH of the solution (at room temperature) was adjusted to 8.51 with 35% hydrochloric acid, and the reaction was stopped by cooling to 22.0°C. The solution after the reaction was obtained as an N-deacetylated heparosan-containing solution. The concentration of N-deacetylated heparosan in this solution was 6.5 g / L. 【0067】 (3) Desalting treatment using a UF membrane The N-deacetylated heparosan-containing solution prepared in (2) was filtered using a membrane filter (TCF-G100-T1MS, 1.0 μm, manufactured by ADVANTEC). 190.2 L of the filtrate was filtered using two UF modules connected in series (SUEZ membrane (GK2540F30), 2.6 m ２ , fractional molecular weight 3.5 × 10 ３The solution was passed through polyethylene glycol (manufactured by SUEZ) for concentration and desalting. The flow conditions were a supply flow rate of 14.17 L / min and a TMP of 1.0 MPa. Concentration was carried out up to 2.18 times the flow rate (87.2 L). Desalting was performed by gradually adding 5.16 times the volume (450 L) of deionized water to the concentrated solution while maintaining a liquid volume of approximately 87 L. Desalting was completed when the electrical conductivity of the solution reached 2.8 mS / cm. After desalting, the solution was concentrated again to approximately 32 g / L using the above UF module, and then the concentrated solution in the apparatus was recovered by passing 4.9 L of water through it. The obtained solution was used as the desalted solution, and the concentration of N-deacetylated heparosan and its electrical conductivity were checked. As a result, the concentration of N-deacetylated heparosan in the desalted solution obtained by the above method was 31.4 g / L, and the electrical conductivity was 5.8 mS / cm. The electrical conductivity per 1 g / L of N-deacetylated heparosan was 0.18 mS / cm. The permeation loss to the filtrate during the desalting process was 3.4%, and the yield of the desalted solution was 92.0%. The cloud point was measured by gradually increasing the temperature of the desalted solution obtained in the above desalting process and visually observing the occurrence of turbidity. The cloud point was found to be 26-27°C. 【0068】 (4) Concentration of desalted solution The desalted solution obtained in (3) was concentrated under reduced pressure at 45°C using an evaporator to obtain a desalted concentrate. The N-deacetylated heparosan concentration of the obtained desalted concentrate was 100.9 g / L, the electrical conductivity was 12.2 mS / cm, the electrical conductivity per 1 g / L of N-deacetylated heparosan was 0.12 mS / cm, and the pH of the solution (at room temperature) was 8.41. The molecular weight (Mw) of N-deacetylated heparosan contained in the desalted concentrate was 16.0 × 10⁻⁶. ３ The degree of deacetylation was 84.8%. The used UF module was regenerated in the same manner as in Example 2(4). When the permeate flow rate was measured after regeneration, it was 2.5 L / min after regeneration compared to 2.6 L / min before use, confirming that it had recovered to more than 80% of the permeate flow rate before use. 【0069】Example 4: Desalting treatment of N-deacetylated heparosan-containing solution using a UF membrane 4 (1) Preparation of N-deacetylated heparosan-containing solution Desalted heparosan solution obtained in Example 2 (1) (15.5 g / L, Mw 149 × 10 ３ 78.64 L of deionized water was added to 97.27 L of the solution, and the temperature was raised to 58.0°C. Then, 4.41 L of 48% sodium hydroxide aqueous solution was added to achieve a final sodium hydroxide concentration of 0.45 mol / L. The solution after the addition of the sodium hydroxide aqueous solution (heparosan concentration 8.5 g / L) was stirred at 58.0°C for 53 hours to carry out the deacetylation reaction of heparosan. Then, the pH (at room temperature) of the solution was adjusted to 8.58 using 35% hydrochloric acid (manufactured by Kanto Chemical Co., Ltd.), and the reaction was stopped by cooling to 25.6°C. The solution after the reaction was obtained as a solution containing N-deacetylated heparosan. The concentration of N-deacetylated heparosan in this solution was 7.8 g / L. 【0070】 (2) Desalting treatment using a UF membrane The N-deacetylated heparosan-containing solution prepared in (1) was filtered using a membrane filter (TCF-G100-T1MS, 1.0 μm, manufactured by ADVANTEC). 215 L of the filtrate was filtered using one UF module (ST-2-2540HM, molecular weight cutoff 10 × 10). ３The solution was passed through polyethersulfone (manufactured by Synder) and subjected to concentration and desalting treatment. The flow conditions were a supply flow rate of 5.25 L / min and a TMP of 0.2 MPa. Concentration was carried out up to 2.26 times the flow volume (95 L). Desalting was performed by gradually adding 3.0 times the volume (285 L) of deionized water to the concentrated solution while maintaining a liquid volume of approximately 95 L. After desalting was completed, the desalted solution in the apparatus was recovered by passing 5.8 L of water through it. The concentration of N-deacetylated heparosan and the electrical conductivity of the obtained desalted solution were checked. As a result, the concentration of N-deacetylated heparosan in the desalted solution obtained by the above method was 12.0 g / L, the electrical conductivity was 2.9 mS / cm, and the electrical conductivity per 1 g / L of N-deacetylated heparosan was 0.24 mS / cm. During the desalination process, the permeation loss to the filtrate was 14.3%, and the yield of the desalination solution was 81.7%. The cloud point was measured by visually observing the occurrence of turbidity while gradually increasing the temperature of the desalination solution obtained in the above desalination process. No cloud point was observed in the range up to 47.3°C. 【0071】 (3) Concentration of desalted solution The desalted solution obtained in (2) was concentrated under reduced pressure at 45°C using an evaporator to obtain a desalted concentrate. The N-deacetylated heparosan concentration of the obtained desalted concentrate was 121.1 g / L, the electrical conductivity was 14.3 mS / cm, the electrical conductivity per 1 g / L of N-deacetylated heparosan was 0.12 mS / cm, and the pH of the solution (at room temperature) was 8.67. The molecular weight (Mw) of N-deacetylated heparosan contained in the desalted concentrate was 22.3 × 10⁻⁶. ３ The degree of deacetylation was 85.5%. The used UF module was regenerated in the same manner as in Example 2(4). When the permeate flow rate was measured after regeneration, it was 2.0 L / min after regeneration compared to 2.5 L / min before use, confirming that it had recovered to more than 80% of the permeate flow rate before use. 【0072】Example 5: Desalting treatment of N-deacetylated heparosan-containing solution using a UF membrane 5 The fraction obtained as permeate during the concentration and hydrolysis treatment in Example 4 (2) was filtered using a membrane filter (TCF-G100-T1MS, 1.0 μm, ADVANTEC). Filtrate 131 L (N-deacetylated heparosan 0.9 g / L, Mw: 8.17 × 10⁻⁶) ３ Two UF modules (SUEZ module (DK2540C50), 2.3m) connected in series ２ The solution was passed through a fractionation vial (molecular weight cutoff 150-300, manufactured by SUEZ) for concentration and desalting. The flow conditions were a supply flow rate of 8.3 L / min and a TMP of 0.8 MPa. Concentration was carried out up to 17.9 times the flow rate (7.3 L). Desalting was performed by gradually adding 75 times the volume (550 L) of deionized water to the concentrated solution while maintaining a liquid volume of approximately 7 L. The solution after desalting was used as the desalted solution, and the concentration and electrical conductivity of N-deacetylated heparosan were checked. As a result, the concentration of N-deacetylated heparosan in 13.9 L of the desalted solution obtained by the above method was 7.4 g / L, the electrical conductivity was 4.1 mS / cm, and the electrical conductivity per 1 g / L of N-deacetylated heparosan was 0.56 mS / cm. The recovery rate of N-deacetylated heparosan during the desalting treatment was 90.4%. Furthermore, the molecular weight (Mw) of N-deacetylated heparosan contained in the desalting solution is 8.82 × 10⁻⁶. ３ The degree of deacetylation was 85.1%. The desalted solution obtained in the above desalting step was centrifuged at 8000 G for 10 minutes, and the cloud point was measured by observing the occurrence of turbidity while gradually increasing the temperature of the supernatant. The cloud point was found to be 32-33°C. The used UF module was regenerated in the same manner as in Example 2 (4). When the permeate flow rate was measured after regeneration, it was 2.4 L / min after regeneration compared to 2.5 L / min before use, confirming that it had recovered to more than 80% of the permeate flow rate before use. 【0073】Example 6: Desalting treatment of N-deacetylated heparosan-containing solution using a UF membrane 6 (1) Preparation of heparosan desalting solution 35% by mass sulfuric acid was added to 1000 L of heparosan culture solution prepared in the same manner as in Comparative Example 1 (1) to adjust the pH to 3.0 at room temperature. Then, 4 L of 20% (v / v) P-2000 (polyglycol, manufactured by Dow Chemical Japan) was added as an antifoaming agent, and a portion of the solution was withdrawn and heated at 70°C for 10 hours while stirring. After heating, it was cooled to below 30°C and the supernatant was collected by centrifugation. The obtained supernatant was microfiltered using a microfiltration membrane (ULW-348, hollow fiber membrane: polyvinylidene fluoride, nominal pore size: 0.45 μm, manufactured by Asahi Kasei Corporation) to obtain a filtrate. The filtrate obtained by this microfiltration is then subjected to an ultrafiltration membrane (RC10PE-3838 / 48, membrane material: regenerated cellulose, molecular weight cutoff: 10 × 10). ３ , 4.8m ２ Desalination was performed by diafiltration using two Alfa Laval diafiltration systems. The operating conditions for the diafiltration system were 23.6–28.1°C and the intermembrane pressure differential was 0.1 MPa. First, the filtrate obtained by microfiltration was concentrated 5.9 times by volume, and then desalination was performed by gradually adding 5 times the volume of deionized water to the concentrated liquid. Next, the obtained desalination solution was heated at 80°C for 27 hours, and then cooled to below 30°C to obtain a heated desalination solution. The heparosan concentration of the obtained heated desalination solution was 20.9 g / L, and the weight-average molecular weight of the heparosan contained in the solution was 30.5 × 10⁻⁶. ３ That was the case. 【0074】(2) Preparation of N-deacetylated heparosan-containing solution 51.9 L of deionized water was added to 87.8 L of the heparosan desalting solution prepared in (1), and the temperature was raised to 25°C. Then, 40.3 L of 25% sodium hydroxide aqueous solution was added to the total volume of the solution so that the final concentration of sodium hydroxide was 1.8 mol / L. The solution after the addition of the sodium hydroxide aqueous solution (heparosan concentration 8.5 g / L) was stirred at 25°C for 72 hours to carry out the N-deacetylation reaction of heparosan. Then, the temperature was cooled to below 25°C, and the pH of the solution was adjusted to 8.31 using 35% hydrochloric acid to stop the reaction. The solution after the reaction was obtained as an N-deacetylated heparosan-containing solution. The N-deacetylated heparosan concentration of the solution was 7.1 g / L, the electrical conductivity was 88.0 mS / cm, and the electrical conductivity per 1 g / L of N-deacetylated heparosan was 12.4 mS / cm. 【0075】 (3) Desalination treatment using a UF membrane The N-deacetylated heparosan-containing solution prepared in (2) was filtered using a membrane filter (TCF-G100-T1MS, 1.0 μm, manufactured by ADVANTEC), and the liquid in the piping was recovered by passing deionized water through it to a 2:1 dilution compared to the volume of liquid before filtration. 410 L of the filtrate was passed through a UF module (SUEZ membrane (GK4040F30), 7.9 m ２ , fractional molecular weight 3.5 × 10 ３A single tube of polyethylene glycol (manufactured by SUEZ) was passed through for concentration and desalting. The flow conditions were a supply flow rate of 1200 L / min and an outlet pressure of 0.9 MPa. Concentration was carried out up to four times the flow volume (103 L). Desalting was performed by gradually adding five times the volume (522 L) of deionized water to the concentrated solution while maintaining a liquid volume of approximately 103 L. Desalting was terminated after confirming that the electrical conductivity of the solution reached 2.8 mS / cm. After desalting was completed, the solution was concentrated again to approximately 36.4 g / L using the above UF module, and then the concentrated solution in the apparatus was recovered by passing 9 L of water through it. The obtained solution was used as the desalted solution, and the concentration of N-deacetylated heparosan and its electrical conductivity were confirmed. As a result, the concentration of N-deacetylated heparosan in the desalted solution obtained by the above method was 27.0 g / L, and the electrical conductivity was 3.71 mS / cm. The electrical conductivity per 1 g / L of N-deacetylated heparosan was 0.14 mS / cm. There was no permeation loss to the filtrate during the desalting process, and the yield of N-deacetylated heparosan was 95.8%. The cloud point was measured by gradually increasing the temperature of the desalted solution obtained in the above desalting process and visually observing the occurrence of turbidity. The cloud point was found to be 29°C. 【0076】 (4) Concentration of desalted solution The desalted solution obtained in (3) was concentrated under reduced pressure at 40°C using an evaporator to obtain a desalted concentrate with a pH (at room temperature) of 8.66. The concentration of N-deacetylated heparosan in the obtained desalted concentrate was 110.3 g / L, the electrical conductivity was 11.58 mS / cm, and the electrical conductivity per 1 g / L of N-deacetylated heparosan was 0.10 mS / cm. Furthermore, the molecular weight (Mw) of N-deacetylated heparosan contained in the desalted concentrate was 31.2 × 10⁻⁶. ３ The degree of deacetylation was 83.0%. 【0077】Table 1 summarizes the concentrations and electrical conductivity of N-deacetylated heparosan obtained in the comparative examples and examples described above. Furthermore, the salt removal rate in the desalting process was calculated as [(electrical conductivity per 1 g / L of N-deacetylated heparosan in the solution before desalting) - (electrical conductivity per 1 g / L of N-deacetylated heparosan in the desalted solution or desalted concentrate) / (electrical conductivity per 1 g / L of N-deacetylated heparosan in the solution before desalting) * 100] and is summarized in Table 1. 【0078】 【0079】 Reference Example 1: Preparation of N-sulfated heparosan-containing desalting solution (1) Preparation of N-deacetylated heparosan-containing solution A desalting solution of N-acetylated heparosan obtained by the same method as in Comparative Example 2 (1) (13.6 g / L, Mw 23.1 × 10 ３ 7.97 L of deionized water was added to 15.5 L of the solution, and the temperature was raised to 56.2°C. Then, 1.53 L of 10 mol / L sodium hydroxide aqueous solution was added so that the final concentration of sodium hydroxide was 0.63 mol / L. The solution after the addition of the sodium hydroxide aqueous solution (heparosan concentration 8.5 g / L) was stirred at 56.2°C to 56.7°C for 40 hours to carry out the deacetylation reaction of N-acetylated heparosan. 12.5 L was withdrawn after 36 hours of reaction. The solution was then cooled to below 47°C, and the pH of the solution (at room temperature) was adjusted to 7.86 using 36% hydrochloric acid to stop the reaction. The solution after the reaction was obtained as an N-deacetylated heparosan-containing solution. The concentration of N-deacetylated heparosan in this solution was 7.5 g / L. 【0080】 (2) Preparation of N-sulfated heparosan-containing solution Approximately 12.5 L of the N-deacetylated heparosan-containing solution obtained in (1) above was heated to 48.0-50.0°C while stirring. Then, 187.50 g of sodium carbonate and 187.48 g of sulfur trioxide trimethylamine complex were dissolved, and the mixture was stirred at 48.0-50.0°C for 49 hours to carry out the N-sulfation reaction of N-acetylated heparosan. After that, the mixture was cooled to below 30°C to terminate the reaction, and the solution obtained after the reaction was obtained as the N-sulfated heparosan-containing solution. 【0081】(3) Desalination treatment using a UF membrane The N-sulfated heparosan-containing solution obtained in (2) above was filtered through a membrane filter (0.45 μm, Merck). 8.6 L of the filtrate was filtered through one UF module (SUEZ membrane (GK2540F30), 2.6 m ２ , fractional molecular weight 3.5 × 10 ３ Desalination was performed by passing the solution through polyethylene glycol (manufactured by SUEZ). The flow conditions were a circulation flow rate of 316.8 L / h and a TMP of 0.9 MPa. Desalination was performed by gradually adding 18.6 times the volume (160 L) of deionized water to the filtered solution while maintaining a liquid volume of approximately 8.6 L. After desalination was completed, the desalination solution in the apparatus was recovered by passing 4 to 5 L of water through it. The concentration of N-sulfated heparosan and the electrical conductivity of the obtained desalination solution were checked. As a result, the concentration of N-sulfated heparosan in the desalination solution obtained by the above method was 4.9 g / L, and the electrical conductivity was 2.04 mS / cm. The yield of the desalination solution was 93.9%. 【0082】 (4) Concentration of desalted solution The desalted solution obtained in (2) was concentrated under reduced pressure at 40°C using an evaporator to obtain a desalted concentrate. The concentration of N-sulfated heparosan in the obtained desalted concentrate was 54.4 g / L. 【0083】 Reference Example 2: Evaluation of the effect of pH conditions on N-deacetylated heparosan-containing solution The effect of pH conditions on N-deacetylated heparosan-containing solution was evaluated. As a comparison, an N-sulfated heparosan-containing solution was used. For the evaluation, the desalted solution obtained in Example 3 was used as the N-deacetylated heparosan-containing solution, and the desalted concentrate obtained in Reference Example 1 was used as the N-sulfated heparosan-containing solution. 【0084】First, the stock solution and diluted solutions (2x or 10x) containing N-deacetylated heparosan were examined at room temperature (around 25°C) when the pH of each solution was gradually lowered using 1 mol / L hydrochloric acid. As a result, it was found that turbidity occurred in the solution as the pH decreased, and N-deacetylated heparosan precipitated as the solution pH decreased. However, it was observed that the intensity of turbidity in the solution sometimes changed reversibly with temperature and sometimes did not, suggesting that the effect of pH may not have been properly evaluated. Since the N-deacetylated heparosan-containing solution used may contain residual defoaming agent added in the upstream process, it is thought that the reversible change in the intensity of turbidity was influenced not by the precipitation of N-deacetylated heparosan, but by turbidity caused by the defoaming agent. 【0085】 Next, while maintaining the pH of each solution, only the temperature conditions were changed, and the temperature at which turbidity caused by the defoaming agent began to occur (cloud point) was visually measured. Subsequently, in order to distinguish between turbidity caused by the defoaming agent and turbidity caused by the precipitation of N-deacetylated heparosan, the pH was gradually lowered using hydrochloric acid while keeping the liquid temperature below the cloud point (15°C or 25°C), and the pH at which precipitation of N-deacetylated heparosan occurred was investigated. The results are shown in Table 2. 【0086】 【0087】 For comparison, a similar evaluation was performed using a solution containing N-sulfated heparosan. The stock solution and diluted solutions (2x or 10x) containing N-sulfated heparosan were examined for precipitation when the solution pH was gradually lowered using 1 mol / L hydrochloric acid while the solution temperature was kept below the cloud point (15°C). As a result, unlike N-deacetylated heparosan, no precipitation occurred in any of the solutions in the pH range of 2 to 6. 【0088】 As shown in Table 2, it was found that precipitation occurred even under high pH conditions of pH 8.0 or higher, as the concentration of N-deacetylated heparosan in the solution increased. Therefore, it was found that maintaining a pH of 6.5 or higher is necessary to stably concentrate N-deacetylated heparosan to high concentrations.

Claims

1. An N-deacetylated heparosan-containing composition having an electrical conductivity of less than 0.9 mS / cm per unit concentration (g / L) of N-deacetylated heparosan.

2. The composition according to claim 1, wherein the pH at 25°C is 6.5 or higher and the concentration of N-deacetylated heparosan is 4 g / L or higher.

3. The composition according to claim 1, wherein the electrical conductivity per unit concentration (g / L) of N-deacetylated heparosan in a solution with an N-deacetylated heparosan concentration of 7.4 g / L or more is 0.56 mS / cm or less.

4. A method for producing an N-deacetylated heparosan-containing composition, comprising the step of desalting a mixture containing N-deacetylated heparosan using an ultrafiltration membrane, wherein the N-deacetylated heparosan-containing composition obtained after desalting is: (1) an N-deacetylated heparosan-containing composition having an electrical conductivity of less than 0.9 mS / cm per unit concentration (g / L) of N-deacetylated heparosan; (2) an N-deacetylated heparosan-containing composition having an electrical conductivity of less than 0.9 mS / cm per unit concentration (g / L) of N-deacetylated heparosan, a pH of 6.5 or higher at 25°C, and a concentration of N-deacetylated heparosan of 4 g / L or higher; (3) A method for an N-deacetylated heparosan-containing composition in which the electrical conductivity per unit concentration (g / L) of N-deacetylated heparosan is 0.56 mS / cm or less in a solution with an N-deacetylated heparosan concentration of 7.4 g / L or more.

5. The molecular weight cutoff of the ultrafiltration membrane is 30 × 10 ３ The method according to claim 4, which is as follows.

6. A method for producing a sulfated heparosan-containing composition, comprising the step of sulfated N-deacetylated heparosan using the composition described in any one of claims 1 to 3.

7. A method for producing a sulfated heparosan-containing composition, comprising the steps of: obtaining a desalted N-deacetylated heparosan-containing composition by the method described in claim 4 or 5; and sulfated N-deacetylated heparosan using the desalted N-deacetylated heparosan-containing composition.

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