Preparation process of healing-promoting glycosaminoglycan in heparin sodium by-product

Abstract

The invention belongs to the technical field of polysaccharide biopharmacy, and particularly relates to a preparation process of healing-promoting glycosaminoglycan in heparin sodium byproducts, which comprises the following steps: step 1, raw material pretreatment: dissolving heparin sodium byproducts in a buffer system, filtering and purifying after the heparin sodium byproducts are completely dissolved, and retaining filtrate; step 2, targeted enzymolysis: adding a compound enzyme into the filtrate in the step 1, reacting for a period of time, heating to 85 DEG C, carrying out inactivation treatment, then carrying out centrifugal treatment, and taking supernate; 3, dynamic ion chromatography: carrying out resin chromatography on the supernate in the step 2, synchronously carrying out equilibrium buffering, and taking eluent; and 4, ethanol grading: adding ethanol into the eluent in the step 3. The high-purity dermatan sulfate is prepared through selective separation, enzymolysis modification and graded purification technologies, impurity protein is subjected to targeted enzymolysis before ethanol extraction, and a theoretical basis is provided for large-scale production.

Description

Technical Field

[0001] The present invention belongs to the technical field of polysaccharide biopharmaceuticals, and particularly relates to a preparation process of a wound-healing glycosaminoglycan from heparin sodium by-products. Background Art

[0002] Heparin sodium is a common anticoagulant drug, mainly extracted from porcine intestinal mucosa or bovine lungs. By-products during its preparation include dermatan sulfate, chondroitin sulfate, proteins, low-molecular-weight heparin, polysaccharides, nucleic acids, and other impurities. Among them, dermatan sulfate has a structure similar to heparin, but its anticoagulant activity is relatively low and it is often separated during the purification process. Directly discarding it will cause waste. Since dermatan sulfate has anticoagulant and anti-inflammatory effects, can promote wound healing and tissue repair, and can be used to treat chronic wounds, ulcers, and certain inflammatory diseases.

[0003] During the process of treating heparin sodium by-products, for the preparation of dermatan sulfate, for example, a Chinese invention with publication number CN100384884C, a method for separating and purifying dermatan sulfate and low-molecular-weight heparin sulfate from heparin sodium by-products, uses ethanol to directly extract dermatan sulfate from heparin sodium by-products.

[0004] When using ethanol extraction, some proteins dissolve in ethanol, resulting in a low purity of the extraction product and making it inconvenient to separate impurity proteins. Therefore, we propose a preparation process of a high-purity wound-healing glycosaminoglycan based on heparin sodium by-products, using targeted enzymatic hydrolysis to remove impurities. Summary of the Invention

[0005] The purpose of the present invention is to provide a preparation process of a wound-healing glycosaminoglycan from heparin sodium by-products, to prepare high-purity dermatan sulfate through selective separation, enzymatic modification, and fractional purification techniques, and to target and enzymatically hydrolyze impurity proteins before ethanol extraction, providing a theoretical basis for large-scale production.

[0006] The technical solutions adopted by the present invention are specifically as follows: A preparation process of a wound-healing glycosaminoglycan from heparin sodium by-products, comprising the following steps: Step 1. Raw material pretreatment: Dissolve the heparin sodium by-products in a buffer system. After complete dissolution, filter and purify, and retain the filtrate; Step 2. Targeted enzymatic hydrolysis: Add a complex enzyme to the filtrate in Step 1. After reacting for a period of time, raise the temperature to 85°C for inactivation treatment, and then perform centrifugation to obtain the supernatant; Step 3. Dynamic ion chromatography: Subject the supernatant in Step 2 to resin chromatography and simultaneously perform equilibrium buffering to obtain the eluate; Step 4. Ethanol fractionation: Add ethanol to the eluate in Step 3. After reaching the target concentration, precipitate and obtain the crude product through freeze-drying; Step 5. Product refinement: Take the crude product from Step 4, filter it, and dry the product to obtain the finished product.

[0007] As an alternative, the solid-liquid ratio of the buffer system in Step 1 is 1:15, and the temperature condition is ≤25°C.

[0008] As an alternative, in Step 1, filtration and purification remove macromolecular impurities through a membrane filtration system, where the molecular weight cut-off is 80 kDa.

[0009] As an alternative, in Step 2, the composite enzyme E-α reacts with the cofactor C7, where the reaction temperature range is 36.5°C to 37.5°C, the reaction time is 4 h to 6 h, and the characteristic peak λ range is monitored in real-time at 225 nm to 232 nm.

[0010] As an alternative, the inactivation treatment time in Step 2 is 10 min, and centrifugation is used to remove denatured proteins.

[0011] As an alternative, the specific process of adding ethanol in Step 2 is as follows: Primary precipitation: Add ethanol to the eluate until the concentration reaches 40%, let it stand at 4°C for 12 h, discard the precipitate, and remove residual heteropolysaccharides; Secondary precipitation: Adjust the supernatant to an ethanol concentration of 65%, let it stand under the same conditions, and collect the precipitate; Tertiary refinement: Dissolve the precipitate, adjust the ethanol concentration to 72%, and freeze-dry the precipitate to obtain the crude product.

[0012] As an alternative, the specific process of filtration in Step 5 is as follows: S1. Ultrafiltration concentration: Feed the crude product into a tangential flow ultrafiltration system with a membrane retention of 10 kDa, concentrate it until the solid content is ≥15%, and take the concentrated solution; S2. Bacteria and pyrogen removal: Pass the concentrated solution through a nanofilter membrane N-0.22 for double-layer redundant filtration.

[0013] The technical effects achieved by the present invention are as follows: Through the preparation process including Steps 1 to 5, based on the by-products (containing dermatan sulfate precursors and impurities) generated during the heparin sodium production process, the present invention prepares high-purity dermatan sulfate through selective separation, enzymatic modification, and fractional purification techniques. Specifically, before ethanol extraction, a specific composite enzyme E-α is used to target and enzymatically hydrolyze impurity proteins to obtain high-purity wound-healing glycosaminoglycans, providing a theoretical basis for large-scale production. Brief Description of the Drawings

[0014] Figure 1 It is a flowchart of a preparation process for wound-healing glycosaminoglycans in a by-product of heparin sodium of the present invention. Detailed implementation manners

[0015] In order to make the purpose and advantages of the present invention clearer and more understandable, the present invention will be specifically described below in conjunction with embodiments. It should be understood that the following text is only used to describe one or several specific implementation manners of the present invention, and does not strictly limit the scope of protection specifically claimed by the present invention.

[0016] Embodiment 1: As Figure 1 shown, a preparation process of a wound-healing glycosaminoglycan in heparin sodium by-products includes the following steps: Step 1. Raw material treatment Input materials: Input heparin sodium by-products as raw materials; Dissolution control: Dissolve in a buffer system (pH 5.8 - 6.2, containing a masking agent), with a solid-liquid ratio of 1:15 and a temperature ≤ 25°C; Among them, the buffer system contains a buffer (sodium dihydrogen phosphate can be selected), salts (sodium chloride), chelating agents (ethylenediaminetetraacetic acid), and stabilizers (glycerol can be selected); Primary purification: The dissolved product is passed through a membrane filtration system F-M01 (cut-off molecular weight 80 kDa) to remove macromolecular impurities, and the filtrate is retained, denoted as ; Step 2. Targeted enzymatic hydrolysis Enzymatic hydrolysis system: Add a composite enzyme E-α (activity unit: 200 U / g raw material) to , and activation requires the cooperation of a cofactor C7 (concentration 0.1 mol / L), where the cofactor C7 contains an organometallic complex and a nitrogen-containing heterocyclic compound; Reaction control: 1) Temperature: 37.5°C, and it is necessary to operate the dynamic temperature control module T-9; 2) Time: 4 h, and the end point is determined by real-time monitoring of the characteristic peak λ = 232 nm (measured by the UV-Vis method); Inactivation treatment: Heat up to 85°C, maintain for 10 min, and then remove the denatured protein by centrifugation using a centrifuge. The supernatant is denoted as E1; Step 3. Dynamic ion chromatography Chromatography medium: Use a proprietary resin RCX-5 (sulfonic acid group / quaternary ammonium group dual-functional modification); Among them, the proprietary resin RCX-5 uses high-crosslinking agarose microspheres with a particle size of 50 μm - 100 μm; Adsorption sequence: A) Sample loading flow rate: 0.8 CV / h (column volume CV = 5 L); B) Equilibrium buffer: Tris-HCl system (containing NaCl gradient G1); Elution logic: C) Gradient range: 0.5 M - 2 M NaCl, slope dynamically adjusted according to on-line conductivity feedback; D) Target peak collection: In the conductivity range of 1.4 mS / cm - 1.6 mS / cm, the corresponding eluent is denoted as DS- ; Step Four: Ethanol fractionation Primary precipitation: Add ethanol to DS- until the concentration reaches 40% (measured by UV-Vis method), let it stand at 4°C for 12 h, discard the precipitate, which can remove residual heteropolysaccharides; Secondary precipitation: Adjust the supernatant to an ethanol concentration of 65% (measured by UV-Vis method), let it stand under the same conditions, collect the precipitate, denoted as DS- ; Tertiary refinement: DS- is redissolved, the ethanol concentration reaches 72% (measured by UV-Vis method), the precipitate is freeze-dried to obtain the crude product, denoted as DS- '; Step Five: Final refinement Ultrafiltration concentration: Feed DS- ' into a tangential flow ultrafiltration system (membrane retention 10 kDa), concentrate to a solid content ≥ 15%; Sterilization and pyrogen removal: Through a nano-filtration membrane N-0.22 (double-layer redundant filtration); Drying: Adopt a low-temperature spray drying procedure (inlet temperature ≤ 80°C, outlet temperature ≤ 45°C) to obtain the finished product.

[0017] Example Two: As Figure 1 shown, a preparation process of a wound-healing glycosaminoglycan from heparin sodium by-products includes the following steps: Step One: Raw material treatment Input material: Input heparin sodium by-products as raw materials; Dissolution control: Dissolve in a buffer system (pH 5.8 - 6.2, containing a masking agent), solid-liquid ratio of 1:15, temperature ≤ 25°C; Among them, the buffer system contains a buffer (sodium hydrogen phosphate can be selected), salts (sodium chloride), chelating agents (ethylenediaminetetraacetic acid), and stabilizers (sucrose can be selected); Primary purification: The dissolved product is passed through a membrane system F-M01 (molecular weight cut-off 80 kDa) to remove macromolecular impurities, and the filtrate is retained, denoted as ; Step 2. Targeted enzymatic hydrolysis Enzymatic hydrolysis system: Add complex enzyme E-α (activity unit: 200 U / g raw material) to , and activation requires the cooperation of cofactor C7 (concentration 0.1 mol / L), where cofactor C7 contains an organometallic complex and a nitrogen-containing heterocyclic compound; Reaction control: 1) Temperature: 37 °C, and the dynamic temperature control module T-9 needs to be operated; 2) Time: 5 h, and the end point is determined by real-time monitoring of the characteristic peak λ = 229 nm (measured by UV-Vis method); Inactivation treatment: Heat up to 85 °C, maintain for 10 min, and then remove the denatured protein by centrifugation using a centrifuge. Record the supernatant as E1; Step 3. Dynamic ion chromatography Chromatographic medium: Use proprietary resin RCX-5 (sulfonic acid group / quaternary ammonium group dual-functional modification); Adsorption sequence: A) Sample loading flow rate: 0.8 CV / h (column volume CV = 5 L); B) Equilibration buffer: Tris-HCI system (containing NaCl gradient G1); Elution logic: C) Gradient range: NaCl 0.5 M - 2 M, and the slope is dynamically adjusted according to the on-line conductivity feedback; D) Target peak collection: In the range of conductivity 1.4 mS / cm - 1.6 mS / cm, and the corresponding eluate is recorded as DS- ; Step 4. Ethanol fractionation Primary precipitation: Add ethanol to DS- until the concentration reaches 40% (measured by UV-Vis method), and let it stand at 4 °C for 12 h. Discard the precipitate, which can remove residual heteropolysaccharides; Secondary precipitation: Adjust the supernatant to an ethanol concentration of 65% (measured by UV-Vis method), let it stand under the same conditions, and collect the precipitate, which is recorded as DS- ; Tertiary purification: DS- is redissolved, and the ethanol concentration reaches 72% (measured by UV-Vis method). The precipitate is freeze-dried to obtain the crude product, which is recorded as DS- '; Step 5. Final purification Ultrafiltration concentration: Feed DS- ' into a tangential flow ultrafiltration system (membrane cut-off 10 kDa), and concentrate it to a solid content ≥ 15%; Sterilization and pyrogen removal: Through a nano-filtration membrane N-0.22 (double-layer redundant filtration); Drying: A low-temperature spray drying process (inlet temperature ≤ 80 °C, outlet temperature ≤ 45 °C) is adopted to obtain the finished product.

[0018] Example 3: As Figure 1 shown, a preparation process of promoting wound healing glycosaminoglycan from heparin sodium by-products includes the following steps: Step 1. Raw material treatment Input of materials: Heparin sodium by-products are input as raw materials; Dissolution control: Dissolve in a buffer system (pH 5.8 - 6.2, containing a masking agent), with a solid-liquid ratio of 1:15 and a temperature ≤ 25 °C; Among them, the buffer system contains a buffer (sodium dihydrogen phosphate and disodium hydrogen phosphate can be selected), salts (sodium chloride), chelating agents (ethylenediaminetetraacetic acid), and stabilizers (glycerol can be selected); Primary purification: The dissolved product is passed through a membrane filtration system F-M01 (cut-off molecular weight 80 kDa) to remove macromolecular impurities, and the filtrate is retained, denoted as ; Step 2. Targeted enzymatic hydrolysis Enzymatic hydrolysis system: Add complex enzyme E-α (activity unit: 200 U / g raw material) to , and activation requires the cooperation of cofactor C7 (concentration 0.1 mol / L), where cofactor C7 contains an organometallic complex and a nitrogen-containing heterocyclic compound; Reaction control: 1) Temperature: 36.5 °C, and the dynamic temperature control module T-9 needs to be operated; 2) Time: 6 h, and the end point is determined by real-time monitoring of the characteristic peak λ = 225 nm (measured by UV-Vis method); Inactivation treatment: Heat up to 85 °C, maintain for 10 min, and then centrifuge to remove denatured proteins through a centrifuge. The supernatant is denoted as E1; Step 3. Dynamic ion chromatography Chromatographic medium: Use a proprietary resin RCX-5 (sulfonic acid group / quaternary ammonium group dual-functional modification); Adsorption sequence: A) Sample loading flow rate: 0.8 CV / h (column volume CV = 5 L); B) Equilibration buffer: Tris-HCI system (containing NaCl gradient G1); Elution logic: C) Gradient range: NaCl 0.5 M - 2 M, and the slope is dynamically adjusted according to the online conductivity feedback; D) Target peak collection: In the conductivity range of 1.4 mS / cm - 1.6 mS / cm, and the corresponding eluent is denoted as DS- ; Step 4. Ethanol fractionation Primary precipitation: Add ethanol to DS- until the concentration reaches 40% (measured by UV-Vis method), let it stand at 4°C for 12 h, discard the precipitate, which can remove residual heteropolysaccharides; Secondary precipitation: Adjust the supernatant to an ethanol concentration of 65% (measured by UV-Vis method), let it stand under the same conditions, collect the precipitate, denoted as DS- ; Tertiary purification: Redissolve DS- and adjust the ethanol concentration to 72% (measured by UV-Vis method). The precipitate is freeze-dried to obtain the crude product, denoted as DS- '; Step 5. Final purification Ultrafiltration concentration: Feed DS- ' into a tangential flow ultrafiltration system with a membrane cut-off of 10 kDa, and concentrate it to a solid content of ≥15%; Sterilization and pyrogen removal: Filter through a nano-filtration membrane N-0.22 in a double-layer redundant manner; Drying: Use a low-temperature spray drying procedure (inlet temperature ≤80°C, outlet temperature ≤45°C) to obtain the finished product.

[0019] The equipment used for the low-temperature spray drying procedure can be the Büchi Mini Spray Dryer B-290, Yamato Scientific Spray Dryer ADL311S, or GEA Mobile Minor™ spray drying system. The equipment configuration is as follows: 1. Core module Atomizer: Dual-fluid nozzle (aperture 0.5 mm - 1.0 mm).

[0020] Heating system: PID temperature control with an accuracy of ±1°C.

[0021] Gas treatment: Built-in filter (0.2 μm).

[0022] 2. Auxiliary functions Online monitoring: Real-time monitoring of temperature, pressure, and humidity.

[0023] Data recording: Support for USB export of the drying curve.

[0024] Safety protection: Overheat automatic shutdown function.

[0025] The above are only alternative embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention. Structures, devices, and operation methods not specifically described and explained in the present invention are implemented by conventional means in the art without special instructions and limitations.

Claims

1. A process for preparing healing-promoting glycosaminoglycans from heparin sodium byproducts, characterized in that: The following steps are involved: Step 1, raw material pretreatment: dissolving the heparin sodium byproduct in a buffer system, filtering and purifying after dissolution, and retaining the filtrate; Step 2, targeted enzymatic hydrolysis: adding a composite enzyme to the filtrate in step 1, reacting for a period of time, heating to 85°C for inactivation, and then centrifuging to obtain a supernatant; Step 3, dynamic ion chromatography: subjecting the supernatant in step 2 to resin chromatography, performing equilibrium buffering simultaneously, and taking the eluate; Step 4, ethanol classification: adding ethanol to the eluate in step 3, and after reaching the target concentration, freeze-drying the precipitate to obtain a crude product; Step 5, product refining: Take the crude product in step 4, filter it, and dry the product to obtain the finished product.

2. The process for preparing the healing-promoting glycosaminoglycan from the byproduct of heparin sodium according to claim 1, characterized in that: The solid-liquid ratio of the buffer system in step 1 is 1:15, and the temperature condition is ≤25°C.

3. The process for preparing the healing-promoting glycosaminoglycan from the byproduct of heparin sodium according to claim 1, characterized in that: In the step 1, the filtration purification removes macromolecular impurities through a membrane filter system, wherein the molecular weight cut-off is 80 kDa.

4. The process for preparing the healing-promoting glycosaminoglycan from the byproduct of heparin sodium according to claim 1, characterized in that: In the step 2, the complex enzyme E-α reacts with the cofactor C7, wherein the reaction temperature ranges from 36.5° C. to 37.5° C., the reaction time is from 4 h to 6 h, and the real-time monitoring characteristic peak λ ranges from 225 nm to 232 nm.

5. The process for preparing the healing-promoting glycosaminoglycan from the byproduct of heparin sodium according to claim 1, characterized in that: The inactivation treatment time in step 2 is 10 minutes, during which the denatured proteins are removed by centrifugation.

6. The process for preparing the healing-promoting glycosaminoglycan from heparin sodium byproduct according to claim 1, characterized in that: The specific process of adding ethanol in the step 2 is as follows: Primary precipitation: add ethanol to the eluate until the concentration reaches 40%, let it stand at 4°C for 12 hours, discard the precipitate, and remove the residual heteropolysaccharides; Secondary precipitation: the supernatant was adjusted to 65% ethanol concentration, allowed to stand under the same conditions, and the precipitate was collected; Tertiary refining: The precipitate is redissolved and adjusted to an ethanol concentration of 72%. The precipitate is freeze-dried to obtain a crude product.

7. The process for preparing the healing-promoting glycosaminoglycan from heparin sodium byproduct according to claim 1, characterized in that: The specific process of filtering in step 5 is as follows: S1. Ultrafiltration concentration: send the crude product into a tangential flow ultrafiltration system with a membrane cutoff of 10 kDa, concentrate it to a solid content ≥ 15%, and take the concentrate; S2. Sterilization and pyrogen removal: The concentrated solution is filtered through a nanofiltration membrane N-0.22 and double-layered redundant filtration.

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