A method for removing endotoxin from gelatin

Abstract

The application discloses a method for removing endotoxin from gelatin. The method comprises the following steps: (1) adding pyrogen-free water to gelatin raw materials, swelling, and then heating to 50-70 DEG C to prepare a gelatin solution with a mass concentration of 3-20%; (2) adding an adsorbent with a mass of 2.5%-100% of the gelatin, adsorbing at 40-70 DEG C for 30-240 min, and then separating the adsorbent from the gelatin solution by filtration or centrifugal separation to obtain a filtrate; (3) further filtering the filtrate by filter paper and thin-layer diatomite to obtain a refined filtrate, diluting the refined filtrate with 4 times the volume of water, ultrafiltering by using a membrane with a molecular weight cut-off of 5000-20000 Da, finally concentrating to a concentration of 10-25%, sterilizing, and drying to obtain gelatin solid with low endotoxin; the adsorbent used in step (2) is an adsorbent with a silica gel carrier, polyethylene imine as a ligand, and a spacer arm grafted or ungrafted, and the spacer arm is hexanediamine. The purified gelatin can be used in the preparation of blood plasma substitutes, tissue engineering scaffolds, implants and absorbable hemostatic materials.

Description

A method for removing endotoxins from gelatin Technical Field

[0001] This invention belongs to the field of gelatin purification and medical gelatin preparation technology. More specifically, it relates to a method for removing endotoxins from gelatin. Background Technology

[0002] Gelatin is a product obtained by purifying collagen from animal skin, bones, tendons, and ligaments through appropriate hydrolysis (acid method, alkaline method, mixed acid-base method, or enzymatic method). The endotoxin content of commercially available pharmaceutical gelatin is generally around 10. 2 ~10 5 Between EU / g, endotoxin levels, once introduced into implantable medical devices along with raw materials and exceeding the threshold, can trigger the release of a series of vasoactive peptides and cytokine mediators, leading to fever, inflammatory response, shock, organ failure, and death. In tissue engineering applications, endotoxins affect cell adhesion, growth, and differentiation on scaffold surfaces. Therefore, when gelatin is used as a blood plasma substitute, tissue engineering scaffold, implant, or in vivo hemostatic material, the endotoxin content must be strictly limited.

[0003] Endotoxin removal methods mainly include non-specific removal and selective separation. Non-specific removal methods include dry heat, chemical methods, activated carbon adsorption, and ultrafiltration. Since most biological materials undergo biological property changes under high temperature and strong acid, strong alkali, or oxidizing conditions, dry heat and chemical methods are mainly suitable for removing pyrogens from production and testing equipment, but not for removing endotoxins from proteins. Activated carbon adsorption and ultrafiltration mainly utilize physical adsorption and molecular weight differences to separate endotoxins from the substance to be purified. They are suitable for separating endotoxins from low molecular weight, low viscosity injectables, but are less effective for separating high molecular weight proteins.

[0004] Gelatin is a high-molecular-weight protein composed of 18 amino acids. During collagen degradation, one or more complex degradation processes occur, including thermal degradation, acid degradation, alkali degradation, or enzymatic degradation. Therefore, gelatin exhibits a polydisperse molecular weight distribution, ranging from 20 kDa to several thousand kDa. Its composition includes both hydrophilic and hydrophobic amino acid residues. In gelatin solutions, various interactions coexist, including hydrogen bonding, hydrophobic interactions, electrostatic interactions, covalent cross-linking, and van der Waals forces. The concentration, pH, temperature, and soluble inorganic salt content of gelatin aqueous solutions have a significant impact on the most crucial aspects of gelatin use: sol-gel conversion characteristics, rheological properties, foaming, and emulsification. Therefore, for the removal of endotoxins from gelatin, it is not only necessary to consider the removal efficiency but, more importantly, to minimize the changes in the molecular weight distribution and amino acid composition of gelatin caused by the endotoxin adsorption process, and to reduce the changes in the properties of gelatin before and after adsorption as much as possible.

[0005] JP2005 / 289841 describes a production method for reducing the endotoxin content in type B gelatin. This method involves treating animal tissues with a calcium hydroxide solution and a quaternary ammonium salt at pH 12 for at least 5 days. Gelatin is extracted from a hot aqueous solution using conventional methods, and the gelatin solution is filtered through a 0.2 μm membrane, resulting in a gelatin product with an endotoxin content of less than 5 EU / g. However, monomeric endotoxins have a particle size of less than 2 nm, and some aggregates have a particle size of tens to 100 nm, which often permeate through the filter membrane into the gelatin solution, increasing the possibility of endotoxin contamination. Furthermore, due to the small pore size of the filter membrane used in the filtration step, high molecular weight components cannot permeate, leading to the loss of the β and γ components in the gelatin, affecting its viscosity properties.

[0006] EP 1829946A1 uses an ultrafiltration membrane with a molecular weight cutoff of 300 kDa to separate endotoxins from gelatin. However, since the molecular weight distribution ranges of gelatin and endotoxins partially overlap, they cannot be effectively separated by ultrafiltration.

[0007] Therefore, there is a need to develop a method that can effectively remove endotoxins from gelatin solutions without significantly altering the properties of the gelatin after removal. Summary of the Invention

[0008] To address the aforementioned problems, one objective of this invention is to provide a method for removing endotoxins from gelatin. This method can effectively remove endotoxins from gelatin and ensure that the endotoxin-removed gelatin exhibits minimal changes in molecular weight distribution, amino acid composition, etc., compared to its state before endotoxin removal.

[0009] Another objective of this invention is to provide an application of low-endotoxin gelatin solid in the preparation of plasma substitutes, tissue engineering scaffolds, implants, absorbable hemostatic materials, etc., so as to expand the application range of gelatin.

[0010] To achieve the first objective mentioned above, the present invention adopts the following technical solution:

[0011] This invention discloses a method for removing endotoxins from gelatin, comprising the following steps:

[0012] (1) Add pyrogen-free raw water to the gelatin raw material, and heat it to 50-70℃ after swelling to prepare a gelatin solution with a mass concentration of 3-20%.

[0013] (2) Add 2.5%-100% of the gelatin mass of adsorbent, adsorb at 40-70℃ for 30-240 min, and then separate the adsorbent from the gelatin solution by filtration or centrifugation to obtain filtrate;

[0014] (3) The filtrate is further filtered with filter paper and thin layer of diatomaceous earth to obtain fine filtrate. No less than 4 times the volume of water is added to dilute the fine filtrate. Ultrafiltration is performed using a membrane with a molecular weight cutoff of 5000-20000 Da. Finally, the filtrate is concentrated to a concentration of 10-25%, sterilized, and dried to obtain gelatin solid with low endotoxin.

[0015] In step (2), the adsorbent used is an adsorbent with silica gel as a carrier, polyethyleneimine as a ligand, and grafted or ungrafted spacer arms, wherein the spacer arms are hexamethylenediamine.

[0016] It should be noted that the gelatin mentioned in this invention refers to acid-processed gelatin, alkali-processed gelatin, enzymatic gelatin, or a mixture of the above types of gelatin.

[0017] Gelatin is a protein that denatures or hydrolyzes under high temperatures and strong acid or alkali conditions. It contains numerous -COOH, -OH, and -NH2 groups, which readily bind to endotoxins via hydrogen bonds. However, compared to ordinary proteins, gelatin exhibits unique sol-gel transition properties. Typically, a 10% gelatin sol has a gel point between 25-30°C. For high-gelatin gels, at concentrations above 0.5% by weight, they primarily exist in a gelled state at room temperature. Furthermore, the molecular weight of gelatin ranges from 5 kDa to several thousand kDa, almost completely covering the molecular weight distribution range of endotoxins, making the isolation of endotoxins from gelatin extremely difficult.

[0018] The endotoxin content in gelatin to be treated is usually above 1000 EU / g, and can even exceed 10000 EU / g, making it impossible to directly contact blood and myelin, thus limiting the application of gelatin. The first removal method of this invention is adsorption. Two silica-based adsorbents for removing endotoxins from gelatin were developed: silica gel as a carrier, polyethyleneimine as a ligand, and adsorbents with grafted or ungrafted spacer arms. In the adsorbent, polyethyleneimine is grafted onto the surface of the silica gel. In a solution system with a pH less than 7, the amino groups of polyethyleneimine are protonated and become positively charged, mainly binding to the negatively charged phospholipid groups in the endotoxin through electrostatic interactions. The endotoxin is adsorbed onto the surface of the silica-based adsorbent. Further separation of the silica-based adsorbent from the gelatin solution is achieved through filtration or chromatography, thereby removing the endotoxin. Of course, to further improve the removal effect, the second removal method combines flocculation and adsorption. First, cationic polyacrylamide flocculant is added, which binds to the negatively charged endotoxins through electrostatic interaction, forming a precipitate. Then, an adsorbent is added for further removal. Using this method, endotoxins in gelatin can be effectively removed, reducing the endotoxin content to below 10 EU / g, or even below 5 EU / g, resulting in gelatin with good biocompatibility. Furthermore, the gelatin before and after removal does not show significant changes in molecular weight distribution or amino acid composition, and the gel strength and viscosity decrease by less than 10%, thus ensuring that the functionality of the gelatin remains unaffected.

[0019] Furthermore, the ungrafted adsorbent with spacer arms is prepared according to the following method:

[0020] Activate silica gel with dilute hydrochloric acid, then add silane coupling agent to the activated silica gel and react for 2-12 hours to obtain amino-grafted silica gel. Then graft ligand polyethyleneimine onto it to obtain the final product.

[0021] Furthermore, the adsorbent grafted with spacer arms is prepared according to the following method:

[0022] Activate silica gel with dilute hydrochloric acid, then add a silane coupling agent to the activated silica gel and react for 2-12 hours to obtain epoxy-grafted silica gel. Then add hexamethylenediamine and react at 60-95℃ for 1-5 hours to obtain silica gel grafted with spacer arms. Finally, graft a ligand polyethyleneimine to obtain the final product.

[0023] Furthermore, the molar ratio of hexamethylenediamine to silica gel grafted with epoxy or amino groups is 0.5-10 mmol / g.

[0024] Furthermore, the silane coupling agent includes, but is not limited to, KH550 or KH560; preferably, the molar mass ratio of the silane coupling agent to the activated silica gel is 0.5-10 mmol / g.

[0025] Furthermore, the specific steps for grafting ligand-modified polyethyleneimine are as follows:

[0026] Using an aqueous pH buffer solution as the dispersion phase, silica gel and glutaraldehyde to be grafted with the ligand were added to the reaction. After the reaction was completed, the mixture was filtered, the solid was washed, and the solid was dispersed again in the aqueous pH buffer solution. Polyethyleneimine was added, and the mixture was reacted at 25-60℃ for 10-24 hours. The mixture was then filtered, washed until the pH of the washing solution remained unchanged, and dried to obtain the final product.

[0027] Furthermore, the molar mass ratio of glutaraldehyde to silica gel to be grafted with the ligand is 1-20 mmol / g.

[0028] Furthermore, the molecular weight of the polyethyleneimine includes, but is not limited to, polyethyleneimine with a molecular weight of 1800, 3000, or 10000.

[0029] Furthermore, the molar mass ratio of the polyethyleneimine monomer to the silica gel is 10-50 mmol / g.

[0030] Furthermore, the adsorbent needs to be pretreated before it is added:

[0031] The adsorbent was soaked in 0.2M HCl for more than 2 hours, washed with pyrogen-free water, filtered, and dried with hot air for later use.

[0032] Furthermore, the purpose of ultrafiltration in this invention is to further remove trace amounts of acrylamide monomer from polyacrylamide, soluble small molecule impurities that may be introduced into the adsorbent, and soluble inorganic salts present in the gelatin raw material. By diluting the solution with at least four times its volume of water and then further concentrating it, the content of small molecule impurities can be effectively reduced, increasing product safety.

[0033] Furthermore, the specific steps for activating the silicone are as follows:

[0034] Add dilute hydrochloric acid to porous silica gel for chromatography with a particle size of 40-200 mesh. The volume (mL) of dilute hydrochloric acid added should be more than 3 times the weight (g) of silica gel. Stir at room temperature for 6-48 hours, and then separate and dry the activated silica gel solid by centrifugation or filtration.

[0035] In one specific embodiment, to further improve the efficiency of endotoxin removal, a flocculation method can be added before step (2) to remove endotoxins from the gelatin solution. This method utilizes the fact that cationic polyacrylamide flocculant contains a large number of positive charges, which combine with negatively charged endotoxins through electrostatic interaction. Diatomaceous earth is used as a flocculant aid, and the cationic polyacrylamide molecular chains can be fixed on the surface of diatomaceous earth. Polymer bridges are formed between the particles, forming aggregates that settle and carry the endotoxins into the sediment, achieving the purpose of removal in one step. The specific steps are as follows:

[0036] Diatomaceous earth is added to the gelatin solution, and after thorough stirring, cationic polyacrylamide flocculant is added. The mixture is filtered to form a flocculent precipitate, and the resulting filtrate is used in step (2). Preferably, the concentration of the flocculant is 0.05-0.5%.

[0037] Furthermore, the endotoxin content in the gelatin raw material is 50-100,000 EU / g. For example, the endotoxin content in the gelatin is 50-200 EU / g, 200-1000 EU / g, 200-100,000 EU / g, 1,000-100,000 EU / g, etc.

[0038] To achieve the second objective mentioned above, the present invention adopts the following technical solution:

[0039] This invention discloses the application of low-endotoxin gelatin solids obtained by the method described above in the preparation of plasma substitutes, tissue engineering scaffolds, implants, and absorbable hemostatic materials, such as gelatin plasma substitutes, gelatin hemostatic sponges, gelatin sutures, gelatin scaffolds, etc.

[0040] The beneficial effects of this invention are as follows:

[0041] This invention utilizes the positive charge of polyethyleneimine grafted onto a silica-based adsorbent to electrostatically interact with the negatively charged phospholipid groups in endotoxins. Endotoxins are adsorbed onto the surface of the silica-based adsorbent, and then further separated from the gelatin solution by filtration or chromatography, thereby removing the endotoxins. To further enhance the removal effect, a cationic polyacrylamide flocculant can be introduced to pre-treat the gelatin solution, ultimately reducing the endotoxin content in the gelatin to 10 EU / g, or even below 5 EU / g, resulting in gelatin with good biocompatibility. The purified gelatin shows an increase of less than 50% in the limits for elements such as Cr, Cd, As, and Pb, meeting the gelatin requirements of the 2020 edition of the Pharmacopoeia, Part II. Furthermore, the molecular weight distribution and protein content of the gelatin do not change significantly before and after purification, and the freezing power and viscosity decrease by less than 10%, ensuring that the functionality of the gelatin remains unaffected. Simultaneously, the purification process does not introduce new impurities, or the residual levels meet the requirements of Pharmacopoeia, Part II.

[0042] The low-endotoxin gelatin solids obtained by this method can be used in the preparation of plasma substitutes, tissue engineering scaffolds, implants, and absorbable hemostatic materials. Attached Figure Description

[0043] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0044] Figure 1 shows the infrared spectra of the intermediate and final products during the synthesis of Example 1 (SG-HMDA-PEI).

[0045] Figure 2 shows the thermogravimetric analysis of intermediate and final products during the synthesis of Example 1 (SG-HMDA-PEI).

[0046] Figure 3 shows the infrared spectra of the intermediate and final products during the synthesis of Example 2 (SG-PEI).

[0047] Figure 4 shows the thermogravimetric analysis of intermediate and final products during the synthesis of Example 2 (SG-PEI).

[0048] Figure 5 shows the high performance liquid chromatograms of gelatin raw material, purified gelatin in Examples 3 and 5.

[0049] Figure 6 shows the cytotoxicity test of the purified gelatin in Example 5. Detailed Implementation

[0050] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments and accompanying drawings, further clarifies the invention. Those skilled in the art should understand that the specific description below is illustrative rather than restrictive and should not be construed as limiting the scope of protection of the present invention.

[0051] In this invention, the endotoxin concentration (i.e., endotoxin content) in gelatin is defined as endotoxin units (EUs) per gram of gelatin. The endotoxin concentration is determined using Escherichia coli O113:H10K as a reference and by testing the endotoxin content in gelatin using the Limulus amebocyte lysate (LAL) reagent dynamic turbidimetric method.

[0052] Example 1: Preparation and Pretreatment of SG-HMDA-PEI

[0053] (1) Silica gel activation: Add 0.1M dilute hydrochloric acid to porous silica gel with a particle size of 150 mesh. The volume (mL) of dilute hydrochloric acid added is 4 times the weight (g) of silica gel. Stir at room temperature for 24 hours, filter to obtain activated silica gel solid, and dry with hot air to obtain powdered activated silica gel (abbreviated as SG).

[0054] (2) Grafting epoxy groups onto the surface of silica gel: Using toluene as the dispersed phase, add the activated silica gel powder from step 1, add silane coupling agent KH560, the molar mass ratio of the silane coupling agent to the activated silica gel powder is 8 mmol / g, react for 4 hours, after the reaction is completed, separate the solid-liquid mixture by filtration, wash the solid with anhydrous ethanol, and dry with hot air to obtain epoxy silica gel (abbreviated as SG-KH560).

[0055] (3) SG-KH560 grafted spacer arm: SG-KH560 was added with aqueous pH buffer as the dispersed phase. The mass-volume ratio of SG-KH560 to the buffer solution was 1:10. Hexamethylenediamine was added with a molar mass ratio of 5 mmol / g to SG-KH560. The reaction temperature was 90℃ and the reaction time was 5 hours. After the reaction was completed, the solid-liquid mixture was separated by filtration. The solid was washed with water until the pH was constant and then dried with hot air to obtain the silica gel grafted spacer arm (abbreviated as SG-KH560-HMDA).

[0056] (4) SG-KH560-HMDA grafted with polyethyleneimine (PEI): Using an aqueous pH buffer as the dispersed phase, SG-KH560-HMDA was added at a mass-to-volume ratio of SG-KH560-HMDA to the buffer solution of 1:10. Glutaraldehyde solution was added at a molar mass ratio of glutaraldehyde to SG-KH560-HMDA of 15 mmol / g. After the reaction, the solid was separated by filtration and washed with water until the pH of the washing solution remained constant and the solution was colorless. The solid was dispersed in an aqueous pH buffer, and polyethyleneimine (molecular weight 1800) was added at a molar mass ratio of polyethyleneimine monomer to silica gel of 30 mmol / g. The reaction was carried out at 50°C for 12 hours. After the reaction, the solid-liquid mixture was separated by filtration, and the solid was washed with a large amount of pure water until the pH of the washing solution remained constant. The solid was dried under hot air to obtain an adsorbent with silica gel as the carrier, hexamethylenediamine as the spacer arm, and polyethyleneimine as the ligand (abbreviated as SG-HMDA-PEI).

[0057] (5) Pretreatment of the adsorbent: Soak SG-HMDA-PEI in 0.2M HCl for more than 2 hours, wash with pyrogen-free water, filter, and dry with hot air.

[0058] The infrared spectrum of the adsorbent is shown in Figure 1. From the infrared spectrum of silica gel, we know that at 1630 cm⁻¹... -1 The peak at this point is Si-O-Si, and after grafting KH560, it reaches 3443 cm⁻¹. -1 The hydroxyl vibration peak weakens at 970 cm⁻¹ -1 The silanol vibration peak disappears at 2939 cm⁻¹. -1 A methylene shoulder peak appears at 1486 cm. -1 The methylene deformation vibration peak is at 959 cm⁻¹. -1 Characteristic peak of epoxy group at 959 cm⁻¹; after grafting hexamethylenediamine (HMDA), 959 cm⁻¹ -1 The characteristic peak of the epoxy group disappears at 2939 cm⁻¹. -1 The methylene shoulder is enhanced, 3443cm. -1 The vibrational peaks at the hydroxyl and amino groups are enhanced; after grafting glutaraldehyde (GA), a peak at 1717 cm⁻¹ appears. -1 C=O stretching peak and 1682 cm⁻¹ -1At C=N, the methylene group is also enhanced; after grafting PEI, 1717C=Ocm -1 The stretching peak disappeared, and the C=N peak was enhanced and blue-shifted to 1684 cm⁻¹. -1 Place.

[0059] The thermogravimetric analysis (TGA) is shown in Figure 2. At 800℃, all organic components in the silica gel had completely decomposed, and the remaining mass was the silica gel component. The TGA shows that the mass of the residual silica gel in each step continuously decreased, which also proves the successful conduct of each grafting reaction.

[0060] Example 2: Preparation and Pretreatment of SG-PEI

[0061] (1) Silica gel activation: Add 0.1M dilute hydrochloric acid to porous silica gel with a particle size of 100 mesh. The volume of dilute hydrochloric acid added is 4 times the weight of silica gel. Stir at room temperature for 24 hours, filter to obtain activated silica gel solid, and dry with hot air to obtain powdered activated silica gel (abbreviated as SG).

[0062] (2) Grafting amino groups onto the surface of silica gel: Using toluene, xylene or anhydrous ethanol as the dispersion phase, add the activated silica gel powder from step 1, and add silane coupling agent KH550 at a concentration of 8 mmol per gram of silica gel, and react for 6 hours (SG-KH550).

[0063] (3) SG-KH550 grafted with polyethyleneimine (PEI): Using an aqueous pH buffer as the dispersed phase, SG-KH550 was added at a mass-to-volume ratio of SG-KH550 to the buffer solution of 1:10. Glutaraldehyde solution was added at a molar mass ratio of glutaraldehyde to SG-KH550 of 15 mmol / g. After the reaction, the mixture was separated by centrifugation or filtration. The solid was washed with water until the pH of the washing solution remained constant and the solution was colorless. The solid was dispersed in an aqueous pH buffer, and polyethyleneimine (molecular weight 3000) was added at a molar mass ratio of polyethyleneimine monomer to silica gel of 25 mmol / g. The reaction was carried out at 50°C for 12 hours. After the reaction, the solid-liquid mixture was separated by filtration, and the solid was washed with a large amount of pure water until the pH of the washing solution remained constant. The solid was dried under hot air to obtain an adsorbent with silica gel as the carrier and polyethyleneimine as the ligand (abbreviated as SG-PEI).

[0064] (4) Pretreatment of the adsorbent: Soak SG-PEI in 0.2M HCl for more than 2 hours, wash with pyrogen-free water, filter, and dry with hot air.

[0065] The infrared spectrum of the adsorbent is shown in Figure 3. Figure 3 shows that after grafting KH550, the 970 cm⁻¹... -1 The silanol vibration peak disappears; 2939 cm⁻¹ -1 A methylene shoulder appeared at the point, and after grafting glutaraldehyde (GA), a peak of 1721 cm⁻¹ appeared. -1The C=O stretching peak at 1685 cm⁻¹ and the peak at 1685 cm⁻¹ -1 At C=N, the methylene peak is also enhanced. After grafting PEI, the peak is 1717 cm⁻¹. -1 The C=O stretching peak disappears, and the C=N peak is enhanced and redshifted to 1681 cm⁻¹. -1 .

[0066] The thermogravimetric analysis (TGA) is shown in Figure 4. At 800℃, all organic components in the silica gel had completely decomposed, and the remaining mass was the silica gel component. The TGA shows that the mass of the residual silica gel portion of the product decreased continuously in each step, which also proves the successful conduct of each grafting reaction.

[0067] Example 3: Removal of endotoxins by flocculation

[0068] (1) Sol: Add pyrogen-free water (endotoxin less than 0.002 EU / mL) to gelatin, and after swelling, heat to 60°C to prepare a 5% gelatin solution.

[0069] (2) Add diatomaceous earth to a 5% gelatin solution at 50% of the weight of the gelatin, and stir thoroughly to mix the gelatin and diatomaceous earth.

[0070] (3) While stirring, add cationic polyacrylamide flocculant to the mixture of gelatin and diatomaceous earth, so that the diatomaceous earth and polyacrylamide in the gelatin solution are fully mixed and form flocculent precipitate, which settles to the bottom of the solution. Filter the supernatant to obtain coarse filtrate.

[0071] (4) The filtrate from step 3 is further filtered with filter paper and a thin layer of diatomaceous earth to obtain a fine filtrate.

[0072] (5) Use a membrane ultrafiltration with a molecular weight cutoff between 5000-20000 Da, add 4 times the volume of water (relative to the fine filtrate in step 4) to dilute the concentrate, and finally concentrate it to a concentration of 20%, sterilize it, and freeze dry it to obtain gelatin solid with low endotoxin.

[0073] The HPLC chromatograms of the gelatin before and after endotoxin removal are shown in Figure 5. The molecular weight distribution data obtained from Figure 5 are shown in Table 1. The molecular weight distribution of the gelatin did not change significantly before and after endotoxin removal. The indicators of the gelatin before and after endotoxin removal are shown in Table 2. The gel strength, viscosity and protein content of the gelatin did not change significantly. The content of ash, Pb, As, Cr and other limited elements all meet the requirements of the 2020 version of gelatin. The endotoxin content was reduced by 67%.

[0074] Example 4: Removal of endotoxins from gelatin using SG-HMDA-PEI adsorbent

[0075] (1) Sol: Add pyrogen-free water (endotoxin less than 0.002 EU / mL) to gelatin, and after swelling, heat to 60°C to prepare a 10% gelatin solution.

[0076] (2) Adjust the pH of the gelatin solution from step 1 to 4.5. While stirring, add the SG-HMDA-PEI adsorbent prepared in Example 1 (which removes pyrogens) to the gelatin solution from step 1. The amount of adsorbent added is 25% of the dry weight of the gelatin. Stir to ensure sufficient contact between the adsorbent and the gelatin solution, and adsorb at 50°C for 240 min. After adsorption, filter or centrifuge to obtain the coarse filtrate.

[0077] (3) Filtrate 2 is further filtered with filter paper and a thin layer of diatomaceous earth to obtain fine filtrate.

[0078] (4) Add 4 times the volume of water (relative to the fine filtrate in step 3) to dilute the fine filtrate, use a membrane with a molecular weight cutoff of 5000-20000 Da for ultrafiltration, finally concentrate to a concentration of 25%, sterilize, and freeze dry to obtain gelatin solid with low endotoxin.

[0079] The indicators of the gelatin before and after endotoxin removal are shown in Table 2.

[0080] Example 5: Combined use of flocculation and adsorption methods to remove endotoxins from gelatin

[0081] (1) Sol: Add pyrogen-free water (endotoxin less than 0.002 EU / mL) to gelatin, and after swelling, heat to 60°C to prepare a 10% gelatin solution.

[0082] (2) Add diatomaceous earth to a 10% gelatin solution at 40% of the weight of the gelatin, and stir thoroughly to mix the gelatin and diatomaceous earth.

[0083] (3) While stirring, add cationic polyacrylamide flocculant to the mixture of gelatin and diatomaceous earth, so that the diatomaceous earth and polyacrylamide in the gelatin solution are fully mixed and form flocculent precipitate, which settles to the bottom of the solution. Filter the supernatant to obtain coarse filtrate.

[0084] (4) Adjust the pH of the coarse filtrate from step 3 to 5.0. While stirring, add the SG-HMDA-PEI adsorbent from Example 1 (which removes pyrogens), stir to ensure full contact between the adsorbent and the filtrate, and adsorb at 50°C for 240 min. After adsorption, filter or centrifuge to obtain the filtrate.

[0085] (5) The filtrate obtained in step 4 is further filtered with filter paper and a thin layer of diatomaceous earth to obtain a fine filtrate.

[0086] (6) Add 4 times the volume of water (relative to the filtrate in step 5) to dilute the filtrate, use a membrane with a molecular weight cutoff of 10000 Da for ultrafiltration, finally concentrate to a concentration of 25%, sterilize, and freeze dry to obtain gelatin solid with low endotoxin.

[0087] The HPLC chromatograms and molecular weight distribution data of the gelatin before and after endotoxin removal are shown in Figure 5 and Table 1. The molecular weight distribution of the gelatin did not change significantly before and after endotoxin removal. The indicators of the gelatin before and after endotoxin removal are shown in Table 2. The gel strength, viscosity, and protein content of the gelatin did not change significantly. The contents of ash, Pb, As, Cr, and other limit elements all met the 2020 requirements for gelatin, and the endotoxin content decreased by 90.5%.

[0088] Table 1. Molecular weight distribution data of gelatin raw materials, purified gelatin in Examples 3 and 5.

[0089]

[0090]

[0091] The cytotoxicity of purified gelatin was tested according to GB16886.5 (the preparation method of the extract was based on GB16886.12). The results showed no cytotoxicity. Specific data are shown in Figure 6. The cell viability of the negative control group was 91.4%, the positive control group was 2.16%, the gelatin raw material group was 94.7%, and the cell viability of the gelatin prepared in this example was 88.2%. According to the cytotoxicity classification results of GB16886.5, this gelatin has no cytotoxicity.

[0092] Example 6: Removal of endotoxins from gelatin using SG-PEI adsorbent

[0093] (1) Sol: Add pyrogen-free water (endotoxin less than 0.002 EU / mL) to gelatin, and after swelling, heat to 50°C to prepare a 3% gelatin solution.

[0094] (2) Adjust the pH of the gelatin solution from step 1 to 4.5. While stirring, add the SG-PEI adsorbent (Example 2) for pyrogen removal to the gelatin solution from step 1. The amount of adsorbent added is 50% of the dry weight of the gelatin. Stir to ensure sufficient contact between the adsorbent and the gelatin solution. Adsorb at 50°C for 240 minutes. After adsorption, filter or centrifuge to obtain the coarse filtrate. (Temperature correct)

[0095] (3) The filtrate from step 2 is further filtered with filter paper and a thin layer of diatomaceous earth to obtain a fine filtrate.

[0096] (4) Add 4 times the volume of water (relative to the fine filtrate in step 3) to dilute the fine filtrate, use a membrane with a molecular weight cutoff of 10000 Da for ultrafiltration, finally concentrate to a concentration of 25%, sterilize, and freeze dry to obtain gelatin solid with low endotoxin.

[0097] The indicators of the gelatin before and after endotoxin removal are shown in Table 2.

[0098] Example 7: Removal of endotoxins from gelatin using a combination of flocculation and adsorption

[0099] (1) Sol: Add pyrogen-free water (endotoxin less than 0.002 EU / mL) to gelatin, and after swelling, heat to 60°C to prepare a 10% gelatin solution.

[0100] (2) Add diatomaceous earth to a 10% gelatin solution at 40% of the weight of the gelatin, and stir thoroughly to mix the gelatin and diatomaceous earth.

[0101] (3) While stirring, add cationic polyacrylamide flocculant to the mixture of gelatin and diatomaceous earth. The concentration of the flocculant is 0.25%. This allows the diatomaceous earth and polyacrylamide in the gelatin solution to mix thoroughly and form flocculent precipitate, which settles to the bottom of the solution. Filter the supernatant to obtain coarse filtrate.

[0102] (4) Adjust the pH of the coarse filtrate from step 3 to 5.0. While stirring, add the SG-PEI adsorbent from Example 2 (which removes pyrogens), stir to ensure full contact between the adsorbent and the filtrate, and adsorb at 50°C for 75 minutes. After adsorption, filter or centrifuge to obtain the filtrate.

[0103] (5) The filtrate obtained in step 4 is further filtered with filter paper and a thin layer of diatomaceous earth to obtain a fine filtrate.

[0104] (6) Add 4 times the volume of water (relative to the fine filtrate in step 5) to dilute the fine filtrate, use a membrane with a molecular weight cutoff of 5000-20000 Da for ultrafiltration, finally concentrate to a concentration of 25%, sterilize, and freeze dry to obtain gelatin solid with low endotoxin.

[0105] The indicators of the gelatin before and after endotoxin removal are shown in Table 2. The gel strength, viscosity and protein content of the gelatin did not change significantly. The content of ash, Pb, As and Cr and other limited elements all met the requirements of the 2020 version of gelatin. The endotoxin content was reduced by 95.2%.

[0106] Example 8: Preparation and Adsorption of SG-PEI

[0107] (1) Silica gel activation: Add 0.1M dilute hydrochloric acid to porous silica gel with a particle size of 100 mesh. The volume of dilute hydrochloric acid added is 4 times the weight of silica gel. Stir at room temperature for 24 hours, filter to obtain activated silica gel solid, and dry with hot air to obtain powdered activated silica gel (abbreviated as SG).

[0108] (2) Grafting amino groups onto the surface of silica gel: Using toluene, xylene or anhydrous ethanol as the dispersed phase, add the activated silica gel powder from step 1, and add silane coupling agent KH550 at a concentration of 0.1 mmol per gram of silica gel, and react for 6 hours (SG-KH550).

[0109] (3) SG-KH550 grafted with polyethyleneimine (PEI): Using an aqueous pH buffer as the dispersed phase, SG-KH550 was added at a mass-to-volume ratio of SG-KH550 to the buffer solution of 1:10. Glutaraldehyde solution was added at a molar mass ratio of 0.2 mmol / g to SG-KH550. After the reaction, the mixture was separated by centrifugation or filtration. The solid was washed with water until the pH of the washing solution remained constant and the solution was colorless. The solid was dispersed in an aqueous pH buffer, and polyethyleneimine (molecular weight 3000) was added at a molar mass ratio of 2 mmol / g to silica gel. The reaction was carried out at 50°C for 12 hours. After the reaction, the solid-liquid mixture was separated by filtration, and the solid was washed with a large amount of pure water until the pH of the washing solution remained constant. The solid was dried under hot air to obtain an adsorbent with silica gel as the carrier and polyethyleneimine as the ligand (abbreviated as SG-PEI).

[0110] (4) Pretreatment of the adsorbent: Soak SG-PEI in 0.2M HCl for more than 2 hours, wash with pyrogen-free water, filter, and dry with hot air.

[0111] (5) Sol: Add pyrogen-free water (endotoxin less than 0.002 EU / mL) to gelatin, and after swelling, heat to 60°C to prepare a 10% gelatin solution.

[0112] (6) Adjust the pH of the gelatin solution from step 5 to 4.5. While stirring, add the adsorbent obtained after removing the pyrogens in step 4. The amount of adsorbent added is 50% of the dry weight of the gelatin. Stir to ensure that the adsorbent and the gelatin solution are in full contact. Adsorb at 50°C for 240 minutes. After the adsorption is complete, filter to obtain the coarse filtrate.

[0113] (7) The coarse filtrate from step 6 is further filtered with filter paper and a thin layer of diatomaceous earth to obtain the fine filtrate.

[0114] (8) Add 4 times the volume of water (relative to the filtrate in step 7) to dilute the filtrate, use a membrane with a molecular weight cutoff of 10000 Da for ultrafiltration, finally concentrate to a concentration of 25%, sterilize, and freeze dry to obtain gelatin solid with low endotoxin.

[0115] The indicators of the gelatin before and after endotoxin removal are shown in Table 2.

[0116] Table 2. Molecular weight distribution data of gelatin raw materials and purified gelatin in each example.

[0117]

[0118]

[0119] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all the implementation methods here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A method for removing endotoxins from gelatin, characterized in that, The process includes the following steps: (1) Adding pyrogen-free raw water to the gelatin raw material, swelling it, and heating it to 50-70°C to prepare a gelatin solution with a mass concentration of 3-20%; (2) Adding an adsorbent of 2.5%-100% of the mass of the gelatin, adsorbing it at 40-70°C for 30-240 minutes, and then separating the adsorbent from the gelatin solution by filtration or centrifugation to obtain a filtrate; (3) Filtering the filtrate further with filter paper and thin-layer diatomaceous earth to obtain a fine filtrate, adding 4 times the volume of water to dilute the fine filtrate, using a membrane with a molecular weight cutoff of 5000-20000 Da for ultrafiltration, finally concentrating it to a concentration of 10-25%, sterilizing it, drying it, and obtaining a gelatin solid with low endotoxin; wherein, the adsorbent used in step (2) is an adsorbent with silica gel as a carrier, polyethyleneimine as a ligand, and grafted or ungrafted spacer arms, wherein the spacer arms are hexamethylenediamine.

2. The method according to claim 1, characterized in that, The ungrafted adsorbent with spacer arms is prepared by the following method: silica gel is activated with dilute hydrochloric acid, then a silane coupling agent is added to the activated silica gel, and the reaction is carried out for 2-12 hours to obtain amino-grafted silica gel, which is then grafted with a ligand polyethyleneimine to obtain the final product.

3. The method according to claim 1, characterized in that, The adsorbent grafted with spacer arms is prepared by the following method: silica gel is activated with dilute hydrochloric acid, then a silane coupling agent is added to the activated silica gel, and the reaction is carried out for 2-12 hours to obtain epoxy-grafted silica gel. Hexamethylenediamine is then added, and the reaction is carried out at 60-95℃ for 1-5 hours to obtain silica gel grafted with spacer arms. Finally, a ligand polyethyleneimine is grafted onto the silica gel to obtain the final product.

4. The method according to claim 3, characterized in that, The molar ratio of hexamethylenediamine to grafted epoxy silica gel is 0.5-10 mmol / g.

5. The method according to claim 2 or 3, characterized in that, The silane coupling agent includes KH550 or KH560.

6. The method according to claim 2 or 3, characterized in that, The molar ratio of the silane coupling agent to the activated silica gel is 0.5-10 mmol / g.

7. The method according to claim 2 or 3, characterized in that, The specific steps for grafting ligands onto polyethyleneimine are as follows: using an aqueous pH buffer as the dispersion phase, add silica gel and glutaraldehyde to be grafted and react. After the reaction is complete, filter, wash the solid, disperse the solid again in an aqueous pH buffer, add polyethyleneimine, react at 25-60℃ for 10-24 hours, filter, wash until the pH of the washing solution remains unchanged, and dry to obtain the final product.

8. The method according to claim 7, characterized in that, The molar ratio of glutaraldehyde to silica gel to be grafted with the ligand is 1-20 mmol / g.

9. The method according to claim 7, characterized in that, The molecular weight of the polyethyleneimine includes 1800, 3000, or 10000.

10. The method according to claim 7, characterized in that, The molar ratio of polyethyleneimine to silica gel is 10-50 mmol / g.

11. The method according to claim 1, characterized in that, The adsorbent needs to be pretreated before it is added: soak the adsorbent in 0.2M HCl for more than 2 hours, wash it with pyrogen-free water, filter it, and dry it with hot air for later use.

12. The method according to claim 1, characterized in that, Before step (2), the gelatin solution is detoxified by flocculation. The specific steps are as follows: add diatomaceous earth to the gelatin solution, stir thoroughly, add cationic polyacrylamide flocculant, filter to form flocculent precipitate, and use the filtrate in step (2).

13. The method according to claim 12, characterized in that, The concentration of the flocculant is 0.05-0.5%.

14. The method according to claim 1, characterized in that, The endotoxin content in the gelatin raw material is 50-100,000 EU / g.

15. The use of a low-endotoxin gelatin solid obtained by the method of any one of claims 1-14 in the preparation of plasma substitutes, tissue engineering scaffolds, implants, and absorbable hemostatic materials.

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