Method for the detection of bacterial endotoxins in a phagel filler

Abstract

The present application provides a kind of detection method of bacterial endotoxin in PHA gel filling agent.The filling agent of the present application comprises PHA microspheres, gel carrier sodium carboxymethyl cellulose, osmotic pressure regulator, pH regulator and water for injection, the present application systematically studies the influence of gel carrier and osmotic pressure regulator on the performance of composition shear viscosity and osmotic pressure, and scientifically selects the excellent and stable gel prescription.The present application accidentally finds that the combination of water and a small amount of dichloromethane can realize the sufficient release of endotoxin, and accurately detect its content.The method of the present application can effectively ensure the safety of human medicine, and has important industrial value.

Description

Technical Field

[0001] This invention relates to the field of microbial detection technology, specifically to a method for detecting bacterial endotoxins in PHA gel fillers. Background Technology

[0002] As humans age, muscle and collagen tissues in the body undergo varying degrees of functional degeneration. Based on this, various fillers have been developed, such as hyaluronic acid. However, hyaluronic acid's effects as a filler are relatively short-lived, requiring frequent injections. To achieve long-lasting filling effects, researchers have attempted to use non-biodegradable materials such as polyvinyl alcohol (PVA) microspheres as fillers. While these fillers offer significantly longer-lasting effects, prolonged retention in the body can release harmful substances, triggering a series of side effects and endangering human health.

[0003] Polyhydroxyalkanoates (PHA) are a novel type of biodegradable polymer material. PHAs are mainly classified into first-generation PHB, second-generation PHBV, third-generation PHBHHx, fourth-generation P34HB, and fifth-generation PHBVHHx. Among them, PHB is an oligomer composed solely of 3-hydroxybutyric acid (3HB), PHBV is a copolymer of 3-hydroxybutyric acid (3HB) and 3-hydroxyvalerate (3HV), PHBHHx (i.e., P3HB-co-3HHx) is a copolymer of 3-hydroxybutyric acid (3HB) and 3-hydroxyhexanoic acid (3HHx), and is also the first short-chain and medium-long-chain PHA copolymer discovered and studied. Compared with PHB, it has good flexibility, and the material properties change with the content of copolymer chain segments. P34HB (i.e., P3HB-co-4HB) is a copolymer of 3-hydroxybutyric acid (3HB) and 4-hydroxybutyric acid (4HB), and PHBVHHx is a copolymer of 3-hydroxybutyric acid (3HB), 3-hydroxyhexanoic acid (3HHx), and 3-hydroxyvalerate (3HV). In addition, there are polyhydroxyhexanoate esters, i.e., PHHx, and polyhydroxyoctanoate esters, PHO, etc. PHA polymers exhibit excellent biocompatibility, allowing cells to grow well on this scaffold, which can also degrade into CO2 and H2O. PHA is one of the most ideal biomedical materials currently available. Developing PHA dermal fillers has significant industrial value and broad application prospects.

[0004] Bacterial endotoxins are a major representative of pyrogens and a significant contaminant in injectable products. To ensure human safety during the development of injectable filler products, strict control over the bacterial endotoxin content is necessary. Summary of the Invention

[0005] One objective of this invention is to provide a method for detecting bacterial endotoxins in PHA gel fillers. The method includes the following steps: mixing PHA gel fillers, an organic solvent, and water, separating the layers, and taking an appropriate amount of the aqueous layer solution for bacterial endotoxin detection. The organic solvent is a haloalkane.

[0006] In a preferred embodiment of the present invention, the volume / volume / weight ratio of water, organic solvent and PHA gel filler in the detection method is 10-30ml:2-10ml:1g, preferably 15-25ml:3-7ml:1g.

[0007] In a preferred embodiment of the present invention, the haloalkane in the detection method is C 1-6 Halogenated alkanes, preferably dichloromethane.

[0008] In a preferred embodiment of the present invention, the endotoxin is determined using a gel electrophoresis method.

[0009] In a preferred embodiment of the present invention, the detection method is as follows: take an appropriate amount of PHA gel filler, add dichloromethane, mix thoroughly, then add water, mix thoroughly, let stand to separate into layers, measure an appropriate amount of the upper layer solution, and determine the endotoxin according to the gel method, wherein the volume / volume / weight ratio of water, dichloromethane and PHA gel filler is 20ml:5ml:1g.

[0010] In a preferred embodiment of the present invention, the filler comprises PHA microspheres, sodium carboxymethyl cellulose, an osmotic pressure regulator, a pH regulator, and water for injection, wherein the weight fraction of sodium carboxymethyl cellulose in the filler is 2%-4%, and the viscosity range is 6700-17000 mPa·s.

[0011] In a preferred embodiment of the present invention, the PHA is a natural or non-natural polyhydroxy fatty acid ester. Preferably, the natural or non-natural polyhydroxy fatty acid ester is selected from any one or a combination of PHB, PHBV, PHBHHx, P34HB, PHBVHHx, PHHx and PHO.

[0012] In a preferred embodiment of the present invention, the PHA is PHBHHx.

[0013] In a preferred embodiment of the present invention, the molecular weight of the PHBHHx microspheres is 50,000 Da to 150,000 Da, and the particle size D50 is 20 μm to 50 μm.

[0014] In a preferred embodiment of the present invention, the weight fraction of PHA microspheres in the PHA gel filler is 28%-32%.

[0015] In a preferred embodiment of the present invention, the osmotic pressure regulator in the PHA gel filler is selected from any one or a combination of glycerol, mannitol, sodium chloride, and potassium chloride, and preferably the osmotic pressure regulator is glycerol.

[0016] In a preferred embodiment of the present invention, the pH adjuster in the PHA gel filler is at least one selected from dihydrogen phosphate, dihydrogen phosphate, dihydrogen phosphate hydrate, and dihydrogen phosphate hydrate; preferably, the pH adjuster is dihydrogen phosphate hydrate and dihydrogen phosphate hydrate; preferably, the dihydrogen phosphate is selected from one or more selected from disodium hydrogen phosphate, dipotassium hydrogen phosphate, and diammonium hydrogen phosphate; preferably, the dihydrogen phosphate is selected from one or more selected from sodium dihydrogen phosphate, potassium dihydrogen phosphate, and ammonium dihydrogen phosphate; preferably, the pH adjuster is disodium hydrogen phosphate hydrate and sodium dihydrogen phosphate hydrate; preferably, the pH adjuster is disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate monohydrate.

[0017] In a preferred embodiment of the present invention, the weight fraction of the osmotic pressure regulator in the PHA gel filler is 0.2%-1.0%.

[0018] In a preferred embodiment of the present invention, the weight fraction of the pH adjuster in the PHA gel filler is 0.5%-0.8%.

[0019] In a preferred embodiment of the present invention, the PHA gel filler comprises PHA microspheres, sodium carboxymethyl cellulose, glycerol, disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate monohydrate, and water for injection, wherein the weight fraction of PHA microspheres in the composition is 28-32%, the weight fraction of sodium carboxymethyl cellulose is 2%-4%, the weight fraction of glycerol is 0.2%-1.0%, and the weight fractions of disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate monohydrate are 0.5%-0.8%. Preferably, the weight fraction of disodium hydrogen phosphate dodecahydrate is 0.5%, and the weight fraction of sodium dihydrogen phosphate monohydrate is 0.12%.

[0020] In a preferred embodiment of the present invention, the preparation method of the PHA gel filler includes the following steps:

[0021] (1) Dissolve the pH adjuster and osmotic pressure adjuster in water for injection, and then add sodium carboxymethyl cellulose to prepare a gel matrix;

[0022] (2) Sterilize the gel matrix and PHA microspheres;

[0023] (3) Mix the PHA microspheres and the gel matrix;

[0024] In a preferred embodiment of the present invention, the preparation method includes the following steps:

[0025] (1) Add water for injection to the mixing tank, and add the weighed pH adjuster and osmotic pressure adjuster in sequence. Stir until dissolved, and slowly add sodium carboxymethyl cellulose while stirring. At the same time, turn on high shear to obtain the gel matrix; (2) Sterilize the gel matrix and PHA microspheres; (3) Add PHA microspheres slowly to the gel matrix while stirring until the materials are mixed evenly; (4) Fill and seal.

[0026] Another objective of this invention is to provide the application of the method for detecting bacterial endotoxins in PHA gel fillers of this invention in the detection of bacterial endotoxins in PHA gel fillers.

[0027] Unless otherwise stated, when this invention relates to percentages between liquids, the percentage is volume / volume percentage; when this invention relates to percentages between liquids and solids, the percentage is volume / weight percentage (ml / g); when this invention relates to percentages between solids and liquids, the percentage is weight / volume percentage (g / ml); the remainder is weight / weight percentage.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] This invention develops a novel filler for PHA injection, systematically studies the effects of gel carriers and osmotic pressure regulators on the composition's shear viscosity and osmotic pressure properties, and scientifically screens to obtain an excellent and stable gel formulation.

[0030] The PHA microspheres in the composition of this invention have good biocompatibility. After being injected into the dermis, they can stimulate collagen proliferation, achieving a long-term filling effect. The carrier, sodium carboxymethyl cellulose, has good viscosity and elasticity, and after being injected into the skin surface, it can achieve a short-term filling function. The filler of this invention has broad application prospects in the field of medical aesthetics.

[0031] This invention scientifically screened solvents for detecting bacterial endotoxins in PHA gel fillers. It was found that increasing the amount of water or using combinations of water and various conventional organic solvents failed to achieve endotoxin detection. However, it was unexpectedly discovered that a combination of water and a small amount of dichloromethane could achieve sufficient release of endotoxins and accurate detection of their content. The method of this invention can effectively ensure the safety of human medication and has significant industrial value. Attached Figure Description

[0032] Figure 1 Photographs showing the appearance of the composition in Example 1;

[0033] Figure 2 Microscopic photographs of the composition of Example 1;

[0034] Figure 3The results are the viscoelasticity test results of the composition in Example 14. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be noted that, unless otherwise specified, the embodiments of the present invention and the features thereof can be arbitrarily combined with each other.

[0036] In this embodiment of the invention, sodium carboxymethyl cellulose was purchased from Ashland Pharmaceuticals, glycerol from Hunan Ercon Pharmaceutical Co., Ltd., disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate monohydrate from Hunan Jiudian Hongyang Pharmaceutical Co., Ltd., horseshoe crab reagent from Zhanjiang Bokang Marine Biology or Zhanjiang Andus, water for bacterial endotoxin testing from Zhanjiang Andus, and bacterial endotoxin working standards from the National Institutes for Food and Drug Control. Unless otherwise specified, PHA in the embodiments is PHBHHx, purchased from Beijing Lanjing Microbial Technology Co., Ltd. The microspheres were prepared using conventional membrane emulsification method, with a molecular weight (Mw) of 50,000 Da to 150,000 Da and a particle size (D50) of 20 μm to 50 μm.

[0037] 1. The appearance of the composition is examined under an illuminance of 1000 lx to 1500 lx; ​​2. Pushing force: used to evaluate the flowability and deformation ability of the cosmetic injection product during the pushing process to ensure its smoothness and safety in clinical use. Low pushing force makes the sample easy to squeeze out, while high pushing force makes the sample difficult to squeeze out. A large difference in pushing force indicates that the sample is unevenly dispersed or has aggregated and concentrated, which will also affect the handiness during injection. Based on clinical experience and the characteristics of this product, the maximum pushing force should not exceed 50 N, and the average pushing force should be within the range of 20 N ± 10 N. Pushing force test method: Take a sample, install the accompanying injection needle, and determine the force according to Appendix A of "YY / T 0962-2021 Cross-linked Sodium Hyaluronate Gel for Plastic Surgery". The machine pushes the syringe piston at a speed of 30 mm / min, and records the minimum pushing force, maximum pushing force, and average pushing force; 3. Shear viscosity: that is, the ratio of shear stress to shear rate under steady flow conditions. Shear viscosity is a measure of the internal friction of liquid molecules and a specific reflection of the viscous flow properties of a substance. Low viscosity means good fluidity, and high viscosity means poor fluidity. The pushing force will also increase accordingly. Based on the use and characteristics of this product, the shear viscosity range is set at 200-600 Pa·s. Test method: At (25±0.2)℃, a rheometer is used at a shear rate of 1s -14. Osmotic pressure: Osmotic pressure close to that of the human body can reduce irritation in clinical applications. The osmotic pressure of the human body is basically 280-310 mOsm / kg, therefore the osmotic pressure range of the composition of this invention should be 200-400 mOsm / kg. Test method: Determined according to the osmolar concentration determination method of 0632 in the Pharmacopoeia of the People's Republic of China (Part IV) (2020 edition); 5. PHA molecular weight: Changes in molecular weight can reflect its in vitro degradation during storage, providing a basis for storage conditions. Determination method: Determined according to the size exclusion chromatography method in 0514 of the Pharmacopoeia of the People's Republic of China (Part IV) (2020 Edition); 6. PHA microsphere particle size: Microspheres that are too small are easily phagocytosed by macrophages, causing inflammatory reactions. Microspheres that are too large affect injectability and needle penetration, and also increase the incidence of adverse reactions such as granulomas and nodules. It is advisable to select a target particle size of 20-50μm. Test method: Determined according to the wet method of the third method (light scattering method) of the Particle Size and Particle Size Distribution Determination Method in 0982 of the Pharmacopoeia of the People's Republic of China (Part IV) (2020 Edition).

[0038] According to the Bacterial Endotoxin Test Method 1143 of the Pharmacopoeia of the People's Republic of China (Part IV) (2020 Edition), the bacterial endotoxin test includes two methods: gel method and photometric method. This invention employs the gel method. The Application Guidelines for the Bacterial Endotoxin Test Method 9251 of the Pharmacopoeia of the People's Republic of China (Part IV) (2020 Edition) stipulate that endotoxin testing should generally be performed using water to dissolve the sample. Examples 16-19 all use the PHA injection gel filler sample prepared according to Example 1.

[0039] Example 1: Preparation of PHA Injectable Filler

[0040] This embodiment describes the preparation of a pilot-scale batch of injectable filler, with a batch size of 8,000 vials. The specific composition is shown in Table 1 below.

[0041] Table 1

[0042]

[0043] Preparation method: (1) Add water for injection to the mixing tank, and add the weighed disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate monohydrate, and glycerol in sequence. Stir until dissolved, and slowly add sodium carboxymethyl cellulose while stirring. At the same time, turn on high shear to obtain the gel matrix; (2) Perform moist heat sterilization and irradiation sterilization on the gel matrix and PHA microspheres respectively; (3) Add PHA microspheres slowly into the gel matrix while stirring until the materials are mixed evenly; (4) Fill and seal.

[0044] Performance tests of the filler composition: maximum extrusion force 20 N, average extrusion force 18 N, shear viscosity 325 Pa·s, pH value 7.2, osmotic pressure 282 mOsmol / kg, the composition is a white gel. Appearance photos and microscopic photos are attached. Figure 1-2 All indicators meet the requirements.

[0045] Examples 2-5: Preparation of PHA Injectable Fillers

[0046] The following formulations differ from Example 1 in that they differ in the content of the osmotic pressure regulator, as detailed in Table 2 below:

[0047] Table 2

[0048]

[0049]

[0050] Note: Except for water for injection, the proportions of all other components are the same as in Example 1.

[0051] The composition was prepared according to Example 1, and the osmotic pressure results are shown in Table 3 below:

[0052] Table 3

[0053] Example Osmotic pressure (mOsmol / kg) Example 2 378 Example 3 340 Example 4 259 Example 5 223

[0054] The results showed that the osmotic pressure of the composition met the requirements when the glycerol content in the composition was in the range of 0.2%-1.0% (w / w).

[0055] Examples 6-9: Preparation of PHA Injectable Fillers

[0056] For the injectable filler of the present invention, sodium carboxymethyl cellulose serves as a gel carrier, which has a significant impact on the dispersion of PHA microspheres and the performance parameters of the composition, such as shear viscosity and extrusion force. This set of examples involves changing the content of sodium carboxymethyl cellulose in the formulation and examining its effect on the composition's performance, as detailed in Table 4 below:

[0057] Table 4

[0058]

[0059] Note: Except for water for injection, the proportions of all other components are the same as in Example 1.

[0060] The results showed that as the sodium carboxymethyl cellulose content in the composition increased, the shear viscosity, maximum extrusion force, average extrusion force, and osmotic pressure of the composition all increased. The sample prepared in Example 6 was easily flowable, and due to its low viscosity, the microsphere gel showed visible microsphere aggregation and uneven dispersion; while the sample prepared in Example 9 had a relatively high maximum extrusion force, making it inconvenient for doctors to use. Therefore, the preferred content of sodium carboxymethyl cellulose in the composition is 2%-4%.

[0061] Examples 10-11: Preparation of PHA Injectable Fillers

[0062] The content of PHA microspheres, a biomaterial, in the formulation was varied, as detailed in Table 5 below:

[0063] Table 5

[0064] PHA microspheres Single dose (mg) Percentage (w / w) Example 10 280 28％ Example 11 320 32％

[0065] Note: Except for water for injection, the proportions of all other components are the same as in Example 1.

[0066] The composition was subjected to performance tests similar to those in Example 1. The results showed that when the proportion of PHA microspheres in the formulation was 28%-32%, all indicators met the requirements.

[0067] Examples 12-15: Preparation of PHA Injectable Fillers

[0068] As mentioned earlier, sodium carboxymethyl cellulose, as a gel carrier, has a significant impact on the properties of the composition. This set of examples studies the effect of using sodium carboxymethyl cellulose at different viscosity levels on the gel formulation properties, as detailed in Table 6 below:

[0069] Table 6

[0070]

[0071] Studies have found that the selection of sodium carboxymethyl cellulose (CMC) with different viscosity levels has a significant impact on the performance of the composition. For example, when CMC-Na from Examples 14 or 15 is used, the shear viscosity of the composition meets normal requirements, while when CMC-Na from Examples 12 or 13 is used, the shear viscosity of the composition is significantly lower. Furthermore, this invention also discovered that sodium carboxymethyl cellulose with different viscosity levels has a significant impact on the viscoelasticity of the composition. Viscoelastic properties are an important indicator for evaluating the supporting performance of a product, and are generally tested by frequency scanning, i.e., oscillating the material at different frequencies under a certain temperature and amplitude to test its viscoelasticity. Based on the product's G' and G' trends, materials can generally be classified into viscoelastic solids, gels, and viscoelastic liquids. The study of this set of examples shows that the compositions of Examples 14 and 15 have superior viscoelastic properties due to the selection of sodium carboxymethyl cellulose with suitable viscosity. Figure 3 The results are the viscoelasticity test results of the composition in Example 14.

[0072] Based on the above studies, it is believed that when sodium carboxymethyl cellulose with a viscosity level of 6700-17000 mPa·s is used, the overall performance of the prepared PHA injection filler is better.

[0073] Example 16: Detection of bacterial endotoxins in PHA injectable gel filler

[0074] Take 1.0g of gel filler and add 10ml-50ml of water for bacterial endotoxin testing. Mix thoroughly and find that the system disperses into flocculent form. After standing, the microspheres float on the upper layer and cannot be used for endotoxin detection.

[0075] Example 17: Detection of bacterial endotoxins in PHA injectable gel filler

[0076] Take 1.0g of each gel filler and add 5mL of acetone, toluene, ethyl acetate, or n-hexane to each, mix thoroughly, and then add 20mL of water for bacterial endotoxin testing to each, mix thoroughly. The results are shown in Table 7 below:

[0077] Table 7

[0078] organic solvents Phenomenon acetone No layering Toluene The mixture separated into layers, but the organic phase remained turbid even after adding 20 mL of the organic phase. Ethyl acetate The mixture separated into layers, but the organic phase remained turbid even after adding 20 mL of the organic phase. n-Hexane The mixture separated into layers, but the organic phase remained turbid even after adding 20 mL of the organic phase. .

[0079] Example 18: Detection of bacterial endotoxins in PHA injectable gel filler

[0080] Take 1.0 g of gel filler, add 5 mL of dichloromethane, mix thoroughly, then add 20 mL of water for bacterial endotoxin testing, mix thoroughly, and allow to stand for separation. Both the upper and lower layers are clear solutions. Take an appropriate amount of the upper aqueous solution and determine the endotoxin content according to the bacterial endotoxin test method 1143 of the Pharmacopoeia of the People's Republic of China (Part IV) (2020 edition). The endotoxin content meets the requirements (limit L = 20 EU / g).

[0081] Example 19: Validation of the Methodology for Detecting Bacterial Endotoxins in PHA Injectable Gel Fillers

[0082] The bacterial endotoxin content was determined using the gel electrophoresis method, and the methodology was validated. Three batches of gel filler samples were selected, and each batch was validated using horseshoe crab reagents from two different manufacturers.

[0083] 1) Maximum effective dilution factor of the test sample:

[0084] MVD = cL / λ

[0085] Where L is the bacterial endotoxin limit of the test sample, c is the concentration of the test sample, and λ is the labeled sensitivity (EU / ml) of the Limulus amebocyte lysate (LAL) reagent in the gel electrophoresis method.

[0086] The bacterial endotoxin limit for the test sample is L = 20 EU / g. This experiment is the first to use a Limulus amebocyte lysate (LAL) reagent with a sensitivity λ of 0.0625 EU / ml, and the minimum effective dilution concentration was calculated to be 3.125 mg / ml.

[0087] 2) Sample pretreatment

[0088] Take 1g of gel filler, add 5ml of dichloromethane solution, mix thoroughly, then add 20ml of water for bacterial endotoxin testing, mix thoroughly, let stand to separate into layers, and record the upper layer as C. 50 The concentration is 50 mg / ml. Take an appropriate amount of the upper layer solution and test it according to the procedure.

[0089] 3) Experimental procedures

[0090] Solutions A, B, C, and D were prepared according to Table 8. The test solutions used were those in which endotoxins were not detected and whose dilution did not exceed the maximum dilution factor. In this experiment, a test solution with a concentration of 3.125 mg / ml was selected.

[0091] Table 8

[0092]

[0093] Note: A is the test solution; B is the interference test series; C is the control series for the labeled sensitivity of the Limulus amebocyte lysate (LAL) reagent; D is the negative control.

[0094] Preparation of solution A: The C obtained from the pretreatment of the sample in step 2) above... 50 The supernatant solution was diluted with water to 3.125 mg / ml for bacterial endotoxin testing.

[0095] Preparation of Solution B: Reconstitute one vial of bacterial endotoxin working standard with 1 ml of endotoxin test water, then vortex mix for 15 minutes to obtain a concentration of 90 EU / ml. Dilute the reconstituted bacterial endotoxin working standard to 2λ, λ, 0.5λ, and 0.25λ using Solution A.

[0096] Preparation of solution C: Reconstitute one vial of bacterial endotoxin working standard with 1 ml of endotoxin test water, then vortex mix for 15 minutes to obtain a concentration of 90 EU / ml. Dilute the reconstituted bacterial endotoxin working standard to 2λ, λ, 0.5λ, and 0.25λ with bacterial endotoxin test water.

[0097] Sample addition: Take 28 Limulus amebocyte lysate (LAL) reagents, add 0.1 ml of bacterial endotoxin test water to each reagent to reconstitute them, and add 0.1 ml of the reconstituted LAL reagents according to the table above.

[0098] Incubation: Gently shake the ampoule containing the added Limulus ampoule to mix the solution thoroughly without generating air bubbles. Seal the ampoule and place it vertically in a constant temperature water bath at 37℃±1℃. Gently cover the ampoule and incubate for 60 minutes±2 minutes. After incubation, slowly invert the ampoule 180° and observe the results: If a gel forms inside the ampoule and does not deform or slip off the ampoule wall, the result is positive; if no gel forms or the formed gel is not firm, deformed, or slips off the ampoule wall, the result is negative.

[0099] The test is valid only if all parallel tubes of solution A and negative control solution D are negative, and the results of series solution C meet the requirements of the Limulus Amebocyte Lysate (LAL) reagent sensitivity verification test. If the results of series solution B meet the requirements of the LAL reagent sensitivity verification test, the test sample is considered to have no interfering effect at that concentration. Otherwise, the test sample is considered to have an interfering effect at that concentration.

[0100] 4) Experimental Results

[0101] Table 9

[0102]

[0103] Note: "+" indicates a positive result, and "-" indicates a negative result.

[0104] Solution B: λ c =antilg(∑X / 4)=0.036

[0105] Solution C: λ c =antilg(∑X / 2)=0.06

[0106] Conclusion: The interference test results meet the requirements.

[0107] The above experiment was repeated using a different manufacturer's horseshoe crab reagent, and the results are shown in Table 10 below:

[0108] Table 10

[0109]

[0110] Solution B: λ c =antilg(∑X / 4)=0.042

[0111] Solution C: λ c =antilg(∑X / 2)=0.06

[0112] The results meet the requirements.

[0113] The test results of the other two batches of test samples also met the requirements for interference experiments and complied with regulations.

[0114] While the embodiments disclosed in this invention are as described above, the content is merely for the purpose of facilitating understanding of the invention and is not intended to limit the invention. Any person skilled in the art to which this invention pertains may make any modifications and variations in the form and details of the implementation without departing from the spirit and scope disclosed herein; however, the scope of patent protection of this invention shall still be determined by the scope defined in the appended claims.

Claims

1. A method for detecting bacterial endotoxins in PHA gel fillers, the method comprising the following steps: PHA gel filler, organic solvent and water are mixed and separated into layers. An appropriate amount of the aqueous layer solution is taken for bacterial endotoxin detection. The organic solvent is a haloalkane.

2. The detection method according to claim 1, characterized in that, The detection method satisfies at least one of the following conditions (1)-(3): (1) The volume / volume / weight ratio of water, organic solvent and PHA gel filler is 10-30ml:2-10ml:1g, preferably 15-25ml:3-7ml:1g; (2) The haloalkane is C 1-6 Halogenated alkanes, preferably dichloromethane; (3) Endotoxin was determined by gel electrophoresis.

3. The detection method according to any one of claims 1-2, characterized in that, The specific detection method is as follows: Take an appropriate amount of PHA gel filler, add dichloromethane, mix thoroughly, then add water, mix thoroughly, let stand to separate into layers, measure an appropriate amount of the upper layer solution, and determine the endotoxin according to the gel method, wherein the volume / volume / weight ratio of water, dichloromethane and PHA gel filler is 20ml:5ml:1g.

4. The detection method according to any one of claims 1-3, characterized in that, The filler comprises PHA microspheres, sodium carboxymethyl cellulose, osmotic pressure regulator, pH regulator and water for injection, wherein the weight fraction of sodium carboxymethyl cellulose in the filler is 2%-4% and the viscosity range is 6700-17000 mPa·s.

5. The detection method according to any one of claims 1-4, characterized in that, The filler satisfies at least one of the following conditions (1)-(6): (1) The PHA is a natural or non-natural polyhydroxy fatty acid ester; preferably, the natural or non-natural polyhydroxy fatty acid ester is selected from any one or a combination of PHB, PHBV, PHBHHx, P34HB, PHBVHHx, PHHx and PHO; preferably, the PHA is PHBHHx. (2) The weight fraction of PHA microspheres in the filler is 28%-32%; (3) The osmotic pressure regulator is selected from any one or a combination of glycerol, mannitol, sodium chloride, and potassium chloride, preferably glycerol; (4) The pH adjuster is at least one of dihydrogen phosphate, dihydrogen phosphate, dihydrogen phosphate hydrate, and dihydrogen phosphate hydrate; preferably, the pH adjuster is dihydrogen phosphate hydrate and dihydrogen phosphate hydrate; preferably, the dihydrogen phosphate is selected from one or more of disodium hydrogen phosphate, dipotassium hydrogen phosphate, and diammonium hydrogen phosphate; preferably, the dihydrogen phosphate is selected from one or more of sodium dihydrogen phosphate, potassium dihydrogen phosphate, and ammonium dihydrogen phosphate; preferably, the pH adjuster is disodium hydrogen phosphate hydrate and sodium dihydrogen phosphate hydrate; preferably, the pH adjuster is disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate monohydrate; (5) The weight fraction of the osmotic pressure regulator in the filler is 0.2%-1.0%; (6) The weight fraction of the pH adjuster in the filler is 0.5%-0.8%.

6. The detection method according to any one of claims 1-5, characterized in that, The filler comprises PHA microspheres, sodium carboxymethyl cellulose, glycerol, disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate monohydrate, and water for injection. The composition contains 28-32% PHA microspheres by weight, 2%-4% sodium carboxymethyl cellulose by weight, 0.2%-1.0% glycerol by weight, and 0.5%-0.8% disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate monohydrate by weight. Preferably, the disodium hydrogen phosphate dodecahydrate is 0.5% by weight, and the sodium dihydrogen phosphate monohydrate is 0.12% by weight.

7. The detection method according to any one of claims 1-6, characterized in that, The preparation method of the PHA gel filler includes the following steps: (1) Dissolve the pH adjuster and osmotic pressure adjuster in water for injection, and then add sodium carboxymethyl cellulose to prepare a gel matrix; (2) Sterilize the gel matrix and PHA microspheres; (3) Mix the PHA microspheres and the gel matrix.

8. The detection method according to any one of claims 1-7, characterized in that, The preparation method of the PHA gel filler includes the following steps: (1) Add water for injection to the solution preparation tank, add the weighed pH adjuster and osmotic pressure adjuster in sequence, stir until dissolved, maintain stirring and slowly add sodium carboxymethyl cellulose while stirring, and turn on high shear at the same time to obtain the gel matrix. (2) Sterilize the gel matrix and PHA microspheres; (3) While stirring, slowly add the PHA microspheres into the gel matrix and stir until the materials are evenly mixed; (4) Filling and sealing.

9. The application of the detection method according to any one of claims 1-8 in the detection of bacterial endotoxins in PHA gel fillers.

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