Method for the detection of bacterial endotoxins in gelatin microcarriers for cell culture

Abstract

This invention discloses a method for detecting bacterial endotoxins in gelatin microcarriers used for cell culture, comprising the following steps: (1) rechecking the sensitivity of the Limulus Amebocyte Lysate (LAL) reagent to ensure that the sensitivity result of the LAL reagent is between 0.5 and 2.0 λ; (2) determining the limit value and minimum dilution concentration of bacterial endotoxins in the gelatin microcarriers; (3) enzymatically digesting the PBS solution of the gelatin microcarriers to be tested with collagenase solution to obtain an enzymatic digest; (4) checking for bacterial endotoxins in the gelatin microcarriers: mixing a magnesium ion regulator and the LAL reagent to obtain a reconstituted LAL reagent, mixing the enzymatic digest obtained in step (3) with the reconstituted LAL reagent to obtain a mixed solution, and using the gelation method to determine whether bacterial endotoxins are present. By combining the pretreatment enzymatic digestion step with the LAL reagent reconstituted with a magnesium ion regulator, the gelatin microcarriers can be effectively enzymatically digested, fully releasing endotoxins, and the interference of phosphate ions can be effectively eliminated by the magnesium ion regulator, thereby improving the specificity and accuracy of the detection.

Description

Technical Field

[0001] This invention relates to the field of microbial detection technology, specifically to a method for detecting bacterial endotoxins in gelatin microcarriers used in cell culture. Background Technology

[0002] Gelatin microcarriers, as commonly used biomaterials, are widely applied in biomedical fields such as cell culture. Bacterial endotoxins are lipopolysaccharide components of the cell walls of Gram-negative bacteria, possessing extremely strong pyrogenic and toxic properties; their content is one of the important indicators for evaluating the safety of biomaterials. Therefore, rigorous bacterial endotoxin testing of gelatin microcarriers is crucial.

[0003] Conventional methods for detecting bacterial endotoxins mainly rely on the Limulus amebocyte lysate (LAL) reagent method specified in the pharmacopoeia, including the gel electrophoresis method, dynamic turbidity method, and dynamic colorimetric method (such as the patent document with publication number CN116047076A). For example, Chinese patent document with publication number CN116930500A discloses an analytical method for determining the content of bacterial endotoxins in hyaluronic acid, which adopts the following steps: (1) adding an enzyme to macromolecular or medium-molecular-weight hyaluronic acid and obtaining hyaluronic acid hydrolysate through enzymatic hydrolysis; (2) diluting the hyaluronic acid hydrolysate with bacterial endotoxin test water to prepare a test solution; (3) diluting the hyaluronic acid hydrolysate with bacterial endotoxin test water and mixing it with an endotoxin standard solution to prepare a positive control solution; (4) detecting the endotoxin content in the test solution and the control solution respectively.

[0004] However, gelatin microcarriers present unique challenges in detection: gelatin itself is a large protein molecule, and its particle size and high viscosity interfere with the reaction between the horseshoe crab reagent and endotoxins when directly used for endotoxin detection; if conventional solvents are used for dissolution, the gelatin microcarriers are difficult to degrade completely, leading to incomplete endotoxin release and affecting detection accuracy. Furthermore, the pretreatment of gelatin microcarriers often requires the use of phosphate-containing reagents such as PBS buffer, which inhibits the coagulating enzyme activity of the horseshoe crab reagent, causing false negative results and further increasing the difficulty of detection. Therefore, establishing a method suitable for gelatin microcarriers that can effectively eliminate interference and accurately detect endotoxin content is of significant practical importance. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for detecting bacterial endotoxins in gelatin microcarriers used in cell culture. By combining a pretreatment enzymatic hydrolysis step with a magnesium ion regulator-reconstituted Limulus amebocyte lysate (LAL) reagent, the gelatin microcarriers can be effectively enzymatically hydrolyzed to fully release endotoxins. Furthermore, the magnesium ion regulator effectively eliminates interference from phosphate ions, thereby improving the specificity and accuracy of the detection.

[0006] A method for detecting bacterial endotoxins in gelatin microcarriers used in cell culture, comprising the following steps: (1) Verify the sensitivity of the Limulus amebocyte lysate (LAL) reagent to ensure that the sensitivity result of the LAL reagent is between 0.5 and 2.0 λ; (2) Determine the limits of bacterial endotoxins and the minimum dilution concentration in gelatin microcarriers; (3) The gelatin microcarriers to be tested were enzymatically hydrolyzed in PBS solution using collagenase solution to obtain the hydrolysate; (4) Examination of bacterial endotoxins in gelatin microcarriers: magnesium ion regulator and horseshoe crab reagent were mixed to obtain reconstituted horseshoe crab reagent. The enzymatic hydrolysate obtained in step (3) was mixed with the reconstituted horseshoe crab reagent to obtain a mixed solution. The gel method was used to determine whether it contained bacterial endotoxins.

[0007] In existing technologies, the use of PBS buffer during enzymatic hydrolysis introduces phosphate ions that bind with metal ions such as calcium and magnesium ions in the horseshoe crab reagent, affecting the activation of coagulation prozyme. Therefore, this invention addresses this by adding a magnesium ion regulator during the reconstitution of the horseshoe crab reagent. This replenishes the free magnesium ions lost due to phosphate complexation, maintaining the balance of metal ions in the horseshoe crab reagent reaction system and thus eliminating the inhibitory effect of phosphate ions on the horseshoe crab reagent. The mixture of the magnesium ion regulator and the horseshoe crab reagent is then mixed with the enzymatically hydrolyzed gelatin microcarriers to determine the endotoxin content in the gelatin microcarriers using a gelation method.

[0008] Preferably, in step (2), the bacterial endotoxin limit in the gelatin microcarrier is less than 40 EU / g gelatin microcarrier.

[0009] Preferably, in step (3), the mass ratio of collagenase to gelatin microcarrier is 1.5~2.5:1 mg / g.

[0010] More preferably, the collagenase is collagenase I, and the mass ratio of collagenase I to gelatin microcarrier is 1.75~2:1 mg / g.

[0011] Preferably, in step (3), before the collagenase solution is mixed with the PBS solution of the gelatin microcarrier to be tested, the collagenase solution is subjected to ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of ≤300 kDa to remove endotoxins while retaining collagenase activity and eliminating interference from endotoxins on subsequent tests.

[0012] Preferably, in step (3), the enzymatic hydrolysis temperature is 32~42 ℃ and the time is 3~12 h.

[0013] Preferably, in step (4), the magnesium ion regulator is magnesium chloride or magnesium sulfate.

[0014] Preferably, in step (4), the ratio of magnesium ion regulator to Limulus amebocyte lysate (LAL) reagent in the reconstituted Limulus amebocyte lysate is 18-22 mg: 0.06 EU.

[0015] Preferably, in step (4), the judgment criteria for the gel method are as follows: if the mixed solution does not solidify and slips when tilted at 180°, the result is negative, indicating that the gelatin microcarrier to be tested does not contain bacterial endotoxin; if the mixed solution solidifies and does not slip when tilted at 180°, the result is positive, indicating that the gelatin microcarrier to be tested contains bacterial endotoxin.

[0016] Preferably, in step (4), before performing the bacterial endotoxin test in the gelatin microcarrier, an interference experiment is first performed on the collagenase solution to verify whether the collagenase solution interferes with the bacterial endotoxin test.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: To address the problems of high interference and low accuracy in the detection of bacterial endotoxins from gelatin microcarriers in existing technologies, this invention provides a creative and unique detection method. This method can efficiently degrade gelatin microcarriers, fully release endotoxins, and effectively eliminate the interference of phosphate ions, thereby improving the specificity and accuracy of the detection method. Attached Figure Description

[0018] Figure 1 and Figure 2 The images show the positive and negative results of the endotoxin detection in this invention, in that order. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to the embodiments, but the implementation of the present invention is not limited to the following embodiments.

[0020] All raw materials used in this invention are commercially available.

[0021] Example 1 (1) Refer to the relevant provisions of the General Chapter 1143 of Part IV of the Chinese Pharmacopoeia 2020 edition, Bacterial Endotoxin Test Method (Gel Method), verify the sensitivity of the horseshoe crab reagent, and determine the limit value of bacterial endotoxin of the test sample and the minimum dilution concentration.

[0022] Upon verification, the sensitivity values ​​of the horseshoe crab reagent used in this invention (purchased from Zhanjiang Bokang Marine Technology Co., Ltd., batch number: 2512012) are all within the range of 0.5 λ to 2.0 λ (λ is the labeled sensitivity of the horseshoe crab reagent), which meets the requirements and can be tested according to the labeled sensitivity value.

[0023] (2) In accordance with the provisions of Section 1143 of the General Chapter of the 2020 edition of the Chinese Pharmacopoeia, the limit value of bacterial endotoxin in gelatin microcarriers was determined to be 40 EU / 1g gelatin microcarrier.

[0024] (3) Weigh collagenase I powder and prepare a collagenase I solution of 1.5 mg / mL. Use an ultrafiltration membrane with a molecular weight cutoff of 300 kDa to perform ultrafiltration treatment and collect the permeate to obtain collagenase I solution. Weigh 1 g of the gelatin microcarrier to be tested (purchased from Zhongke Junda), add 9 mL of PBS buffer at pH 7.2, then add 1 mL of collagenase I solution, and place it at 37 ℃ for 12 hours to obtain the enzymatic hydrolysate.

[0025] (4) Interference experiments were conducted according to General Chapter 1143 of Part IV of the 2020 edition of the Chinese Pharmacopoeia. The initial concentration C of the collagenase solution was 1 mg / mL, L = the standard for endotoxin in collagenase < 5 EU / mg, and λ = the specification of the horseshoe crab reagent used was 0.06 EU / mL. Based on the formulas for the maximum dilution factor (MVD) and the minimum effective concentration (MVC) of the sample in the pharmacopoeia: The calculations showed that the maximum dilution factor for the collagenase sample was 83.33 times, and the minimum effective concentration of collagenase was 0.012 mg / mL.

[0026] Dilute 1 mg / mL collagenase solution to the minimum effective concentration of the sample, 0.012 mg / mL. This concentration is the detection concentration for the interference experiment (collagenase solution concentration B in Table 1). Mix the 0.012 mg / mL collagenase sample with four gradient concentrations of standard solution: 2λ, λ, 0.5λ, and 0.25λ. These four concentrations are the interference verification solution B. Add the spiked sample and the sample mixture. Use water (without pyrogen) for testing as the negative solution D. Dilute the standard to four concentrations: 2λ, λ, 0.5λ, and 0.25λ. This solution is the collagenase positive control group C. Group A is the sample solution. Confirm whether the endotoxin content in group A meets the requirements. Judgment criteria: The test is valid only when 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. When the results of series solution B are between 0.5λ and 2λ (inclusive), the test sample is considered to have no interference effect at that concentration. In other cases, it is considered that the test sample has an interfering effect at that concentration.

[0027] Table 1: Results of the interference experiment with collagenase I solution (5) Examination of bacterial endotoxins in gelatin microcarriers: 0.1 mL of MgCl2·6H2O (purchased from Zhanjiang Bokang Marine Technology Co., Ltd., concentration of 20 mg / mL) and each Limulus amebocyte lysate reagent (0.06 EU / mL) were mixed to obtain reconstituted Limulus amebocyte lysate reagent. 0.1 mL of the enzymatic hydrolysate obtained in step (3) was mixed with the reconstituted Limulus amebocyte lysate reagent to obtain a mixed solution. The solution was kept at 37 °C for 60 minutes and observed to see if a gel was formed.

[0028] Example 4 The detection method is the same as in Example 1, except that the concentration of collagenase I solution is replaced with 1.75 mg / mL and the concentration of MgCl2·6H2O is 18 mg / mL.

[0029] Example 5 The detection method is the same as in Example 4, except that the concentration of MgCl2·6H2O is 20 mg / mL.

[0030] Example 6 The detection method is the same as in Example 4, except that the concentration of MgCl2·6H2O is 22 mg / mL.

[0031] Example 7 The detection method is the same as in Example 1, except that the concentration of collagenase I solution is replaced with 2.0 mg / mL.

[0032] Comparative Example 1 The detection method is the same as in Example 1, except that the concentration of collagenase I solution is replaced with 1 mg / mL.

[0033] Comparative Example 2 The detection method is the same as in Example 1, except that the concentration of collagenase I solution is replaced with 3 mg / mL.

[0034] Comparative Example 3 The detection method is the same as in Example 3, except that the concentration of MgCl2·6H2O is 16 mg / mL.

[0035] Comparative Example 4 The detection method is the same as in Example 3, except that the concentration of MgCl2·6H2O is 24 mg / mL.

[0036] Comparative Example 5 The detection method is the same as in Example 1, except that the enzymatic digestion step is not performed.

[0037] Comparative Example 6 The detection method is the same as in Example 1, except that the horseshoe crab reagent was not reconstituted using MgCl2·6H2O.

[0038] Comparative Example 7 The detection method is the same as in Example 1, except that collagenase I solution is replaced with collagenase II solution, the concentration remains the same, and the enzymatic hydrolysis time is extended to 14 h.

[0039] The interference experiment procedure was the same as in Example 1, and the specific results are shown in the table below.

[0040] Table 2: Results of the interference experiment with collagenase II solution Comparative Example 8 The detection method is the same as in Example 1, except that collagenase I solution is replaced with collagenase III solution, the concentration remains the same, and the enzymatic hydrolysis time is extended to 15 h.

[0041] The interference experiment procedure was the same as in Example 1, and the specific results are shown in the table below.

[0042] Table 3: Results of the interference experiment with collagenase III solution Test Example 1: Gel Formation Results and Endotoxin Detection Rate Endotoxin tests were performed on Examples 1-5, Comparative Examples 1-8, the blank control group (Limulus amebocyte lysate (LAL) reagent + product only), and the positive control group (LAL reagent + endotoxin standard), with each group tested in triplicate. The endotoxin detection rate was calculated using the following formula: Endotoxin detection rate .

[0043] Table 4 shows the detection results of Examples 1-5 and Comparative Examples 1-8 (sample number n=3). Test Example 2: The effect of collagenase filtration on test results The results of degradation tests on the same batch of microcarriers using collagenase I solutions that have undergone ultrafiltration and those that have not are shown in the table below.

[0044] Table 5: Effects of ultrafiltration and non-ultrafiltration collagenase I solutions on detection results Collagenase I solution that has not undergone ultrafiltration may have a high endotoxin content, which may significantly interfere with the experimental results. Collagenase I solution that has undergone ultrafiltration will produce normal experimental results.

[0045] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting bacterial endotoxins in gelatin microcarriers used in cell culture, characterized in that, Includes the following steps: (1) Verify the sensitivity of the Limulus amebocyte lysate (LAL) reagent to ensure that the sensitivity result of the LAL reagent is between 0.5 and 2.0 λ; (2) Determine the limits of bacterial endotoxins and the minimum dilution concentration in gelatin microcarriers; (3) The gelatin microcarriers to be tested were enzymatically hydrolyzed in PBS solution using collagenase solution to obtain the hydrolysate; (4) Examination of bacterial endotoxins in gelatin microcarriers: magnesium ion regulator and horseshoe crab reagent were mixed to obtain reconstituted horseshoe crab reagent. The enzymatic hydrolysate obtained in step (3) was mixed with the reconstituted horseshoe crab reagent to obtain a mixed solution. The gel method was used to determine whether it contained bacterial endotoxins.

2. The method for detecting bacterial endotoxins in gelatin microcarriers for cell culture according to claim 1, characterized in that, In step (2), the bacterial endotoxin limit in the gelatin microcarrier is less than 40 EU / 1g gelatin microcarrier.

3. The method for detecting bacterial endotoxins in gelatin microcarriers for cell culture according to claim 1, characterized in that, In step (3), the mass ratio of collagenase to gelatin microcarrier is 1.5~2.5:1 mg / g.

4. The method for detecting bacterial endotoxins in gelatin microcarriers for cell culture according to claim 3, characterized in that, The collagenase is collagenase I, and the mass ratio of collagenase I to gelatin microcarrier is 1.75~2:1 mg / g.

5. The method for detecting bacterial endotoxins in gelatin microcarriers for cell culture according to claim 1, characterized in that, In step (3), before the collagenase solution is mixed with the PBS solution of the gelatin microcarrier to be tested, the collagenase solution is subjected to ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of ≤300 kDa to remove endotoxins while retaining collagenase activity and eliminating the interference of endotoxins on subsequent tests.

6. The method for detecting bacterial endotoxins in gelatin microcarriers for cell culture according to claim 1, characterized in that, In step (3), the enzymatic hydrolysis temperature is 32~42 ℃ and the time is 3~12 h.

7. The method for detecting bacterial endotoxins in gelatin microcarriers for cell culture according to claim 1, characterized in that, In step (4), the magnesium ion regulator is magnesium chloride or magnesium sulfate.

8. The method for detecting bacterial endotoxins in gelatin microcarriers for cell culture according to claim 1, characterized in that, In step (4), the ratio of magnesium ion regulator to Limulus amebocyte lysate (LAL) reagent in the reconstituted Limulus amebocyte lysate reagent is 18-22 mg: 0.06 EU.

9. The method for detecting bacterial endotoxins in gelatin microcarriers for cell culture according to claim 1, characterized in that, In step (4), the judgment criteria for the gel method are as follows: if the mixed solution does not solidify and slips when tilted at 180°, the result is negative, indicating that the gelatin microcarrier to be tested does not contain bacterial endotoxin; if the mixed solution solidifies and does not slip when tilted at 180°, the result is positive, indicating that the gelatin microcarrier to be tested contains bacterial endotoxin.

10. The method for detecting bacterial endotoxins in gelatin microcarriers for cell culture according to claim 1, characterized in that, In step (4), before performing the bacterial endotoxin test in the gelatin microcarrier, an interference experiment is first conducted on the collagenase solution to verify whether the collagenase solution interferes with the bacterial endotoxin test.

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