Method for relieving low endotoxin recovery rate

Abstract

The invention discloses a method for relieving low endotoxin recovery rate. The method comprises the following steps: (1) rechecking sensitivity of tachypleus amebocyte lysate / verifying reliability of a standard curve; (2) determining key parameters of retention time research according to a production process; (3) determining the non-interference dilution ratio of the sample; (4) carrying out retention time research; (5) determining that the sample has an LER phenomenon; and (6) a method for relieving the low endotoxin recovery rate: treating the sample with human serum albumin, and carrying out retention time research again to confirm that the LER phenomenon is eliminated. And (7) confirming that the low endotoxin recovery rate phenomenon is relieved, and calculating the recovery rate of the endotoxin. The method has the following beneficial effects: 1, shielded endotoxin in a sample can be detected; 2, time and labor are saved; cost is low, operation is easy, and precision is higher than that of rabbit experiments; 4, a sample with a low endotoxin recovery rate phenomenon is solved, and clinical safety and effectiveness are guaranteed.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for alleviating low endotoxin recovery rate. Background Art

[0002] "Low Endotoxin Recovery" (LER) refers to the phenomenon that when using the bacterial endotoxin test method (Limulus amebocyte lysate method) specified in the pharmacopoeia to detect bacterial endotoxins, the known concentration of standard endotoxin added to certain sterile biological product samples cannot be detected. According to the definition in Technical Report No. 82 written by the Parenteral Drug Association (PDA), LER refers to the phenomenon that "the recovery rate of the added standard endotoxin in the sample is less than 50% as time goes by when tested by Limulus amebocyte lysate method". This will result in endotoxins being unable to be detected when the drug is released, allowing drugs with potential pyrogen substances to flow into the market and be used by patients. The potential pyrogenic risks brought by LER, especially LER caused by the characteristics of biological products, deserve attention.

[0003] In 2013, the US FDA began to require applicants to provide LER research data when submitting a biologics license application (BLA) to prove that the bacterial endotoxin test method in USP Chapter 85 can detect endotoxins spiked into the product to avoid the potential pyrogenic risk of the product caused by LER. If the endotoxin test method cannot accurately detect, the rabbit pyrogen test is required as a substitute.

[0004] In the "Biologics FAQ" published by the European Medicines Agency (EMA) in 2023, it is mentioned that for preparations containing surfactants (such as polysorbates) and chelating agents (such as EDTA, citrate, phosphate, histidine), LER study data must be submitted when submitting marketing authorization applications. This shows that regulators are paying more and more attention to LER.

[0005] In my country's drug regulatory laws and regulations, the "Production Quality Control of Downstream Processes" in the "Guide to Pharmaceutical GMP" (2nd Edition) "Sterile Preparations" Volume 2 "Biological Products (Monoclonal Antibody) Cell Therapy Products" lists relevant information about low endotoxin recovery, but no specific regulatory requirements are made. The 2025 edition of the "Chinese Pharmacopoeia" is expected to include relevant requirements for low endotoxin recovery rates.

[0006] Although in recent years, with the in-depth study of LER phenomenon, people have a better understanding of its definition, mechanism, influencing factors, etc., but there are still a lot of LER phenomena that are difficult to solve. Summary of the invention

[0007] The above technical purpose of the present invention is achieved through the following technical scheme: A method for alleviating low endotoxin recovery rate, comprising the following steps:

[0008] (1) Re-check of sensitivity of horseshoe crab reagent / reliability verification of standard curve

[0009] The gel method uses 0.25-0.03EU / ml of gel method horseshoe crab reagent, and the sensitivity verification test of horseshoe crab reagent is carried out according to the Chinese Pharmacopoeia. The linear range is recommended to be 10-0.01EU / ml or 50-0.005EU / ml.

[0010] (2) Determine the key parameters of holding time study based on the production process: temperature and time

[0011] Look for high risk factors for LER in the production process, i.e. steps where surfactants and chelating agents are present at the same time.

[0012] (3) Determine the non-interfering dilution factor of the sample or determine the method to eliminate interference of the sample

[0013] According to the interference test of bacterial endotoxin test method in Chinese Pharmacopoeia, determine the concentration of sample without interference. When other methods are used to guess the interference, the method should be verified.

[0014] (4) Conduct retention time research

[0015] Add bacterial endotoxins to the endotoxin test water and samples in advance to a concentration of 10EU / ml / 20EU / ml,

[0016] Determine the temperature according to step (2), and take samples in sequence on the 1st, 2nd, 3rd, 4th, 7th, 14th and 28th days, without exceeding the time determined in step (2).

[0017] After each sampling, test the endotoxin test water and sample with added endotoxin: dilute the sample with endotoxin test water to the non-interference dilution factor determined in step 3 for testing. Alternatively, use other validated methods to treat the sample before testing.

[0018] Calculate the recovery rate of endotoxin. (You can also examine 1h, 2h, 4h, 8h and other time points according to actual conditions)

[0019] (5) Determine whether the sample has LER phenomenon

[0020] When the endotoxin recovery rate was lower than 50% at two consecutive time points, the sample was judged to have low endotoxin recovery.

[0021] (6) Method to alleviate low endotoxin recovery rate: Treat the sample with human albumin and conduct a retention time study again to confirm that the LER phenomenon has been eliminated.

[0022] Add bacterial endotoxins to the endotoxin test water and samples in advance to a concentration of 10EU / ml-20EU / ml.

[0023] Determine the temperature according to step (2), and take samples in sequence on the 1st, 2nd, 3rd, 4th, 7th, 14th and 28th days, without exceeding the time determined in step (2).

[0024] After each sampling, the endotoxin test water and the sample to which endotoxin has been added are tested: the sample is diluted with human albumin solution to the non-interfering dilution factor determined in step (3) for testing.

[0025] The concentration of the human albumin solution is 0.01%-1%. Preferably, the concentration of the human albumin solution is 0.1%.

[0026] If the test results are still disturbed by dilution with human albumin, the sample can be pre-treated by ultrafiltration using an ultrafiltration centrifuge tube. The molecular weight of the ultrafiltration membrane should be between 3000 and 30000 Daltons, preferably 20000 Daltons. After ultrafiltration, the sample is diluted with human albumin dilution solution.

[0027] (7) Confirm that the low endotoxin recovery rate phenomenon has been alleviated: calculate the endotoxin recovery rate. (You can also examine the time points of 1h, 2h, 4h, 8h, etc. according to actual conditions).

[0028] In the above, steps 1-5 are confirmation of the low endotoxin recovery phenomenon, step 6 is a method for alleviating the low endotoxin recovery rate, which is the core content of the present invention, and step 7 is confirmation that the low endotoxin recovery phenomenon has been alleviated.

[0029] Preferably, in step (1), the method for verifying the reliability of the standard curve includes a kinetic turbidity method, a kinetic colorimetric method, a recombinant factor C method, and a recombinant cascade reagent method.

[0030] In summary, the present invention has the following beneficial effects:

[0031] 1After treatment with this protocol, the masked endotoxin in the sample (recovery rate <50%) can be detected again (recovery rate >50%).

[0032] 2 This treatment scheme saves time and effort. It only requires the use of a specific solution to dilute the sample, without the need to use the currently common complex technical means such as adding divalent cations, adding dispersants, surfactants, organic solvents, refrigeration, etc.

[0033] 3. At present, most samples still cannot solve the problem of low endotoxin recovery even if all the common methods mentioned above are used. The only option is to use the rabbit pyrogen method to check the pyrogen contamination in the samples. The rabbit pyrogen method is far inferior to the bacterial endotoxin test method in terms of cost, operation difficulty and experimental precision.

[0034] 4. The problem of low endotoxin recovery rate has been solved. The bacterial endotoxin test method can truly detect the endotoxin in the sample, providing a guarantee for clinical safety and effectiveness. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below in conjunction with the embodiments:

[0036] Example 1

[0037] Research on variety "IMGN853"

[0038] The monoclonal antibody variety IMGN853 has been shown in preliminary studies to have low endotoxin recovery.

[0039] Instruments and reagents

[0040] The microplate reader (model: Multiskan FC / ET) was purchased from Thermo Scientific.

[0041] The ELISA reader (model: ELX808) was purchased from BIOTEK, USA.

[0042] The fully automatic bacterial endotoxin detection system (model: Robot 100) was purchased from Zhanjiang Andus Biological Co., Ltd.

[0043] National standard for bacterial endotoxin (150800-201601, 9000EU / tube), produced by China Food and Drug Inspection Institutes.

[0044] Dynamic colorimetric horseshoe crab reagent (10-0.01EU·mL-1, 1.25mL / tube) was purchased from Zhanjiang Andus Biological Co., Ltd.

[0045] Human albumin (20%, 50ml) 20180625, Boya Biopharmaceutical Co., Ltd.

[0046] Confirmation of low endotoxin recovery

[0047] Verification of the reliability of the standard curve and adjustment of the instrument sensitivity

[0048] According to the "Chinese Pharmacopoeia" 1143 Bacterial Endotoxin Test Method 2 Photometric Method, Dynamic Colorimetric Method Operation Requirements, the reliability of the standard curve was verified. Detection conditions: Dynamic colorimetric method horseshoe crab reagent, standard curve range 10-0.01EU / ml, sample and horseshoe crab reagent addition volume per well 100μL, detection wavelength 405nm, preset OD value 0.1. The result equation is as follows:

[0049] logC=-3.01lgT+5.83, r=1.000.

[0050] According to the requirements of the recombinant C factor method in the guidance of the application of bacterial endotoxin test method in the Chinese Pharmacopoeia 9251, the instrument sensitivity adjustment experiment was carried out before the experiment. The settings were 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, etc. The results showed that when the sensitivity was 80, lgΔCFU was closest to 3.5.

[0051] Does not interfere with determination of dilution factor

[0052] According to the "Chinese Pharmacopoeia" 1143 Bacterial Endotoxin Test Method 2 Photometric Method, Dynamic Colorimetric Method Operation Requirements, explore the sample concentration without interference. Detection conditions: Dynamic colorimetric method horseshoe crab reagent, standard curve range 10-0.01EU / ml, sample and horseshoe crab reagent addition volume per well 100μL, detection wavelength 405nm, preset OD value 0.1. Sample dilution multiples are 20 times, 200 times, 2000 times, 20000 times. The results are as follows:

[0053] Does not interfere with determination of dilution factor

[0054]

[0055] Duration study

[0056] The duration study was conducted with reference to PDA Technical Report No. 82. Sample processing conditions: Take the international standard endotoxin and add it to the sample so that the final concentration of the sample is 10EU / ml. Also add the international standard to the endotoxin test water so that the final concentration of the sample is 10EU / ml as a recovery control.

[0057] The company has verified that this sample does not produce low endotoxin recovery under low temperature conditions, and endotoxin recovery will occur after being placed at room temperature for only 24 hours. Therefore, the experimental conditions are set to be stored at room temperature of 25℃ for 14 days. Samples are taken on the 1st, 2nd, 3rd, 4th, 7th, and 14th days to test the endotoxin content, and the results are observed and recorded. As follows:

[0058] Duration of sample study

[0059]

[0060] It can be seen that low endotoxin recovery began to appear on the first day and was confirmed on the second day (low endotoxin recovery was confirmed when the endotoxin recovery rate was less than 50% at two consecutive time points). In order to eliminate the difference in detection methods, the recombinant C factor method was used for detection on the fourth day.

[0061] Resolving the phenomenon of low endotoxin recovery

[0062] Referring to PDA Technical Report No. 82, we tried to alleviate the low endotoxin recovery phenomenon. We diluted the samples to be tested with endotoxin international standard products using tris buffer, PBS buffer, and human albumin diluent (0.1%) as diluents to solve the low endotoxin recovery phenomenon. The final result was that using human albumin diluent (0.1%) as a diluent, the endotoxin recovery rate could reach 83.59%, which met the requirement of 50%-200%.

[0063] Detection conditions: Dynamic colorimetric method with horseshoe crab reagent, standard curve range 10-0.01EU / ml, sample and horseshoe crab reagent loading volume 100μL per well, detection wavelength 405nm, preset OD value 0.1. Dilute with 0.1% human albumin solution, sample dilution multiple 200 times, sample endotoxin content on days 1, 2, 3, 4, 7, 14, observe and record the results. The results are as follows:

[0064] Duration study of processed samples

[0065]

[0066] It can be seen that the low endotoxin recovery phenomenon has been alleviated since day 1. Therefore, the low endotoxin recovery rate problem of this sample can be solved by using this method.

[0067] In this example: the monoclonal antibody variety IMGN853, the endotoxin detection method should be: take this product, dilute it 200 times with 0.1% human albumin solution diluent, and then check it according to the law (1143 bacterial endotoxin test method).

[0068] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A method to alleviate low endotoxin recovery rate, It is characterized in that The following steps are involved: (1) Re-check of sensitivity of horseshoe crab reagent / reliability verification of standard curve The gel method uses 0.25-0.03EU / ml of the gel method horseshoe crab reagent, and the horseshoe crab reagent sensitivity verification test is carried out according to the Chinese Pharmacopoeia; Photometric method / recombinant C factor method / recombinant cascade reagent method are tested for the reliability of the standard curve according to the Chinese Pharmacopoeia; (2) Determine the key parameters of holding time research, temperature and time, based on the production process In the production process, look for high risk factors for LER in steps where surfactants and chelating agents appear at the same time; (3) Determine the non-interfering dilution multiple of the sample or determine the method for eliminating interference of the sample. According to the interference test of the bacterial endotoxin test method in the Chinese Pharmacopoeia, determine the non-interfering concentration of the sample. When other methods are used to guess the interference, the method should be verified; (4) Conduct retention time research Add bacterial endotoxin to the endotoxin test water and samples in advance to a concentration of 10EU / ml / 20EU / ml, etc., determine the temperature according to step (2), and take samples on the 1st, 2nd, 3rd, 4th, 7th, 14th, and 28th days without exceeding the time determined in step (2). After each sampling, test the endotoxin test water and samples to which endotoxin has been added: dilute the samples with the endotoxin test water to the non-interference dilution multiple determined in step (3) for testing, or use other verified methods to treat the samples for testing; and then calculate the recovery rate of endotoxin; (5) Determine whether the sample has LER phenomenon When the endotoxin recovery rate of endotoxin at two consecutive time points is less than 50%, the sample is judged to have low endotoxin recovery; (6) Alleviate low endotoxin recovery: Treat samples with human albumin and conduct retention time studies again to confirm that the LER phenomenon has been eliminated; Add bacterial endotoxins to the endotoxin test water and samples in advance to a concentration of 10EU / ml-20EU / ml; Determine the temperature according to step (2), and take samples on the 1st, 2nd, 3rd, 4th, 7th, 14th, and 28th day respectively without exceeding the time determined in step (2); After each sampling, the endotoxin test water and the sample to which endotoxin is added are tested: the sample is diluted with human albumin solution to the non-interference dilution multiple determined in step (3) for testing; wherein the concentration of the human albumin solution is 0.01%-1%; if the test result still has interference after the human albumin dilution, the sample can be pre-treated by ultrafiltration using an ultrafiltration centrifuge tube, and after ultrafiltration, the sample is diluted with human albumin dilution solution; (7) Confirm that the low endotoxin recovery phenomenon has been alleviated: calculate the endotoxin recovery rate.

2. A method for alleviating low endotoxin recovery rate according to claim 1, It is characterized in that In step (1), the methods for verifying the reliability of the standard curve include kinetic turbidity method, kinetic colorimetric method, recombinant factor C method, and recombinant cascade reagent method.

3. A method for alleviating low endotoxin recovery rate according to claim 1, It is characterized in that In step (1), the linear range is 10-0.01EU / ml or 50-0.005EU / ml.

4. A method for alleviating low endotoxin recovery rate according to claim 1, It is characterized in that The concentration of the human albumin solution in step (6) is 0.1%.

5. A method for alleviating low endotoxin recovery rate according to claim 1, It is characterized in that The molecular weight of the ultrafiltration membrane in step (6) should be selected between 3000 and 30000 Daltons.

6. A method for alleviating low endotoxin recovery rate according to claim 1, It is characterized in that The molecular weight of the ultrafiltration membrane in step (6) should be 20,000 Daltons.

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