A method for rapid detection of anti-rhGH neutralizing antibodies that eliminates residual drug and serum matrix interference

Abstract

This invention discloses a method for rapidly detecting anti-rhGH neutralizing antibodies, which eliminates interference from residual drugs and serum matrix. The method includes the following steps: 1. Sample acid dissociation; 2. Addition of alkaline solution and biotinylated rhGH; 3. Capture of the biotinylated rhGH-neutralizing antibody complex using a streptomycin-coated plate; 4. Elution of the neutralizing antibody; 5. Reaction of the eluted neutralizing antibody solution with rhGH working solution; 6. Co-incubation of the neutralizing antibody and rhGH reaction solution with cells for approximately 5 hours; 7. Addition of luciferase substrate for fluorescence detection. This method is highly sensitive, has good drug resistance, and is time-efficient, effectively solving the interference problems caused by residual drugs and serum matrix in existing neutralizing antibody detection methods.

Description

Technical Field

[0001] This invention relates to a method for rapidly detecting anti-rhGH neutralizing antibodies that can eliminate interference from residual drugs and serum matrix, and belongs to the field of analytical technology. Background Technology

[0002] Human growth hormone (HGH) is a non-glycosylated peptide composed of 191 amino acids, secreted by eosinophilic cells in the anterior pituitary gland of the human brain. It is one of the most important endocrine hormones during growth and development, stimulating metabolism and promoting the maturation of tissues, organs, bones, and the entire human body. GH exerts its effects by binding to GH receptors (GHR), stimulating target cell growth and inducing the production of insulin-like growth factor (IGF). 1) It acts on multiple target organs, including the liver, skeletal muscle, and fat. Unlike steroid hormones, GH does not produce adverse reactions similar to glucocorticoids or sex hormones.

[0003] hypothalamus Pituitary dysfunction leading to insufficient GH synthesis or secretion can cause growth, development, and metabolic disorders. Childhood growth hormone deficiency (GHD) is primarily characterized by short stature and growth retardation. Adult growth hormone deficiency clinically manifests as abnormal body composition, abnormal matrix metabolism, and increased cardiovascular risk. GH replacement therapy is currently the main treatment for GHD. Studies have shown that daily use of recombinant human GH can increase the rate of height growth in GHD patients and effectively improve body composition. Although GH treatment is well-established, daily injections are inconvenient, leading to poor adherence and reduced efficacy. Long-acting growth hormone is produced through genetic engineering, fusion modification, or special chemical modifications to slow its metabolism and prolong its half-life, thereby reducing the injection frequency from once daily to once weekly or even once a week.

[0004] Immunogenicity testing is an essential part of the biotechnology drug development process. According to FDA guidelines, drug immunogenicity assessment is a holistic risk assessment that continues throughout the entire biotechnology drug development process, up to and including post-marketing. Antidote neutralizing antibodies can lead to reduced or lost efficacy and altered pharmacokinetics, directly impacting the success or failure of new drug development.

[0005] Currently, there are no published reports on the detection of anti-rhGH neutralizing antibodies in human serum. Two patents have been found, one of which is "A method for detecting rhGH neutralizing antibodies." This patent provides a method for detecting rhGH neutralizing antibodies in human serum, the core of which includes: inactivating the serum sample to be tested, negative control, and positive control samples; mixing the inactivated samples with rhGH working solution and incubating; then co-incubating the mixture with cells for 48 hours, followed by live cell detection. The main drawback of this method is that it cannot effectively remove the interference of residual drug on the detection. rhGH is used to treat growth retardation in children, administered via subcutaneous injection once daily, with a recommended dose of 0.033-0.050 mg / kg body weight / day, continuing until epiphyseal closure. During treatment, both rhGH and anti-rhGH neutralizing antibodies may coexist in the child's serum. Residual rhGH in the biological sample can form complexes with anti-drug neutralizing antibodies, thus interfering with the detection of neutralizing antibodies. If detection methods cannot effectively remove interference from drug residues (and drug resistance issues), false negative results may occur. This can not only mislead drug development but also pose potential risks to clinical trial participants and patients using the new drug after its market launch. Therefore, drug resistance issues in anti-drug neutralizing antibody assays have become a key problem to be solved in current drug immunogenicity research. Furthermore, various components in serum (such as complement, heterophilic antibodies, high concentrations of non-specific proteins, lipids, and even components released from hemolysis) can also interfere with the detection of neutralizing antibodies. In addition, assays based on cell proliferation principles typically require several cell growth cycles to observe differences between groups, often necessitating several days of culture, which is time-consuming.

[0006] Another patent is for a "method for detecting anti-PEG-rhGH neutralizing antibodies," the main steps of which include: heating a biological sample (serum), adding acetic acid solution to the heated sample, adding a neutralizing reagent, adding PEG-rhGH working solution, incubating with cells for approximately 70 hours, and detecting anti-PEG-rhGH neutralizing antibodies in the sample. The sensitivity of this invention is approximately 0.8 μg / mL. Although this invention can eliminate interference from endogenous GH, the content of endogenous GH is inherently low (the normal GH range is 0.06 μg / mL). (At 5.0 ng / mL), its actual interference is limited. More importantly, this method cannot solve the problem of detecting biological samples caused by high concentrations of residual drugs. In addition, this invention is also designed based on the principle of cell proliferation, resulting in a long detection time. Summary of the Invention

[0007] To address the shortcomings of the existing technologies, this invention provides a method that can effectively remove high concentrations of residual drugs and serum matrix interference, and can rapidly detect anti-rhGH neutralizing antibodies in human serum. This method aims to solve the technical problems of insufficient drug resistance, significant serum interference, and long detection time in current detection methods.

[0008] The first technical solution provided by this invention is a detection system for anti-rhGH neutralizing antibodies in human serum, wherein the detection system contains biotin-labeled rhGH, streptavidin, glycine, HEK293-GHR-nanoLuc cells, and luciferase substrate.

[0009] In some embodiments, the detection system further includes a positive control sample, which is an anti-human growth hormone antibody.

[0010] The second technical solution provided by the present invention is a reagent kit, which includes the detection system described in the first technical solution.

[0011] The third technical solution provided by the present invention is an in vitro detection method for anti-rhGH neutralizing antibodies in human serum. The method involves using the detection system described in the first technical solution or the kit described in the second technical solution to detect anti-rhGH neutralizing antibodies in human serum in vitro.

[0012] In some implementations, the following steps are included: (1) Sample acid dissociation: Add the sample and acetic acid solution to a 96-well plate at a certain volume ratio, and incubate with shaking for a certain time; the sample includes negative control sample, positive control sample and serum to be tested; (2) Add alkaline solution and biotinylated rhGH: Add biotinylated rhGH and Tris to the 96-well plate from step (1) and incubate with shaking for a certain period of time; (3) Capture biotinylated rhGH-neutralizing antibody complex: Add the liquid from step (2) to a 96-well plate coated with streptavidin and incubate with shaking for a certain period of time; (4) Elution of neutralizing antibodies: (3) After rinsing the 96-well plate, add glycine solution, shake and incubate for a certain time to obtain neutralizing antibody eluent; (5) Mix the eluted neutralizing antibody solution with rhGH working solution and incubate: Aspirate the acid washing solution from 4), add 1 M Tris solution; then add rhGH diluted with cell culture medium and incubate for a certain period of time; (6) Add cell incubation: Add the mixed liquid in (5) to HEK293-GHR-nanoLuc cells that have been plated 1 day in advance, and incubate the cells in a 37°C, 5% CO2 incubator for a certain period of time; (7) Detection of fluorescence: Add luciferase substrate (Nano Glo Luciferase Assay System) to the cells in (6), incubate at room temperature for 2 minutes, detect fluorescence intensity with a microplate reader, calculate the inhibition rate of the sample, and determine whether there is anti-rhGH neutralizing antibody in the sample.

[0013] In some embodiments, in step (1), the final concentration of the acetic acid is 240 mM; the volume ratio of the sample to the acetic acid solution is 1:5.

[0014] In some implementations, in step (2), the concentration of biotin-labeled rhGH is 2 mg / mL.

[0015] In some implementations, the final concentration of glycine in step (5) is 100 mM.

[0016] In some implementations, in step (6), all samples are added to 100 μL of cell culture medium containing 40 ng / mL rhGH.

[0017] In some embodiments, in step (7), the volume ratio of cell supernatant to luciferase substrate is 1:1.

[0018] The fourth technical solution provided by this invention is the application of the detection system described in the first technical solution or the kit described in the second technical solution in the in vitro detection of anti-rhGH neutralizing antibodies in human serum.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention has the following beneficial effects: 1. This invention removes non-neutralizing antibody components from serum through steps such as sample acid dissociation, solid-phase capture, and washing, thus solving the interference of serum matrix on the detection of neutralizing antibodies.

[0020] 2. In this invention, the resistance level of the positive control antibody LPC (300 ng / mL) was 1.65 μg / mL, and the resistance level of HPC (3000 ng / mL) was 7.86 μg / mL. This method can also accurately quantify the anti-rhGH neutralizing antibodies present in serum samples with rhGH residues.

[0021] 3. The method of the present invention has a sensitivity of 0.243 μg / mL, which is better than the sensitivity requirement of 0.5~2 μg / mL for the detection of neutralizing antibodies in the United States Pharmacopeia, showing higher detection sensitivity.

[0022] 4. The method of the present invention is quick; after the sample is added to the cells, it can be detected after incubation for about 5 hours. Detailed Implementation

[0023] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0024] Raw materials used in the examples:

[0025] Example 1: Sensitivity Assessment The study was conducted by two analysts over a period of at least two days, examining at least six independently formulated sensitivity curves.

[0026] Sample preparation: An analytical batch includes a negative control (NC), LPC, HPC, and the sample to be tested. All samples used are replicates (n=2).

[0027] Individual serum samples were collected and stored at ≤-60℃.

[0028] Negative control: Ten blank serum samples from healthy individuals from different sources were mixed as negative control samples, which complied with the ethical approval of Liaocheng Second People's Hospital.

[0029] Positive control: The positive control antibody (0.5 mg / mL) was diluted with the negative control sample to prepare HPC samples with concentrations of 3000 ng / mL and LPC samples with concentrations of 300 ng / mL.

[0030] Sensitivity samples: Positive control antibodies were serially diluted using negative control samples, with concentrations ranging from 31.25 to 1000 ng / mL.

[0031] The specific testing steps are as follows: (1) Sample acid dissociation: Add 50 μL of negative control sample, positive control sample and test sample to a 96-well plate, then add 200 μL of 240 mM acetic acid, shake and incubate for 30 min to obtain acid hydrolysate.

[0032] (2) Add alkaline solution and biotin-labeled rhGH: Add 10 μL of diluted biotinylated rhGH (10 μg / mL) and 100 μL of 1M Tris to the sample wells of the 96-well plate containing the acid hydrolysate from step (1) for neutralization, shake and incubate for 60 min to obtain the acid-neutralized solution.

[0033] (3) Capture biotinylated rhGH-neutralizing antibody complex: Add the acidification and neutralization solution from step (2) to a 96-well plate coated with streptavidin and incubate with shaking for 30 min; (4) Elution of neutralizing antibody (NAb): After rinsing the 96-well plate from step (3), add 50 μL of 100 mM glycine solution, shake and incubate for 45 min to obtain neutralizing antibody eluent; (5) Mix the eluted NAb with rhGH working solution and incubate: add 50 μL of 1M Tris to neutralize the elution solution from step (4); then add 100 μL of cell culture medium containing 1 μg / mL rhGH, mix well and incubate for 30 min. (6) Cell plating: HEK293-GHR-nanoluc cells (4×10⁻⁶) 4 ( / well) Spread the plate one day in advance and incubate overnight in a 37℃, 5% CO2 incubator; (7) Adding cells: Aspirate 180 μL of cell supernatant and add the same volume of the mixture from step (5). Place the cells in a 37°C, 5% CO2 incubator for about 5 hours. (8) Detection: Add the detection reagent (Nano Glo Luciferase Assay) to the sample well, incubate at room temperature for 2 minutes, detect the fluorescence intensity with an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the inhibition rate.

[0034] Inhibition rate = (1 - positive control serum / negative control) × 100.

[0035] Result determination: Criterion determination: Two analysts tested 36 blank human serum samples on different days, performing a total of 10 batches of tests. The critical value was calculated using the neutralization rate: Neutralization rate (%) = (1 - RLUR) * 100, where RLUR = individual serum signal response value / mean response value of negative control samples. The critical value was used to determine the positivity or positivity of neutralizing antibodies in the samples; a neutralization rate ≥ the critical value indicated a positive neutralizing antibody test, and a neutralization rate < the critical value indicated a negative neutralizing antibody test.

[0036] Table 1. Results of Sensitivity Experiment

[0037] Sensitivity calculation formula: mean sensitivity + 2.015 * SD.

[0038] The results are shown in Table 1. The neutralization rate increased in a concentration-dependent manner with increasing concentration of the positive control antibody in the samples. When the positive antibody concentration was 250 ng / mL, the neutralization rate of all samples was higher than the critical value of 25.64%, and the samples were judged to be positive for neutralizing antibodies. When the positive antibody concentration was 125 ng / mL, the neutralization rate of all samples was lower than the critical value of 25.64%, and the samples were judged to be negative for neutralizing antibodies. The sensitivity of the six sensitivity curves was calculated using the intercept method, and the average value was then used to calculate the sensitivity using a formula. The sensitivity was 0.243 μg / mL. This method's sensitivity is lower than the sensitivity (0.8 μg / mL) in the patent "Method for Detecting Anti-PEG-rhGH Neutralizing Antibodies," and also lower than the USP's sensitivity requirement of 0.5–2 μg / mL for neutralizing antibody detection.

[0039] Example 2: Drug Resistance Study To investigate drug resistance at two concentration levels, the intercept method was used to calculate the maximum drug concentration that the positive control antibody could tolerate at that concentration.

[0040] Drug-resistant samples: rhGH was serially diluted using the negative control sample from Example 1, and then mixed with 3000 ng / mL neutralizing antibody (HPC) and 300 ng / mL neutralizing antibody (LPC), respectively, and incubated at room temperature for 1 hour. Detection was performed according to the method in Example 1.

[0041] Table 2 Results of drug resistance test

[0042] The results are shown in Table 2. In samples containing positive control antibodies at both LPC and HPC concentration levels, the neutralization rate decreased in a concentration-dependent manner with increasing rhGH concentration. At the HPC level, the neutralization rate was 35.48% at a concentration of 5000 ng / mL, higher than the critical value of 25.64%. At a concentration of 10000 ng / mL, the neutralization rate was 18.43%, lower than the critical value of 25.64%. The maximum resistant concentration was calculated to be 7886 ng / mL using the intercept method, indicating that this method can tolerate residual drug interference even at concentrations up to 7886 ng / mL in biological samples, thus classifying them as positive. At the LPC level, the neutralization rate was 30.79% at a concentration of 1250 ng / mL, higher than the critical value of 25.64%. At a concentration of 2500 ng / mL, the neutralization rate was 14.57%, lower than the critical value of 25.64%. The maximum drug resistance concentration was calculated to be 1647 ng / mL using the intercept method, indicating that the method can still tolerate the interference of residual drugs when the residual drug concentration in biological samples reaches a maximum of 1647 ng / mL, and thus it is judged as positive.

[0043] Example 3: Precision Examination Referring to the detection method in Example 1, the analytical batch includes 6 sets of system suitability samples (NC, LPC, HPC). The analytical batch acceptance criteria are: the %CV of the neutralization rate of LPC and HPC does not exceed 30%, and the neutralization rate satisfies HPC > LPC ≥ critical value.

[0044] Experimental results: Intra-batch precision: The neutralization rates (%CV) of LPC and HPC samples ranged from 0.33 to 10.07, and the neutralization rates satisfied HPC > LPC > critical value; Inter-batch precision: The neutralization rates of LPC and HPC samples were 89.16% and 51.79%, respectively; the neutralization rates (%CV) were 10.08% and 1.16%, respectively, and the neutralization rates satisfied HPC > LPC > critical value.

[0045] Example 4: Specificity Study Referring to the detection method in Example 1, 10,000 and 100,000 ng / mL IgG were added to the negative control sample and LPC sample, respectively. Analytical batch acceptance criteria: the neutralization rate of the negative control sample was less than the critical value, and the neutralization rate of the LPC sample was greater than the critical value.

[0046] Table 3. Results of Specificity Examination

[0047] The results are shown in Table 3. The neutralization rates of the negative control samples with 10,000 and 100,000 ng / mL IgG were -8.20% and 11.27%, respectively, both less than the critical value of 25.64%, and were therefore judged as negative for neutralizing antibodies. The neutralization rates of the LPC samples with 10,000 and 100,000 ng / mL IgG were 44.49% and 39.97%, respectively, both greater than the critical value of 25.64%, and were therefore judged as positive for neutralizing antibodies. The above data show that the method has good specificity.

[0048] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A detection system for anti-rhGH neutralizing antibodies in human serum, characterized in that, The detection system contains biotin-labeled rhGH, streptavidin, glycine, HEK293-GHR-nanoLuc cells, and luciferase substrate.

2. The detection system according to claim 1, characterized in that, The detection system also contains a positive control sample, which is an anti-human growth hormone antibody.

3. A reagent kit, characterized in that, The kit comprises the detection system according to any one of claims 1 to 2.

4. An in vitro detection method for anti-rhGH neutralizing antibodies in human serum, characterized in that, The method involves in vitro detection of anti-rhGH neutralizing antibodies in human serum using the detection system described in any one of claims 1 to 2 or the kit described in claim 3.

5. The detection method according to claim 4, characterized in that, The method includes the following steps: (1) Sample acid dissociation: Add the sample and acetic acid solution to a 96-well plate at a certain volume ratio, and incubate with shaking for a certain time; the sample includes negative control sample, positive control sample and serum to be tested; (2) Add alkaline solution and biotinylated rhGH: Add biotinylated rhGH and Tris to the 96-well plate from step (1) and incubate with shaking for a certain period of time; (3) Capture biotinylated rhGH-neutralizing antibody complex: Add the liquid from step (2) to a 96-well plate coated with streptavidin and incubate with shaking for a certain period of time; (4) Elution of neutralizing antibodies: (3) After rinsing the 96-well plate, add glycine solution, shake and incubate for a certain time to obtain neutralizing antibody eluent; (5) Mix the eluted neutralizing antibody solution with rhGH working solution and incubate: Aspirate the acid wash solution from (4) and add 1 M Tris solution; then add rhGH diluted with cell culture medium and incubate for a certain period of time; (6) Add cell incubation: Add the mixed liquid in (5) to HEK293-GHR-nanoLuc cells that have been plated 1 day in advance, and incubate the cells in a 37°C, 5% CO2 incubator for a certain period of time; (7) Detecting fluorescence: Add luciferase substrate to the cells in (6), incubate at room temperature for 2 minutes, detect fluorescence intensity with an enzyme-linked immunosorbent assay (ELISA) reader, calculate the inhibition rate of the sample, and determine whether there is anti-rhGH neutralizing antibody in the sample.

6. The detection method according to claim 5, characterized in that, In step (1), the final concentration of acetic acid is 240 mM; the volume ratio of the sample to the acetic acid solution is 1:

5.

7. The detection method according to claim 5, characterized in that, In step (2), the concentration of biotin-labeled rhGH is 2 mg / mL.

8. The detection method according to claim 5, characterized in that, In step (5), the final concentration of glycine is 100 mM.

9. The detection method according to claim 5, characterized in that, In step (7), the volume ratio of cell supernatant to luciferase substrate is 1:

1.

10. The use of the detection system according to any one of claims 1 to 2 or the kit according to claim 3 in the in vitro detection of anti-rhGH neutralizing antibodies in human serum.

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