Pancreatic cancer vaccine performance test system, device and method

Abstract

The invention discloses a pancreatic cancer vaccine performance test system, device and method, and relates to the technical field of biological medicine detection.The system comprises an anti-adsorption storage unit, a light-shielding transmission unit, a buffer solution stabilization unit, an interference correction detection unit and the like. The equipment comprises an intelligent injector, a light-proof camera obscura device and a micro-fluidic detection chip. The method comprises the steps of standardized redissolution, whole-process light-proof storage, specific detection and validity period judgment. Through the technical means of coating the inner wall of the injector with a specific coating, designing a unique light shielding structure, optimizing a buffer solution and a detection system and the like, the LNP adsorption rate is remarkably reduced, illumination damage is reduced, the buffer solution is stabilized, and a T cell response detection value is corrected. The precision and reliability of pancreatic cancer vaccine performance testing are effectively improved, powerful support is provided for vaccine quality control and clinical application, and innovativeness, practicability and good application prospects are achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical testing technology, and in particular to a performance testing system, equipment and method for a pancreatic cancer vaccine. Background Art

[0002] In the process of pancreatic cancer vaccine development and application, the performance test of the vaccine after reconstitution is crucial. Traditional ordinary polypropylene syringes can easily cause 12-18% of LNP to adsorb to the container wall within 6 hours, resulting in a decrease in the effective dose of mRNA and a falsely high immunogenicity test result. Before clinical vaccination, the vaccine was exposed to the clinic fluorescent light (400-500 nm blue light, illumination > 300 lux) for 3 hours, and the mRNA pyrimidine dimer increased by 23%, seriously affecting the stability of the vaccine. After storage at 4°C for 72 hours, 25% of the histidine in the reconstitution buffer was converted to histamine, interfering with the judgment of particle size and stability. In addition, high concentrations of CA199 in the serum of pancreatic cancer patients can non-specifically bind to the vaccine LNP, resulting in an underestimation of the T cell response detected in vitro by 20-40%. Traditional tests use healthy human serum and cannot truly reflect the performance of the vaccine. These problems need to be solved urgently.

[0003] In view of this, this application is hereby filed. Summary of the invention

[0004] The purpose of the present invention is to provide a performance testing system, device and method for pancreatic cancer vaccine to solve the problems mentioned in the above background technology.

[0005] In order to solve the above technical problems, the present invention provides a pancreatic cancer vaccine performance testing system, comprising: Anti-adsorption storage unit: A polypropylene syringe with a 50-80 nm polysulfonated betaine coating on the inner wall by atomic layer deposition (ALD) process. After molecular dynamics simulation optimization, the LNP (lipid nanoparticle) adsorption sites were reduced by 92%, and the adsorption rate of BNT122 (autogene cevumeran) was reduced from 15% to 2.1% 6 hours after reconstitution; Light-proof transmission unit: dark box syringe with integrated optical fiber, 2 mm thick light-shielding layer, light transmittance <0.01%@400-500 nm, simulation display of cumulative blue light exposure <5 lux・min from reconstitution to injection, and supporting hospital clinic light monitoring log; Buffer stabilization unit: Histidine-sucrose buffer pre-loaded with 0.1 U / mL histidine oxidase, pH 6.5±0.1, histamine generation <5 μM after 72 hours of storage at 4°C, HPLC detection spectrum shows that the degradation rate drops from 25% to 4.3%; Interference correction detection unit: The detection system contains 10 nM anti-CA199 nanoantibody with affinity constant KD=2.3 nM. In simulated serum with CA199 equal to 1500 U / mL, the BNT122-specific T cell response was corrected from 58% to 79%, and the flow cytometry comparison data were used.

[0006] Furthermore, the preparation process of the polysulfonated betaine coating in the anti-adsorption storage unit is: the precursor 3-(methacrylamido)propyltrimethoxysilane is cyclically deposited by the ALD process, and cured at 50°C for 2 hours to form a cross-linked network structure. AFM shows that the surface roughness is less than 5 nm, and the coating shedding rate is less than 0.5% after immersion in physiological saline for 7 days.

[0007] Furthermore, it also includes: an AR glasses unit, which recognizes the batch number of the vial through a camera and displays in real time: ① thawing countdown, accurate to seconds; ② expiration date prediction based on adsorption rate and light data, with an error of <±2 hours, and AI model R²=0.92; ③ nurse operation score, such as a red prompt when the injection speed deviates from 0.3 mL / s.

[0008] Furthermore, it also includes: a blockchain unit that records each vaccine’s: ① reconstitution time, accurate to seconds; ② cumulative light exposure value, uploaded in real time by a photoresistor; ③ raw data detection, generation of a PDF certificate with a timestamp, and hash values ​​of vaccine-related data stored in the blockchain for hospitals to scan and verify.

[0009] A performance testing device for a pancreatic cancer vaccine, comprising: Intelligent syringe device: including a 6 mL PP syringe with a zwitterionic coating on the inner wall, the coating contains quaternary ammonium salt and sulfonic acid groups, zeta potential -5±2 mV, with a capillary flow groove at the bottom, depth 0.3 mm, residual liquid <2 μL, weighing method actual measurement: 1.8 μL remains after 5.98 mL of the complex solution is extracted; electric injection gun, injection speed 0.3 mL / s, error ±0.05 mL / s, built-in 40 kHz ultrasonic vibrator, amplitude 5 μm, eliminate bubbles, laser particle size analyzer detects bubble diameter <50 μm; Light-proof dark box device: including a light-shielding shell made of polyurethane, containing photochromic nanoparticles, with a magnetic adsorption seal at the opening and closing, and only the needle tip is exposed through a silicone photoresist film with a thickness of 0.1 mm and a light transmittance of <0.1%. The UV-sensitive ink on the shell turns red after being exposed to light >100 lux for 10 minutes; Microfluidic detection chip: including 2 cm×2 cm PDMS chip, integrated with: ① mRNA integrity FET sensor, detection limit 1 pg / mL, ② NTA particle size analyzer, 100-150 nm range, ③ T cell ELISpot microwells containing anti-CA199 antibodies, antibody coating amount per well 1 μg, overall detection time ≤45 minutes, and supporting detection process record sheet.

[0010] Furthermore, the microfluidic detection chip integrates a Fluo-4NW fluorescent probe with an excitation wavelength of 488 nm. When the histamine concentration is greater than 10 μM, the fluorescence intensity is greater than 5000 au, triggering a buzzer alarm on the device and locking the syringe plunger.

[0011] Furthermore, the light-shielding shell contains spiropyran photochromic molecules at a concentration of 0.5wt%, which release vitamin E derivatives when exposed to 400-500 nm light, with a release amount of 10 μg / cm², and mRNA integrity is retained by 92% after 6 hours of 500 lux light exposure.

[0012] A method for testing the performance of a pancreatic cancer vaccine comprises the following steps: Standardized reconstitution: The vaccine was transferred from -80 ℃ to a 2-8 ℃ refrigerator for thawing. The temperature recorder showed an average thawing time of 118 minutes. The vaccine was reconstituted using a diluent containing histidine oxidase. The injection gun was injected at a constant speed of 0.3 mL / s, and the ultrasonic vibrator was started for 30 seconds to defoam. Storage in the dark during the whole process: transfer to a dark box syringe within 10 minutes after reconstitution, refrigerate at 2-8℃, the temperature and humidity recorder should show fluctuation of ±0.5℃, the shell should be opened for less than 15 seconds during each operation, and the number of openings and closings should be recorded by a photoresistor; Specific detection: Add 10 nM anti-CA199 nanoantibody to artificial serum containing 1500 U / mL CA199 and 500 ng / mL MUC1 to detect IFN-γ secretion of CD8+T cells. ELISpot counts > 50 spots / 10^6 cells are effective. At the same time, the proportion of CD127+ memory T cells is detected, ≥ 30%; Determination of validity period: The product is deemed invalid if any of the following conditions is met: ① LNP adsorption rate > 3%, detected by quartz crystal microbalance; ② mRNA pyrimidine dimer > 15%, HPLC peak area ratio; ③ Corrected T cell response < 70% of the baseline value, where the baseline value is the detection value at 0 hours after reconstitution.

[0013] Furthermore, the artificial serum also contains 20 pg / mL TGF-β and 1% pancreatic stellate cell conditioned medium to construct 3D tumor microtissues with a diameter of 200 μm, and detect the T cell penetration depth, which is effective when ≥50 μm, simulating the immunosuppressive microenvironment of pancreatic cancer.

[0014] Furthermore, the T cell response correction coefficient K=1.2+0.0002×(CA199-500), applicable to 500-5000U / mL, was verified by sera from 100 pancreatic cancer patients, and the correlation between the corrected detection value and the in vivo T cell clone expansion was R=0.89 (p<0.001).

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. Accurate detection: Through the zwitterion-coated syringe, the LNP adsorption rate is reduced to <3%, ensuring the stability of the effective dose of mRNA and making the immunogenicity test more accurate. This effectively avoids the vaccine dose deviation caused by LNP adsorption, lays a solid foundation for accurately evaluating the immune effect of the vaccine, and greatly improves the credibility of the vaccine performance evaluation. The dark box syringe achieves full blue light exposure of <5 lux・min, and mRNA photodegradation is reduced by 80%, ensuring the stability of the vaccine, thereby maintaining the effective ingredients of the vaccine and ensuring that the expected immune effect can be exerted during clinical use. 2. Efficient and convenient: The intelligent injection gun and microfluidic detection chip standardize the reconstitution operation, shorten the detection time to ≤45 minutes, and greatly improve the test efficiency. This not only reduces the operation time cost of medical staff, but also allows medical institutions to obtain vaccine performance data faster, accelerate the process of vaccine testing to clinical application, and provide strong guarantee for the timely use of vaccines. 3. Clinical adaptation: Add anti-CA199 nano-antibody detection system, calibrate T cell response detection value, fit the actual situation of pancreatic cancer patients, and provide a reliable basis for clinical decision-making. It helps doctors to formulate personalized treatment plans based on more accurate test results, improve the effectiveness of pancreatic cancer vaccines in clinical treatment, and improve patients' treatment effects and prognosis. 4. Reliable data: The blockchain traceability module records the data of the entire process and generates a PDF certificate with a time stamp to ensure that the data cannot be tampered with, thereby enhancing the traceability of vaccine quality. This ensures that every link from vaccine production to use can be traced, making it easier for regulatory authorities to supervise and manage, improving the quality control level of the entire vaccine industry, and enhancing the public's trust in vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The schematic diagram of a performance test system for a pancreatic cancer vaccine is shown below; Figure 2 A schematic diagram of a performance test device for a pancreatic cancer vaccine; Figure 3 A flow chart of a performance testing method for a pancreatic cancer vaccine. DETAILED DESCRIPTION

[0017] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0018] See also Figure 1-3 The present invention provides a technical solution: a performance testing system, device and method for pancreatic cancer vaccine, comprising: 1. Preparation of anti-adsorption syringe 1.ALD coating process Equipment: Picosun R-200 ALD equipment; Parameters: Precursor 3-(methacrylamido)propyltrimethoxysilane, deposition temperature 50 °C, 50 cycles, forming a 65 nm coating (measured by ellipsometer); Post-treatment: Curing at 50 °C for 2 hours to form a cross-linked network (AFM showed a roughness of 4.2 nm).

[0019] 2. Coating verification Adsorption test: The adsorption of lipid nanoparticles (LNPs) in the BNT122 vaccine at a concentration of 100 μg / mL was monitored for 6 hours using a quartz crystal microbalance (QCM); Stability: After immersion in saline for 7 days, EDS spectrum showed that the coating elements accounted for 98.7% (with a shedding rate of 0.5%). As shown in Table 1 below:

[0020] Table 1: Comparison of experimental data between examples and comparative examples In the present invention, ALD precisely controls thickness: the number of cycles accurately controls the coating thickness (±5nm), which is 4 times more uniform than the spraying process; zwitterion dual antibody: quaternary ammonium salt (anti-negatively charged LNP) + sulfonic acid group (anti-positively charged protein), the adsorption sites are reduced by 92% (molecular dynamics simulation); clinical value: the loss of each vaccine due to adsorption is less than 2.1 μg (based on 100 μg / dose), avoiding immune failure caused by insufficient dosage.

[0021] 2. Standardized reconstitution operation 1. Thawing control Equipment: Haier HYC-680 refrigerator (2-8 ℃, ±0.5 ℃ fluctuation); Operation: Thawing time of cryotube (5 mL) is 118 ± 5 minutes (temperature recorder model: Onset HOBO UX120-011); 2. Intelligent injection Equipment: XYZ-2025 electric injection gun (accuracy ±0.05 mL / s); Parameters: 0.3 mL / s uniform injection rate, 40 kHz ultrasonic vibration head (amplitude 5 μm) debubbling for 30 seconds; Residue: Capillary flow channel + vacuum suction, residual liquid 1.8±0.2 μL (weighed by Mettler balance MS105DU). As shown in Table 2 below:

[0022] Table 2: Comparison of experimental data between examples and comparative examples In the present invention: Ultrasonic frequency matching: 40 kHz corresponds to LNP particle size (100-150 nm), and the bubble crushing efficiency is 3 times that of traditional oscillation; Capillary guide groove: 0.3 mm depth design, 89% less residual than ordinary syringes (traditional residual 16 μL); Clinical value: 98% of visible bubbles are eliminated, avoiding false high doses caused by LNP encapsulation, and the correlation between test results and in vivo efficacy is improved by 28%.

[0023] 3. Implementation steps for full-process light-proof storage 1. Dark box syringe Material: polyurethane + photochromic nanoparticles (0.5wt% spiropyran); Transmittance: <0.01%@400-500 nm (UV-Vis spectrophotometer Lambda 950); Photoresist film: Silicone film thickness 0.1 mm, needle exposed length 5 mm; 2. Light monitoring Sensor: Vishay VEML6070 (accuracy 0.1lux, 400-500 nm band); Cumulative exposure: <5 lux·min from reconstitution to injection (actual measurement in hospital clinic: average 3.2 lux·min). As shown in Table 3 below:

[0024] Table 3: Comparison of experimental data between examples and comparative examples In the present invention: Photochromic repair: light triggers the release of vitamin E (10 μg / cm²), which repairs 42% more light damage than aluminum foil wrapping; Needle exposure control: only 5 mm needle is exposed, reducing 97% of light path contact compared to traditional syringes; Clinical value: mRNA photodegradation is reduced by 80%, avoiding the underestimation of T cell response caused by light (actual measurement correction 34%).

[0025] Patient-specific testing 1. Preparation of artificial serum CA199: 1500 U / mL (75th percentile value for pancreatic cancer patients); Anti-CA199 nanobody: 10 nM (KD = 2.3 nM, Abcam ab289321); 3D microtissue: pancreatic stellate cells + tumor-associated macrophages, diameter 200±20 μm.

[0026] 2. Testing equipment ELISpot chip: 8 × 12 wells, each well coated with 1 μg of anti-IFN-γ antibody (BD551085); Flow cytometer: BD FACSCanto II, CD127+CD8+ cell gating. As shown in Table 3 below:

[0027] Table 2: Comparison of experimental data between examples and comparative examples In the present invention: Nano-antibody penetrability: 15 kDa molecular weight is easier to bind to free CA199 in serum than traditional antibodies (150 kDa); 3D microtissue simulation: immunosuppressive environment containing TGF-β, closer to the body than 2D culture (penetration depth difference of 33 μm); Clinical value: correcting 40% of detection underestimation and avoiding vaccine abandonment due to false negatives (saving costs of 270,000 yuan / 10,000 doses).

[0028] 5. Determination of validity period 1. Multi-parameter acquisition LNP adsorption rate: QCM detection (Maxtek 27 MHz), >3% alarm; mRNA integrity: Agilent 2100 electrophoresis (RIN>7.0); Correction factor: K=1.2+0.0002×(CA199-500) (500-5000 U / mL).

[0029] 2. Blockchain records Consortium chain: Hyperledger Fabric, block interval 15 minutes; Data items: thawing time (accurate to seconds), cumulative light value (accuracy of 0.1 lux), ELISpot original image. As shown in Table 4 below (data of 100 postoperative patients):

[0030] Table 4: Comparison of experimental data between examples and comparative examples In the present invention: four-dimensional fusion algorithm: adsorption rate + mRNA + T cells + CA199, which improves the prediction accuracy by 82% compared with a single indicator; blockchain cannot be tampered with: the light-sensing chip is directly connected to the chain to avoid human modification (such as automatic recall of a batch due to excessive light); clinical value: the risk of recurrence caused by misjudgment is reduced by 86%.

[0031] 6. Comparison of cost and efficiency of the whole process: Refer to Table 5 below:

[0032] Table 5: Comparison table of cost and efficiency of the whole process in the present invention.

[0033] In summary: The present invention achieves the functions of reducing LNP adsorption rate, reducing the effect of light on the vaccine, stabilizing the pH of the buffer, and correcting the T cell response detection value by coating the inner wall of the syringe with a zwitterionic polymer coating, designing a dark box syringe, adding histidine oxidase to the buffer, and adding anti-CA199 nanoantibodies to the detection system. These technical means effectively solve the key problems in the performance test of the pancreatic cancer vaccine after reconstitution, and improve the accuracy, reliability and applicability of the performance test of the pancreatic cancer vaccine.

Claims

1. A performance testing system for a pancreatic cancer vaccine, characterized in that: include: Anti-adsorption storage unit: A polypropylene syringe with a 50-80 nm polysulfonated betaine coating on the inner wall by atomic layer deposition. The adsorption rate of BNT122 dropped from 15% to 2.1% in 6 hours after reconstitution; Light-proof transmission unit: dark box syringe with integrated optical fiber, simulating the cumulative blue light exposure from reconstitution to injection <5 lux・min; Buffer stabilization unit: Histidine-sucrose buffer pre-loaded with 0.1 U / mL histidine oxidase, histamine generation <5 μM after 72 hours of storage at 4°C; Interference correction detection unit: The detection system containing 10 nM anti-CA199 nanoantibody corrected the BNT122-specific T cell response from 58% to 79% in simulated serum with CA199 equal to 1500 U / mL.

2. The pancreatic cancer vaccine performance testing system according to claim 1, characterized in that: The preparation process of the polysulfonated betaine coating in the anti-adsorption storage unit is as follows: the precursor 3-(methacrylamido)propyltrimethoxysilane is cyclically deposited by the ALD process, cured at 50°C for 2 hours, and the coating shedding rate is less than 0.5% after being immersed in physiological saline for 7 days.

3. The pancreatic cancer vaccine performance testing system according to claim 1, characterized in that: Also includes: The AR glasses unit uses a camera to identify the batch number of the vial and displays the thawing countdown in real time; Prediction of shelf life based on adsorption rate and light exposure data.

4. The pancreatic cancer vaccine performance testing system according to claim 1, characterized in that: Also includes: The blockchain unit records each vaccine’s: reconstitution time; Accumulated value of light exposure; detection raw data.

5. A performance testing device for pancreatic cancer vaccine, characterized in that: include: Smart syringe device: including a 6 mL PP syringe with a zwitterionic coating on the inner wall, a capillary flow groove at the bottom, and a residual liquid of <2 μL; an electric injection gun with an injection speed of 0.3 mL / s and a built-in 40 kHz ultrasonic vibrator to eliminate bubbles; Light-proof dark box device: including a light-shielding shell made of polyurethane, with a magnetic adsorption seal at the opening and closing, and only the needle tip is exposed through the silicone photoresist film. The UV-sensitive ink on the shell turns red after being exposed to light >100 lux for 10 minutes; Microfluidic detection chip: includes a 2 cm×2 cm PDMS chip, which integrates: mRNA integrity FET sensor; NTA particle size analyzer; T cell ELISpot microwell containing anti-CA199 antibody.

6. The performance testing device for a pancreatic cancer vaccine according to claim 5, characterized in that: The microfluidic detection chip integrates the Fluo-4NW fluorescent probe. When the histamine concentration is greater than 10 μM and the fluorescence intensity is greater than 5000 au, the device buzzes and an alarm is triggered, locking the syringe plunger.

7. The performance testing device for a pancreatic cancer vaccine according to claim 5, characterized in that: The light-shielding shell contains spiropyran photochromic molecules, which release vitamin E derivatives when exposed to 400-500 nm light, and the mRNA integrity is retained by 92% after 6 hours of 500 lux light exposure.

8. A method for testing the performance of a pancreatic cancer vaccine, characterized in that: The following steps are involved: Standardized reconstitution: The vaccine is transferred from -80 ℃ to a 2-8 ℃ refrigerator for thawing, and reconstituted with a diluent containing histidine oxidase. The injection gun is injected at a constant speed of 0.3 mL / s, and the ultrasonic vibrator is started for 30 seconds to defoam. Storage in the dark during the whole process: transfer to a dark box syringe within 10 minutes after reconstitution, refrigerate at 2-8 ℃, and keep the shell open for less than 15 seconds during each operation; Specificity detection: 10 nM anti-CA199 nanoantibody was added to artificial serum containing 1500 U / mL CA199 and 500 ng / mL MUC1 to detect IFN-γ secretion of CD8+ T cells and the proportion of CD127+ memory T cells; Determination of validity period: The product will be deemed invalid if any of the following conditions is met: LNP adsorption rate > 3%; mRNA pyrimidine dimer > 15%; corrected T cell response < 70% of the baseline value.

9. The method for testing the performance of a pancreatic cancer vaccine according to claim 8, characterized in that: The artificial serum also contains 20 pg / mL TGF-β and 1% pancreatic stellate cell conditioned medium to construct 3D tumor microtissues, detect T cell penetration depth, and simulate the immunosuppressive microenvironment of pancreatic cancer.

10. The method for testing the performance of a pancreatic cancer vaccine according to claim 8, characterized in that: The T cell response correction coefficient K=1.2+0.0002×(CA199-500) was verified by sera from 100 pancreatic cancer patients, and the correlation between the corrected detection value and the in vivo T cell clone expansion R=0.89.

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