Performance testing system, device and method for a pancreatic cancer vaccine

Abstract

The present invention discloses a performance test system, device and method for a pancreatic cancer vaccine, which relates to the technical field of biomedical detection. This system includes units such as anti-adsorption storage, light-shielded transmission, buffer stabilization and interference correction detection. The device includes an intelligent syringe, a light-shielded dark box device and a microfluidic detection chip. The method covers steps such as standardized reconstitution, full-process light-shielded storage, specific detection and expiration date determination. By means of technical measures such as coating a specific coating on the inner wall of the syringe, designing a unique light-shielded structure, optimizing the buffer and detection system, etc., the LNP adsorption rate is significantly reduced, light damage is reduced, the buffer is stabilized, and the T cell response detection value is corrected. The present invention effectively improves the accuracy and reliability of the performance test of the pancreatic cancer vaccine, provides strong support for vaccine quality control and clinical application, and has innovation, practicability and good application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical detection, and specifically to a performance testing system, device and method for a pancreatic cancer vaccine. Background Art

[0002] During the research and development and application of pancreatic cancer vaccines, the performance testing of the reconstituted vaccine is crucial. In traditional ordinary polypropylene syringes, 12 - 18% of the LNP is adsorbed on the container wall within 6 hours, resulting in a decrease in the effective dose of mRNA and an overestimated immunogenicity test result. Before clinical vaccination, when the vaccine is exposed to the fluorescent light in the consulting room (blue light of 400 - 500 nm, illuminance > 300 lux) for 3 hours, the mRNA pyrimidine dimers increase by 23%, seriously affecting the vaccine stability. After histidine in the reconstitution buffer is stored at 4°C for 72 hours, 25% of it is converted into 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 in in vitro detection by 20 - 40%. Traditional tests use healthy human serum and cannot truly reflect the vaccine performance. These problems need to be solved urgently.

[0003] In view of this, the present application is specifically proposed. Summary of the Invention

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

[0005] To solve the above technical problems, a performance testing system for a pancreatic cancer vaccine provided by the present invention includes:

[0006] Anti-adsorption storage unit: a polypropylene syringe with an inner wall coated with a 50 - 80 nm poly(sulfobetaine) coating by atomic layer deposition (ALD) process. After optimization by molecular dynamics simulation, the LNP (lipid nanoparticle) adsorption sites are reduced by 92%, and the adsorption rate of BNT122 (autogene cevumeran) after reconstitution drops from 15% to 2.1% within 6 hours.

[0007] Light-shielding transmission unit: a dark-box syringe integrated with optical fibers, with a 2 mm thick light-shielding layer and a light transmittance < 0.01% @ 400 - 500 nm. Simulation shows that the cumulative blue light exposure from reconstitution to injection is < 5 lux·min, and it is equipped with a hospital consulting room light monitoring log.

[0008] Buffer stability unit: a histidine-sucrose buffer pre-loaded with 0.1 U / mL histidine oxidase, with a pH of 6.5 ± 0.1. The histamine generation amount is < 5 μM after storage at 4°C for 72 hours, and the HPLC detection spectrum shows that the degradation rate drops from 25% to 4.3%.

[0009] 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.

[0010] 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.

[0011] 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.

[0012] 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.

[0013] A performance testing device for a pancreatic cancer vaccine, comprising:

[0014] 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;

[0015] 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;

[0016] Microfluidic detection chip: including a 2 cm × 2 cm PDMS chip, integrated with: ① mRNA integrity FET sensor with a detection limit of 1 pg / mL, ② NTA particle size analyzer with a range of 100 - 150 nm, ③ T cell ELISpot microwells containing anti-CA199 antibody with an antibody coating amount of 1 μg per well, the overall detection time ≤ 45 minutes, and a supporting detection process record form.

[0017] Further, the microfluidic detection chip integrates a Fluo-4NW fluorescent probe with an excitation wavelength of 488 nm. When the histamine concentration > 10 μM, the fluorescence intensity > 5000 a.u., triggering the device to beep an alarm and lock the syringe push rod.

[0018] Further, the light-shielding housing contains a spiropyran photochromic molecule with a concentration of 0.5 wt%. When exposed to light of 400 - 500 nm, it releases vitamin E derivatives with a release amount of 10 μg / cm², and 92% of the mRNA integrity is retained after 6 hours of 500 lux light exposure.

[0019] A performance testing method for a pancreatic cancer vaccine includes the following steps:

[0020] Standardized reconstitution: The vaccine is transferred from -80 °C to a 2 - 8 °C refrigerator for thawing. The temperature recorder shows an average thawing time of 118 minutes. A diluent containing histidine oxidase is used for reconstitution, and the liquid injection gun injects at a constant speed of 0.3 mL / s while starting the ultrasonic vibration head for 30 seconds to defoam.

[0021] Full-process light-shielded storage: It is transferred to a dark box syringe within 10 minutes after reconstitution and stored at 2 - 8 °C. The temperature and humidity recorder shows a fluctuation of ±0.5 °C. The opening time of the housing is < 15 seconds each time of operation, and the opening and closing times are recorded by a photoresistor.

[0022] Specific detection: In an artificial serum containing 1500 U / mL CA199 and 500 ng / mL MUC1, 10 nM anti-CA199 nanobody is added to detect the secretion of IFN-γ by CD8+ T cells. An ELISpot count > 50 spot / 10^6 cells is considered effective, and at the same time, the proportion of CD127+ memory T cells is detected, ≥ 30%.

[0023] Validity determination: It is judged to be invalid if any of the following conditions are met: ① LNP adsorption rate > 3%, detected by a quartz crystal microbalance; ② mRNA pyrimidine dimers > 15%, HPLC peak area ratio; ③ The corrected T cell response < 70% of the baseline value, and the baseline value is the detection value at 0 hours after reconstitution.

[0024] Furthermore, the artificial serum also contains 20 pg / mL TGF-β and 1% pancreatic stellate cell conditioned medium to construct a 3D tumor microtissue with a diameter of 200 μm. The penetration depth of T cells is detected, and ≥50 μm is considered effective to simulate the immunosuppressive microenvironment of pancreatic cancer.

[0025] Furthermore, the T cell response correction coefficient K = 1.2 + 0.0002 × (CA199 - 500), applicable to 500 - 5000 U / mL. It has been verified by the sera of 100 pancreatic cancer patients. The correlation R between the corrected test value and the in vivo T cell clonal expansion is 0.89 (p < 0.001).

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] 1. Precise detection: Through the zwitterionic-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 precise. 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 vaccine performance evaluation. The dark box syringe achieves a total blue light exposure of <5 lux·min throughout the process, reducing mRNA photodegradation by 80%, ensuring vaccine stability, thus maintaining the active ingredients of the vaccine and ensuring that it can exert the expected immune effect during clinical use.

[0028] 2. High efficiency and convenience: The intelligent liquid injection gun and microfluidic detection chip standardize the reconstitution operation, shortening the detection time to ≤45 minutes and greatly improving the test efficiency. This not only reduces the operation time cost of medical staff but also enables medical institutions to obtain vaccine performance data faster, accelerating the process from vaccine detection to clinical application and providing strong guarantee for the timely use of vaccines.

[0029] 3. Clinical adaptation: The detection system with anti-CA199 nanobody corrects the T cell response test value, conforms to the actual situation of pancreatic cancer patients, and provides a reliable basis for clinical decision-making. It helps doctors formulate personalized treatment plans based on more accurate test results, improves the effectiveness of pancreatic cancer vaccines in clinical treatment, and improves the treatment effect and prognosis of patients.

[0030] 4. Reliable data: The blockchain traceability module records the whole-process data and generates a time-stamped PDF certificate to ensure the data cannot be tampered with, enhancing the traceability of vaccine quality. This makes every link from vaccine production to use traceable, facilitating supervision by regulatory authorities, improving the quality control level of the entire vaccine industry, and enhancing the public's trust in vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a schematic block diagram of the performance test system of a pancreatic cancer vaccine;

[0032] Figure 2 It is a schematic block diagram of the principle of a performance test device for a pancreatic cancer vaccine;

[0033] Figure 3 It is a flowchart of a performance test method for a pancreatic cancer vaccine. Detailed implementation manners

[0034] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0035] Please refer to Figures 1-3 , the present invention provides a technical solution: a performance test system, device and method for a pancreatic cancer vaccine, including:

[0036] I. Preparation of an anti-adsorption syringe

[0037] 1. ALD coating process

[0038] Equipment: Picosun R-200 ALD equipment;

[0039] Parameters: The precursor is 3-(methacryloylamino)propyltrimethoxysilane, the deposition temperature is 50 °C, and the cycle is 50 times to form a 65 nm coating (measured by ellipsometer);

[0040] Post-treatment: Cure at 50 °C for 2 hours to form a cross-linked network (AFM shows a roughness of 4.2 nm).

[0041] 2. Coating verification

[0042] Adsorption test: Use a quartz crystal microbalance (QCM) to monitor the adsorption of lipid nanoparticles (LNP) in the BNT122 vaccine with a concentration of 100 μg / mL for 6 hours;

[0043] Stability: Immerse in physiological saline for 7 days, and EDS energy spectrum shows that the coating element accounts for 98.7% (the shedding rate is 0.5%). As shown in Table 1 below:

[0044]

[0045] Table 1: Comparison of experimental data between the example and the comparative example

[0046] In the present invention, precise thickness control by ALD: The coating thickness is precisely controlled by the number of cycles (±5 nm), and the uniformity is improved by 4 times compared with the spraying process; zwitterionic dual anti - 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 adsorption loss of each vaccine is <2.1 μg (calculated according to 100 μg / dose), avoiding immune failure caused by insufficient dose.

[0047] II. Standardized reconstitution operation

[0048] 1. Thawing control

[0049] Equipment: Haier HYC - 680 refrigerator (2 - 8 °C, ±0.5 °C fluctuation);

[0050] Operation: The thawing time of the cryopreservation tube (5 mL) is 118 ± 5 minutes (temperature recorder model: Onset HOBO UX120 - 011);

[0051] 2. Intelligent liquid injection

[0052] Equipment: XYZ - 2025 electric liquid injection gun (accuracy ±0.05 mL / s);

[0053] Parameters: Inject at a constant speed of 0.3 mL / s, and defoam with a 40 kHz ultrasonic head (amplitude 5 μm) for 30 seconds;

[0054] Residue: Capillary diversion groove + vacuum suction, the residual liquid is 1.8 ± 0.2 μL (weighed by Mettler Toledo MS105DU balance). As shown in Table 2 below:

[0055]

[0056] Table 2: Comparison of experimental data between examples and comparative examples

[0057] In the present invention: Ultrasonic frequency matching: 40 kHz corresponds to the LNP particle size (100 - 150 nm), and the bubble - breaking efficiency is 3 times that of traditional oscillation; Capillary diversion groove: 0.3 mm depth design, the residue is reduced by 89% compared with ordinary syringes (traditional residue is 16 μL); Clinical value: Eliminate 98% of visible bubbles, avoid dose over - estimation caused by LNP encapsulation, and the correlation between detection results and in - vivo efficacy is increased by 28%.

[0058] III. Implementation steps for full - process light - protected storage

[0059] 1. Light - proof syringe

[0060] Material: Polyurethane + photochromic nanoparticles (0.5wt% spiropyran);

[0061] Light transmittance: < 0.01% @ 400 - 500 nm (UV-Vis spectrophotometer Lambda 950);

[0062] Photoresist film: Silicone film thickness 0.1 mm, needle exposed length 5 mm;

[0063] 2. Light exposure monitoring

[0064] Sensor: Vishay VEML6070 (accuracy 0.1 lux, 400 - 500 nm band);

[0065] Cumulative exposure: Reconstituted to < 5 lux·min during the entire injection process (measured in a hospital consulting room: average 3.2 lux·min). As shown in Table 3 below:

[0066]

[0067] Table 3: Comparison of experimental data between examples and comparative examples

[0068] In the present invention: Photochromic repair: Light triggers the release of vitamin E (10 μg / cm²), repairing 42% more light damage than aluminum foil wrapping; Needle exposure control: Only 5 mm of the needle is exposed, reducing the optical path contact by 97% compared to traditional syringes; Clinical value: mRNA photodegradation is reduced by 80%, avoiding the underestimation of T cell response caused by light exposure (measured correction 34%).

[0069] IV. Patient-specific detection

[0070] 1. Preparation of artificial serum

[0071] CA199: 1500 U / mL (75th percentile value of pancreatic cancer patients);

[0072] Anti-CA199 nanobody: 10 nM (KD = 2.3 nM, Abcam ab289321);

[0073] 3D microtissue: Pancreatic stellate cells + tumor-associated macrophages, diameter 200 ± 20 μm.

[0074] 2. Detection equipment

[0075] ELISpot chip: 8 × 12 wells, each well coated with 1 μg of anti-IFN-γ antibody (BD551085);

[0076] Flow cytometer: BD FACSCanto II, CD127+CD8+ cell gating. As shown in Table 3 below:

[0077]

[0078] Table 2: Comparison of experimental data between examples and comparative examples

[0079] In the present invention: Nanobody penetrability: The 15 kDa molecular weight binds more easily to free CA199 in serum than the traditional antibody (150 kDa); 3D microtissue simulation: An immunosuppressive environment containing TGF-β is closer to in vivo than 2D culture (the penetration depth difference is 33 μm); Clinical value: Correct 40% of the underestimated detections, avoid vaccine waste caused by false negatives (save 270,000 yuan per 10,000 doses).

[0080] V. Validity period determination

[0081] 1. Multi-parameter acquisition

[0082] LNP adsorption rate: Detected by QCM (Maxtek 27 MHz), an alarm is given when > 3%;

[0083] mRNA integrity: Agilent 2100 electrophoresis (RIN > 7.0);

[0084] Calibration coefficient: K = 1.2 + 0.0002×(CA199 - 500) (500 - 5000 U / mL).

[0085] 2. Blockchain recording

[0086] Consortium blockchain: Hyperledger Fabric, block interval of 15 minutes;

[0087] Data items: Thawing time (accurate to seconds), cumulative light value (0.1 lux accuracy), original ELISpot image. As shown in Table 4 below (data of 100 postoperative patients):

[0088]

[0089] Table 4: Comparison of experimental data between examples and comparative examples

[0090] In the present invention: Four-dimensional fusion algorithm: Adsorption rate + mRNA + T cells + CA199, the prediction accuracy is improved by 82% compared with single-index prediction; Blockchain immutability: The photosensitive chip is directly connected to the chain to avoid manual modification (e.g., a certain batch is automatically recalled due to excessive light exposure); Clinical value: The recurrence risk caused by misjudgment is reduced by 86%.

[0091] VI. Comparison of the whole process cost and efficiency:

[0092] Refer to Table 5 below:

[0093]

[0094] Table 5: Comparison table of the whole process cost and efficiency in the present invention.

[0095] In summary, the present invention realizes the functions of reducing the LNP adsorption rate, reducing the influence of light on the vaccine, stabilizing the pH of the buffer solution, and calibrating the detection value of T cell response by means of technical measures such as coating the inner wall of the syringe with an amphoteric ion polymer coating, designing a dark box syringe, adding histidine oxidase to the buffer solution, and adding anti-CA199 nanoantibodies to the detection system. These technical measures 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: Including: Anti-adsorption storage unit: A polypropylene syringe with an inner wall coated with a 50 - 80 nm poly(sulfobetaine) coating by atomic layer deposition. After reconstitution of BNT122, the adsorption rate decreases from 15% to 2.1% after 6 hours. The preparation process of the poly(sulfobetaine) coating in the anti-adsorption storage unit is as follows: The precursor 3-(methacryloylamino)propyltrimethoxysilane is deposited by ALD process in cycles, cured at 50 °C for 2 hours, and the coating shedding rate is < 0.5% after being soaked in physiological saline for 7 days; Light-shielding transmission unit: A dark box syringe integrated with an optical fiber. Simulation shows that the cumulative blue light exposure during the whole process from reconstitution to injection is < 5 lux·min; Buffer stabilization unit: A histidine-sucrose buffer pre-loaded with 0.1 U / mL histidine oxidase, and the histamine production amount is < 5 μM after storage at 4 °C for 72 hours; Interference correction detection unit: A detection system containing 10 nM anti-CA199 nanobody. In a simulated serum with CA199 equal to 1500 U / mL, the specific T cell response of BNT122 is corrected from 58% to 79%.

2. The performance testing system of a pancreatic cancer vaccine according to claim 1, characterized in that: Also including: AR glasses unit, which identifies the vial batch number through a camera and displays in real time: thawing countdown; Prediction of the expiration date based on the adsorption rate and light data.

3. The performance testing system of a pancreatic cancer vaccine as claimed in claim 1, characterized in that: Also including: Blockchain unit, recording for each vaccine: reconstitution time; Cumulative light exposure value; detection raw data.

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