A method for preparing and purifying high purity radioiodinated antigen

Abstract

The application discloses a preparation and purification method of high-purity radioiodine-labeled antigen. The method comprises the following steps: pre-activating carrier-free sodium iodine[125I] solution in an orthogonal solvation sensitization buffer containing lithium sulfate, 18-crown-6 and beta-cyclodextrin; adding antigen and chloramine-T for a labeling reaction; and performing magnetic separation and purification by using a suspension containing adamantane-modified magnetic microspheres and quaternary ammonium salt-modified strong anion exchange magnetic microspheres after the reaction. The orthogonal solvation effect is used to enhance the nucleophilic activity of iodine ions, and the amount of oxidizing agent is reduced to reduce protein damage; impurities are quickly removed through the double mechanisms of electrostatic adsorption and host-guest recognition. The application has the advantages of high labeling efficiency, high radiochemical purity of the product and complete retention of biological activity.

Description

Technical Field

[0001] This invention relates to the field of nuclear medicine and radioimmunoassay technology, specifically to a method for preparing and purifying high-purity radioiodine-labeled antigens. Background Technology

[0002] Radiolabeled iodine antigens are core reagents in radioimmunoassay (RIA), and their labeling rate and radiochemical purity directly determine the detection sensitivity and accuracy of the results. Among existing labeling techniques, the chloramine-T method is widely used due to its fast reaction speed and high utilization rate. The basic principle of this method is to use an oxidant to oxidize radioactive iodide ions into electrophilic active species, which then replace hydrogen atoms on tyrosine or histidine residues in protein molecules.

[0003] However, in conventional aqueous reaction systems, iodide anions are strongly hydrated by water molecules, forming a stable hydrated layer that hinders their conversion into active electrophilic reagents. To overcome this hydration barrier and achieve the desired labeling efficiency, existing techniques typically employ methods such as increasing the concentration of chloramine-T or extending the reaction time. This strong oxidizing environment leads to non-specific oxidative damage to antigen proteins, particularly disrupting oxidatively sensitive tryptophan and methionine residues and disulfide bonds, causing conformational changes or polymerization of the protein, thereby reducing the immunoreactivity and specific binding capacity of the labeled antigen.

[0004] Furthermore, the purification process after the labeling reaction is another crucial step affecting the quality of the final product. Currently, gel column chromatography (such as Sephadex columns) is commonly used to remove unreacted free iodine and oxidants. This method mainly relies on gravity or low-pressure flow for molecular sieving, and the separation process is time-consuming. During the long elution period, the labeled product is continuously exposed to residual oxidants and a highly radioactive environment, which can easily induce radiodecomposition effects, leading to a decrease in radiochemical purity over time. Existing purification methods cannot achieve rapid cutoff of oxidants and radioactive impurities at the moment of reaction termination, limiting the preparation of highly active and stable labeled antigens. Summary of the Invention

[0005] The technical problem solved by this invention is that, in the conventional chloramine-T method for radioactive iodine labeling, water molecules solubilize iodide anions, resulting in low efficiency of nucleophilic substitution reaction. High concentrations of oxidants or long reaction times are usually required, which can cause oxidative damage, breakage or aggregation of antigen proteins, leading to loss of immune activity. At the same time, column chromatography purification methods are time-consuming and cannot remove oxidants immediately at the reaction endpoint, resulting in unstable product quality.

[0006] To address the above problems, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for preparing and purifying high-purity radioactive iodine-labeled antigens, employing the following technical solution: A method for preparing and purifying high-purity radioactive iodine-labeled antigen includes the following steps: Step S1: Add lithium sulfate, 18-crown ether-6 and β-cyclodextrin to phosphate buffer and mix well to prepare orthogonal solvation sensitization buffer; Step S2: Add carrier-free sodium iodide [125I] solution to orthogonal solvation sensitization buffer and incubate for radionuclide preactivation; Step S3: Add the antigen to be labeled and chloramine-T aqueous solution to the pre-activated solution to carry out the labeling reaction; Step S4: After the reaction is complete, add the composite functional magnetic separation suspension and shake to mix. Remove impurities from the supernatant through magnetic separation and collect the supernatant. The composite functional magnetic separation suspension contains adamantane-modified magnetic microspheres and quaternary ammonium salt-modified strong anion exchange magnetic microspheres.

[0007] By employing the above technical solution, the orthogonal solvation effect is utilized to enhance reaction activity, combined with a dual magnetic separation mechanism, to achieve labeling and purification at low oxidant concentrations. The specific mechanism is as follows: First, an orthogonal solvation system was constructed using lithium sulfate and 18-crown ether-6. Lithium ions dissociated from lithium sulfate in aqueous solution competitively bind to free water molecules in the system, reducing the activity of water molecules and creating a locally water-depleted microenvironment. Under this environment, 18-crown ether-6 specifically complexes sodium ions in radioactive sodium iodide, disrupting the ion lattice energy of sodium iodide. Furthermore, due to the weakened shielding effect of water molecules, iodide anions transform from a hydrated state to a bare state, enhancing their nucleophilic attack capability.

[0008] Second, the amount of oxidant used is reduced to protect protein activity. Due to the increased nucleophilic activity of iodide anions, the activation energy required for the labeling reaction is lowered, and even a trace amount of chloramine-T can initiate the electrophilic substitution reaction. Low-dose oxidants avoid excessive oxidation of disulfide bonds, tryptophan, and tyrosine residues in the antigen protein, thus preserving the antigen's biological activity.

[0009] Third, in-situ magnetic purification using a dual mechanism. After the reaction, the composite functional magnetic separation suspension is added. Quaternary ammonium salt-modified strong anion exchange magnetic microspheres adsorb and remove residual unreacted iodide ions and anion byproducts generated by chloramine-T through electrostatic interactions. Meanwhile, adamantane-modified magnetic microspheres utilize host-guest recognition to specifically bind β-cyclodextrin and hydrophobic organic impurities encapsulated by β-cyclodextrin in the solution, forming supramolecular complexes. The synergistic effect of the two types of microspheres rapidly completes oxidant cutoff and impurity removal, preventing secondary damage to proteins during separation.

[0010] Preferably, in the orthogonal solvation sensitization buffer, the concentration of lithium sulfate is 0.8-1.5 mol / L, the concentration of 18-crown ether-6 is 1.0-5.0 mmol / L, the concentration of β-cyclodextrin is 2.0-8.0 mmol / L, and the pH of the phosphate buffer is 7.2-7.6.

[0011] By adopting the above technical solution, the lithium salt concentration is controlled in the range of 0.8-1.5 mol / L, providing sufficient ionic strength to strip the hydration layer, while avoiding protein salting-out precipitation due to excessive salt concentration; the concentration ratio of crown ether to cyclodextrin ensures effective complexation of sodium ions and matching of subsequent purification capacity.

[0012] Preferably, in step S3, the concentration of the chloramine-T aqueous solution is 10-50 μg / mL, and the labeling reaction time is 30-60 seconds; in step S2, the incubation time is 45-90 seconds.

[0013] By adopting the above technical solution, labeling can be completed under conditions of low concentration of oxidant and short reaction time, reducing the risk of protein exposure to an oxidizing environment.

[0014] Preferably, in the composite functional magnetic separation suspension, the volume ratio of adamantane-modified magnetic microspheres to quaternary ammonium salt-modified strong anion exchange magnetic microspheres is 1:2 to 2:1; the total concentration of the composite functional magnetic separation suspension is 20 mg / mL.

[0015] By adopting the above technical solution and adjusting the ratio of the two types of functional microspheres, a balanced removal of ionic and hydrophobic impurities in the reaction system is achieved, ensuring the radiochemical purity of the final product.

[0016] Preferably, the adamantane-modified magnetic microspheres are prepared by a method comprising the following steps: dispersing amino-modified magnetic nanospheres in N,N-dimethylformamide, adding 1-adamantane carboxylic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, reacting at room temperature for 12-24 hours, washing and resuspending; wherein the molar ratio of 1-adamantane carboxylic acid to amino groups on the surface of the magnetic nanospheres is 4:1 to 20:1.

[0017] By adopting the above technical solution, adamantane groups are grafted onto the surface of magnetic beads using an amide condensation reaction. The set molar ratio ensures sufficient modification of the surface sites, enabling the adamantane-modified magnetic microspheres to bind β-cyclodextrin.

[0018] Preferably, the quaternary ammonium salt modified strong anion exchange magnetic microspheres are prepared by a method comprising the following steps: dispersing magnetic nanospheres rich in silanol groups on the surface in toluene, adding a (3-trimethoxysilylpropyl)trimethylammonium chloride solution, refluxing at 110°C for 6-12 hours, washing and resuspending; wherein, 0.2-1.0 mL of (3-trimethoxysilylpropyl)trimethylammonium chloride solution is added for every 100 mg of magnetic nanospheres.

[0019] By adopting the above technical solution, a quaternary ammonium salt positive charge layer is constructed on the surface of magnetic beads using a silane coupling agent, enabling the quaternary ammonium salt modified strong anion exchange magnetic microspheres to have anion exchange capacity under physiological pH conditions, and to adsorb and remove free radioactive iodine ions and oxidant anions.

[0020] Preferably, in step S4, the oscillation mixing time is 10-30 seconds, and the magnetic separation settling time is 30-60 seconds.

[0021] By adopting the above technical solution, the rapid response characteristics of magnetic separation are utilized to complete the separation process in a short time, shortening the operation time and blocking the time-dependent radiolysis effect.

[0022] Secondly, the present invention provides a composite functional magnetic separation suspension, which adopts the following technical solution: A composite functional magnetic separation suspension is composed of a mixture of adamantane-modified magnetic microsphere suspension and quaternary ammonium salt-modified strong anion exchange magnetic microsphere suspension; the adamantane-modified magnetic microspheres are Fe3O4@SiO2 magnetic microspheres with 1-adamantane carboxylic acid grafted on their surface; the quaternary ammonium salt-modified strong anion exchange magnetic microspheres are Fe3O4@SiO2 magnetic microspheres with (3-trimethoxysilylpropyl)trimethylammonium chloride grafted on their surface.

[0023] By employing the above technical solution, two types of magnetic microspheres with different adsorption mechanisms are combined in the same system. The adamantane-modified magnetic microspheres target cyclodextrins and cyclodextrin inclusion complexes, while the quaternary ammonium salt-modified strong anion exchange magnetic microspheres target small molecule anions. This combined design allows a single reagent to simultaneously address the issues of residual organic impurities, oxidant removal, and free isotope scavenging, simplifying the purification process.

[0024] Preferably, the volume ratio of the adamantane-modified magnetic microsphere suspension to the quaternary ammonium salt-modified strong anion exchange magnetic microsphere suspension is 1:2 to 2:1.

[0025] By adopting the above technical solution, the proportion of adsorption medium is adjusted according to the estimated amount of impurities generated in the reaction system to ensure adsorption capacity and avoid incomplete purification due to adsorption saturation.

[0026] Thirdly, the present invention provides a method for preparing a composite functional magnetic separation suspension, which adopts the following technical solution: A method for preparing a composite functional magnetic separation suspension includes: placing adamantane-modified magnetic microsphere suspension with a concentration of 20 mg / mL and a quaternary ammonium salt-modified strong anion exchange magnetic microsphere suspension in a container in a certain proportion, vortexing and mixing for 60 seconds, and then sonicating for 30 seconds.

[0027] By adopting the above technical solution, through physical mixing and ultrasonic dispersion, it is ensured that the two types of microspheres are evenly distributed in the suspension and do not pre-aggregate, so that each microsphere can independently perform its adsorption function during use, and maintain the uniformity and stability of the composite functional magnetic separation suspension system.

[0028] This invention provides a method for preparing and purifying high-purity radioactive iodine-labeled antigens. It has the following beneficial effects: 1. This invention constructs an orthogonal solvation system of lithium sulfate and 18-crown ether-6, utilizing the hydration capacity of lithium ions to reduce the activity of water molecules. Combined with the complexation effect of 18-crown ether-6 on sodium ions, this promotes the transformation of iodide anions from a hydrated state to a naked state, thereby enhancing the nucleophilic reactivity of iodide ions. This sensitization mechanism allows the labeling reaction to be completed with only a low concentration of chloramine-T and a short reaction time, avoiding oxidative damage to the active site of the antigen protein caused by high doses of oxidants, thus ensuring the bioactivity and immunoreactivity of the labeled product.

[0029] 2. This invention employs a composite functional magnetic separation suspension containing adamantane-modified magnetic microspheres and quaternary ammonium salt-modified strong anion exchange magnetic microspheres. Through a dual mechanism of electrostatic adsorption and host-guest recognition, it can simultaneously remove unreacted radioactive iodide ions, oxidant anions, and β-cyclodextrin impurities in a short time. Compared to column chromatography purification, magnetic separation shortens the contact time between the product and radioactive impurities and oxidants, blocks the radiodecomposition effect, and improves the radiochemical purity of the final product.

[0030] 3. The lithium sulfate used in this invention does not competitively complex with 18-crown ether-6, ensuring the independent synergistic operation of the salt effect and phase transfer catalysis. Combined with the auxiliary effect of β-cyclodextrin, a stable reaction microenvironment is constructed. Experimental results show that, under different process scale-up conditions or fluctuations in feed concentration, the reaction system can still maintain a high labeling rate and radiochemical purity, exhibiting good process stability and repeatability. Attached Figure Description

[0031] Figure 1 This is a process diagram for the preparation of the present invention. Detailed Implementation

[0032] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0034] Lithium sulfate, anhydrous grade, purity ≥99.0%; 18-Crown ether-6, chemical name 1,4,7,10,13,16-hexaoxane, purity ≥99%; β-Cyclodextrin, purity ≥98%, water content ≤14%; Chloramine-T, chemical name N-chloro-p-toluenesulfonamide sodium salt trihydrate, ACS grade; Sodium iodide [125I] solution, without carrier, radioactive concentration 3.7 GBq / mL, dissolved in 10^-5 mol / L sodium hydroxide solution, radiochemical purity ≥99%; Bovine serum albumin, component V, lyophilized powder, molecular weight approximately 66.5 kDa; Mouse-derived IgG monoclonal antibody, purified by protein A affinity chromatography, with a purity ≥98% and a concentration of 5 mg / mL; 1-Adamantane carboxylic acid, purity ≥99%; (3-Trimethoxysilylpropyl)trimethylammonium chloride, 50% methanol solution. Aminated magnetic nanospheres, matrix of silica-coated iron(III) oxide (Fe3O4@SiO2), surface modified with amino groups (-NH2), average particle size 200 nm, saturation magnetization ≥40 emu / g, amino loading approximately 0.5 mmol / g; Hydroxylated magnetic nanospheres are based on a silica-coated iron(III) oxide matrix (Fe3O4@SiO2) with a surface rich in silanol groups (-Si-OH). The average particle size is 200 nm and the saturation magnetization is ≥40 emu / g.

[0035] To fully support the scope of the claims regarding the purification medium, particularly concerning the degree of surface modification of the magnetic microspheres and the mixing ratio of the two types of microspheres, three groups of nine preparation examples are provided below. Preparation examples 1-3 involve adamantane-modified magnetic microspheres, covering different grafting densities; preparation examples 4-6 involve quaternary ammonium salt-modified magnetic microspheres, covering different ion exchange capacities; and preparation examples 7-9 involve the preparation of composite suspensions, covering different mixing ratios.

[0036] Preparation Example 1: This preparation example provides an adamantane-modified magnetic microsphere (Ad-MNPs-1), including the following steps: 100 mg of aminated magnetic nanospheres were suspended in 5 mL of anhydrous N,N-dimethylformamide and ultrasonically dispersed for 10 minutes to ensure homogeneity. 0.2 mmol of 1-adamantanecarboxylic acid was added to the suspension, followed by 0.4 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.4 mmol of N-hydroxysuccinimide. The mixture was shaken continuously at 600 rpm for 12 hours at room temperature. After the reaction, the supernatant was removed by magnetic separation and washed three times each with N,N-dimethylformamide, anhydrous ethanol, and deionized water. Finally, the nanospheres were resuspended in 5 mL of 0.01 M phosphate buffer (pH 7.4) and the final concentration was adjusted to 20 mg / mL. The mixture was then stored at 4 °C.

[0037] Preparation Example 2: This preparation example provides an adamantane-modified magnetic microsphere (Ad-MNPs-2), including the following steps: 100 mg of aminated magnetic nanospheres were suspended in 5 mL of anhydrous N,N-dimethylformamide and ultrasonically dispersed for 10 minutes to ensure homogeneity. 0.5 mmol of 1-adamantanecarboxylic acid was added to the suspension, followed by 1.0 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 1.0 mmol of N-hydroxysuccinimide. The mixture was continuously shaken at 600 rpm for 18 hours at room temperature. After the reaction, the supernatant was removed by magnetic separation and washed three times each with N,N-dimethylformamide, anhydrous ethanol, and deionized water. Finally, the nanospheres were resuspended in 5 mL of 0.01 M phosphate buffer (pH 7.4) and the final concentration was adjusted to 20 mg / mL. The mixture was then stored at 4 °C.

[0038] Preparation Example 3: This preparation example provides an adamantane-modified magnetic microsphere (Ad-MNPs-3), including the following steps: 100 mg of aminated magnetic nanospheres were suspended in 5 mL of anhydrous N,N-dimethylformamide and ultrasonically dispersed for 10 minutes to ensure homogeneity. 1.0 mmol of 1-adamantanecarboxylic acid was added to the suspension, followed by 2.0 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 2.0 mmol of N-hydroxysuccinimide. The mixture was continuously shaken at 600 rpm for 24 hours at room temperature. After the reaction, the supernatant was removed by magnetic separation, and the nanospheres were washed three times each with N,N-dimethylformamide, anhydrous ethanol, and deionized water. Finally, the nanospheres were resuspended in 5 mL of 0.01 M phosphate buffer (pH 7.4) and the final concentration was adjusted to 20 mg / mL. The mixture was then stored at 4 °C.

[0039] Preparation Example 4: This preparation example provides a quaternary ammonium salt modified strong anion exchange magnetic microsphere (QA-MNPs-1), including the following steps: 100 mg of hydroxylated magnetic nanospheres were suspended in 10 mL of anhydrous toluene and ultrasonically dispersed for 10 minutes. Under nitrogen protection, 0.2 mL of (3-trimethoxysilylpropyl)trimethylammonium chloride solution was added dropwise to the suspension. The mixture was heated to 110 °C and refluxed for 6 hours. After the reaction was completed, the mixture was cooled to room temperature, and the supernatant was removed by magnetic separation. The nanospheres were washed three times each with toluene, anhydrous ethanol, and deionized water. Finally, the nanospheres were resuspended in 5 mL of deionized water and the final concentration was adjusted to 20 mg / mL. The mixture was then stored at 4 °C.

[0040] Preparation Example 5: This preparation example provides a quaternary ammonium salt modified strong anion exchange magnetic microsphere (QA-MNPs-2), including the following steps: 100 mg of hydroxylated magnetic nanospheres were suspended in 10 mL of anhydrous toluene and ultrasonically dispersed for 10 minutes. Under nitrogen protection, 0.5 mL of (3-trimethoxysilylpropyl)trimethylammonium chloride solution was added dropwise to the suspension. The mixture was heated to 110 °C and refluxed for 8 hours. After the reaction was completed, the mixture was cooled to room temperature, and the supernatant was removed by magnetic separation. The nanospheres were washed three times each with toluene, anhydrous ethanol, and deionized water. Finally, the nanospheres were resuspended in 5 mL of deionized water and the final concentration was adjusted to 20 mg / mL. The mixture was then stored at 4 °C.

[0041] Preparation Example 6: This preparation example provides a quaternary ammonium salt modified strong anion exchange magnetic microsphere (QA-MNPs-3), including the following steps: 100 mg of hydroxylated magnetic nanospheres were suspended in 10 mL of anhydrous toluene and ultrasonically dispersed for 10 minutes. Under nitrogen protection, 1.0 mL of (3-trimethoxysilylpropyl)trimethylammonium chloride solution was added dropwise to the suspension. The mixture was heated to 110 °C and refluxed for 12 hours. After the reaction was completed, the mixture was cooled to room temperature, and the supernatant was removed by magnetic separation. The nanospheres were washed three times each with toluene, anhydrous ethanol, and deionized water. Finally, the nanospheres were resuspended in 5 mL of deionized water and the final concentration was adjusted to 20 mg / mL. The mixture was stored at 4 °C.

[0042] Preparation Example 7: This preparation example provides a composite functional magnetic separation suspension (CMS-1), including the following steps: Take 2 mL of the Ad-MNPs-2 suspension prepared in Preparation Example 2 and 1 mL of the QA-MNPs-2 suspension prepared in Preparation Example 5, and place them in a centrifuge tube; vortex and mix for 60 seconds at room temperature, and then sonicate for 30 seconds to prevent microsphere aggregation, thus obtaining a composite functional magnetic separation suspension with a volume ratio of 2:1.

[0043] Preparation Example 8: This preparation example provides a composite functional magnetic separation suspension (CMS-2), including the following steps: Take 1.5 mL of the Ad-MNPs-2 suspension prepared in Preparation Example 2 and 1.5 mL of the QA-MNPs-2 suspension prepared in Preparation Example 5, and place them in a centrifuge tube; vortex and mix at room temperature for 60 seconds, then sonicate for 30 seconds to prevent microsphere aggregation, thus obtaining a composite functional magnetic separation suspension with a volume ratio of 1:1.

[0044] Preparation Example 9: This preparation example provides a composite functional magnetic separation suspension (CMS-3), including the following steps: Take 1 mL of the Ad-MNPs-2 suspension prepared in Preparation Example 2 and 2 mL of the QA-MNPs-2 suspension prepared in Preparation Example 5, and place them in a centrifuge tube; vortex and mix for 60 seconds at room temperature, and then sonicate for 30 seconds to prevent microsphere aggregation, thus obtaining a composite functional magnetic separation suspension with a volume ratio of 1:2.

[0045] The following are embodiments of the present invention. Embodiment 1 represents a preferred embodiment of the present invention (central parameters), Embodiments 2 and 3 represent boundary conditions with different process parameters, and Embodiment 4 demonstrates the feasibility of process scale-up.

[0046] Example 1: This embodiment provides a method for preparing and purifying high-purity radioactive iodine-labeled antigens, using a preferred combination of intermediate parameters, and includes the following steps: Step 1: Preparation of orthogonal solvation sensitization buffer Lithium sulfate, 18-crown ether-6, and β-cyclodextrin were added to 0.05 M phosphate buffer (pH 7.4), stirred to dissolve, and filtered to prepare a sensitizing buffer. The concentrations of lithium sulfate, 18-crown ether-6, and β-cyclodextrin were 1.0 mol / L, 3.0 mmol / L, and 6.0 mmol / L, respectively.

[0047] Step 2: Pre-activation of radionuclides Take a polypropylene reaction tube and add 50 μL of the sensitization buffer prepared in step one; then add 5 μL of carrier-free sodium iodide [125I] solution (radioactivity of about 1.0 mCi), gently shake to mix, and incubate at room temperature of 25°C for 60 seconds to allow iodine ions to complete the naked anionization conversion with the assistance of lithium salt.

[0048] Step 3: Biphasic Control of Labeling Reaction Add 10 μL of a 2 mg / mL mouse IgG monoclonal antibody solution to the reaction tube; then add 5 μL of a 20 μg / mL fresh chloramine-T aqueous solution to start the reaction; react at room temperature for 45 seconds.

[0049] Step 4: Reaction Termination and In-situ Purification When the reaction reaches 45 seconds, immediately add 150 μL of the composite functional magnetic separation suspension (CMS-2, volume ratio 1:1) prepared in Example 8 to the reaction tube; vortex vigorously for 15 seconds to allow the adamantane magnetic beads to capture the β-cyclodextrin inclusion complex, while the quaternary ammonium salt magnetic beads adsorb the residual free iodine; then place the reaction tube on the magnetic separation rack and let it stand for 30 seconds. After the solution becomes clear, aspirate the supernatant to obtain the high-purity radiolabeled iodine antigen.

[0050] Example 2: This embodiment provides a method for preparing and purifying high-purity radioactive iodine-labeled antigens, using a parameter combination of low salt concentration and high ligand concentration, including the following steps: Step 1: Preparation of orthogonal solvation sensitization buffer Lithium sulfate, 18-crown ether-6, and β-cyclodextrin were added to 0.05 M phosphate buffer (pH 7.2), stirred to dissolve, and filtered to prepare a sensitizing buffer. The concentrations of lithium sulfate, 18-crown ether-6, and β-cyclodextrin were 0.8 mol / L, 5.0 mmol / L, and 8.0 mmol / L, respectively.

[0051] Step 2: Pre-activation of radionuclides Take a polypropylene reaction tube and add 50 μL of the sensitization buffer prepared in step one; then add 10 μL of carrier-free sodium iodide [125I] solution (radioactivity of about 2.0 mCi), gently shake to mix, and incubate at room temperature of 20°C for 90 seconds.

[0052] Step 3: Biphasic Control of Labeling Reaction Add 15 μL of a 5 mg / mL mouse IgG monoclonal antibody solution to the reaction tube; then add 5 μL of a 50 μg / mL fresh chloramine-T aqueous solution to start the reaction; react at room temperature for 60 seconds.

[0053] Step 4: Reaction Termination and In-situ Purification When the reaction reaches 60 seconds, immediately add 200 μL of the composite functional magnetic separation suspension (CMS-1, volume ratio 2:1) prepared in Example 7 to the reaction tube; vortex vigorously for 20 seconds; then place the reaction tube on the magnetic separation rack and let it stand for 45 seconds. After the solution becomes clear, aspirate the supernatant to obtain the high-purity radioactive iodine-labeled antigen.

[0054] Example 3: This embodiment provides a method for preparing and purifying high-purity radioactive iodine-labeled antigens, using a parameter combination of high salt concentration and low ligand concentration, including the following steps: Step 1: Preparation of orthogonal solvation sensitization buffer Lithium sulfate, 18-crown ether-6, and β-cyclodextrin were added to 0.05 M phosphate buffer (pH 7.6), stirred to dissolve, and filtered to prepare a sensitizing buffer. The concentrations of lithium sulfate, 18-crown ether-6, and β-cyclodextrin were 1.5 mol / L, 1.0 mmol / L, and 2.0 mmol / L, respectively.

[0055] Step 2: Pre-activation of radionuclides Take a polypropylene reaction tube and add 50 μL of the sensitization buffer prepared in step one; then add 5 μL of carrier-free sodium iodide [125I] solution (radioactivity of about 0.5 mCi), gently shake to mix, and incubate at room temperature of 25°C for 45 seconds.

[0056] Step 3: Biphasic Control of Labeling Reaction Add 10 μL of bovine serum albumin (BSA) solution with a concentration of 1 mg / mL to the reaction tube; then add 5 μL of fresh chloramine-T aqueous solution with a concentration of 10 μg / mL to start the reaction; react for 30 seconds at room temperature.

[0057] Step 4: Reaction Termination and In-situ Purification When the reaction reaches 30 seconds, immediately add 100 μL of the composite functional magnetic separation suspension (CMS-3, volume ratio 1:2) prepared in Example 9 to the reaction tube; vortex vigorously for 10 seconds; then place the reaction tube on the magnetic separation rack and let it stand for 30 seconds. After the solution becomes clear, aspirate the supernatant to obtain the high-purity radioactive iodine-labeled antigen.

[0058] Example 4: This embodiment provides a method for preparing and purifying high-purity radioactive iodine-labeled antigen, and verifies the feasibility of a scale-up system (1 mL level), including the following steps: Step 1: Preparation of orthogonal solvation sensitization buffer Lithium sulfate, 18-crown ether-6, and β-cyclodextrin were added to 0.05 M phosphate buffer (pH 7.4), stirred to dissolve, and filtered to prepare a sensitizing buffer. The concentrations of lithium sulfate, 18-crown ether-6, and β-cyclodextrin were 1.2 mol / L, 2.5 mmol / L, and 5.0 mmol / L, respectively.

[0059] Step 2: Pre-activation of radionuclides Take a 5mL glass reaction bottle and add 1.0mL of the sensitization buffer prepared in step one; then add 50μL of carrier-free sodium iodide [125I] solution (radioactivity of about 10mCi), stir magnetically to mix well, and incubate at room temperature of 25°C for 60 seconds.

[0060] Step 3: Biphasic Control of Labeling Reaction Add 200 μL of a 2 mg / mL mouse IgG monoclonal antibody solution to the reaction flask; then add 50 μL of a 25 μg / mL fresh chloramine-T aqueous solution to start the reaction; stir the reaction at room temperature for 45 seconds.

[0061] Step 4: Reaction Termination and In-situ Purification After the reaction was complete, immediately add 2.0 mL of the composite functional magnetic separation suspension (CMS-2, volume ratio 1:1) prepared in Example 8 to the reaction flask; shake vigorously for 30 seconds; then place the reaction flask on a magnetic separation rack and let it stand for 60 seconds, and aspirate the supernatant. Transfer the supernatant to an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa, centrifuge at 4000 rpm for 5 minutes, wash once with 0.01 M PBS buffer, and collect the retentate to obtain high-purity radioiodine-labeled antigen.

[0062] To fully highlight the synergistic effect of the "orthogonal solvation system," "phase transfer catalysis," "supramolecular protection," and "magnetic in-situ purification" in the technical solution of this invention, six comparative examples were designed below. Comparative Example 1 is a blank control lacking all enhancing components; Comparative Examples 2-4 are controls lacking a single key component; Comparative Example 5 is a counter-evidence experiment to verify the orthogonality of ions; and Comparative Example 6 is a control to verify the superiority of the purification method.

[0063] Comparative Example 1: This comparative example represents the conventional low-concentration chloramine-T labeling method. The differences from Example 1 are: the buffer prepared in step one is only ordinary 0.05M phosphate-buffered saline (PBS), without lithium sulfate, 18-crown ether-6, and β-cyclodextrin; and due to the lack of an adsorption medium, the purification method in step four is replaced by the traditional Sephadex G-25 gel column chromatography, eluting and collecting the protein peak. All other steps and parameters are the same.

[0064] Comparative Example 2: This comparative example aims to verify the key role of the "orthogonal solvation regulator (lithium sulfate)". The difference from Example 1 is that lithium sulfate is not added to the buffer solution in step one; it only contains 18-crown ether-6 and β-cyclodextrin. All other steps and parameters are the same.

[0065] Comparative Example 3: This comparative example aims to verify the key role of the phase transfer catalyst (18-crown ether-6). The difference from Example 1 is that 18-crown ether-6 is not added to the buffer solution in step one; it only contains lithium sulfate and β-cyclodextrin. All other steps and parameters are the same.

[0066] Comparative Example 4: This comparative example aims to verify the protective effect of the supramolecular trap (β-cyclodextrin) on protein activity and its impact on purification. The differences from Example 1 are: β-cyclodextrin is not added to the buffer in step one, only lithium sulfate and 18-crown ether-6 are present; and because β-cyclodextrin is absent in the system, only quaternary ammonium salt-modified magnetic microspheres (QA-MNPs-2) are used in step four to remove free iodine, and adamantane magnetic beads cannot be used to remove hydrophobic oxidation byproducts. All other steps and parameters are the same.

[0067] Comparative Example 5: This comparative example aims to verify the "orthogonality" of the ions, demonstrating that lithium salts not complexed by crown ethers must be used. The difference from Example 1 is that potassium sulfate (K₂SO₄) was used in step one instead of lithium sulfate (Li₂SO₄), while maintaining the same molar concentration. All other steps and parameters remain the same.

[0068] (Note: Potassium ions (K+) have an extremely high binding constant with 18-crown ether-6, which competitively inhibits the binding of crown ethers to sodium ions, theoretically leading to reaction failure.) Comparative Example 6: This comparative example aims to verify the impact of "in-situ magnetic separation" on efficiency and product quality. The difference from Example 1 is that, in step four, a composite functional magnetic separation suspension is not added; instead, a conventional Sephadex G-50 gel column is used for centrifugal column chromatography purification for 3 minutes. All other steps and parameters remain the same.

[0069] Test Example 1: Radiochemical Performance Indicators and Feasibility Test of Preparation Process This test evaluates the labeling efficiency and radiochemical purity of the final products in the examples and comparative examples. The labeling rate was measured by aspirating the reaction solution before the reaction termination step, and the radiochemical purity was measured by aspirating the supernatant or eluent after the purification step. The test employed instantaneous thin-layer chromatography (TLC), using ITLC-SG silica gel strips as the stationary phase and 85% methanol aqueous solution as the developing solvent. During the experiment, 1 μL of the sample was spotted at the bottom of the chromatographic strip, allowed to air dry, and then developed in a developing tank. In this chromatographic system, radioiodine-labeled proteins remained at the origin, while unreacted free iodide ions and small iodide molecules migrated to the front with the developing solvent. After development, the radioactivity distribution of the chromatographic strip was measured using a radiometric thin-layer scanner, and the percentage of the radioactivity count at the origin relative to the total radioactivity count of the chromatographic strip was calculated, which represents the labeling rate or radiochemical purity of the sample. Detailed test data for each example and comparative example are shown in Table 1.

[0070] Table 1. Results of radioactive iodine labeling rate and radiochemical purity of products in each experimental group

[0071] In Examples 1 to 4, under different lithium sulfate concentrations, 18-crown ether-6 dosages, and reaction system scales, the labeling rate remained above 90%, and the final product had a radiochemical purity exceeding 97%. This indicates that the reaction system exhibits process stability within the set parameter range, and that the lithium salt concentration can effectively regulate the aqueous phase environment within the range of 0.8-1.5 mol / L, thereby activating iodide ions in conjunction with the crown ether.

[0072] Comparative Examples 2 and 3 show that the labeling rate drops significantly to the 30%-40% range when either lithium sulfate or 18-crown ether-6 is absent. When crown ether is used alone, it cannot effectively expose iodide ions due to the strong hydration of water molecules with ions. When high-concentration lithium salt is used alone, although the water activity is reduced, the nucleophilic substitution reaction kinetics remain slow due to the lack of a phase transfer catalyst. Only when both are present simultaneously can a highly active reaction microenvironment be constructed in the aqueous phase through the dual action of lithium salt competing for water molecules and crown ether complexing cations.

[0073] In Comparative Example 5, replacing lithium sulfate with potassium sulfate resulted in the lowest labeling rate (27.9%). This is because the potassium ion radius matches the cavity size of the 18-crown ether-6, allowing potassium ions to competitively occupy the crown ether cavity. This inhibits the complexation of sodium ions from radioactive sodium iodide by the crown ether, thus blocking the phase transfer catalytic process. The results conversely confirm that the reaction system must select a lithium salt that does not complex with the crown ether to ensure the independent operation of the salt effect and the catalytic effect.

[0074] Regarding purification efficiency, Comparative Example 6 used conventional gel column chromatography. Although the labeling rate was comparable to that of Example 1, the radiochemical purity of the final product (93.7%) was lower than that of Example 1 (99.1%). The column chromatography separation process was time-consuming, and residual oxidants and radionuclides continued to contact the protein during separation, leading to radiodegradation or oxidative damage to some products. In contrast, the magnetic separation used in Example 1 completed oxidant removal and impurity separation within 1 minute, reducing the time the protein was exposed to adverse environments, thus achieving higher radiochemical purity.

[0075] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing and purifying high-purity radioactive iodine-labeled antigen, characterized in that, Includes the following steps: Step S1: Add lithium sulfate, 18-crown ether-6 and β-cyclodextrin to phosphate buffer and mix well to prepare orthogonal solvation sensitization buffer; Step S2: Add carrier-free sodium iodide [125I] solution to the orthogonal solvation sensitization buffer and incubate for radionuclide preactivation; Step S3: Add the antigen to be labeled and chloramine-T aqueous solution to the pre-activated solution to carry out the labeling reaction; Step S4: After the reaction is complete, add the composite functional magnetic separation suspension and shake to mix. Remove impurities from the supernatant through magnetic separation and collect the supernatant. The composite functional magnetic separation suspension contains adamantane-modified magnetic microspheres and quaternary ammonium salt-modified strong anion exchange magnetic microspheres.

2. The method for preparing and purifying high-purity radioactive iodine-labeled antigen according to claim 1, characterized in that, In the orthogonal solvation sensitization buffer, the concentration of lithium sulfate is 0.8-1.5 mol / L, the concentration of 18-crown ether-6 is 1.0-5.0 mmol / L, and the concentration of β-cyclodextrin is 2.0-8.0 mmol / L; the pH value of the phosphate buffer is 7.2-7.

6.

3. The method for preparing and purifying high-purity radioactive iodine-labeled antigen according to claim 1, characterized in that, In step S3, the concentration of the chloramine-T aqueous solution is 10-50 μg / mL, and the labeling reaction time is 30-60 seconds; in step S2, the incubation time is 45-90 seconds.

4. The method for preparing and purifying high-purity radioactive iodine-labeled antigen according to claim 1, characterized in that, In the composite functional magnetic separation suspension, the volume ratio of adamantane-modified magnetic microspheres to quaternary ammonium salt-modified strong anion exchange magnetic microspheres is 1:2 to 2:1; the total concentration of the composite functional magnetic separation suspension is 20 mg / mL.

5. The method for preparing and purifying high-purity radioactive iodine-labeled antigen according to claim 1, characterized in that, The adamantane-modified magnetic microspheres were prepared by a method comprising the following steps: Amino-modified magnetic nanospheres were dispersed in N,N-dimethylformamide, and 1-adamantanecarboxylic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added. The mixture was reacted at room temperature for 12-24 hours, washed and resuspended. The molar ratio of 1-adamantane carboxylic acid to the amino groups on the surface of the magnetic nanospheres is 4:1 to 20:

1.

6. The method for preparing and purifying high-purity radioactive iodine-labeled antigen according to claim 1, characterized in that, The quaternary ammonium salt-modified strong anion exchange magnetic microspheres were prepared by a method comprising the following steps: Magnetic nanospheres with silanol-rich surfaces were dispersed in toluene, and a solution of (3-trimethoxysilylpropyl)trimethylammonium chloride was added. The mixture was refluxed at 110°C for 6-12 hours, washed, and resuspended. For every 100 mg of the magnetic nanospheres, 0.2-1.0 mL of the (3-trimethoxysilylpropyl)trimethylammonium chloride solution is added.

7. The method for preparing and purifying high-purity radioactive iodine-labeled antigen according to claim 1, characterized in that, In step S4, the oscillation mixing time is 10-30 seconds, and the magnetic separation settling time is 30-60 seconds.

8. A composite functional magnetic separation suspension, characterized in that, It is composed of a mixture of adamantane-modified magnetic microsphere suspension and quaternary ammonium salt-modified strong anion exchange magnetic microsphere suspension; The adamantane-modified magnetic microspheres are Fe3O4@SiO2 magnetic microspheres with 1-adamantane carboxylic acid grafted onto their surface. The quaternary ammonium salt modified strong anion exchange magnetic microspheres are Fe3O4@SiO2 magnetic microspheres with (3-trimethoxysilylpropyl)trimethylammonium chloride grafted onto their surface.

9. A composite functional magnetic separation suspension according to claim 8, characterized in that, The volume ratio of the adamantane-modified magnetic microsphere suspension to the quaternary ammonium salt-modified strong anion exchange magnetic microsphere suspension is 1:2 to 2:

1.

10. The method for preparing the composite functional magnetic separation suspension according to claim 8 or 9, characterized in that, include: A suspension of adamantane-modified magnetic microspheres with a concentration of 20 mg / mL and a suspension of quaternary ammonium salt-modified strong anion exchange magnetic microspheres were placed in a container in a certain proportion, vortexed for 60 seconds, and then sonicated for 30 seconds.

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