A diagnostic reagent marker protective agent and its application

Abstract

The present invention provides a diagnostic reagent marker protective agent and its application, belonging to the field of medical technology. The diagnostic reagent marker protective agent of the present invention comprises the following components: a buffer with an ionic strength of 30 to 80 mmol / L, 1 g / L to 5 g / L protein, 5 g / L to 35 g / L sugar, 25 g / L to 45 g / L alcohol, and 0.001% to 0.01% preservative. By selecting specific components, the present invention provides a diagnostic reagent marker protective agent that can improve the stability of the diagnostic reagent marker in the freeze-dried state and the reconstituted state; when used in the freeze-drying process of the diagnostic reagent marker, it can improve the stability of the diagnostic reagent marker during the freeze-drying process, the product qualification rate, and the consistency between batches.

Description

Technical Field

[0001] The invention relates to a diagnostic reagent marker protective agent and application thereof, belonging to the technical field of medicine. Background Art

[0002] A marker is a combination of fluorescein, radioisotopes, enzymes, ferritin, colloidal gold, rare earth elements, and chemiluminescent (or bioluminescent) agents, typically used as a tracer to label antigens or antibodies, nucleic acid probes, and cells. Once a reaction (e.g., an antigen-antibody immune reaction, a biotin-avidin reaction, a nucleic acid probe hybridization reaction, or a target cell-effector cell killing reaction) occurs, the tracer signal in the final product can be measured with an instrument to determine the presence and / or level of the analyte in the reaction system.

[0003] Markers, a crucial component of diagnostic reagents, contain bioactive substances such as enzymes, antigens, and antibodies. They are particularly temperature-sensitive, highly unstable, and prone to cross-reaction. These characteristics result in a short lifecycle for liquid diagnostic reagents, requiring low-temperature storage and full cold chain transportation. This results in a short shelf life, high costs, and unmanageable risks. The stability of biological reagents is a key factor in this industry, and freeze-drying technology is an ideal option. However, current freeze-drying processes still face the following challenges: 1) The complex formulation of bioactive substance protectants leads to large batch-to-batch variability; 2) Immature freeze-drying processes often result in unstable product structure, a fluffy appearance, shrinkage, and even gel-like formation; and significant delamination, leading to high scrap rates and poor intra-batch consistency. Furthermore, freeze-drying cycles are long, resulting in low production capacity and high energy consumption. Therefore, in freeze-drying marker markers for diagnostic reagents, a simple and stable freeze-drying protectant and a rational freeze-drying process are needed to improve product stability, product quality, batch consistency, and production capacity. Summary of the Invention

[0004] The purpose of the present invention is to provide a diagnostic reagent marker protective agent and its application. The protective agent of the present invention can improve the stability of the diagnostic reagent marker in the freeze-dried state and the re-dissolved state, and at the same time improve the stability, product qualification rate, batch consistency and production capacity of the diagnostic reagent marker during the freeze-drying process.

[0005] To achieve the above-mentioned purpose, the technical solution adopted by the present invention is: a diagnostic reagent marker protective agent, including a buffer with an ionic strength of 30 to 80 mmol / L, 1 g / L to 5 g / L protein, 5 g / L to 35 g / L sugar, 25 g / L to 45 g / L alcohol and 0.001% to 0.01% preservative.

[0006] Research has shown that when using freeze-drying technology to improve the stability of diagnostic reagent markers, the existing technology still suffers from problems such as poor stability, large batch-to-batch variation, and low product qualification rate due to the lack of a protective agent or the overly complex formulation of the protective agent. After extensive experiments, the inventors of this application discovered that a diagnostic reagent marker protective agent comprising a buffer with an ionic strength of 30 to 80 mmol / L, 1 g / L to 5 g / L protein, 5 g / L to 35 g / L sugar, 25 g / L to 45 g / L alcohol, and a preservative accounting for 0.001% to 0.01% of the protective agent mass improves the stability of the diagnostic reagent marker in the freeze-dried state and the reconstituted state through the interaction between the various components, thereby improving the stability of the diagnostic reagent marker during the freeze-drying process, the product qualification rate, the batch consistency, and the production capacity.

[0007] As a preferred embodiment of the diagnostic reagent marker protective agent of the present invention, the buffer includes at least one of bis(2-hydroxyethyl)amino(trihydroxymethyl)methane (Bis-Tris), 4-hydroxyethylpiperazineethanesulfonic acid, 3-morpholinepropanesulfonic acid, triethanolamine, tris(hydroxymethyl)aminomethane, barbital sodium, 1,4-piperazinediethanesulfonic acid, tris(hydroxymethyl)methylglycine, 2-morpholinoethanesulfonic acid, phthalic acid, glycine, borax, sodium carbonate, sodium bicarbonate, sodium hydroxide, hydrochloric acid, potassium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, citric acid and sodium citrate.

[0008] As a preferred embodiment of the diagnostic reagent marker protective agent of the present invention, the buffer comprises 3-morpholinepropanesulfonic acid and triethanolamine. The inventors of the present application have discovered through extensive experiments that when 3-morpholinepropanesulfonic acid and triethanolamine are selected as the buffer, the synergistic effect between 3-morpholinepropanesulfonic acid and triethanolamine improves the activity of the diagnostic reagent marker.

[0009] As a preferred embodiment of the diagnostic reagent marker protective agent of the present invention, the protein comprises at least one of whey protein, milk powder, and bovine serum albumin. The inventors of this application have found through extensive experiments that the activity of the diagnostic reagent marker is better preserved when bovine serum albumin (BSA) is used. More preferably, when BSA is selected, the activity of the diagnostic reagent marker is best preserved when it meets the following quality requirements: a purity of at least 99% and the absence of fatty acids, bovine IgG, bovine viruses, and proteases.

[0010] In a preferred embodiment of the diagnostic reagent marker preservative of the present invention, the sugar comprises at least one of sucrose, lactose, maltose, galactose, glucose, and trehalose. The inventors of this application have found through extensive experiments that the activity of the diagnostic reagent marker is better preserved when trehalose is used. More preferably, the activity of the diagnostic reagent marker is best preserved when the selected trehalose meets the following quality requirements: a purity of at least 98% and a heavy metal content of no more than 5 ppm.

[0011] As a preferred embodiment of the diagnostic reagent marker preservative of the present invention, the alcohol comprises at least one of sorbitol, mannitol, xylitol, maltitol, polyethylene glycol, and lactitol. The inventors of this application have discovered through extensive experiments that the activity of the diagnostic reagent marker is better preserved when mannitol is selected. More preferably, the activity of the diagnostic reagent marker is best preserved when the selected mannitol meets the following quality requirements: a free acid content (calculated as acetic acid) of less than 0.0025%, a heavy metal content (calculated as copper) of less than 0.0005%, and a reducing sugar content of less than 0.04%.

[0012] In a preferred embodiment of the diagnostic reagent marker preservative of the present invention, the preservative includes at least one of sodium azide, thimerosal, gentamicin, and ProClin 300. The inventors of this application have discovered through extensive experiments that the synergistic effect between thimerosal and ProClin 300 improves the preservation of the activity of the diagnostic reagent marker.

[0013] The present invention also provides the use of the diagnostic reagent marker protective agent in preparing a diagnostic reagent marker freeze-dried product.

[0014] The present invention also provides a method for preparing a lyophilized product of a diagnostic reagent marker, comprising the following steps:

[0015] (1) diluting the marker mother solution with the diagnostic reagent marker protective agent to obtain a marker semi-finished product;

[0016] (2) freeze-drying the semi-finished marker product described in step (1) to obtain the freeze-dried product of the diagnostic reagent marker.

[0017] As a preferred embodiment of the preparation method of the freeze-dried product of the diagnostic reagent marker of the present invention, the freeze-drying is specifically as follows: the marker semi-finished product is cooled to a temperature of -45 to -35°C within 1 to 5 hours in a semi-pressed state and kept warm for 1 to 3 hours; after the rear box is pre-cooled to a temperature of -50 to -40°C for 5 to 35 minutes, the pre-vacuum time is 25 to 35 minutes, and when the vacuum in the front box is 10 to 30 Pa, the sublimation process is entered; the temperature is raised to a temperature of -30 to -20°C within 2 to 11 hours, and kept warm at this temperature for 2 to 13 hours; the temperature is raised to a temperature of 0°C within 2 to 11 hours, and kept warm at this temperature for 2 to 8 hours; the temperature is raised to a temperature of 10 to 15°C within 1.5 to 2 hours, and kept warm at this temperature for 1 to 2 hours; the temperature is raised to 25°C within 1 to 2 hours, and kept warm at this temperature for 3 to 8 hours, and then a negative pressure sealing and pressing is performed.

[0018] Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a diagnostic reagent marker protective agent through the selection of specific components, and the diagnostic reagent marker protective agent can improve the stability of the diagnostic reagent marker in the freeze-dried state and the re-dissolved state; and when applied to the freeze-drying process of the diagnostic reagent marker, the stability of the diagnostic reagent marker during the freeze-drying process, the product qualification rate, and the consistency between batches can be improved. DETAILED DESCRIPTION

[0019] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0020] Examples 1 to 27, Comparative Examples 1 to 7

[0021] The component types and contents of the diagnostic reagent marker protective agents of Examples 1 to 27 and Comparative Examples 1 to 7 are shown in Table 1.

[0022] Table 1

[0023]

[0024]

[0025]

[0026] Effect Examples

[0027] Europium-labeled hepatitis B virus surface antigen was diluted with the diagnostic reagent marker protective agent of Example 1 to obtain a semi-finished product of equal concentration. The semi-finished product was then packaged into flat-bottomed lyophilized powder pharmaceutical glass bottles at 1.5 ml / L concentrations and lyophilized. The control group was stored in a -20°C refrigerator, while the experimental group was stored in a 37°C incubator for 6 days. The europium-labeled hepatitis B virus surface antigen in the control and experimental groups was then used to measure the fluorescence values ​​of hepatitis B virus surface antibody calibrators A (0 mIU / mL) and F (640 mIU / mL). The results are shown in Table 2.

[0028] Table 2

[0029]

[0030] As can be seen from Table 2, in Examples 1 to 5, the effects of the protective agents added with different buffers are different. Among them, the protective agent of Example 1 has a smaller fluorescence value result (marker background) when detecting calibrator A in the experimental group, and a smaller decrease in the fluorescence value when detecting calibrator F than other buffers (marker activity is better preserved). The change rate of the fluorescence value of calibrator A and the change rate of the fluorescence value of calibrator F show that the protective effect of the protective agent of Example 1 is the best.

[0031] Example 28

[0032] The diagnostic reagent marker protective agent of Example 1 was used to dilute horseradish peroxidase-labeled human hepatitis B virus surface antigen into semi-finished products of equal concentrations. The semi-finished products were then packaged into flat-bottomed freeze-dried powder pharmaceutical glass bottles at 0.5, 1.0, 1.5, and 2.0 ml / L concentrations, and lyophilized using the three protocols shown in Tables 3 to 5. The lyophilization results are shown in Table 6. As can be seen from Table 6, the protocol in Table 5 can lyophilize the marker into qualified lyophilized products in aliquots of 0.5 to 2.0 ml / L.

[0033] Table 3 Scheme 1

[0034]

[0035] Table 4 Scheme 2

[0036]

[0037] Table 5 Scheme 3

[0038]

[0039] Table 6

[0040]

[0041] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A diagnostic reagent marker protective agent, characterized in that The method comprises the following components: a buffer with an ionic strength of 30 to 80 mmol / L, 1 g / L to 5 g / L protein, 5 g / L to 35 g / L sugar, 25 g / L to 45 g / L alcohol, and 0.001% to 0.01% preservative; The buffer is 3-morpholinepropanesulfonic acid and triethanolamine; The protein is bovine serum albumin; The sugar is trehalose; The alcohol is mannitol; The preservatives are thimerosal and ProClin 300.

2. Use of the diagnostic reagent marker protective agent according to claim 1 in preparing a diagnostic reagent marker lyophilized product.

3. A method for preparing a lyophilized product of a diagnostic reagent marker, characterized in that: The following steps are involved: (1) diluting the marker mother solution with the diagnostic reagent marker protective agent according to claim 1 to obtain a marker semi-finished product; (2) freeze-drying the semi-finished marker product described in step (1) to obtain the freeze-dried product of the diagnostic reagent marker.

4. The method for preparing a lyophilized product of a diagnostic reagent marker according to claim 3, characterized in that: The freeze-drying process is as follows: the semi-finished product of the marker is cooled to a temperature of -45 to -35°C in a semi-pressed state within 1 to 5 hours and kept warm for 1 to 3 hours; after the rear box is pre-cooled to a temperature of -50 to -40°C for 5 to 35 minutes, the pre-vacuum time is 25 to 35 minutes, and when the vacuum in the front box is 10 to 30 Pa, the sublimation process is entered; the temperature is increased to a temperature of -30 to -20°C within 2 to 11 hours, and kept warm at this temperature for 2 to 13 hours; the temperature is increased to a temperature of 0°C within 2 to 11 hours, and kept warm at this temperature for 2 to 8 hours; the temperature is increased to a temperature of 10 to 15°C within 1.5 to 2 hours, and kept warm at this temperature for 1 to 2 hours; the temperature is increased to 25°C within 1 to 2 hours, and kept warm at this temperature for 3 to 8 hours, and then negative pressure sealing and plugging are performed.

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