Apolipoprotein A-I and transferrin composite quality control product and preparation method thereof

Abstract

By systematically screening the protective agent of the composite quality control product, the optimal protective agent type and concentration of apolipoprotein A-I and transferrin are determined, so that the stability of the apolipoprotein A-I and the transferrin is improved, the degradation rate of the apolipoprotein A-I and the transferrin is reduced, and the composite quality control product can be stored for 4 weeks under an accelerated condition, so that the degradation rate of the apolipoprotein A-I and the transferrin is reduced. The contents of apolipoprotein A-I and transferrin are only slightly reduced; by optimizing a freeze-drying process, the appearance of the composite quality control product after freeze-drying, the stability of the composite quality control product after redissolution and the uniformity of the quality control product are improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of in vitro diagnosis, and in particular relates to a composite quality control product of apolipoprotein AI and transferrin and a preparation method thereof. Background Art

[0002] Acute cerebral infarction is a common cerebrovascular disease in elderly patients. It is caused by ischemia and hypoxia caused by cerebral blood circulation disorders, which leads to localized ischemic necrosis or softening of brain tissue. It has a sudden onset, critical condition, rapid progression, high morbidity, high disability rate and high mortality rate, and seriously threatens the life safety of patients. At the onset of acute cerebral infarction, serum apolipoprotein AI (APO-AI) and transferrin levels are significantly reduced. Apolipoprotein AI and transferrin can be used clinically to make early predictions of acute cerebral infarction. Combined detection of apolipoprotein AI and transferrin can evaluate and predict the severity and prognosis of acute cerebral infarction in the elderly.

[0003] Protein quality control products are used to test and verify measurement precision, measurement accuracy, analytical reagents, analytical instrument deviations, etc. Regular use of quality control products can evaluate the correctness of laboratory equipment, reagents, and personnel operations. By judging whether the test results are within the quality control range, potential risks can be identified and corrected in a timely manner to provide the accuracy of laboratory tests. Protein quality control products are usually divided into liquid and freeze-dried forms. The storage conditions of liquid quality control products are relatively strict, usually requiring a storage condition of -15°C to -20°C, with relatively poor stability, and more demanding transportation conditions. Freeze-dried powder quality control products can be stored at 2°C to 8°C, with stable performance, and more relaxed transportation and storage conditions.

[0004] Since apolipoprotein AI and transferrin are unstable, there is no apolipoprotein AI and transferrin composite quality control product on the market. Therefore, it is of great significance to select the appropriate quality control form according to the characteristics of apolipoprotein AI and transferrin to stabilize the protein performance and achieve the purpose of long-term storage. Summary of the invention

[0005] To solve the above technical problems, the first aspect of the present invention provides a composite quality control product of apolipoprotein AI and transferrin, the composite quality control product comprising apolipoprotein AI, transferrin, a protective agent, a surfactant, and a preservative, wherein the protective agent comprises sugar alcohol, albumin, disaccharides, and amino acids, and the remaining component is an artificial matrix composed of apolipoprotein AI and transferrin antigen; the concentration of apolipoprotein AI is 2g / L~3g / L, the concentration of transferrin is 2.5g / L~3.5g / L, the concentration of mannitol is 10g / L~100g / L, the concentration of albumin is 10g / L~100g / L, the concentration of sugar is 10g / L~100g / L, the concentration of amino acid is 10g / L~100g / L, the volume concentration of surfactant is 0.001%~1%, and the volume concentration of preservative is 1%~10%.

[0006] As a preferred embodiment, the albumin is bovine serum albumin.

[0007] As a preferred embodiment, the sugar is a disaccharide, and the disaccharide is one or more of sucrose, trehalose, lactose, and maltose.

[0008] As a preferred embodiment, the sugar is a monosaccharide, and the monosaccharide is one or more of glucose and fructose.

[0009] As a preferred embodiment, the amino acids are one or more of glycine, alanine, phenylalanine and arginine.

[0010] As a preferred embodiment, the sugar alcohol is one or more of sorbitol, mannitol, xylitol, inositol and tert-butyl alcohol.

[0011] As a preferred embodiment, the surfactant is polysorbate 80.

[0012] As a preferred embodiment, the preservative is one or more of Proclin300 and Proclin950.

[0013] The second aspect of the present invention provides a method for preparing a composite quality control product of apolipoprotein AI and transferrin, comprising the following steps:

[0014] S1: Add apolipoprotein AI, transferrin, protective agent, surfactant, and preservative into a preparation container, wherein the protective agent includes sugar alcohol, albumin, disaccharide, and amino acid, and the remaining component is an artificial matrix composed of apolipoprotein AI and transferrin antigen; stir and dissolve to make the concentration of apolipoprotein AI 2g / L-3g / L, the concentration of transferrin 2.5g / L-3.5g / L, the concentration of mannitol 10g / L-100g / L, the concentration of albumin 10g / L-100g / L, the concentration of sugar 10g / L-100g / L, the concentration of amino acid 10g / L-100g / L, the volume concentration of surfactant 0.001%-1%, and the volume concentration of preservative 1%-10%;

[0015] S2: Adjust the pH to 7.35-7.45 with hydrochloric acid or sodium hydroxide and dispense into 1 ml bottles;

[0016] S3: After packaging, the samples are freeze-dried. The specific procedures of the freeze-drying process are as follows:

[0017] S31, cool to 4℃~6℃, keep for 25min~35min;

[0018] S32, cool to -4℃~-6℃, keep for 25min~35min;

[0019] S33, cooling to -35~-45℃, maintaining for 160min~200min;

[0020] S34, control the temperature at -35 to -45°C, the vacuum degree at 3Pa to 7Pa, and maintain for 1600min to 2000min;

[0021] S35, control the vacuum degree to 3Pa~7Pa, raise the temperature to -5℃~-15℃, and maintain for 100min~140min;

[0022] S36, control the vacuum degree to 3Pa~7Pa, raise the temperature to 3℃~7℃, and maintain for 100min~140min;

[0023] S37, control the vacuum degree to 3Pa-7Pa, raise the temperature to 15-25°C, and maintain for 500-700 minutes;

[0024] S38, capping and sealing.

[0025] As a preferred embodiment, the temperature of step S2 is 20°C±0.5°C.

[0026] As a preferred solution, the cooling time in step S32 is 5 min to 15 min.

[0027] As a preferred solution, the cooling time in step S33 is 30 min to 40 min.

[0028] As a preferred solution, the heating time of step S35 is 120 min to 180 min.

[0029] As a preferred solution, the heating time of step S36 is 90 min to 600 min.

[0030] As a preferred solution, the heating time of step S37 is 90 min to 600 min.

[0031] The present invention determines the optimal protective agent type and concentration of apolipoprotein AI and transferrin by systematically screening the protective agent of the composite quality control product, thereby improving the stability of apolipoprotein AI and transferrin, reducing the degradation rate of apolipoprotein AI and transferrin, and the composite quality control product is stored for 4 weeks under accelerated conditions, and the content of apolipoprotein AI and transferrin only slightly decreases; the present invention improves the appearance of the composite quality control product after freeze-drying, the stability after reconstitution, and the uniformity of the quality control product by optimizing the freeze-drying process. Due to good stability, the stringent requirements of transportation and storage on the environment are reduced, and transportation and storage are convenient. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a comparison chart of the change trend of apolipoprotein AI in Example 6;

[0033] Figure 2 This is a comparison chart of the transferrin variation trend in Example 6;

[0034] Figure 3 This is a comparison chart of the change trend of apolipoprotein AI in Example 13;

[0035] Figure 4 This is a comparison chart of the transferrin variation trend in Example 13;

[0036] Figure 5 This is a comparison chart of the change trend of apolipoprotein AI in Example 19;

[0037] Figure 6 This is a comparison chart of the transferrin change range trend in Example 19. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical scheme and advantages of the present invention more clearly and completely understood, the technical scheme of the present invention and the technical effects that can be achieved are described in detail below through embodiments. Obviously, the described embodiments are only examples of a part of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without making creative work belong to the scope of protection of the present invention.

[0039] Example 1

[0040] The preparation process of apolipoprotein AI and transferrin composite quality control product includes the following steps:

[0041] S1: In a clean preparation container, add sucrose, mannitol, glycine, bovine serum albumin (BSA), Proclin 300, apolipoprotein AI and transferrin, and stir until completely dissolved.

[0042] The concentration of sucrose reaches 20 g / L, the concentration of mannitol reaches 20 g / L, the concentration of glycine reaches 20 g / L, the concentration of bovine serum albumin (BSA) reaches 20 g / L, the concentration of Proclin300 reaches 1% (V / V), the concentration of apolipoprotein AI reaches 2.5 g / L, the concentration of transferrin reaches 3 g / L, and the rest is an artificial matrix component composed of apolipoprotein AI and transferrin antigen;

[0043] S2: Adjust the pH to 7.35-7.45 (20℃±0.5℃) with hydrochloric acid or sodium hydroxide, and pack in 1ml / bottle;

[0044] S3: After packaging, the samples are freeze-dried, and the specific freeze-drying procedure is as follows: the room temperature is reduced to 5°C and maintained for 30 minutes; the temperature is reduced to -5°C during the cooling process for 10 minutes and maintained for 30 minutes; the temperature is reduced to -40°C during the cooling process for 35 minutes and maintained for 180 minutes; the temperature is maintained at -40°C for 1800 minutes, and the vacuum degree is 5Pa; the temperature is increased to -10°C during the heating process for 150 minutes and maintained for 120 minutes, and the vacuum degree is 5Pa; the temperature is increased to 5°C during the heating process for 75 minutes and maintained for 120 minutes, and the vacuum degree is 5Pa; the temperature is increased to 20°C during the heating process for 75 minutes and maintained for 600 minutes, and the vacuum degree is 5Pa.

[0045] Example 2

[0046] The preparation process of apolipoprotein AI and transferrin composite quality control product includes the following steps:

[0047] S1: In a clean preparation container, add sucrose, mannitol, alanine, bovine serum albumin (BSA), Proclin 300, apolipoprotein AI and transferrin, and stir until completely dissolved.

[0048] The concentration of sucrose reaches 20 g / L, the concentration of mannitol reaches 20 g / L, the concentration of alanine reaches 20 g / L, the concentration of bovine serum albumin (BSA) reaches 20 g / L, the concentration of Proclin300 reaches 1% (V / V), the concentration of apolipoprotein AI reaches 2.5 g / L, the concentration of transferrin reaches 3 g / L, and the rest is an artificial matrix component composed of apolipoprotein AI and transferrin antigen;

[0049] S2: Adjust the pH to 7.35-7.45 (20℃±0.5℃) with hydrochloric acid or sodium hydroxide, and pack in 1ml / bottle;

[0050] S3: After packaging, the samples are freeze-dried, and the specific freeze-drying procedure is as follows: the room temperature is reduced to 5°C and maintained for 30 minutes; the temperature is reduced to -5°C during the cooling process for 10 minutes and maintained for 30 minutes; the temperature is reduced to -40°C during the cooling process for 35 minutes and maintained for 180 minutes; the temperature is maintained at -40°C for 1800 minutes, and the vacuum degree is 5Pa; the temperature is increased to -10°C during the heating process for 150 minutes and maintained for 120 minutes, and the vacuum degree is 5Pa; the temperature is increased to 5°C during the heating process for 75 minutes and maintained for 120 minutes, and the vacuum degree is 5Pa; the temperature is increased to 20°C during the heating process for 75 minutes and maintained for 600 minutes, and the vacuum degree is 5Pa.

[0051] Example 3

[0052] The preparation process of apolipoprotein AI and transferrin composite quality control product includes the following steps:

[0053] S1: In a clean preparation container, add sucrose, mannitol, phenylalanine, bovine serum albumin (BSA), Proclin 300, apolipoprotein AI and transferrin, and stir until completely dissolved.

[0054] The concentration of sucrose reaches 20 g / L, the concentration of mannitol reaches 20 g / L, the concentration of phenylalanine reaches 20 g / L, the concentration of bovine serum albumin (BSA) reaches 20 g / L, the concentration of Proclin300 reaches 1% (V / V), the concentration of apolipoprotein AI reaches 2.5 g / L, the concentration of transferrin reaches 3 g / L, and the rest is an artificial matrix component composed of apolipoprotein AI and transferrin antigen;

[0055] S2: Adjust the pH to 7.35-7.45 (20℃±0.5℃) with hydrochloric acid or sodium hydroxide, and pack in 1ml / bottle;

[0056] S3: After packaging, the samples are freeze-dried, and the specific freeze-drying procedure is as follows: the room temperature is reduced to 5°C and maintained for 30 minutes; the temperature is reduced to -5°C during the cooling process for 10 minutes and maintained for 30 minutes; the temperature is reduced to -40°C during the cooling process for 35 minutes and maintained for 180 minutes; the temperature is maintained at -40°C for 1800 minutes, and the vacuum degree is 5Pa; the temperature is increased to -10°C during the heating process for 150 minutes and maintained for 120 minutes, and the vacuum degree is 5Pa; the temperature is increased to 5°C during the heating process for 75 minutes and maintained for 120 minutes, and the vacuum degree is 5Pa; the temperature is increased to 20°C during the heating process for 75 minutes and maintained for 600 minutes, and the vacuum degree is 5Pa.

[0057] Example 4

[0058] The preparation process of apolipoprotein AI and transferrin composite quality control product includes the following steps:

[0059] S1: In a clean preparation container, add sucrose, mannitol, arginine, bovine serum albumin (BSA), Proclin 300, apolipoprotein AI and transferrin, and stir until completely dissolved.

[0060] The concentration of sucrose reaches 20 g / L, the concentration of mannitol reaches 20 g / L, the concentration of arginine reaches 20 g / L, the concentration of bovine serum albumin (BSA) reaches 20 g / L, the concentration of Proclin300 reaches 1% (V / V), the concentration of apolipoprotein AI reaches 2.5 g / L, the concentration of transferrin reaches 3 g / L, and the rest is an artificial matrix component composed of apolipoprotein AI and transferrin antigen;

[0061] S2: Adjust the pH to 7.35-7.45 (20℃±0.5℃) with hydrochloric acid or sodium hydroxide, and pack in 1ml / bottle;

[0062] S3: After packaging, the samples are freeze-dried, and the specific freeze-drying procedure is as follows: the room temperature is reduced to 5°C and maintained for 30 minutes; the temperature is reduced to -5°C during the cooling process for 10 minutes and maintained for 30 minutes; the temperature is reduced to -40°C during the cooling process for 35 minutes and maintained for 180 minutes; the temperature is maintained at -40°C for 1800 minutes, and the vacuum degree is 5Pa; the temperature is increased to -10°C during the heating process for 150 minutes and maintained for 120 minutes, and the vacuum degree is 5Pa; the temperature is increased to 5°C during the heating process for 75 minutes and maintained for 120 minutes, and the vacuum degree is 5Pa; the temperature is increased to 20°C during the heating process for 75 minutes and maintained for 600 minutes, and the vacuum degree is 5Pa.

[0063] Example 5

[0064] The preparation process of apolipoprotein AI and transferrin composite quality control product includes the following steps:

[0065] S1: In a clean preparation container, add sucrose, mannitol, bovine serum albumin (BSA), Proclin 300, apolipoprotein AI and transferrin, and stir until completely dissolved.

[0066] The concentration of sucrose reaches 20 g / L, the concentration of mannitol reaches 20 g / L, the concentration of bovine serum albumin (BSA) reaches 20 g / L, the concentration of Proclin300 reaches 1% (V / V), the concentration of apolipoprotein AI reaches 2.5 g / L, the concentration of transferrin reaches 3 g / L, and the rest is an artificial matrix component composed of apolipoprotein AI and transferrin antigen;

[0067] S2: Adjust the pH to 7.35-7.45 (20℃±0.5℃) with hydrochloric acid or sodium hydroxide, and pack in 1ml / bottle;

[0068] S3: After packaging, the samples are freeze-dried, and the specific freeze-drying procedure is as follows: the room temperature is reduced to 5°C and maintained for 30 minutes; the temperature is reduced to -5°C during the cooling process for 10 minutes and maintained for 30 minutes; the temperature is reduced to -40°C during the cooling process for 35 minutes and maintained for 180 minutes; the temperature is maintained at -40°C for 1800 minutes, and the vacuum degree is 5Pa; the temperature is increased to -10°C during the heating process for 150 minutes and maintained for 120 minutes, and the vacuum degree is 5Pa; the temperature is increased to 5°C during the heating process for 75 minutes and maintained for 120 minutes, and the vacuum degree is 5Pa; the temperature is increased to 20°C during the heating process for 75 minutes and maintained for 600 minutes, and the vacuum degree is 5Pa, to obtain the composite quality control product.

[0069] Example 6

[0070] The main difference between the protective agents in Examples 1 to 5 is the different amino acids. The quality control products prepared in Examples 1 to 5 were placed at 37°C for accelerated stability testing.

[0071] 1. Accelerated stability test method: The quality control products prepared in Examples 1 to 5 were placed in a drying oven at 37° C. for 4 weeks. Three bottles of composite quality control products were randomly selected each week to measure the apolipoprotein AI and transferrin content. Each bottle was measured 3 times and the average value was calculated.

[0072] 2. Detection reagents: Apolipoprotein AI assay kit from Mindray, transferrin assay kit from Mindray.

[0073] 3. Testing instrument: Mindray BS-820 fully automatic biochemical analyzer.

[0074] 4. Detection method: Use Mindray transferrin calibrator and lipid calibrator for calibration. After successful calibration, use Mindray biochemical composite fixed value quality control to verify whether the measured value is within the quality control range. After successful verification, add 1ml of deionized water to the prepared apolipoprotein AI and transferrin composite quality control, mix for 30 minutes, and then measure the content of apolipoprotein AI and transferrin in the composite quality control. Calculate the difference between the first test result and the test results at each time point, and the percentage of the difference to the first test result is the range of change. The closer the range of change of the quality control product is to 0, the more stable the quality control product is, and the acceptable standard is a range of change of ±10%.

[0075] 5. Test results and analysis: The change range at each time point is shown in Table 1 and Figure 1 , Figure 2 The main purpose is to explore the protective effects of formulas 1-5 on freeze-dried apolipoprotein AI and transferrin. The vertical axis is the type of amino acids, and the horizontal axis is the change range after four weeks of acceleration at 37 degrees. Figure 1 Apolipoprotein AI, Figure 2 For transferrin.

[0076] From Table 1 and Figure 1 , Figure 2 It can be seen that the composite quality control product was observed at 37°C for four weeks. In Example 1, the maximum change of apolipoprotein AI within four weeks was 5.21%, and the maximum change of transferrin within four weeks was 1.04%; in Example 2, the maximum change of apolipoprotein AI within four weeks was 9.10%, and the maximum change of transferrin within four weeks was -4.57%. In Example 3, the maximum change of apolipoprotein AI within four weeks was 3.84%, and the maximum change of transferrin within four weeks was -4.10%. In Example 4, the maximum change of apolipoprotein AI within four weeks was 14.84%, and the maximum change of transferrin within four weeks was -7.67%. In Example 5, the maximum change of apolipoprotein AI within four weeks was 11.11%, and the maximum change of transferrin within four weeks was -2.72%. It can be concluded that the formulas of Example 1 and Implementation 3, namely glycine and phenylalanine, have a good protective effect on apolipoprotein AI and transferrin. However, after freeze drying using the formulation of Example 1, the surface of the product did not crack, but only shrank slightly, with a gap between the product and the packaging container. When freeze drying using the formulation of Example 3, cracks appeared on the surface of the product. It can be concluded that glycine has the best freeze drying effect on apolipoprotein AI and transferrin.

[0077] Table 1: Accelerated stability variation of quality control products in Examples 1 to 5

[0078]

[0079]

[0080] Example 7

[0081] The preparation process of apolipoprotein AI and transferrin composite quality control product includes the following steps:

[0082] S1: In a clean preparation container, add glucose, mannitol, glycine, bovine serum albumin (BSA), Proclin 300, apolipoprotein AI and transferrin, and stir until completely dissolved.

[0083] The concentration of glucose reaches 20 g / L, the concentration of mannitol reaches 20 g / L, the concentration of glycine reaches 20 g / L, the concentration of bovine serum albumin (BSA) reaches 20 g / L, the concentration of Proclin300 reaches 1% (V / V), the concentration of apolipoprotein AI reaches 2.5 g / L, the concentration of transferrin reaches 3 g / L, and the rest is an artificial matrix component composed of apolipoprotein AI and transferrin antigen;

[0084] S2: Adjust the pH to 7.35-7.45 (20℃±0.5℃) with hydrochloric acid or sodium hydroxide, and pack in 1ml / bottle;

[0085] S3: After packaging, the samples are freeze-dried, and the specific freeze-drying procedure is as follows: the room temperature is reduced to 5°C and maintained for 30 minutes; the temperature is reduced to -5°C during the cooling process for 10 minutes and maintained for 30 minutes; the temperature is reduced to -40°C during the cooling process for 35 minutes and maintained for 180 minutes; the temperature is maintained at -40°C for 1800 minutes, and the vacuum degree is 5Pa; the temperature is increased to -10°C during the heating process for 150 minutes and maintained for 120 minutes, and the vacuum degree is 5Pa; the temperature is increased to 5°C during the heating process for 75 minutes and maintained for 120 minutes, and the vacuum degree is 5Pa; the temperature is increased to 20°C during the heating process for 75 minutes and maintained for 600 minutes, and the vacuum degree is 5Pa.

[0086] Example 8

[0087] The preparation process of apolipoprotein AI and transferrin composite quality control product includes the following steps:

[0088] S1: In a clean preparation container, add trehalose, mannitol, glycine, bovine serum albumin (BSA), Proclin 300, apolipoprotein AI and transferrin, and stir until completely dissolved.

[0089] The concentration of trehalose reaches 20 g / L, the concentration of mannitol reaches 20 g / L, the concentration of glycine reaches 20 g / L, the concentration of bovine serum albumin (BSA) reaches 20 g / L, the concentration of Proclin300 reaches 1% (V / V), the concentration of apolipoprotein AI reaches 2.5 g / L, the concentration of transferrin reaches 3 g / L, and the rest is an artificial matrix component composed of apolipoprotein AI and transferrin antigen;

[0090] S2: Adjust the pH to 7.35-7.45 (20℃±0.5℃) with hydrochloric acid or sodium hydroxide, and pack in 1ml / bottle;

[0091] S3: After packaging, the samples are freeze-dried, and the specific freeze-drying procedure is as follows: the room temperature is reduced to 5°C and maintained for 30 minutes; the temperature is reduced to -5°C during the cooling process for 10 minutes and maintained for 30 minutes; the temperature is reduced to -40°C during the cooling process for 35 minutes and maintained for 180 minutes; the temperature is maintained at -40°C for 1800 minutes, and the vacuum degree is 5Pa; the temperature is increased to -10°C during the heating process for 150 minutes and maintained for 120 minutes, and the vacuum degree is 5Pa; the temperature is increased to 5°C during the heating process for 75 minutes and maintained for 120 minutes, and the vacuum degree is 5Pa; the temperature is increased to 20°C during the heating process for 75 minutes and maintained for 600 minutes, and the vacuum degree is 5Pa.

[0092] Example 9

[0093] The preparation process of apolipoprotein AI and transferrin composite quality control product includes the following steps:

[0094] S1: In a clean preparation container, add fructose, mannitol, glycine, bovine serum albumin (BSA), Proclin 300, apolipoprotein AI and transferrin, and stir until completely dissolved.

[0095] The concentration of fructose reaches 20 g / L, the concentration of mannitol reaches 20 g / L, the concentration of glycine reaches 20 g / L, the concentration of bovine serum albumin (BSA) reaches 20 g / L, the concentration of Proclin300 reaches 1% (V / V), the concentration of apolipoprotein AI reaches 2.5 g / L, the concentration of transferrin reaches 3 g / L, and the rest is an artificial matrix component composed of apolipoprotein AI and transferrin antigen;

[0096] S2: Adjust the pH to 7.35-7.45 (20℃±0.5℃) with hydrochloric acid or sodium hydroxide, and pack in 1ml / bottle;

[0097] S3: After packaging, the samples are freeze-dried, and the specific freeze-drying procedure is as follows: the room temperature is reduced to 5°C and maintained for 30 minutes; the temperature is reduced to -5°C during the cooling process for 10 minutes and maintained for 30 minutes; the temperature is reduced to -40°C during the cooling process for 35 minutes and maintained for 180 minutes; the temperature is maintained at -40°C for 1800 minutes, and the vacuum degree is 5Pa; the temperature is increased to -10°C during the heating process for 150 minutes and maintained for 120 minutes, and the vacuum degree is 5Pa; the temperature is increased to 5°C during the heating process for 75 minutes and maintained for 120 minutes, and the vacuum degree is 5Pa; the temperature is increased to 20°C during the heating process for 75 minutes and maintained for 600 minutes, and the vacuum degree is 5Pa.

[0098] Example 10

[0099] The preparation process of apolipoprotein AI and transferrin composite quality control product includes the following steps:

[0100] S1: In a clean preparation container, add lactose, mannitol, glycine, bovine serum albumin (BSA), Proclin 300, apolipoprotein AI and transferrin, and stir until completely dissolved.

[0101] The concentration of lactose reaches 20 g / L, the concentration of mannitol reaches 20 g / L, the concentration of glycine reaches 20 g / L, the concentration of bovine serum albumin (BSA) reaches 20 g / L, the concentration of Proclin300 reaches 1% (V / V), the concentration of apolipoprotein AI reaches 2.5 g / L, the concentration of transferrin reaches 3 g / L, and the rest is an artificial matrix component composed of apolipoprotein AI and transferrin antigen;

[0102] S2: Adjust the pH to 7.35-7.45 (20℃±0.5℃) with hydrochloric acid or sodium hydroxide, and pack in 1ml / bottle;

[0103] S3: After packaging, the samples are freeze-dried, and the specific freeze-drying procedure is as follows: the room temperature is reduced to 5°C and maintained for 30 minutes; the temperature is reduced to -5°C during the cooling process for 10 minutes and maintained for 30 minutes; the temperature is reduced to -40°C during the cooling process for 35 minutes and maintained for 180 minutes; the temperature is maintained at -40°C for 1800 minutes, and the vacuum degree is 5Pa; the temperature is increased to -10°C during the heating process for 150 minutes and maintained for 120 minutes, and the vacuum degree is 5Pa; the temperature is increased to 5°C during the heating process for 75 minutes and maintained for 120 minutes, and the vacuum degree is 5Pa; the temperature is increased to 20°C during the heating process for 75 minutes and maintained for 600 minutes, and the vacuum degree is 5Pa.

[0104] Embodiment 11

[0105] The preparation process of apolipoprotein AI and transferrin composite quality control product includes the following steps:

[0106] S1: In a clean preparation container, add maltose, mannitol, glycine, bovine serum albumin (BSA), Proclin 300, apolipoprotein AI and transferrin, and stir until completely dissolved.

[0107] The concentration of maltose reaches 20 g / L, the concentration of mannitol reaches 20 g / L, the concentration of glycine reaches 20 g / L, the concentration of bovine serum albumin (BSA) reaches 20 g / L, the concentration of Proclin300 reaches 1% (V / V), the concentration of apolipoprotein AI reaches 2.5 g / L, the concentration of transferrin reaches 3 g / L, and the rest is an artificial matrix component composed of apolipoprotein AI and transferrin antigen;

[0108] S2: Adjust the pH to 7.35-7.45 (20℃±0.5℃) with hydrochloric acid or sodium hydroxide, and pack in 1ml / bottle;

[0109] S3: After packaging, the samples are freeze-dried, and the specific freeze-drying procedure is as follows: the room temperature is reduced to 5°C and maintained for 30 minutes; the temperature is reduced to -5°C during the cooling process for 10 minutes and maintained for 30 minutes; the temperature is reduced to -40°C during the cooling process for 35 minutes and maintained for 180 minutes; the temperature is maintained at -40°C for 1800 minutes, and the vacuum degree is 5Pa; the temperature is increased to -10°C during the heating process for 150 minutes and maintained for 120 minutes, and the vacuum degree is 5Pa; the temperature is increased to 5°C during the heating process for 75 minutes and maintained for 120 minutes, and the vacuum degree is 5Pa; the temperature is increased to 20°C during the heating process for 75 minutes and maintained for 600 minutes, and the vacuum degree is 5Pa.

[0110] Example 12

[0111] The preparation process of apolipoprotein AI and transferrin composite quality control product includes the following steps:

[0112] S1: In a clean preparation container, add mannitol, glycine, bovine serum albumin (BSA), Proclin 300, apolipoprotein AI and transferrin, and stir until completely dissolved.

[0113] The concentration of mannitol reaches 20 g / L, the concentration of glycine reaches 20 g / L, the concentration of bovine serum albumin (BSA) reaches 20 g / L, the concentration of Proclin300 reaches 1% (V / V), the concentration of apolipoprotein AI reaches 2.5 g / L, the concentration of transferrin reaches 3 g / L, and the rest is an artificial matrix component composed of apolipoprotein AI and transferrin antigen;

[0114] S2: Adjust the pH to 7.35-7.45 (20℃±0.5℃) with hydrochloric acid or sodium hydroxide, and pack in 1ml / bottle;

[0115] S3: After packaging, the samples are freeze-dried, and the specific freeze-drying procedure is as follows: the room temperature is reduced to 5°C and maintained for 30 minutes; the temperature is reduced to -5°C during the cooling process for 10 minutes and maintained for 30 minutes; the temperature is reduced to -40°C during the cooling process for 35 minutes and maintained for 180 minutes; the temperature is maintained at -40°C for 1800 minutes, and the vacuum degree is 5Pa; the temperature is increased to -10°C during the heating process for 150 minutes and maintained for 120 minutes, and the vacuum degree is 5Pa; the temperature is increased to 5°C during the heating process for 75 minutes and maintained for 120 minutes, and the vacuum degree is 5Pa; the temperature is increased to 20°C during the heating process for 75 minutes and maintained for 600 minutes, and the vacuum degree is 5Pa.

[0116] Example 13

[0117] The main difference between the protective agents in Example 1 and Examples 7 to 12 is the different carbohydrates. The quality control products prepared in Example 1 and Examples 7 to 12 were placed at 37° C. for accelerated stability testing.

[0118] 1. Accelerated stability test method: The quality control products prepared in Example 1 and Examples 7 to 12 were placed in a drying oven at 37° C. The observation period was 4 weeks. Three bottles of composite quality control products were randomly selected each week to measure the content of apolipoprotein AI and transferrin. Each bottle was measured 3 times and the average value was calculated.

[0119] 2. Detection reagents: Apolipoprotein AI assay kit from Mindray, transferrin assay kit from Mindray.

[0120] 3. Testing instrument: Mindray BS-820 fully automatic biochemical analyzer.

[0121] 4. Detection method: Use Mindray transferrin calibrator and lipid calibrator for calibration. After successful calibration, use Mindray biochemical composite fixed value quality control to verify whether the measured value is within the quality control range. After successful verification, add 1 ml of deionized water to the prepared apolipoprotein AI and transferrin composite quality control, mix for 30 minutes, and then measure the content of apolipoprotein AI and transferrin in the composite quality control. Calculate the amplitude of change, and the calculation method of the amplitude of change is the same as that of Example 6.

[0122] 5. Test results and analysis: The change range at each time point is shown in Table 2 and Figure 3 , Figure 4 .

[0123] From Table 2 and Figure 3 , Figure 4It can be seen that within four weeks of 37-degree acceleration, the maximum change of apolipoprotein AI in Example 1 within four weeks is 5.21%; the maximum change of transferrin within four weeks is 1.04%. In Example 7, the maximum change of apolipoprotein AI within four weeks is 21.62%, and the maximum change of transferrin within four weeks is 3.58%. In Example 8, the maximum change of apolipoprotein AI within four weeks is 11.30%, and the maximum change of transferrin within four weeks is 1.30%. In Example 9, the maximum change of apolipoprotein AI within four weeks is 27.38%, and the maximum change of transferrin within four weeks is 4.93%. In Example 10, the maximum change of apolipoprotein AI within four weeks is 10.34%, and the maximum change of transferrin within four weeks is 2.53%. In Example 11, the maximum change of apolipoprotein AI within four weeks is 9.69%, and the maximum change of transferrin within four weeks is 2.15%. In Example 12, the maximum change range of apolipoprotein AI within four weeks was 9.93%, and the maximum change range of transferrin within four weeks was -1.46%. Therefore, it can be concluded that the formula of Example 1, that is, sucrose, has a better protective effect on apolipoprotein AI and transferrin.

[0124] Table 2: Accelerated stability variation of quality control products of Examples 1 and Examples 7 to 12

[0125]

[0126]

[0127] Embodiment 14

[0128] The preparation process of apolipoprotein AI and transferrin composite quality control product includes the following steps:

[0129] S1: In a clean preparation container, add mannitol, glycine, bovine serum albumin (BSA), Proclin 950, polysorbate 80, apolipoprotein AI and transferrin, and stir until completely dissolved.

[0130] The concentration of mannitol reaches 20 g / L, the concentration of glycine reaches 100 g / L, the concentration of bovine serum albumin (BSA) reaches 20 g / L, the concentration of Proclin950 reaches 1% (V / V), the concentration of polysorbate 80 reaches 0.02% (V / V), the concentration of apolipoprotein AI reaches 2.5 g / L, the concentration of transferrin reaches 3 g / L, and the rest is an artificial matrix component composed of apolipoprotein AI and transferrin antigen;

[0131] S2: Adjust the pH to 7.35-7.45 (20℃±0.5℃) with hydrochloric acid or sodium hydroxide, and pack in 1ml / bottle;

[0132] S3: After packaging, the samples are freeze-dried, and the specific freeze-drying procedure is as follows: the room temperature is reduced to 5°C and maintained for 30 minutes; the temperature is reduced to -5°C during the cooling process for 10 minutes and maintained for 30 minutes; the temperature is reduced to -40°C during the cooling process for 35 minutes and maintained for 120 minutes; the temperature is increased to -30°C during the heating process for 10 minutes and maintained for 120 minutes; the temperature is reduced to -40°C during the cooling process for 10 minutes and maintained for 120 minutes; the temperature remains unchanged at -40°C and maintained for 1800 minutes, and the vacuum degree is 5Pa; the temperature is increased to -10°C during the heating process for 150 minutes and maintained for 120 minutes, and the vacuum degree is 5Pa; the temperature is increased to 5°C during the heating process for 75 minutes and maintained for 120 minutes, and the vacuum degree is 5Pa; the temperature is increased to 20°C during the heating process for 75 minutes and maintained for 600 minutes, and the vacuum degree is 5Pa.

[0133] Embodiment 15

[0134] The preparation process of apolipoprotein AI and transferrin composite quality control product includes the following steps:

[0135] S1: In a clean preparation container, add sucrose, mannitol, glycine, bovine serum albumin (BSA), Proclin 950, polysorbate 80, apolipoprotein AI and transferrin, and stir until completely dissolved.

[0136] The concentration of sucrose reaches 20 g / L, the concentration of mannitol reaches 20 g / L, the concentration of glycine reaches 100 g / L, the concentration of bovine serum albumin (BSA) reaches 20 g / L, the concentration of Proclin950 reaches 1% (V / V), the concentration of polysorbate 80 reaches 0.02% (V / V), the concentration of apolipoprotein AI reaches 2.5 g / L, the concentration of transferrin reaches 3 g / L, and the rest is an artificial matrix component composed of apolipoprotein AI and transferrin antigen;

[0137] S2: Adjust the pH to 7.35-7.45 (20℃±0.5℃) with hydrochloric acid or sodium hydroxide, and pack in 1ml / bottle;

[0138] S3: After packaging, the samples are freeze-dried, and the specific freeze-drying procedure is as follows: the room temperature is reduced to 5°C and maintained for 30 minutes; the temperature is reduced to -5°C during the cooling process for 10 minutes and maintained for 30 minutes; the temperature is reduced to -40°C during the cooling process for 35 minutes and maintained for 120 minutes; the temperature is increased to -30°C during the heating process for 10 minutes and maintained for 120 minutes; the temperature is reduced to -40°C during the cooling process for 10 minutes and maintained for 120 minutes; the temperature remains unchanged at -40°C and maintained for 1800 minutes, and the vacuum degree is 5Pa; the temperature is increased to -10°C during the heating process for 150 minutes and maintained for 120 minutes, and the vacuum degree is 5Pa; the temperature is increased to 5°C during the heating process for 75 minutes and maintained for 120 minutes, and the vacuum degree is 5Pa; the temperature is increased to 20°C during the heating process for 75 minutes and maintained for 600 minutes, and the vacuum degree is 5Pa.

[0139] Example 16

[0140] The preparation process of apolipoprotein AI and transferrin composite quality control product includes the following steps:

[0141] S1: In a clean preparation container, add sucrose, mannitol, glycine, bovine serum albumin (BSA), Proclin 950, polysorbate 80, apolipoprotein AI and transferrin, and stir until completely dissolved.

[0142] The concentration of sucrose reaches 50 g / L, the concentration of mannitol reaches 20 g / L, the concentration of glycine reaches 100 g / L, the concentration of bovine serum albumin (BSA) reaches 20 g / L, the concentration of Proclin950 reaches 1% (V / V), the concentration of polysorbate 80 reaches 0.02% (V / V), the concentration of apolipoprotein AI reaches 2.5 g / L, the concentration of transferrin reaches 3 g / L, and the rest of the composition is an artificial matrix component composed of apolipoprotein AI and transferrin antigen;

[0143] S2: Adjust the pH to 7.35-7.45 (20℃±0.5℃) with hydrochloric acid or sodium hydroxide, and pack in 1ml / bottle;

[0144] S3: After packaging, the samples are freeze-dried, and the specific freeze-drying procedure is as follows: the room temperature is reduced to 5°C and maintained for 30 minutes; the temperature is reduced to -5°C during the cooling process for 10 minutes and maintained for 30 minutes; the temperature is reduced to -40°C during the cooling process for 35 minutes and maintained for 120 minutes; the temperature is increased to -30°C during the heating process for 10 minutes and maintained for 120 minutes; the temperature is reduced to -40°C during the cooling process for 10 minutes and maintained for 120 minutes; the temperature remains unchanged at -40°C and maintained for 1800 minutes, and the vacuum degree is 5Pa; the temperature is increased to -10°C during the heating process for 150 minutes and maintained for 120 minutes, and the vacuum degree is 5Pa; the temperature is increased to 5°C during the heating process for 75 minutes and maintained for 120 minutes, and the vacuum degree is 5Pa; the temperature is increased to 20°C during the heating process for 75 minutes and maintained for 600 minutes, and the vacuum degree is 5Pa.

[0145] Embodiment 17

[0146] The preparation process of apolipoprotein AI and transferrin composite quality control product includes the following steps:

[0147] S1: In a clean preparation container, add sucrose, mannitol, glycine, bovine serum albumin (BSA), Proclin 950, polysorbate 80, apolipoprotein AI and transferrin, and stir until completely dissolved.

[0148] The concentration of sucrose reaches 100 g / L, the concentration of mannitol reaches 20 g / L, the concentration of glycine reaches 100 g / L, the concentration of bovine serum albumin (BSA) reaches 20 g / L, the concentration of Proclin950 reaches 1% (V / V), the concentration of polysorbate 80 reaches 0.02% (V / V), the concentration of apolipoprotein AI reaches 2.5 g / L, the concentration of transferrin reaches 3 g / L, and the rest is an artificial matrix component composed of apolipoprotein AI and transferrin antigen;

[0149] S2: Adjust the pH to 7.35-7.45 (20℃±0.5℃) with hydrochloric acid or sodium hydroxide, and pack in 1ml / bottle;

[0150] S3: After packaging, the samples are freeze-dried, and the specific freeze-drying procedure is as follows: the room temperature is reduced to 5°C and maintained for 30 minutes; the temperature is reduced to -5°C during the cooling process for 10 minutes and maintained for 30 minutes; the temperature is reduced to -40°C during the cooling process for 35 minutes and maintained for 120 minutes; the temperature is increased to -30°C during the heating process for 10 minutes and maintained for 120 minutes; the temperature is reduced to -40°C during the cooling process for 10 minutes and maintained for 120 minutes; the temperature remains unchanged at -40°C and maintained for 1800 minutes, and the vacuum degree is 5Pa; the temperature is increased to -10°C during the heating process for 150 minutes and maintained for 120 minutes, and the vacuum degree is 5Pa; the temperature is increased to 5°C during the heating process for 75 minutes and maintained for 120 minutes, and the vacuum degree is 5Pa; the temperature is increased to 20°C during the heating process for 75 minutes and maintained for 600 minutes, and the vacuum degree is 5Pa.

[0151] Embodiment 18

[0152] The preparation process of apolipoprotein AI and transferrin composite quality control product includes the following steps:

[0153] S1: In a clean preparation container, add sucrose, mannitol, glycine, bovine serum albumin (BSA), Proclin 950, polysorbate 80, apolipoprotein AI and transferrin, and stir until completely dissolved.

[0154] The concentration of sucrose reaches 150 g / L, the concentration of mannitol reaches 20 g / L, the concentration of glycine reaches 100 g / L, the concentration of bovine serum albumin (BSA) reaches 20 g / L, the concentration of Proclin950 reaches 1% (V / V), the concentration of polysorbate 80 reaches 0.02% (V / V), the concentration of apolipoprotein AI reaches 2.5 g / L, the concentration of transferrin reaches 3 g / L, and the rest is an artificial matrix component composed of apolipoprotein AI and transferrin antigen;

[0155] S2: Adjust the pH to 7.35-7.45 (20℃±0.5℃) with hydrochloric acid or sodium hydroxide, and pack in 1ml / bottle;

[0156] S3: After packaging, the samples are freeze-dried, and the specific freeze-drying procedure is as follows: the room temperature is reduced to 5°C and maintained for 30 minutes; the temperature is reduced to -5°C during the cooling process for 10 minutes and maintained for 30 minutes; the temperature is reduced to -40°C during the cooling process for 35 minutes and maintained for 120 minutes; the temperature is increased to -30°C during the heating process for 10 minutes and maintained for 120 minutes; the temperature is reduced to -40°C during the cooling process for 10 minutes and maintained for 120 minutes; the temperature remains unchanged at -40°C and maintained for 1800 minutes, and the vacuum degree is 5Pa; the temperature is increased to -10°C during the heating process for 150 minutes and maintained for 120 minutes, and the vacuum degree is 5Pa; the temperature is increased to 5°C during the heating process for 75 minutes and maintained for 120 minutes, and the vacuum degree is 5Pa; the temperature is increased to 20°C during the heating process for 75 minutes and maintained for 600 minutes, and the vacuum degree is 5Pa.

[0157] Embodiment 19

[0158] The main difference between the protective agents in Examples 14 to 18 is the different sucrose concentrations. The quality control products prepared in Examples 14 to 18 were placed at 37°C for accelerated stability testing.

[0159] 1. Accelerated stability test method: The quality control products prepared in Examples 14 to 18 were placed in a drying oven at 37° C. for 4 weeks. Three bottles of composite quality control products were randomly selected each week to measure the apolipoprotein AI and transferrin content. Each bottle was measured 3 times and the average value was calculated.

[0160] 2. Detection reagents: Apolipoprotein AI assay kit from Prebna, transferrin assay kit from Prebna.

[0161] 3. Testing instrument: Mindray BS-820 fully automatic biochemical analyzer.

[0162] 4. Detection method: Use Prebac multi-protein calibrator and apolipoprotein calibrator for calibration. After successful calibration, use Mindray Biochemical composite fixed value quality control to verify whether the measured value is within the quality control range. After successful verification, add 1 ml of deionized water to the prepared apolipoprotein AI and transferrin composite quality control, mix for 30 minutes, and then measure the content of apolipoprotein AI and transferrin in the composite quality control. Calculate the amplitude of change, and the calculation method of the amplitude of change is the same as that of Example 6.

[0163] 5. Test results and analysis: The change range at each time point is shown in Table 3 and Figure 5 , Figure 6 . Figure 5 , Figure 6 The main purpose is to explore the protective effect of the formula on freeze-drying of apolipoprotein AI and transferrin. Figure 5 Apolipoprotein AI, Figure 6 For transferrin.

[0164] From Table 3, Figure 5 , Figure 6 It can be seen that, after four weeks of accelerated observation at 37°C, the apolipoprotein AI of Example 14 has a maximum change of 5.44% within four weeks; the maximum change of transferrin within four weeks is -0.37%. In Example 15, the maximum change of apolipoprotein AI within four weeks is 5.80%, and the maximum change of transferrin within four weeks is 2.17%. In Example 16, the maximum change of apolipoprotein AI within four weeks is 4.25%, and the maximum change of transferrin within four weeks is -2.15%. In Example 17, the maximum change of apolipoprotein AI within four weeks is 11.07%, and the maximum change of transferrin within four weeks is -4.60%. In Example 18, the maximum change of apolipoprotein AI within four weeks is 14.51%, and the maximum change of transferrin within four weeks is 7.30%. When the sucrose concentration is too high, the change increases, and the appearance of the freeze-dried product becomes more fragmented.

[0165] The formulations with similar accelerated stability at 37 degrees were screened out, and the appearance of the freeze-dried quality control products was analyzed to select the formulations that were complete and uniform without cracking and shrinkage. After comparison, the formulation of Example 15 best met the target requirements. After four weeks of acceleration at 37 degrees, the changes in apolipoprotein AI and transferrin were less than 6%, and the appearance was cake-shaped and not cracked.

[0166] Table 3: Accelerated stability variation of quality control products of Examples 14 to 18

[0167]

[0168]

[0169] Example 20 Homogeneity Test

[0170] 1. Detection reagents: Apolipoprotein AI assay kit (immunoturbidimetric method) from Prebna, transferrin assay kit from Prebna; Apolipoprotein AI assay kit from Mecon, transferrin assay kit from Mecon; Apolipoprotein AI assay kit from Jiuqiang Biological, transferrin assay kit from Jiuqiang Biological.

[0171] 2. Testing instruments: Mindray BS-820 fully automatic biochemical analyzer, Hitachi 3500 fully automatic biochemical analyzer.

[0172] 3. Detection method: Add 1 ml of deionized water to the freeze-dried quality control prepared in Example 15 for redissolution. First, use the apolipoprotein AI calibrator and the transferrin calibrator for calibration. After successful calibration, use the apolipoprotein AI and transferrin control products provided with the kit to verify whether the measured value is within the quality control range. After successful verification, perform a homogeneity test.

[0173] 4. Homogeneity test method: Randomly select 10 bottles of self-made quality control products from the same batch, number them 1 to 10, and measure each packaging unit 3 times in the following order. Measurement order: 1, 3, 5, 7, 9, 2, 4, 6, 8, 10, 10, 9, 8, 7, 6, 5,

[0174] 4, 3, 2, 1, 2, 4, 6, 8, 10, 1, 3, 5, 7, 9. Record the measurement results and calculate F, Sbb, Sr, CV within the bottle, and CV between bottles according to the following formulas (see Table 4 and Table 5).

[0175] Table 4 Homogeneity experimental formula

[0176]

[0177] Table 5 Meaning of letters in the homogeneity formula

[0178]

[0179] 5. Test results and analysis: The test results are shown in Tables 6 to 18. From Tables 6 to 18, it can be concluded that the CVs of apolipoprotein AI between bottles and within bottles are less than 4%, and the results of transferrin between bottles and within bottles are less than 2%, indicating that the composite quality control product has good intra-bottle repeatability, small inter-bottle differences, and good uniformity.

[0180] Table 6 Homogeneity test results of the Prebacter Apolipoprotein AI Assay Kit on the Mindray BS-820 fully automatic biochemical analyzer (g / L)

[0181]

[0182] Table 7 Homogeneity test results of the Jiuqiang Apolipoprotein AI Assay Kit in Mindray BS-820 fully automatic biochemical analyzer (g / L)

[0183]

[0184]

[0185] Table 8 Homogeneity test results of the Meikang apolipoprotein AI assay kit on the Mindray BS-820 fully automatic biochemical analyzer (g / L)

[0186]

[0187] Table 9 Homogeneity test results of the Prebacter Apolipoprotein AI Assay Kit on the Hitachi 3500 fully automatic biochemical analyzer (g / L)

[0188]

[0189]

[0190] Table 10 Homogeneity test results of the Jiuqiang Apolipoprotein AI assay kit on the Hitachi 3500 fully automatic biochemical analyzer (g / L)

[0191]

[0192] Table 11 Homogeneity test results of the Mecon Apolipoprotein AI Assay Kit on the Hitachi 3500 fully automatic biochemical analyzer (g / L)

[0193]

[0194]

[0195] Table 12 Homogeneity test results of using the Prebacter transferrin assay kit on the Mindray BS-820 fully automatic biochemical analyzer (g / L)

[0196]

[0197] Table 13 Homogeneity test results of using the Jiuqiang transferrin assay kit on Mindray BS-820 fully automatic biochemical analyzer (g / L)

[0198]

[0199]

[0200] Table 14 Homogeneity test results of the transferrin assay kit using the Mindray BS-820 fully automatic biochemical analyzer (g / L)

[0201]

[0202] Table 15 Homogeneity test results of using the Prebacter transferrin assay kit on the Hitachi 3500 fully automatic biochemical analyzer (g / L)

[0203]

[0204] Table 16 Homogeneity test results of using the Jiuqiang transferrin assay kit on the Hitachi 3500 fully automatic biochemical analyzer (g / L)

[0205]

[0206]

[0207] Table 17 Homogeneity test results of the transferrin assay kit using the Hitachi 3500 fully automatic biochemical analyzer (g / L)

[0208]

[0209] Table 18 Statistical summary of uniformity test results

[0210]

[0211] Example 21 Opening stability test

[0212] 1. Stability test method after opening: Place the opened composite quality control product in a 2℃~8℃ refrigerator and test it on the 3rd day.

[0213] 2. Detection reagents: Apolipoprotein AI assay kit (immunoturbidimetric method) from Prebna, transferrin assay kit from Prebna; Apolipoprotein AI assay kit from Mecon, transferrin assay kit from Mecon; Apolipoprotein AI assay kit from Jiuqiang Biological, transferrin assay kit from Jiuqiang Biological.

[0214] 3. Testing instruments: Mindray BS-820 fully automatic biochemical analyzer, Hitachi 3500 fully automatic biochemical analyzer.

[0215] 4. Detection method: Add 1 ml of deionized water to the freeze-dried quality control prepared in Example 15 for reconstitution. First, use the Prebac apolipoprotein AI calibrator and multiple protein calibrators for calibration. After successful calibration, use the Prebac multiple biochemical quality control level 1 to verify whether the measured value is within the quality control range. After successful verification, perform an unsealed stability test.

[0216] 5. Test results and analysis

[0217] The test results are shown in Table 19. Table 19 shows that after 7 days of opening, the maximum change of APOA-I is 4.6%, and the change of transferrin is -4.0%, both less than 5%, indicating that the prepared apolipoprotein AI and transferrin control products can be stored for 7 days after opening and refrigeration.

[0218] Table 19 Unsealed stability results

[0219]

[0220]

[0221] After verification by multiple detection platforms including Hitachi 3500 and Mindray BS820, reagents from Mindray, Jiuqiang, Prebacter, and Meikang, the composite quality control product of apolipoprotein AI and transferrin prepared in Example 15 has a low change range after accelerated stabilization, and the uniformity between bottles and within bottles is small. It can be refrigerated and stored for 7 days after opening. At the same time, the appearance of the quality control product is uniform and complete. Therefore, the quality control product prepared by the present invention has excellent performance and meets the relevant technical requirements.

[0222] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A composite quality control product of apolipoprotein AI and transferrin, characterized in that: The composite quality control product includes apolipoprotein AI, transferrin, a protective agent, a surfactant, and a preservative. The protective agent includes sugar alcohol, albumin, sugar, and amino acid, and the remaining component is an artificial matrix composed of apolipoprotein AI and transferrin antigen; the concentration of apolipoprotein AI is 2g / L-3g / L, the concentration of transferrin is 2.5g / L-3.5g / L, the concentration of mannitol is 10g / L-100g / L, the concentration of albumin is 10g / L-100g / L, the concentration of sugar is 10g / L-100g / L, the concentration of amino acid is 10g / L-100g / L, the volume concentration of surfactant is 0.001%-1%, and the volume concentration of preservative is 1%-10%.

2. The composite quality control product according to claim 1, characterized in that: The albumin is bovine serum albumin.

3. The composite quality control product according to claim 2, characterized in that: The sugar is a disaccharide, and the disaccharide is one or more of sucrose, trehalose, lactose, and maltose.

4. The composite quality control product according to claim 2, characterized in that: The sugar is a monosaccharide, and the monosaccharide is one or more of glucose and fructose.

5. The composite quality control product according to claim 3, characterized in that: The amino acid is one or more of glycine, alanine, phenylalanine and arginine.

6. The composite quality control product according to claim 5, characterized in that: The surfactant is polysorbate 80.

7. The composite quality control product according to claim 6, characterized in that: The preservative is one or more of Proclin300 and Proclin950.

8. The method for preparing the composite quality control product according to claim 1, characterized in that: The steps include: S1: Add apolipoprotein AI, transferrin, protective agent, surfactant, and preservative into a preparation container, wherein the protective agent includes sugar alcohol, albumin, disaccharide, and amino acid, and the remaining component is an artificial matrix composed of apolipoprotein AI and transferrin antigen; stir and dissolve to make the concentration of apolipoprotein AI 2g / L-3g / L, the concentration of transferrin 2.5g / L-3.5g / L, the concentration of mannitol 10g / L-100g / L, the concentration of albumin 10g / L-100g / L, the concentration of sugar 10g / L-100g / L, the concentration of amino acid 10g / L-100g / L, the volume concentration of surfactant 0.001%-1%, and the volume concentration of preservative 1%-10%; S2: Adjust the pH to 7.35-7.45 with hydrochloric acid or sodium hydroxide and dispense in 1 ml / bottle; S3: After packaging, the sample is freeze-dried. The freeze-drying process is as follows: S31, cool to 4℃~6℃, keep for 25min~35min; S32, cool to -4℃~-6℃, keep for 25min~35min; S33, cooling to -35~-45℃, maintaining for 160min~200min; S34, control the temperature at -35 to -45°C, the vacuum degree at 3Pa to 7Pa, and maintain for 1600min to 2000min; S35, control the vacuum degree to 3Pa~7Pa, raise the temperature to -5℃~-15℃, and maintain for 100min~140min; S36, control the vacuum degree to 3Pa~7Pa, raise the temperature to 3℃~7℃, and maintain for 100min~140min; S37, control the vacuum degree to 3Pa-7Pa, raise the temperature to 15-25°C, and maintain for 500-700 minutes; S38, capping and sealing.

9. The preparation method according to claim 8, characterized in that: The cooling time of step S32 is 5min to 15min, the cooling time of step S33 is 30min to 40min, the heating time of step S35 is 120min to 180min, the heating time of step S36 is 90min to 600min, and the heating time of step S37 is 90min to 600min.

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