Active human insulin solution standard material with interchangeability and preparation method and application thereof

Abstract

The application discloses a kind of active human insulin solution standard substance with interchangeability and its preparation method and application, belong to chemical metrology analytical detection technical field.The preparation method provided in the application includes the purification of human insulin, self-assembly of human insulin, confirmation of insulin self-assembly complex structure, the purification of active human insulin, determination of immunological affinity active concentration and specific activity, preparation of active human insulin solution standard substance with interchangeability.The above preparation method provided in the application can be used for the calibration of human insulin routine in vitro diagnostic system, the development of in vitro diagnostic detection reagent and method performance verification, and for the value traceability of human insulin detection result in serum, ensure the accuracy and comparability of clinical detection result, protect public health and life safety.

Description

Technical Field

[0001] The present application belongs to the technical field of chemometric analysis and detection, and in particular relates to an interchangeable active human insulin solution standard substance, a preparation method and application thereof. Background Art

[0002] In the "Terminology of Metrology (4th Edition)", "interchangeability" is an abbreviation of the term "interchangeability of standard (reference) materials" and is defined as "the characteristics of a reference material by the closeness of the relationship between the measurement results of a given amount of the material and the relationship between the results obtained using a measurement procedure applicable to the material and the measurement results of other specified materials". Interchangeability can be simply understood as: to what extent a certified reference material (CRM) simulates the characteristics of a typical set of fresh clinical samples for a specific measurement method and a specified measurement quantity. An interchangeable CRM (RM) exhibits similar behavior to routine samples when different measurement procedures are applied. For an interchangeable CRM / RM, the ratio of its measurement results must be the same as the ratio of fresh routine samples.

[0003] In vitro diagnostics (IVDs) play a crucial role in modern medicine. According to literature, 60-70% of medical decisions rely on IVD results. The accuracy of IVD results directly impacts the correct diagnosis and effective treatment of diseases. Interchangeability is crucial for ensuring the traceability of IVD results and is a prerequisite for effective calibration, trueness verification, and trueness-based proficiency testing. Renowned standardization expert Greg Miller has stated that interchangeability is central to the success of standardization and harmonization efforts throughout laboratory medicine.

[0004] To ensure the accuracy and comparability of in vitro diagnostic (IVD) results, establishing a traceability chain for IVD results has been proven both theoretically and practically to be an effective approach. The use of non-interchangeable reference materials for calibration disrupts the traceability chain, thus defeating the intended calibration and standardization objectives. Reflecting this in experimental data, the use of non-interchangeable reference materials to calibrate IVD systems can produce unintended biases, which are then propagated through the traceability chain to the final IVD results, resulting in inaccurate and non-comparable results. For example, calibrating an IVD system with a non-interchangeable cardiac troponin reference material still produces significant variability in results, defeating the purpose of standardization. Furthermore, because non-interchangeable reference materials can produce unintended biases when calibrating IVD systems, they cannot be used for trueness verification or trueness-based proficiency testing.

[0005] There are many reasons for non-interchangeability, including differences in the matrix, inconsistency between the measured value in the standard substance and the fresh clinical sample, inconsistency in the measured value at the microscopic level, etc. Therefore, in order to ensure that the developed standard substances are interchangeable, real human samples are usually used as standard material raw materials to develop matrix standard substances to ensure that the developed standard substances have the expected interchangeability. The international standard ISO 17511:2020 In vitro diagnostic medical devices—Requirements for establishing metrological traceability of values ​​assigned to calibrators, trueness control materials and human samples stipulates 6 ways to establish the traceability of in vitro diagnostic results, among which the methods that can be fully traced back to SI units are as follows Figure 1 As shown in the figure, reference materials at the M1 and M2 levels are purity reference materials and are generally not interchangeable. Therefore, the measurement value must be transferred to interchangeable matrix reference materials at the M3 level, and then further transferred to the manufacturer's master calibrator. Because the measurement value transfer procedure P4 used by manufacturers is often a routine measurement procedure, only interchangeable reference materials can be used for calibration of routine measurement procedures. For other traceability methods, interchangeability of reference materials at the M3 level is also required; otherwise, the measurement value traceability transfer chain will be broken.

[0006] However, the development and use of matrix reference materials are now difficult, especially protein-based in vitro diagnostic marker matrix reference materials. Protein-based in vitro diagnostic markers account for a significant proportion of in vitro diagnostics, and many major diseases such as tumor, diabetes, cardiovascular and cerebrovascular diseases, and neurodegenerative diseases are diagnosed by protein markers. The lack of protein-based in vitro diagnostic marker matrix reference materials with interchangeability has become a constraint factor for the standardization of protein-based in vitro diagnostic marker results. Since the raw materials of matrix reference materials are generally derived from real human samples, the raw materials are limited and difficult to obtain, especially low-value and high-value real human samples under abnormal pathological conditions. Adding the standard material may also lead to non-interchangeability, so the value level of the matrix reference material is often limited and cannot be used for calibration of the entire clinical reportable range. Protein-based in vitro diagnostic markers often exist in the form of protein complexes or aggregates in vivo, and pure proteins often exist in the form of monomers. Directly adding protein pure products to serum to attempt to change the value level may lead to non-interchangeability of the added standard material due to inconsistencies in the measured values. Proteins may have glycosylation structures, and inconsistencies in the microstate of glycosylation of the same protein in the standard material and the real clinical sample may lead to non-interchangeability. The preparation and preservation processes of the standard material such as freeze-drying may also cause the disaggregation of protein aggregates in serum, leading to the interchangeability of the standard material. C-reactive protein is a typical example. At the same time, even if isotope dilution mass spectrometry, a metrological reference method, is used to accurately quantify the protein in the matrix, significant differences are still found in international comparisons, which is difficult to meet the requirements of the total error of clinical practice. These unfavorable factors make it difficult to develop protein-based in vitro diagnostic marker matrix reference materials with interchangeability, and the number of such materials is very limited, making it difficult to meet the current needs of protein-based in vitro diagnostic result standardization.

[0007] Therefore, it is necessary to find a simple and easy way to develop low-cost, interchangeable, wide-range, and high-accuracy protein-based in vitro diagnostic marker standard materials to meet the requirements of in vitro diagnostic result standardization. SUMMARY

[0008] To overcome the above-mentioned defects in the prior art, the present application provides a human insulin solution standard material with interchangeability, a preparation method and application thereof, aiming to fill the gap in related technologies and ensure the accuracy and effectiveness of human insulin clinical test results. The innovative idea of the present application is to find the crux of the problem by analyzing the reasons for the non-interchangeability of protein pure solution standard materials, and then to regulate and evaluate the interchangeability of human insulin solution standard materials through structure adjustment and confirmation, determination of physicochemical concentration and immunoaffinity activity concentration, etc., to overcome the non-interchangeability factors, and then to develop human insulin pure solution standard materials traceable to SI units and with interchangeability, realizing the wide-range calibration of conventional in vitro diagnostic systems.

[0009] In order to achieve the above-mentioned invention objectives, this application provides the following technical solutions:

[0010] In one aspect, the present application provides a method for preparing an interchangeable active human insulin solution standard substance, comprising the following steps:

[0011] (1) Purification of human insulin: Human insulin with a purity of 90-98% is selected and purified by reverse-phase high performance liquid chromatography and molecular sieves to obtain high-purity human insulin raw material;

[0012] (2) Self-assembly of human insulin: The high-purity human insulin raw material was dissolved in a 1×PBS solution containing zinc ions and allowed to stand to obtain an insulin self-assembly complex;

[0013] (3) Confirmation of the structure of the insulin self-assembled complex: The insulin self-assembled complex is subjected to circular dichroism and mass spectrometry spectrum scanning analysis. At the same time, a human insulin solution prepared with a PBS solution without zinc ions and having the same concentration as that in step (2) is subjected to circular dichroism and mass spectrometry spectrum scanning analysis as a comparison. Whether the self-assembled complex is formed is determined based on the scanning results. If not, repeat step (2);

[0014] (4) Purification of active human insulin: A high performance liquid chromatography coupled with surface plasmon resonance (SPR) spectroscopy apparatus was constructed, and the insulin self-assembled complex confirmed in step (3) was subjected to online separation and online SPR spectroscopy detection using a molecular sieve column. Fractions with a signal slope increase of >30 degrees on the sensorgram were collected and freeze-dried to obtain active human insulin raw material;

[0015] (5) Determination of immunoaffinity concentration and specific activity: Determine the immunoaffinity concentration and specific activity of the active human insulin raw material. If the specific activity is ≥ 0.9, proceed to the next step; otherwise, re-prepare from step (1):

[0016] (6) Preparation of interchangeable active human insulin solution standard substance: The active human insulin raw material in step (4) is prepared into solutions with a concentration range of 10 to 1000 pmol / L using a diluent, and the solutions are divided and sealed to form interchangeable active human insulin solution standard substance candidates. The candidates are then subjected to a uniformity test, a stability test, a standard value determination, an uncertainty assessment, and an interchangeability evaluation in sequence to obtain interchangeable active human insulin solution standard substances.

[0017] Optionally, in step (1), the human insulin comprises protein sequences as shown in SEQ ID No. 1 and SEQ ID No. 2.

[0018] Optionally, in step (1), the conditions of the reverse-phase high-performance liquid chromatography include: using a sodium sulfate buffer solution with a concentration of 0.1 to 0.4 mol / L: acetonitrile in a ratio of 82:18, or acetonitrile: water in a ratio of 50:50 as the mobile phase, and using a C4, C8 or C18 chromatographic column for separation to separate human insulin and A21-deamino human insulin, and collecting the human insulin fraction for the next step of molecular sieve purification.

[0019] Optionally, in step (1), the molecular sieve conditions include: using PBS and water as mobile phases, respectively, further purifying with a molecular sieve strain, monitoring the peak time by absorbance at 280 nm, separating insulin aggregates from monomeric insulin, collecting fractions within the monomeric insulin peak time period, and freeze-drying to obtain high-purity human insulin raw material.

[0020] Optionally, in step (2), the source of zinc ions includes zinc acetate.

[0021] Optionally, in step (2), the final concentration of the high-purity human insulin raw material is 0.01-1 mg / mL, and the molar ratio of insulin to zinc ions in the solution is 6:1-6:60.

[0022] Optionally, in step (2), the final concentration of the high-purity human insulin raw material is independently selected from any value among 0.01 mg / mL, 0.02 mg / mL, 0.05 mg / mL, 0.08 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 0.8 mg / mL, 1 mg / mL, or a range between any two of them, and the molar ratio of insulin to zinc ion in the solution is independently selected from any value among 6:1, 6:5, 6:10, 6:20, 6:30, 6:40, 6:50, 6:60, or a range between any two of them.

[0023] Optionally, in step (2), the standing temperature is 4 to 10° C., and the standing time is 4 to 48 hours.

[0024] Optionally, in step (2), the standing temperature is independently selected from any value among 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, or a range between any two of them, and the standing time is independently selected from any value among 4h, 10h, 15h, 20h, 30h, 40h, 48h, or a range between any two of them.

[0025] Optionally, in step (3), the scanning optical path of the circular dichroism spectrum is 0.1 to 1 cm, the scanning wavelength range is 200 to 400 nm, and the scanning speed is 10 to 100 nm / min.

[0026] Optionally, in step (3), the scanning optical path of the circular dichroism spectrum is independently selected from any value among 0.1 cm, 0.2 cm, 0.4 cm, 0.6 cm, 0.8 cm, 1 cm, or a range value between any two of them, the scanning wavelength range is independently selected from any value among 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, or a range value between any two of them, and the scanning speed is independently selected from any value among 10 nm / min, 20 nm / min, 40 nm / min, 50 nm / min, 60 nm / min, 80 nm / min, 100 nm / min, or a range value between any two of them.

[0027] Optionally, in step (3), the judgment criterion is: if the circular dichroism peak moves toward higher wavelengths and becomes flat, and a peak with a larger molecular weight appears on the mass spectrometer spectrum, a self-assembled complex is formed; otherwise, no self-assembled complex is formed.

[0028] Optionally, in step (4), when constructing a high performance liquid chromatography coupled with surface plasmon resonance (SPR) spectroscopy device, 1×PBST is used as the mobile phase and running buffer, sodium hydroxide is used as the regeneration solution, and the human insulin monoclonal antibody is coupled to a gold foil surface with carboxymethyl cellulose as a detector for the SPR signal.

[0029] Optionally, the molar concentration of the sodium hydroxide is 0.5 to 20 mmol / L.

[0030] Optionally, the molar concentration of the sodium hydroxide is independently selected from any value among 0.5mmol / L, 1mmol / L, 5mmol / L, 10mmol / L, 15mmol / L, 20mmol / L, or any range value therebetween.

[0031] Optionally, in step (5), the determination of the immunoaffinity activity concentration and specific activity includes:

[0032] S1. Prepare active human insulin solution by using 1×PBST solution to prepare active human insulin solution, and measure the immunoaffinity activity concentration c of the active human insulin solution. raw,active ;

[0033] S2. Take the prepared active human insulin solution, weigh its mass and add isotope-labeled amino acids thereto, concentrate by centrifugation or blow dry with nitrogen, then add concentrated hydrochloric acid, pass nitrogen protection and seal, hydrolyze, determine the content of the above amino acids in the hydrolyzate by isotope dilution mass spectrometry, and calculate the physicochemical concentration C of the active human insulin solution based on the protein sequence of human insulin selected in step (1). raw,chem ;

[0034]

[0035] Where C AA is the concentration of amino acids in insulin hydrolysate determined by isotope dilution mass spectrometry; M AA is the molecular weight of the amino acid; N AA is the number of the amino acid contained in insulin; M insulin is the molecular weight of insulin;

[0036] S3. Calculate the purity of active human insulin raw material according to the following formula:

[0037]

[0038] Where, P raw is the purity of active human insulin raw material, m raw is the mass of active human insulin raw material, m solvent The mass of 1×PBST solution, C raw,chem is the physicochemical concentration of active human insulin solution;

[0039] S4. Using a density meter to measure the density of the prepared active human insulin solution, the physicochemical concentration c of the active human insulin solution was determined. raw,chem (Unit: mg / g) and immunoaffinity activity concentration c raw,active , and then calculate the specific activity of the two according to the following formula:

[0040]

[0041] Where R is the specific activity of active human insulin raw material, c raw,active is the immunoaffinity activity concentration of active human insulin solution, c raw,chem is the physicochemical concentration of active human insulin solution, and ρ is the density of active human insulin solution.

[0042] Optionally, in step S1, the physicochemical concentration of the active human insulin solution is 0.01-1 mg / mL, and the immunoaffinity activity concentration c of the active human insulin solution is raw,active Measured by surface plasmon resonance spectroscopy.

[0043] Optionally, in step S1, the physicochemical concentration of the active human insulin solution is independently selected from any value among 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, or any range between two values.

[0044] Optionally, in step S2, the isotope comprises 13 C. 15At least one of N and D, the amino acids include at least one of proline, valine, leucine, isoleucine, and phenylalanine, and the number of isotope-labeled atoms is ≥3.

[0045] Optionally, in step S2, the molar concentration of the concentrated hydrochloric acid is 6 to 8 mol / L, the hydrolysis temperature is 110 to 150° C., and the hydrolysis time is 24 to 96 h.

[0046] Optionally, in step S2, the molar concentration of the concentrated hydrochloric acid is independently selected from any value among 6 mol / L, 7 mol / L, 8 mol / L, or a range between any two of them, the hydrolysis temperature is independently selected from any value among 110°C, 120°C, 130°C, 140°C, 150°C, or a range between any two of them, and the hydrolysis time is independently selected from any value among 24h, 36h, 48h, 60h, 72h, 84h, 96h, or a range between any two of them.

[0047] Optionally, in step (6), the diluent is a bovine serum albumin solution, the mass fraction of the bovine serum albumin solution is 5-8%, the high performance liquid chromatography purity of the bovine serum albumin solution is greater than 99%, and the bovine serum albumin solution further contains 0-500 mmol / L zinc acetate, and the zinc acetate is of analytical grade or above.

[0048] Optionally, in step (6), the standard value determination is performed according to the following formula:

[0049]

[0050] Where c CRM Indicates the certified value of active human insulin solution standard substance, unit is pmol / L; m STD Indicates the mass of weighed active human insulin raw material, in g; P raw Indicates the purity of active human insulin raw materials, unit is g / g; M insulin Indicates the molar mass of human insulin in g / mol; V STD Indicates the volume of the volumetric flask used to prepare the solution, in L.

[0051] Optionally, in step (6), the uncertainty includes the uncertainty introduced in the determination process, the uncertainty introduced by the uniformity of the standard substance, the uncertainty introduced by the long-term stability, the combined standard uncertainty of the determination result, and the expanded standard uncertainty of the determination result.

[0052] Optionally, in step (6), the solution of each concentration level is packaged and sealed in brown ampoule bottles, with each bottle containing 10 to 5000 μL. The packaged active human insulin solution standard substance candidate is labeled and stored in a refrigerator at -70°C. Each packaging unit is numbered consecutively according to the packaging order, and the number of packaging units is P, thereby forming an interchangeable active human insulin solution standard substance candidate.

[0053] Optionally, in step (6), the uniformity test includes:

[0054] Use the randbetween function in Excel to randomly select and test the consecutively numbered packaging units. When P≤200, the number of packaging units m to be selected is not less than 11; when 200 <P≤500时,抽取分装单元数量m不少于15个;当500<P≤1000时,抽取分装单元数量m不少于25个;当分装单元P> When the number of subpackaging units is 1000, the number m of subpackaging units extracted is not less than 30. The m subpackaging units extracted are renumbered and arranged in sequence according to the order of extraction, and the uniformity of the insulin concentration of the active human insulin solution standard substance in the above-mentioned subpackaging units is tested by fluorescence or chemiluminescence immunoassay. During the test, the insulin concentration of the active human insulin solution standard substance in each subpackaging unit extracted is first tested in the above-mentioned order, and then the above-mentioned order is disrupted, and the insulin concentration of the active human insulin solution standard substance in each subpackaging unit extracted is tested for the second time. The above operation is repeated until the insulin concentration of the active human insulin solution standard substance in each subpackaging unit extracted is tested for the nth time, where n is an integer greater than or equal to 3, thereby obtaining n test results, and calculating and counting them according to the following formula: uniformity of insulin concentration of the active human insulin solution standard substance in the subpackaging unit;

[0055] When m candidate standard substances are sampled, and the above test method is used with n repeatability conditions, m groups of precision measurement data are obtained as follows:

[0056] No. 1: x 11 ,x 12 ......x 1n ,average value

[0057] Number 2: x 21 ,x 22 ......x 2n ,average value ......

[0059] No. m:x m1 ,x m2 ......xmn ,average value

[0060] average value:

[0061] Statistic N: N = m·n Formula (4)

[0062] The sum of squares of differences between groups:

[0063] The sum of squares of within-group differences:

[0064] Between-group degrees of freedom: v1 = m-1; within-group degrees of freedom: v2 = Nm

[0065]

[0066] Statistics F:

[0067] Among them S1 2 is the between-group variance, S2 2 is the within-group variance. According to the degrees of freedom (v1, v2) and the given significance level α = 0.05, F is obtained from the table. α The critical value The F value calculated by formula (9) and F α For comparison, if F <F α If there is no significant difference between the groups, the insulin concentration of the active human insulin solution standard substance in the packaging unit is uniform. Otherwise, the insulin concentration of the active human insulin solution standard substance in the packaging unit is uneven.

[0068] Optionally, in step (6), the stability test includes:

[0069] The duration of the long-term stability test is not less than 6 months. During the long-term stability test period, according to the principle of dense at the beginning and sparse at the end, select not less than 5 time points k. At each time point, extract at least 2 sub-packaging units of the active human insulin solution standard substance stored at a temperature of -70°C in step (6). The insulin concentration of the active human insulin solution standard substance in each sub-packaging unit is tested three times by fluorescence or luminescence immunoassay, and then the arithmetic mean of these measurement results is calculated to obtain the arithmetic mean Y of the measurement results at each time point. i , the arithmetic mean value Y of the above measurement results i The corresponding measurement time is fitted according to the following linear model:

[0070] Y i =b0+bX formula (10)

[0071] In formula (10), b0 represents the intercept, b represents the regression coefficient (slope); X represents the test time, unit (month); Y i It represents the arithmetic mean of the insulin concentration in the active human insulin solution standard substance at each time point.

[0072] The regression coefficient (slope) and intercept are calculated according to formula (11) and formula (12):

[0073]

[0074] In formula (11) and formula (12), X i Indicates the detection time at the i-th time point, in months; Y i represents the arithmetic mean of the insulin concentration determination results of the active human insulin solution standard substance in the above-mentioned packaging unit at the i-th time point; X represents the average detection time of all time points, in months; Y represents the arithmetic mean of the insulin concentration determination results of the active human insulin solution standard substance in the above-mentioned packaging unit at all time points; k represents the number of time points.

[0075] Calculate the s value according to formula (13):

[0076]

[0077] The meanings of the symbols in formula (13) are the same as those in formula (10), formula (11) and formula (12).

[0078] Calculate the value of s(b) according to formula (14):

[0079]

[0080] Look up the t distribution table and get t 0.95,k-2 The value of

[0081] If |b|<t 0.95,k-2 ·s(b), it indicates that the slope is not significant and no instability is observed, indicating that the active human insulin solution standard substance is long-term stable; otherwise, it indicates that the active human insulin solution standard substance is unstable.

[0082] The duration of the short-term stability test is not less than 7 days. During the short-term stability test period, according to the principle of dense at the beginning and sparse at the end, select not less than 5 time points k. At each time point, take out at least two subpackaging units from step (6), place them in a temperature range of -20°C to 40°C for inspection, extract the active human insulin solution standard substance in the above subpackaging units, and use fluorescence or luminescence immunoassay to repeat the insulin concentration of the active human insulin solution standard substance in each subpackaging unit for 3 times. Calculate the arithmetic mean of these measurement results to obtain the arithmetic mean Y of the measurement results at each time point. i , fit the arithmetic mean of the above measurement results and the corresponding measurement time according to the following linear model:

[0083] Y i =b0+bX formula (15)

[0084] In formula (15), b0 represents the intercept; b represents the regression coefficient (slope); X represents the test time, unit (day); Y i It represents the arithmetic mean of the insulin concentration in the active human insulin solution standard substance at each time point.

[0085] The regression coefficient (slope) and intercept are calculated according to formula (16) and formula (17):

[0086]

[0087] In formula (16) and formula (17), X i Y represents the detection time at the i-th time point, in days; i represents the arithmetic mean of the insulin concentration determination results of the active human insulin solution standard substance in the above packaging unit at the i-th time point; It represents the average detection time of all time points, in days; represents the arithmetic mean of the insulin concentration determination results of the active human insulin solution standard substance in the above packaging unit at all time points; k represents the number of time points.

[0088] Calculate the s value according to formula (18):

[0089]

[0090] The meanings of the symbols in formula (18) are the same as those in formula (15), formula (16) and formula (17).

[0091] Calculate the value of s(b) according to formula (19):

[0092]

[0093] Look up the t distribution table and get t 0.95,k-2 The value of

[0094] If |b|<t 0.95,k-2 ·s(b), it indicates that the slope is not significant and no instability is observed, indicating that the active human insulin solution standard substance is short-term stable; otherwise, it indicates that the active human insulin solution standard substance is unstable.

[0095] Optionally, in step (6), the uncertainty assessment includes:

[0096] Mathematical model based on uncertainty assessment:

[0097] The uncertainty of the determination results of active human insulin solution standard substance comes from the uncertainty introduced in the determination process, the uncertainty introduced by the homogeneity of the standard substance, and the uncertainty introduced by long-term stability.

[0098] ①Uncertainty introduced in the process of setting the value:

[0099] According to formula (20), taking into account the influence of the solvent blank detection limit, the uncertainty of the preparation results of the active human insulin solution standard substance can be calculated using formula (21):

[0100]

[0101] Where u r，cCRM It represents the relative uncertainty of the preparation result of active human insulin solution standard material, dimensionless;

[0102] It represents the relative uncertainty of weighing the mass of active human insulin raw material, dimensionless;

[0103] It represents the relative uncertainty of the volume of the volumetric flask when preparing the active human insulin solution standard substance, dimensionless;

[0104] It represents the relative uncertainty of the purity of active human insulin raw materials, dimensionless;

[0105] It represents the relative uncertainty of the molar mass of human insulin, dimensionless;

[0106] u r，LOD It represents the detection limit of fluorescence or chemiluminescence immunoassay method, dimensionless.

[0107] ②Uncertainty component introduced by uniformity

[0108] The uncertainty introduced by the homogeneity of the standard substance is equal to the standard deviation of the homogeneity between bottles. When the mean square between groups is greater than the mean square within groups, the calculation is performed according to formula (22):

[0109]

[0110] When the between-group mean square is smaller than the within-group mean square, calculate according to formula (23):

[0111]

[0112] ③Uncertainty component introduced by stability

[0113] The long-term stability of the reference material is calculated according to formula (24):

[0114] u lts =s k，lts ·t Formula (24)

[0115] ④The combined standard uncertainty of the fixed value result

[0116] The combined standard uncertainty of the determination result of the active human insulin solution standard substance is calculated according to formula (25):

[0117]

[0118] ⑤ Expanded standard uncertainty of the fixed value result

[0119] According to formula (26), with coverage factor k = 2, the expanded uncertainty of the determination result of the active human insulin solution standard substance is calculated as follows:

[0120] U=ku c Formula (26)

[0121] Therefore, the determination result of the active human insulin solution standard substance can be expressed as:

[0122] c CRM ±U

[0123] Where c CRM is the certified value, and U is the expanded uncertainty of the certified value;

[0124] Finally, we obtained the uniformity, stability and determination results of the active human insulin solution standard substance.

[0125] In a second aspect, the present application provides an interchangeable active human insulin solution standard substance, which is prepared by any of the preparation methods described above.

[0126] In a third aspect, the present application provides the use of the interchangeable active human insulin solution standard substance as an interchangeable standard substance.

[0127] Compared with the prior art, the present application includes the following beneficial effects:

[0128] (1) The insulin solution standard material regulated by the method of the present application has interchangeability between commonly used luminescence immunoassay systems, and can be used for calibration of these conventional chemiluminescence immunoassay systems. Therefore, it is no longer necessary to develop complex matrix standard materials to ensure interchangeability, thereby reducing the development cost of the interchangeable protein standard material, and at the same time, a wide range of calibration standard materials can be easily prepared according to the requirements of the calibration range, so that the interchangeability standard material is no longer restricted by the limited number of abnormal value serum matrix raw materials.

[0129] (2) The active human insulin solution standard material provided by the present application replaces the previous matrix standard material, shortens the value traceability chain of ISO 17511:2020, and helps to reduce the measurement uncertainty of the value transmission terminal. The uncertainty of the interchangeable active human insulin solution standard material valued by the method of the present application is much smaller than the uncertainty level of the determination of human insulin in complex matrix such as serum by isotope dilution mass spectrometry, and can meet the current requirements of the total error of human insulin clinical test items, thereby providing a new idea for the development of interchangeable standard materials, and helping to carry out the standardization of protein in vitro diagnosis results. BRIEF DESCRIPTION OF DRAWINGS

[0130] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0131] Figure 1 Value traceability transmission schematic diagram for ISO 17511:2020 which can be completely traced to SI unit;

[0132] Figure 2 Cross-linking of disulfide bonds between the two sequences of human insulin SEQ ID No. 1 and SEQ ID No. 2 of the present application, and within the sequence (Note: A-Chain represents SEQ ID No. 1; B-Chain represents SEQ ID No. 2);

[0133] Figure 3 Uncertainty source schematic diagram of the active human insulin solution standard material of the present application;

[0134] Figure 4 Different insulin and Zn 2+Circular dichroism spectra of solutions with different ratios (Note: I represents insulin, Z represents Zn 2+ );

[0135] Figure 5 For this application, different insulin and Zn 2+ Mass photometric spectra of solutions of the ratio;

[0136] Figure 6 Sensorgram for determination of concentration of immunoaffinity activity of active insulin raw material for this application;

[0137] Figure 7 Long-term stability results of active human insulin solution standard material (level 1) for this application;

[0138] Figure 8 Long-term stability results of active human insulin solution standard material (level 2) for this application;

[0139] Figure 9 Long-term stability results of active human insulin solution standard material (level 3) for this application;

[0140] Figure 10 The short-term stability results of the active human insulin solution standard material (level 1) at 4°C for this application;

[0141] Figure 11 The short-term stability results of the active human insulin solution standard material (level 2) at 4°C for this application;

[0142] Figure 12 The short-term stability results of the active human insulin solution standard material (level 3) at 4°C for this application;

[0143] Figure 13 The short-term stability results of the active human insulin solution standard material (level 1) at room temperature for this application;

[0144] Figure 14 The short-term stability results of the active human insulin solution standard material (level 2) at room temperature for this application;

[0145] Figure 15 The short-term stability results of the active human insulin solution standard material (level 3) at room temperature for this application;

[0146] Figure 16 This is the result of the interchangeability evaluation of active human insulin solution standard material according to the CLSI EP14 standard for this application. DETAILED DESCRIPTION

[0147] The application will be further described in conjunction with specific examples. The following description is merely exemplary of the application and is in no way intended to limit the application, as described herein is one preferred embodiment, however, any skilled person familiar with the art will be able to make minor changes or modifications to the disclosed technical content without departing from the scope of the technical solutions of the application, and all equivalent embodiments are equivalent to the equivalent embodiments, and all belong to the scope of the technical solutions.

[0148] Unless otherwise specified, the raw materials in the examples of the application are purchased through commercial channels and are directly used without any special treatment.

[0149] Unless otherwise specified, the analysis methods in the examples all use the conventional settings of instruments or equipment and conventional analysis methods.

[0150] Example 1

[0151] The preparation steps of the active human insulin solution standard material with interchangeability provided by the application are as follows:

[0152] I. Purification of human insulin

[0153] The human insulin sequence described in the application has a protein sequence as shown in SEQ ID No. 1 and SEQ ID No. 2:

[0154] SEQ ID No. 1: GIVEQCCTSICSLYQLENYCN

[0155] SEQ ID No. 2: FVNQHLCGSHLVEALYLVCGERGFFYTPKT

[0156] The cross-linking of disulfide bonds between the sequences of SEQ ID No. 1 and SEQ ID No. 2, and within the sequences is as shown in Figure 2

[0157] The raw material is a commercially available human insulin biochemical reagent from Sigm-Aldrich Company, which has a HPLC purity of 97.5%. Due to the low purity, it cannot be directly used for the development of standard materials. In order to obtain a high-purity human insulin standard material candidate, the commercially available human insulin is further purified by reverse phase high performance liquid chromatography and molecular sieve. The purification conditions used are as follows:

[0158] Mobile phase A: 0.2 mol / L sodium sulfate buffer: acetonitrile (82:18)

[0159] Mobile phase B: acetonitrile: water (50:50)

[0160] The flow phase gradient is shown in Table 1.

[0161] ​Table 1 Reversed-phase HPLC mobile phase gradient for insulin purification

[0162] Time / min A％ B％ 0 78 22 36 78 22 61 33 67 67 33 67 70 78 22 80 78 22

[0163] Chromatographic column: Vydac C8, 250 mm × 4.6 mm

[0164] Detection wavelength: 220nm

[0165] Flow rate: 1 mL / min

[0166] Column temperature: 40°C

[0167] Injection volume: 100 μL

[0168] The above chromatographic conditions effectively separate human insulin from A21-desaminated human insulin, and the human insulin chromatographic peak is collected to complete purification. The collected human insulin fraction is further purified using molecular sieves using PBS and water as the mobile phase, respectively. The peak elution time is monitored by absorbance at 280 nm to separate insulin aggregates from monomeric insulin. The specific separation conditions are as follows:

[0169] Injection volume: 100 μL

[0170] Chromatographic column: TSKGel 2000SWxl gel exclusion chromatography column

[0171] Gradient: Isocratic elution

[0172] Flow rate: 0.5 mL / min

[0173] Analysis time: 35 minutes

[0174] Detection wavelength: 280nm

[0175] Sample concentration: 1.0 mg / mL

[0176] Mobile phase: 1× PBS and water

[0177] The fractions within the monomeric insulin peak period were collected and freeze-dried (cold trap temperature -70°C, vacuum degree 1 Pa, freeze-drying for 48 hours) to obtain high-purity human insulin raw material.

[0178] 2. Self-assembly of human insulin

[0179] The purified high-purity human insulin raw material was dissolved in a 1×PBS solution containing zinc acetate. The insulin concentration was 0.02 mg / mL and the molar ratio of insulin to zinc ions in the solution was 6:60. The solution was placed in a 4°C refrigerator for self-assembly for 24 hours.

[0180] 3. Confirmation of the structure of insulin self-assembly complex

[0181] The self-assembled complex of insulin prepared in step 2 in the presence of zinc ions was subjected to circular dichroism and mass spectrometry scanning analysis, wherein the circular dichroism scanning path length was 1 cm, the scanning wavelength range was 200-400 nm, and the scanning speed was 50 nm / min. The mass spectrometer was scanned according to the default conditions. At the same time, the circular dichroism spectrum and mass spectrometry spectrum of the human insulin solution of the same concentration prepared in PBS without zinc ions were scanned for comparison. The results are shown in FIG. Figure 4 and Figure 5 shown.

[0182] Depend on Figure 4 It can be seen that when there is no Zn in the solution 2+ When the solution contains Zn 2 + When the concentration gradually increased, the circular dichroism spectrum of the insulin solution changed, the peak intensity decreased and became flat. These changes indicate that the concentration of Zn 2+ Insulin forms aggregates under the action of Figure 5 It can be seen that as the proportion of zinc ions in the solution increases, the peak of the spectrum gradually shifts to the right, proving that the scanning mass of the compound is also increasing, indicating that in the presence of Zn 2+ Insulin aggregates were formed in the presence of

[0183] 4. Purification of Active Human Insulin

[0184] A high-performance liquid chromatography coupled with surface plasmon resonance (SPR) spectroscopy was constructed, using 1× PBST as the mobile phase and running buffer, 0.5 mmol / L sodium hydroxide as the regeneration solution, and human insulin monoclonal antibodies coupled to a gold foil surface with carboxymethyl cellulose as the detector for the SPR signal. The liquid phase separation conditions were as follows:

[0185] Injection volume: 100 μL

[0186] Chromatographic column: TSKGel 2000SWxl gel exclusion chromatography column

[0187] Gradient: Isocratic elution

[0188] Flow rate: 0.5 mL / min

[0189] Analysis time: 35 minutes

[0190] Sample concentration: 1.0 mg / mL

[0191] Mobile phase: 1×PBST

[0192] The insulin complex is subjected to online separation and online SPR spectrum detection using a molecular sieve column, and fractions with a signal slope rising by more than 30 degrees on the sensorgram are collected and freeze-dried to obtain active insulin raw materials.

[0193] 5. Determination of immunoaffinity concentration and specific activity

[0194] Weigh 1 mg of the active insulin raw material from Step 4 into a 50 mL volumetric flask. Add 1× PBST to a volumetric volume of 0.02 mg / mL of active human insulin solution. While preparing, place the volumetric flask on a balance and weigh 50096 mg of the 1× PBST solution. Determine the immunoaffinity activity concentration of the active human insulin solution from Step 4 using a surface plasmon resonance spectrometer. Set the instrument temperature to 25°C, and the sample chamber temperature to 25°C. Three pre-start cycles were set as background noise references to avoid the influence of baseline drift on experimental accuracy. The flow rate was 30 μL / min. Two blank controls were set to remove the background interference of sample buffer: the sample buffer was injected at flow rates of 5 and 100 μL / min, the contact time was 36 s, the dissociation time was 120 s, and the regeneration flow rate was 30 μL / min for 30 s. The flow rates of each sample were set at 5 and 100 μL / min respectively. After the experiment, the insulin immunoaffinity activity concentration c was calculated using the instrument's built-in analysis software. raw,active , the result is 0.021mg / mL, the sensor graph is as follows Figure 6 shown.

[0195] Take 20 μL of the prepared active human insulin solution, weigh it and add 0.01 mg / g of 13 C isotope-labeled proline, valine, leucine, isoleucine, and phenylalanine (with at least 3 isotope-labeled atoms), were centrifuged, concentrated, and dried. 500 μL of 8 mol / L concentrated hydrochloric acid was added, nitrogen was purged, the container was sealed, and the product was hydrolyzed in a 110°C oven for 48 hours. After hydrolysis, the content of the above amino acids in the hydrolyzate was determined by isotope dilution mass spectrometry using national standards for proline, valine, leucine, isoleucine, and phenylalanine. The physicochemical concentration of the purified active human insulin solution was calculated based on the protein sequences shown in SEQ ID No. 1 and SEQ ID No. 2. raw,chem :

[0196]

[0197] Where C AA is the concentration of amino acids in insulin hydrolysate determined by isotope dilution mass spectrometry; M AA is the molecular weight of the amino acid; N AAis the number of the amino acid contained in insulin; M insulin is the molecular weight of insulin.

[0198] Then calculate the purity P of active human insulin raw material according to formula (1): raw , the result is 0.857g / g.

[0199]

[0200] The density of the prepared active human insulin solution was measured using a density meter (unit: g / mL). raw,chem (Unit: mg / g) and the results of immunoaffinity activity concentration c raw,active (Unit: mg / mL), and the specific activity R of the two was calculated according to formula (2), and the result was 0.95.

[0201]

[0202] VI. Preparation of Interchangeable Active Human Insulin Solution Standard

[0203] A 7% bovine serum albumin (BSA) solution containing 200 mmol / L zinc acetate was prepared as a diluent. The BSA used had a high performance liquid chromatography purity of 99.2%, and the zinc acetate used was of analytical grade.

[0204] Under the condition of ensuring the ambient temperature at 20±2℃, accurately weigh 1.0195mg of the active human insulin raw material in step 4, dissolve it with the prepared solvent, and then transfer it to a 10mL volumetric flask to make up the volume to obtain the insulin mother solution. According to the weighed mass, the purity of human insulin and the volume of the volumetric flask, the insulin concentration of the mother solution is 1.504×10 7 pmol / L, the calculation process is as follows:

[0205]

[0206] Where C hINS，0 Indicates the concentration of No. 0 active human insulin solution, m hINS represents the measured mass of active human insulin raw material, q hINS Indicates the purity of active human insulin raw materials, V indicates the volume of the volumetric flask used to prepare the solution, M hINS Indicates the molecular weight of human insulin.

[0207] Continue to pipette 1.000mL of the mother solution, transfer it to a 100mL volumetric flask and dilute to volume to obtain solution 1. Based on the volume pipetted, the concentration of insulin in the mother solution and the volume of the volumetric flask, the concentration of insulin in solution 1 was calculated to be 1.504×10 5pmol / L, the calculation process is as follows:

[0208]

[0209] Where C hINS，1 Indicates the concentration of human insulin in solution 1#, C hINS，0 It represents the concentration of No. 0 active human insulin solution, V0 represents the volume of No. 0 active human insulin solution transferred, and V1 represents the volume of the volumetric flask used to prepare No. 1 solution.

[0210] Continue to pipette 95.5 μL of solution 1, transfer it to a 100 mL volumetric flask, and dilute to volume to obtain solution 2. Based on the volume transferred, the insulin concentration in solution 1, and the volume of the volumetric flask, the insulin concentration in solution 2 is calculated to be 143.67 pmol / L. The calculation process is as follows:

[0211]

[0212] Where C hINS，2 Indicates the concentration of human insulin in solution 2#, C hINS，1 represents the concentration of human insulin in 1# solution, V1 represents the volume of 1# solution transferred, and V2 represents the volume of the volumetric flask used to prepare 2# solution.

[0213] Use a pipette to transfer 174.8 μL of solution 1# to a 250 mL volumetric flask and dilute to volume to obtain solution 3#. Based on the transferred volume, the insulin concentration in solution 1#, and the volume of the volumetric flask, the insulin concentration in solution 3# is calculated to be 105.19 pmol / L. The calculation process is as follows:

[0214]

[0215] Where C hINS，3 Indicates the concentration of human insulin in solution 3#, C hINS，1 represents the concentration of human insulin in 1# solution, V1 represents the volume of 1# solution transferred, and V3 represents the volume of the volumetric flask used to prepare 3# solution.

[0216] Use a pipette to transfer 74.4 μL of solution 1# to a 250 mL volumetric flask and dilute to volume to obtain solution 4#. Based on the transferred volume, the insulin concentration in solution 1#, and the volume of the volumetric flask, the insulin concentration in solution 4# is calculated to be 44.77 pmol / L. The calculation process is as follows:

[0217]

[0218] Where C hINS，4 Indicates the concentration of human insulin in solution 4#, ChINS，1 C1 represents the concentration of human insulin in the 1# solution, V1 represents the volume of the 1# solution removed, and V4 represents the volume of the volumetric flask used when preparing the 4# solution.

[0219] The 2#, 3#, and 4# solutions described above are three levels of active human insulin solution standard material candidates. The solutions are thoroughly mixed, and the solutions are divided into clean brown ampoules under shaking, with 500 μL in each unit, 200 units accumulated for each level, and stored in a -70°C refrigerator.

[0220] Seven, homogeneity and stability test of the standard material

[0221] 1. Homogeneity test of the active human insulin solution standard material

[0222] The randbetween function in Excel is used to randomly extract the continuously numbered divided units (P). When P≤200, the number of extracted divided units m is not less than 11; when 200

[0223] When m standard material candidates are extracted, m sets of precision measurement data are obtained under the condition of repeatability n times according to the test method as follows:

[0224] Number 1: x 11 ,x 12 ... x 1n , average value

[0225] Number 2: x 21 ,x 22 ... x 2n , average value ......

[0227] No. m:x m1 ,x m2 ......x mn ,average value

[0228] average value:

[0229] Statistic N: N = m·n Formula (4)

[0230] The sum of squares of differences between groups:

[0231] The sum of squares of within-group differences:

[0232] Between-group degrees of freedom: v1 = m-1; within-group degrees of freedom: v2 = Nm

[0233]

[0234] Statistics F:

[0235] Among them S1 2 is the between-group variance, S2 2 is the within-group variance. According to the degrees of freedom (v1, v2) and the given significance level α = 0.05, F is obtained from the table. α The critical value The F value calculated by formula (9) and F α For comparison, if F <F α If there is no significant difference between the groups, the insulin concentration of the active human insulin solution standard substance in the packaging unit is uniform. Otherwise, the insulin concentration of the active human insulin solution standard substance in the packaging unit is uneven.

[0236] Using the Beijing Leadman chemiluminescence immunoassay system, 11 bottles of standard material candidates were randomly selected from each level of the packaged active human insulin solution standard material. Each bottle sample was analyzed three times. The homogeneity test results are shown in Tables 2 to 4.

[0237] Table 2 Homogeneity test of active human insulin solution standard material (level 1)

[0238]

[0239] Table 3 Homogeneity test of active human insulin solution standard material (level 2)

[0240]

[0241]

[0242] Table 4 Homogeneity test of active human insulin solution standard material (level 3)

[0243]

[0244]

[0245] Therefore, after the homogeneity test, the F values ​​calculated for the three levels of active human insulin solution standard substances are all less than the F critical value, and therefore the three levels of active human insulin solution standard substances prepared are all homogeneous.

[0246] When performing the homogeneity test, the injection volume of the chemiluminescence immunoassay is 30 μL each time, so the minimum sampling volume of the three-level active human insulin solution standard material is 30 μL.

[0247] 2. Long-term stability test of active human insulin solution standard substance

[0248] The duration of the long-term stability test is not less than 6 months. During the long-term stability test period, according to the principle of dense at the beginning and sparse at the end, select not less than 5 time points k. At each time point, extract the active human insulin solution standard substance from at least 2 packaging units stored at -70°C in step 6, and perform three repeated tests on the insulin concentration of the active human insulin solution standard substance in each packaging unit using a fluorescence or luminescence immunoassay. Then calculate the arithmetic mean of these measurement results to obtain the arithmetic mean Y of the measurement results at each time point. i , the arithmetic mean value Y of the above measurement results i The corresponding measurement time is fitted according to the following linear model:

[0249] Y i =b0+bX formula (10)

[0250] In formula (10), b0 represents the intercept, b represents the regression coefficient (slope); X represents the test time, unit (month); Y i It represents the arithmetic mean of the insulin concentration in the active human insulin solution standard substance at each time point.

[0251] The regression coefficient (slope) and intercept are calculated according to formula (11) and formula (12):

[0252]

[0253]

[0254] In formula (11) and formula (12), X i Indicates the detection time at the i-th time point, in months; Y i represents the arithmetic mean of the insulin concentration determination results of the active human insulin solution standard substance in the above packaging unit at the i-th time point; It represents the average detection time of all time points in months; represents the arithmetic mean of the insulin concentration determination results of the active human insulin solution standard substance in the above packaging unit at all time points; k represents the number of time points.

[0255] Calculate the s value according to formula (13):

[0256]

[0257] The meanings of the symbols in formula (13) are the same as those in formula (10), formula (11) and formula (12).

[0258] Calculate the value of s(b) according to formula (14):

[0259]

[0260] Look up the t distribution table and get t 0.95,k-2 The value of

[0261] If |b|<t 0.95,k-2 ·s(b), it indicates that the slope is not significant and no instability is observed, indicating that the active human insulin solution standard substance is long-term stable; otherwise, it indicates that the active human insulin solution standard substance is unstable.

[0262] The stability test was conducted at 0, 1, 2, 4 and 6 months using chemiluminescence immunoassay. Three packages were sampled each time and each package was tested twice in parallel. The long-term stability test results of the standard substances of human insulin solution with different levels of activity are shown in Tables 5 to 7, and the trend graph is shown in Figures 7 to 9 shown.

[0263] Table 5 Long-term stability study results of active human insulin solution standard substance (level 1) (pmol / L)

[0264]

[0265]

[0266] The data in Table 5 are fitted into a straight line, with x representing time and y representing the characteristic value of the standard substance. The slope is k = 0.161206897 and the intercept is b = 143.0008621.

[0267] The standard deviation of the straight line can be calculated by the following formula:

[0268]

[0269] Taking the square root s = 1.096218684, the uncertainty of the slope is calculated by the following formula:

[0270]

[0271] The t-distribution factor for 2 degrees of freedom and p = 0.95 (95% confidence level) is equal to 3.18, since

[0272] |k| = 0.161206897 < t 0.95,n-2 • s(k) = 0.724293007

[0273] Therefore, the active human insulin solution reference material candidate (level 1) is stable at -70°C.

[0274] Table 6 Results of long-term stability study of active human insulin solution reference material (level 2) (pmol / L)

[0275] Time / month 1 2 3 average 0 102.5 103.4 107.9 104.6 1 107.9 103.8 106.3 106.0 2 106.5 104.5 106.4 105.8 4 105.5 102.4 108.0 105.3 6 107.4 103.5 105.5 105.5

[0276] The data in Table 6 are fitted into a straight line with x representing time and y representing the characteristic value of the reference material, then the slope k = 0.046551724 and the intercept b = 105.3189655.

[0277] The standard deviation of the straight line can be calculated by the following formula:

[0278]

[0279] Taking the square root s = 0.611480209, the uncertainty of the slope is calculated by the following formula:

[0280]

[0281] The t-distribution factor for 2 degrees of freedom and p = 0.95 (95% confidence level) is equal to 3.18, since

[0282] |k| = 0.046551724 < t 0.95,n-2 • s(k) = 0.404016868

[0283] Therefore, the active human insulin solution reference material (level 2) is stable at -70°C.

[0284] Table 7 Results of long-term stability study of active human insulin solution reference material (level 3) (pmol / L)

[0285] Time / month 1 2 3 average 0 45.8 43.4 44.7 44.6 1 44.2 46.2 46.1 45.5 2 46.3 44.5 44.7 45.2 4 43.6 46.2 44.6 44.8 6 43.7 45.5 43.9 44.4

[0286] The data in Table 7 are fitted into a straight line, with x representing time and y representing the characteristic value of the standard substance. The slope is k = -0.094827586 and the intercept is b = 45.14655172.

[0287] The standard deviation of the straight line can be calculated as follows:

[0288]

[0289] Take the square root s = 0.44398923, and calculate the uncertainty of the slope using the following formula:

[0290]

[0291] The t-distribution factor with 2 degrees of freedom and p = 0.95 (95% confidence level) is equal to 3.18, since

[0292] |k|=0.094827586<t 0.95,n-2 s(k)=0.293352321

[0293] Therefore, the active human insulin solution standard material (level 3) is stable at -70°C.

[0294] 3. Short-term stability test of insulin concentration in active human insulin solution standard material

[0295] The duration of the short-term stability test is no less than 7 days. During the short-term stability test period, according to the principle of close coverage at the beginning and sparse coverage at the end, no less than 5 time points k are selected. At each time point, at least two subpackaging units are removed from the above step 2 and placed in a temperature range of -20°C to 40°C for inspection. The active human insulin solution standard substance in the above subpackaging units is extracted and the insulin concentration of the active human insulin solution standard substance in each subpackaging unit is tested three times using a fluorescence or luminescence immunoassay. The arithmetic mean of these measurement results is calculated to obtain the arithmetic mean Y of the measurement results at each time point. i , fit the arithmetic mean of the above measurement results and the corresponding measurement time according to the following linear model:

[0296] Y i =b0+bX formula (15)

[0297] In formula (15), b0 represents the intercept; b represents the regression coefficient (slope); X represents the test time, unit (day); Y i It represents the arithmetic mean of the insulin concentration in the active human insulin solution standard substance at each time point.

[0298] The regression coefficient (slope) and intercept are calculated according to formula (16) and formula (17):

[0299]

[0300] In formula (16) and formula (17), X i Y represents the detection time at the i-th time point, in days; i represents the arithmetic mean of the insulin concentration determination results of the active human insulin solution standard substance in the above packaging unit at the i-th time point; It represents the average detection time of all time points, in days; represents the arithmetic mean of the insulin concentration determination results of the active human insulin solution standard substance in the above packaging unit at all time points; k represents the number of time points.

[0301] Calculate the s value according to formula (18):

[0302]

[0303] The meanings of the symbols in formula (18) are the same as those in formula (15), formula (16) and formula (17).

[0304] Calculate the value of s(b) according to formula (19):

[0305]

[0306] Look up the t distribution table and get t 0.95,k-2 The value of

[0307] If |b|<t 0.95,k-2 ·s(b), it indicates that the slope is not significant and no instability is observed, indicating that the active human insulin solution standard substance is short-term stable; otherwise, it indicates that the active human insulin solution standard substance is unstable.

[0308] The short-term stability of candidate standard substances was tested using the Beijing Leadman chemiluminescence immunoassay. The short-term stability test temperatures were 4°C, 25°C, and 40°C, respectively. The test time at each temperature was 7 days. The table below lists the average results of the stability test of 3 samples taken at different intervals.

[0309] (1) Short-term stability test at 4°C. The results are shown in Tables 8 to 10. Figures 10 to 12 shown.

[0310] Table 8 Results of short-term stability study of active human insulin solution standard substance (level 1) at 4°C (pmol / L)

[0311] Time / day 1 2 3 average 0 145.1 144.7 141.4 143.7 1 140.2 140.7 138.8 139.9 3 140.5 141.4 140.2 140.7 5 138.5 139.1 141.3 139.6 7 139.1 135.0 141.9 138.7

[0312] The data in Table 8 are fitted into a straight line, with x representing time and y representing the characteristic value of the standard substance. The slope is k = -0.531097561 and the intercept is b = 142.2195122.

[0313] The standard deviation of the straight line can be calculated as follows:

[0314]

[0315] Take the square root s = 1.346140221, and the uncertainty of the slope is calculated using the following formula:

[0316]

[0317] The t-distribution factor with 2 degrees of freedom and p = 0.95 (95% confidence level) is equal to 3.18, since

[0318] |k|=0.531097561<t 0.95,n-2 s(k)=0.74802216

[0319] Therefore, the active human insulin solution standard substance candidate (level 1) is stable at 4°C. Table 9 Results of short-term stability study of active human insulin solution standard substance (level 2) at 4°C (pmol / L)

[0320] Time / day 1 2 3 average 0 107.4 104.8 103.6 105.3 1 104.7 108.5 107.2 106.8 3 104.6 108.9 106.7 106.7 5 105.6 109.6 104.7 106.6 7 106.5 107.6 108.6 107.6

[0321] The data in Table 9 are fitted into a straight line, with x representing time and y representing the characteristic value of the standard substance. The slope is k = 0.228658537 and the intercept is b = 105.8682927.

[0322] The standard deviation of the straight line can be calculated as follows:

[0323]

[0324] Take the square root s = 0.584539983, and calculate the uncertainty of the slope using the following formula:

[0325]

[0326] The t-distribution factor with 2 degrees of freedom and p = 0.95 (95% confidence level) is equal to 3.18, since

[0327] |k|=0.228658537<t 0.95,n-2 s(k)=0.324816727

[0328] Therefore, the slope is not significant because the insulin (human) solution standard material (level 2) for calibration of the fully automatic closed luminescent immunoassay analyzer is stable at 4°C.

[0329] Table 10 Results of short-term stability study of active human insulin solution standard substance (level 3) at 4°C (pmol / L)

[0330] Time / day 1 2 3 average 0 45.0 44.9 45.6 45.2 1 43.7 44.6 45.0 44.4 3 44.6 45.5 44.9 45.0 5 44.0 43.0 44.0 43.7 7 43.9 44.4 43.5 43.9

[0331] The data in Table 10 are fitted into a straight line, with x representing time and y representing the characteristic value of the standard substance. The slope is k = -0.17804878 and the intercept is b = 45.0097561.

[0332] The standard deviation of the straight line can be calculated as follows:

[0333]

[0334] Take the square root s = 0.480345404, and the uncertainty of the slope is calculated using the following formula:

[0335]

[0336] The t-distribution factor with 2 degrees of freedom and p = 0.95 (95% confidence level) is equal to 3.18, since

[0337] |k|=0.17804878<t 0.95,n-2 s(k)=0.266917964

[0338] Therefore, the slope is insignificant, and thus the active human insulin solution standard material (level 3) is stable at 4°C.

[0339] (2) Short-term stability test at room temperature. The results are shown in Tables 11 to 13. Figures 13 to 15 shown.

[0340] First, short-term stability tests at room temperature were conducted at 0, 1, 3, 5, and 7 days. The results showed that the stability of active human insulin solution standard substances at multiple levels at room temperature was less than 1 day. Therefore, the stability test was changed to be conducted at hours, and short-term stability tests were conducted at 0, 1, 2, 4, and 6 hours respectively.

[0341] Table 11 Results of room temperature stability study of active human insulin solution standard substance (level 1) (pmol / L)

[0342] Time / hour 1 2 3 average 0 140.3 147.1 143.7 143.7 1 143.5 146.0 142.2 143.9 2 147.5 146.2 141.4 145.0 4 145.1 143.2 142.5 143.6 6 139.4 143.2 140.5 141.0

[0343] The data in Table 11 are fitted into a straight line, with x representing time and y representing the characteristic value of the standard substance. The slope is k = -0.449137931 and the intercept is b = 144.6077586.

[0344] The standard deviation of the straight line can be calculated as follows:

[0345]

[0346] Take the square root s = 1.156428807, and calculate the uncertainty of the slope using the following formula:

[0347]

[0348] The t-distribution factor with 2 degrees of freedom and p = 0.95 (95% confidence level) is equal to 3.18, since

[0349] |k|=0.449137931<t 0.95,n-2 s(k)=0.764075007

[0350] Therefore, the slope is insignificant, and the active human insulin solution standard material (level 1) is stable at room temperature for 6 hours.

[0351] Table 12 Results of room temperature stability study of active human insulin solution standard substance (level 2) (pmol / L)

[0352] Time / hour 1 2 3 average 0 105.2 106.7 103.7 105.2 1 101.8 104.7 104.1 103.5

[0353]

[0354] The data in Table 12 are fitted into a straight line, with x representing time and y representing the characteristic value of the standard substance. The slope is k = -0.185344828 and the intercept is b = 104.2818966.

[0355] The standard deviation of the straight line can be calculated as follows:

[0356]

[0357] Taking the square root s = 0.966612167, the uncertainty of the slope is calculated using the following formula:

[0358]

[0359] The t-distribution factor with 2 degrees of freedom and p = 0.95 (95% confidence level) is equal to 3.18, since

[0360] |k|=0.185344828<t 0.95,n-2 s(k)=0.63865946

[0361] Therefore, the slope is insignificant, and the active human insulin solution standard material (level 2) is stable at room temperature for 6 hours.

[0362] Table 13 Results of room temperature stability study of active human insulin solution standard substance (level 3) (pmol / L)

[0363] Time / hour 1 2 3 average 0 44.8 44.6 45.0 44.8 1 45.8 45.3 45.3 45.5 2 44.9 44.6 45.5 45.0 4 45.3 45.1 45.9 45.4 6 44.7 44.5 44.4 44.5

[0364] The data in Table 13 are fitted into a straight line, with x representing time and y representing the characteristic value of the standard substance. The slope is k = -0.061206897 and the intercept is b = 45.19913793.

[0365] The standard deviation of the straight line can be calculated as follows:

[0366]

[0367] Taking the square root s = 0.449105113, the uncertainty of the slope is calculated using the following formula:

[0368]

[0369] The t-distribution factor with 2 degrees of freedom and p = 0.95 (95% confidence level) is equal to 3.18, since

[0370] |k|=0.061206897<t 0.95,n-2 s(k)=0.296732484

[0371] Therefore, the slope is insignificant, and the active human insulin solution standard material (level 3) is stable at room temperature for 6 hours.

[0372] (3) Short-term stability test at 40°C. The results are shown in Tables 14 to 16.

[0373] Table 14 Stability test results of active human insulin solution standard substance (level 1) at 40°C (pmol / L)

[0374] Time / day 1 2 3 average 0 145.1 144.7 141.4 143.7 1 99.2 99.2 97.0 98.4

[0375] The stability test data at day 1 and day 0 in Table 14 were subjected to a Student t-test of the two groups' means. The mean difference test showed p = 0.0001, indicating that the difference between the short-term stability test data at day 1 and day 0 was highly significant. Therefore, the active human insulin solution standard substance was unstable at 40°C.

[0376] Table 15 Stability test results of active human insulin solution standard substance (level 2) at 40°C (pmol / L)

[0377] Time / day 1 2 3 average 0 107.4 104.8 103.6 105.3 1 71.4 70.0 78.1 73.2

[0378] The stability test data for day 1 and day 0 in Table 15 were subjected to a Student's t-test, and the difference in means was p = 0.0003. Therefore, there is a significant difference between the short-term stability test data for day 1 and day 0, indicating that the active human insulin solution standard substance (level 2) is unstable at 40°C.

[0379] Table 16 Stability test results of active human insulin solution standard substance (level 3) at 40°C (pmol / L)

[0380] Time / day 1 2 3 average 0 45.8 43.4 44.7 44.6 1 12.7 19.5 23.3 18.5

[0381] The stability test data for day 1 and day 0 in Table 16 were subjected to a Student t-test of the two group means. The mean difference test showed p = 0.001, indicating that there was a significant difference between the short-term stability test data for day 1 and day 0. Therefore, the active human insulin solution standard substance (level 3) was unstable at 40°C.

[0382] 8. Determination of Standard Value of Active Human Insulin Solution Standard Substance

[0383] The standard values ​​of active human insulin solution standard substances at various concentration levels were calculated according to formula (20).

[0384]

[0385] In formula (20), c CRM Indicates the certified value of active human insulin solution standard substance, unit is pmol / L; m STD Indicates the mass of weighed active human insulin raw material, in g; P raw Indicates the purity of active human insulin raw materials, unit is g / g; M insulin Indicates the molar mass of human insulin in g / mol; V STD Indicates the volume of the volumetric flask used to prepare the solution, in L.

[0386] The values ​​of the active human insulin solution standard substance at three levels were calculated and summarized in Table 17.

[0387] Table 17 Determination results of three levels of active human insulin solution standard substances (pmol / L)

[0388] serial number Level 1 Level 2 Level 3 Fixed value results 143.67 105.19 44.77

[0389] IX. Uncertainty Assessment of the Determination Results of Active Human Insulin Solution Reference Material

[0390] According to the uncertainty assessment mathematical model (Formula (20)), the uncertainty of the determination result of the active human insulin solution standard substance comes from the uncertainty introduced in the determination process, the uncertainty introduced by the homogeneity of the standard substance, and the uncertainty introduced by the long-term stability. The specific sources of uncertainty are as follows: Figure 3 shown.

[0391] 1. Uncertainty introduced in the process of setting the value

[0392] (1) When preparing insulin mother solution, accurately weigh 1.0195 mg of active human insulin raw material, dissolve it in the prepared solvent, and then transfer it to a 10 mL volumetric flask to make up the volume. The concentration can be calculated using the following formula:

[0393]

[0394] Where c hINS Indicates the concentration of human insulin in the mother solution, m hINS Indicates the weighed mass of active human insulin raw material, P hINS Indicates the purity of active human insulin raw materials, V indicates the volume of the volumetric flask used to prepare the mother solution, M hINS Indicates the molecular weight of human insulin.

[0395] According to this model, taking into account the influence of the solvent blank detection limit, the uncertainty of the insulin mother solution concentration preparation result can be calculated by the following formula:

[0396]

[0397] Where, It represents the relative uncertainty of the preparation result of insulin mother solution concentration, dimensionless;

[0398] It represents the relative uncertainty of the quality of insulin purity standard material, dimensionless;

[0399] u r,V It represents the relative uncertainty of the volume of insulin mother solution, dimensionless;

[0400] u r,P It represents the relative uncertainty introduced by the standard substance of insulin purity, dimensionless;

[0401] It represents the relative uncertainty of the molar mass (molecular weight) of insulin, dimensionless;

[0402] It represents the relative uncertainty introduced by the detection limit of the solution blank verification method, which is dimensionless.

[0403] ① Take the balance for weighing the standard substance. According to the balance verification certificate, when the weighing value is 1 mg, the indication error is 0.0002 mg, the eccentricity error is -0.0014 mg, and the repeatability is 0.0008 mg. All of these are considered uniformly distributed. The uncertainty introduced by one weighing is:

[0404]

[0405] When weighing the standard substance, one is an empty pan and the other is gross weight. Because each weighing is an independent observation result, the standard uncertainty introduced by weighing the mass of the purity standard substance is:

[0406]

[0407] ② According to the calibration certificate of the volumetric flask, the U of the 10mL volumetric flask r =1% (k=2), so the standard uncertainty is U r / 2=0.5%; the temperature fluctuation range of the laboratory is ±2℃, and the volume expansion coefficient of the solvent water is 2.1×10 -2 (%·℃ -1 ). Using a rectangular distribution, the volume uncertainty caused by the difference between the calibration temperature and the operating temperature of the volumetric flask is: The uncertainty introduced by the 10mL volumetric flask used in this research is 0.00004% for the six times of volume determination. The uncertainty introduced by the synthetic 10mL volumetric flask is:

[0408]

[0409] ③The determination result of the purity standard substance of insulin is (0.857±0.024)g / g. Therefore, the relative standard uncertainty introduced by the purity of the insulin standard substance is:

[0410] u r,P =0.024 / 2 / 0.857×100%=1.4%

[0411] ④Based on the molecular formula of insulin C 257 H 383 N 65 O 77 S6, calculate the uncertainty introduced by the insulin molecular weight according to the following formula:

[0412]

[0413] Where, It represents the relative uncertainty of the molecular weight of insulin, dimensionless;

[0414] N i It represents the number of atoms of each element in an insulin molecule, dimensionless;

[0415] u i It represents the uncertainty of the relative atomic mass of each atom in the insulin molecule, in units of u;

[0416] M hINS Indicates the molecular weight of insulin, unit u.

[0417] According to the IUPAC International Atomic Weights Table, the relative atomic masses and uncertainties of the five atoms are:

[0418] C: 12.0107±0.0008;

[0419] H: 1.00794 ± 0.00007;

[0420] O: 15.9994 ± 0.0003;

[0421] N: 14.00674 ± 0.00007;

[0422] S:32.065±0.005.

[0423] therefore:

[0424]

[0425] ⑤ When performing the solvent blank test, the chemiluminescence immunoassay method was used. The detection limit of this method was 0.09 pmol / L, and the concentration of the prepared mother solution was 1.504×10 7 pmol / L, considering uniform distribution, the uncertainty component introduced by the method detection limit is:

[0426]

[0427] Therefore, the relative uncertainty of the insulin stock solution preparation result is:

[0428]

[0429] (2) Preparation of 1# insulin solution

[0430] During preparation, continue to pipette 1.000mL of the mother solution, transfer it to a 100mL volumetric flask and dilute to volume to obtain solution 1. Therefore, the mathematical model for uncertainty assessment is:

[0431]

[0432] Where c 1# Indicates the concentration of 1# insulin solution, unit is pmol / L;

[0433] c0 represents the molar concentration of insulin mother solution, in pmol / L;

[0434] V0 represents the volume of insulin stock solution, in mL;

[0435] V represents the volume of solution 1#, in mL.

[0436] Therefore, according to the uncertainty assessment mathematical model, and taking into account the uncertainty introduced by the detection limit of the solvent blank method, the uncertainty of the concentration result of solution 1# can be calculated by the following formula:

[0437]

[0438] Where u r,c1# It represents the relative uncertainty of the concentration result of 1# insulin solution, dimensionless;

[0439] It represents the relative uncertainty of the result of insulin mother solution concentration, dimensionless;

[0440] It represents the relative uncertainty of the volume of insulin mother solution, dimensionless;

[0441] u r,V It represents the relative uncertainty of the volume of 1# insulin solution, dimensionless;

[0442] It represents the uncertainty introduced by the detection limit of the solvent blank method and is dimensionless.

[0443] ① According to the calibration certificate of the 1mL pipette at the 1mL point, the permissible error of the capacity is ±1%, so the relative standard uncertainty is The volume uncertainty caused by the difference between the calibration temperature and the operating temperature is 0.024%. The relative uncertainty introduced by the 1mL pipette is:

[0444]

[0445] ② According to the calibration certificate of the volumetric flask, the correction value at the 100mL point is -0.03mL. Considering the relative uncertainty according to the rectangular distribution, The volume uncertainty caused by the difference between the calibration temperature and the operating temperature is 0.024%, and the uncertainty introduced by the six volume repeatability is 0.0001%. The uncertainty introduced by the 100mL volumetric flask is:

[0446]

[0447] ③ When performing the solvent blank test, the chemiluminescence immunoassay method was used. The detection limit of this method was 0.09 pmol / L, and the concentration of the prepared 1# was 1.504×10 5pmol / L, the uncertainty component introduced by the method detection limit was considered as uniformly distributed:

[0448]

[0449] Therefore, the uncertainty of the 1# solution was:

[0450]

[0451] (3) Preparation of human insulin solution (level 1)

[0452] When preparing the human insulin solution (level 1), a 200 L pipette was used to transfer the appropriate volume of the 1# solution into a 100 mL volumetric flask to complete the preparation process, so the uncertainty evaluation mathematical model was:

[0453]

[0454] In the formula, c 1# represents the concentration of the 1# insulin solution, with units of pmol / L;

[0455] c1 represents the molar concentration of the insulin solution (level 1), with units of pmol / L;

[0456] V 1# represents the volume of the 1# insulin solution, with units of mL;

[0457] V represents the volume of the insulin solution (level 1), with units of mL.

[0458] Therefore, according to the uncertainty evaluation mathematical model, considering the uncertainty introduced by the solvent blank method detection limit, the uncertainty of the concentration result of the insulin solution (level 1) can be calculated by the following formula:

[0459]

[0460] In the formula, represents the relative uncertainty of the concentration result of the insulin solution (level 1), dimensionless;

[0461] represents the relative uncertainty of the concentration result of the insulin 1# solution, dimensionless;

[0462] represents the relative uncertainty of the volume of the insulin 1# solution, dimensionless;

[0463] u r,V represents the relative uncertainty of the volume of the insulin solution (level 1), dimensionless;

[0464] It represents the uncertainty introduced by the detection limit of the solvent blank method and is dimensionless.

[0465] ① According to the calibration certificate of the 200μL pipette at the 200μL point, the permissible error of the capacity is ±1.5%, so the relative standard uncertainty is The volume uncertainty caused by the difference between the calibration temperature and the operating temperature is 0.024%. The relative uncertainty introduced by the 200μL pipette is:

[0466]

[0467] ② According to the calibration certificate of the volumetric flask, the correction value at the 100mL point is -0.03mL. Considering the relative uncertainty according to the rectangular distribution, The volume uncertainty caused by the difference between the calibration temperature and the operating temperature is 0.024%, and the uncertainty introduced by the six volume repeatability is 0.0001%. The uncertainty introduced by the 100mL volumetric flask is:

[0468]

[0469] ③ When performing the solvent blank test, the chemiluminescence immunoassay method was used. The detection limit of this method is 0.09 pmol / L. The prepared insulin solution (level 1) is 143.67 pmol / L. Considering the uniform distribution, the uncertainty component introduced by the method detection limit is:

[0470]

[0471] Therefore, the uncertainty of the insulin solution (level 1) is:

[0472]

[0473] (4) Preparation of human insulin solution (level 2)

[0474] When preparing human insulin solution (Level 2), a 200 μL pipette is used to transfer an appropriate volume of solution 1 into a 250 mL volumetric flask to complete the preparation process. Therefore, the uncertainty assessment mathematical model is:

[0475]

[0476] Where c 1# Indicates the concentration of 1# insulin solution, unit is pmol / L;

[0477] c2 represents the molar concentration of the insulin solution (level 2), in pmol / L;

[0478] V 1# Indicates the volume of 1# insulin solution, unit is mL;

[0479] V represents the volume of the insulin solution (level 2) in mL.

[0480] Therefore, according to the uncertainty assessment mathematical model, and taking into account the uncertainty introduced by the detection limit of the solvent blank method, the uncertainty of the concentration result of the insulin solution (level 2) can be calculated by the following formula:

[0481]

[0482] Where, It represents the relative uncertainty of the concentration result of insulin solution (level 2), dimensionless;

[0483] It represents the relative uncertainty of the concentration result of insulin 1# solution, dimensionless;

[0484] It represents the relative uncertainty of the volume of insulin 1# solution, dimensionless;

[0485] u r,V represents the relative uncertainty of the volume of insulin solution (level 2), dimensionless;

[0486] It represents the uncertainty introduced by the detection limit of the solvent blank method and is dimensionless.

[0487] ① According to the calibration certificate of the 200μL pipette at the 200μL point, the permissible error of the capacity is ±1.5%, so the relative standard uncertainty is The volume uncertainty caused by the difference between the calibration temperature and the operating temperature is 0.024%. The relative uncertainty introduced by the 200μL pipette is:

[0488]

[0489] ② According to the calibration certificate of the volumetric flask, the correction value at the 250mL point is -0.13mL. Considering the relative uncertainty according to the rectangular distribution, The volume uncertainty caused by the difference between the calibration temperature and the operating temperature is 0.024%, and the uncertainty introduced by the six volume repeatability is 0.0001%. The uncertainty introduced by the synthetic 250mL volumetric flask is:

[0490]

[0491] ③ When performing the solvent blank test, the chemiluminescence immunoassay method was used. The detection limit of this method is 0.09 pmol / L. The prepared insulin solution (level 2) is 105.19 pmol / L. Considering the uniform distribution, the uncertainty component introduced by the method detection limit is:

[0492]

[0493] Therefore, the uncertainty of the insulin solution (level 2) is:

[0494]

[0495] (5) Preparation of human insulin solution (level 3)

[0496] When preparing human insulin solution (Level 3), a 200 μL pipette is used to transfer an appropriate volume of solution 1 into a 250 mL volumetric flask to complete the preparation process. Therefore, the uncertainty assessment mathematical model is:

[0497]

[0498] Where c 1# Indicates the concentration of 1# insulin solution, unit is pmol / L;

[0499] c3 represents the molar concentration of the insulin solution (level 3), in pmol / L;

[0500] V 1# Indicates the volume of 1# insulin solution, unit is mL;

[0501] V represents the volume of the insulin solution (level 3) in mL.

[0502] Therefore, according to the uncertainty assessment mathematical model, and taking into account the uncertainty introduced by the detection limit of the solvent blank method, the uncertainty of the concentration result of the insulin solution (level 3) can be calculated by the following formula:

[0503]

[0504] Where, It represents the relative uncertainty of the concentration result of insulin solution (level 3), dimensionless;

[0505] It represents the relative uncertainty of the concentration result of insulin 1# solution, dimensionless;

[0506] It represents the relative uncertainty of the volume of insulin 1# solution, dimensionless;

[0507] u r,Vrepresents the relative uncertainty of the volume of insulin solution (level 3), dimensionless;

[0508] It represents the uncertainty introduced by the detection limit of the solvent blank method and is dimensionless.

[0509] ① According to the calibration certificate of the 200μL pipette at the 200μL point, the permissible error of the capacity is ±1.5%, so the relative standard uncertainty is The volume uncertainty caused by the difference between the calibration temperature and the operating temperature is 0.024%. The relative uncertainty introduced by the 200μL pipette is:

[0510]

[0511] ② According to the calibration certificate of the volumetric flask, the correction value at the 250mL point is -0.13mL. Considering the relative uncertainty according to the rectangular distribution, The volume uncertainty caused by the difference between the calibration temperature and the operating temperature is 0.024%, and the uncertainty introduced by the six volume repeatability is 0.0001%. The uncertainty introduced by the synthetic 250mL volumetric flask is:

[0512]

[0513] ③ When performing the solvent blank test, the chemiluminescence immunoassay method was used. The detection limit of this method is 0.09 pmol / L. The prepared insulin solution (level 3) is 44.77 pmol / L. Considering the uniform distribution, the uncertainty component introduced by the method detection limit is:

[0514]

[0515] Therefore, the uncertainty of the insulin solution (level 3) is:

[0516]

[0517] Therefore, the uncertainty of the determination process of active human insulin solution standard materials at various levels is shown in Table 18.

[0518] Table 18 Uncertainty introduced in the determination process of active human insulin solution standard substance

[0519]

[0520] 2. Uncertainty component introduced by uniformity

[0521] According to JJF1343-2022, the uncertainty introduced by the homogeneity of the reference material is equal to the standard deviation of the homogeneity between bottles. When the mean square between groups is greater than the mean square within groups, the following formula is used for calculation:

[0522]

[0523] When the between-group mean square is less than the within-group mean square, it is calculated according to the following formula:

[0524]

[0525] The uncertainty components introduced by the homogeneity of the three levels of active human insulin solution standard materials are calculated as shown in Table 19.

[0526] Table 19 Uncertainty components introduced by homogeneity of three levels of active human insulin solution standard materials pmol / L

[0527] level 1 2 3 <![CDATA[u bb ]]> 1.26 0.52 0.24

[0528] 3. Uncertainty components introduced by stability

[0529] The long-term stability of standard substances is calculated according to the following formula:

[0530] u sts =s k,sts ·t

[0531] The uncertainty components introduced by the stability of the three levels of active human insulin solution standard substances are calculated as shown in Table 20.

[0532] Table 20 Uncertainty components introduced by the stability of three levels of active human insulin solution standard substances (pmol / L)

[0533] level 1 2 3 <![CDATA[u lts ]]> 1.37 0.76 0.55

[0534] 4. Uncertainty of the fixed value result

[0535] The uncertainty components introduced by the setting process, uniformity and stability are synthesized to obtain the uncertainty of the setting result, as shown in Table 21.

[0536] Table 21 Uncertainty of the determination results of three levels of human insulin solution standard substances (pmol / L)

[0537]

[0538] Therefore, the results of the determination of the three levels of active human insulin solution standard substances are shown in Table 22:

[0539] Table 22 Determination results of three levels of active human insulin solution standard substances pmol / L

[0540] serial number Level 1 Level 2 Level 3 Fixed value results 143.7±6.3 105.2±4.2 44.8±2.0

[0541] 10. Interchangeability Evaluation of Active Human Insulin Solution Reference Material

[0542] 39 single serum samples covering the concentration range of the active human insulin solution standard substance prepared in step six were collected. These samples were centrifuged within 24 hours after blood collection and stored in a -70°C refrigerator. Together with the active human insulin solution standard substances of various levels prepared in step six, the insulin concentrations of clinical samples and standard substances were measured on 6 common domestic and foreign chemiluminescence immunoassay systems. These test systems include Siemens, Beckman, Roche, Abbott, Mindray and Antu. Each sample was measured twice in parallel. The results were averaged and evaluated according to the CLSI EP14 standard. The results are as follows: Figure 16 shown.

[0543] Depend on Figure 16 It can be seen that the three insulin solution standard substances prepared from active insulin (the figure also shows the active human insulin solution standard substance not described in detail in this example) are all within the 95% prediction interval determined by the patient sample results, indicating that the insulin solution standard substances at these three levels have good interchangeability.

[0544] In summary, the insulin solution standard material after regulation using the method of the present application has interchangeability between commonly used luminescent immunoassay systems and can be used for the calibration of these conventional chemiluminescent immunoassay systems. Therefore, there is no need to develop complex matrix standard materials to ensure interchangeability, thereby reducing the development cost of interchangeable protein standard materials, and at the same time, a wide range of calibration standard materials can be easily prepared according to the requirements of the calibration range, so that interchangeable standard materials are no longer restricted by the limited number of sources of abnormal value serum matrix raw materials. At the same time, since the solution standard material replaces the previous matrix standard material, the traceability transmission chain of ISO 17511:2020 is shortened, which helps to reduce the measurement uncertainty of the measurement value transmission terminal. The uncertainty of the interchangeable active human insulin solution standard material determined by the method of the present application is much smaller than the current uncertainty level of human insulin in complex matrices such as serum by isotope dilution mass spectrometry, and can meet the requirements of the total error of current human insulin clinical test items, thereby providing a new idea for the development of interchangeable standard materials and contributing to the standardization of protein in vitro diagnostic results.

[0545] The above descriptions are merely a few embodiments of the present application and do not constitute any form of limitation to the present application. Although the present application discloses the preferred embodiments as above, they are not intended to limit the present application. Any technical personnel familiar with the present profession, without departing from the scope of the technical solution of the present application, using the technical content disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. A method for preparing an interchangeable active human insulin solution standard substance, characterized in that: The steps include: (1) Purification of human insulin: Human insulin with a purity of 90-98% was selected and purified by reverse-phase high performance liquid chromatography and molecular sieve separation to obtain high-purity human insulin raw material; (2) Self-assembly of human insulin: The high-purity human insulin raw material was dissolved in a 1×PBS solution containing zinc ions and allowed to stand to obtain an insulin self-assembly complex; (3) Confirmation of the structure of the insulin self-assembled complex: The insulin self-assembled complex is subjected to circular dichroism and mass spectrometry spectrum scanning analysis. At the same time, a human insulin solution prepared with a PBS solution without zinc ions and having the same concentration as that in step (2) is subjected to circular dichroism and mass spectrometry spectrum scanning analysis as a comparison. Whether the self-assembled complex is formed is determined based on the scanning results. If not, repeat step (2); (4) Purification of active human insulin: A high performance liquid chromatography coupled with surface plasmon resonance (SPR) spectroscopy device was constructed, and human insulin monoclonal antibodies were coupled to a gold foil surface with carboxymethyl cellulose as a detector for the SPR signal. The insulin self-assembled complex confirmed in step (3) was separated online and detected by online SPR spectroscopy using a molecular sieve column. The fractions with a signal slope increase of more than 30 degrees on the sensorgram were collected and freeze-dried to obtain the active human insulin raw material; (5) Determination of immunoaffinity activity concentration and specific activity: Determine the immunoaffinity activity concentration and specific activity of the active human insulin raw material. If the specific activity is ≥ 0.9, proceed to the next step; otherwise, re-prepare from step (1); (6) Preparation of interchangeable active human insulin solution standard substance: The active human insulin raw material in step (4) is prepared into solutions with a concentration range of 10 to 1000 pmol / L using a diluent, wherein the diluent is a bovine serum albumin solution, and the bovine serum albumin solution also contains 200 to 500 mmol / L of zinc acetate. The solutions are packaged and sealed to form interchangeable active human insulin solution standard substance candidates. The candidates are then subjected to a uniformity test, a stability test, a standard value determination, an uncertainty assessment, and an interchangeability evaluation in sequence to obtain interchangeable active human insulin solution standard substances.

2. The method for preparing an interchangeable active human insulin solution standard substance according to claim 1, characterized in that: In step (1), the human insulin comprises protein sequences as shown in SEQ ID No. 1 and SEQ ID No.

2.

3. The method for preparing an interchangeable active human insulin solution standard substance according to claim 1, characterized in that: In step (1), the conditions of the reversed-phase high-performance liquid chromatography include: using a sodium sulfate buffer solution with a concentration of 0.1-0.4 mol / L: acetonitrile in a ratio of 82:18 or acetonitrile: water in a ratio of 50:50 as the mobile phase, and performing separation on a C4, C8 or C18 chromatographic column to separate human insulin and A21-desaminated human insulin, and collecting the human insulin fraction for the next step of molecular sieve purification.

4. The method for preparing an interchangeable active human insulin solution standard substance according to claim 1, characterized in that: In step (1), the molecular sieve conditions include: using PBS and water as mobile phases, respectively, further purifying with a molecular sieve column, monitoring the peak time by absorbance at 280 nm, separating insulin aggregates from monomeric insulin, collecting fractions within the monomeric insulin peak time period, and freeze-drying to obtain high-purity human insulin raw material.

5. The method for preparing an interchangeable active human insulin solution standard substance according to claim 1, characterized in that: In step (2), the source of the zinc ions includes zinc acetate.

6. The method for preparing an interchangeable active human insulin solution standard substance according to claim 1, characterized in that: In step (2), the final concentration of the high-purity human insulin raw material is 0.01-1 mg / mL, and the molar ratio of insulin to zinc ions in the solution is 6:1-6:

60.

7. The method for preparing an interchangeable active human insulin solution standard substance according to claim 1, characterized in that: In step (2), the standing temperature is 4-10° C., and the standing time is 4-48 hours.

8. The method for preparing an interchangeable active human insulin solution standard substance according to claim 1, characterized in that: In step (3), the scanning optical path of the circular dichroism spectrum is 0.1~1 cm, the scanning wavelength range is 200~400 nm, and the scanning speed is 10~100 nm / min.

9. The method for preparing an interchangeable active human insulin solution standard substance according to claim 1, characterized in that: In step (3), the judgment criteria are: if the circular dichroism peak moves toward a higher wavelength and becomes flat, and a peak with a larger molecular weight appears on the mass spectrometer spectrum, then a self-assembled complex is formed; otherwise, no self-assembled complex is formed.

10. The method for preparing an interchangeable active human insulin solution standard substance according to claim 1, characterized in that: In step (4), when constructing a high performance liquid chromatography coupled with surface plasmon resonance (SPR) spectroscopy, 1×PBST was used as the mobile phase and running buffer, sodium hydroxide was used as the regeneration solution, and human insulin monoclonal antibody was coupled to a gold foil surface with carboxymethyl cellulose as a detector for the SPR signal.

11. The method for preparing an interchangeable active human insulin solution standard substance according to claim 10, characterized in that: The molar concentration of the sodium hydroxide is 0.5-20 mmol / L.

12. The method for preparing an interchangeable active human insulin solution standard substance according to claim 1, characterized in that: In step (5), the determination of the immunoaffinity activity concentration and specific activity includes: S1. Prepare active human insulin solution by using 1×PBST solution to prepare active human insulin solution, and measure the immunoaffinity activity concentration c of the active human insulin solution. raw,active ; S2. Take the prepared active human insulin solution, weigh its mass and add isotope-labeled amino acids into it, concentrate it by centrifugation or blow it dry with nitrogen, then add concentrated hydrochloric acid, protect it with nitrogen and seal it, and hydrolyze it. Determine the content of the above amino acids in the hydrolyzate by isotope dilution mass spectrometry, and calculate the physicochemical concentration C of the active human insulin solution based on the protein sequence of human insulin selected in step (1). raw,chem ; ， Where C AA is the concentration of amino acids in insulin hydrolysate determined by isotope dilution mass spectrometry; M AA is the molecular weight of the amino acid; N AA is the number of the amino acid contained in insulin; M insulin is the molecular weight of insulin; S3. Calculate the purity of active human insulin raw material according to the following formula: ， Where, P raw is the purity of active human insulin raw material, m raw is the mass of active human insulin raw material, m solvent The mass of 1×PBST solution, C raw,chem is the physicochemical concentration of active human insulin solution; S4. Use a density meter to measure the density of the prepared active human insulin solution. According to the measured physicochemical concentration c of the active human insulin solution, raw,chem , unit: mg / g, and immunoaffinity activity concentration c raw,active , and then calculate the specific activity of the two according to the following formula: ， Where R is the specific activity of active human insulin raw material, c raw,active is the immunoaffinity activity concentration of active human insulin solution, c raw,chem is the physicochemical concentration of active human insulin solution, and ρ is the density of active human insulin solution.

13. The method for preparing an interchangeable active human insulin solution standard substance according to claim 12, characterized in that: In step S1, the physicochemical concentration of the active human insulin solution is 0.01-1 mg / mL, and the immunoaffinity activity concentration c of the active human insulin solution is raw,active Measured by surface plasmon resonance spectroscopy.

14. The method for preparing an interchangeable active human insulin solution standard substance according to claim 12, characterized in that: In step S2, the isotopes include 13 C. 15 At least one of N and D, the amino acids include at least one of proline, valine, leucine, isoleucine, and phenylalanine, and the number of isotope-labeled atoms is ≥3.

15. The method for preparing an interchangeable active human insulin solution standard substance according to claim 12, characterized in that: In step S2, the molar concentration of the concentrated hydrochloric acid is 6-8 mol / L, the hydrolysis temperature is 110-150° C., and the hydrolysis time is 24-96 h.

16. The method for preparing an interchangeable active human insulin solution standard substance according to claim 1, characterized in that: In step (6), the diluent is a bovine serum albumin solution, the mass fraction of the bovine serum albumin solution is 5-8%, the high performance liquid chromatography purity of the bovine serum albumin solution is greater than 99%, and the bovine serum albumin solution further contains 200-500 mmol / L of zinc acetate, and the zinc acetate is of analytical grade or higher.

17. The method for preparing an interchangeable active human insulin solution standard substance according to claim 1, characterized in that: In step (6), the standard value is determined according to the following formula: ， Where c CRM Indicates the certified value of active human insulin solution standard substance, unit is pmol / L; m STD Indicates the mass of weighed active human insulin raw material, in g; P raw Indicates the purity of active human insulin raw materials, unit is g / g; M insulin Indicates the molar mass of human insulin in g / mol; V STD Indicates the volume of the volumetric flask used to prepare the solution, in L.

18. The method for preparing an interchangeable active human insulin solution standard substance according to claim 1, characterized in that: In step (6), the uncertainty includes the uncertainty introduced in the determination process, the uncertainty introduced by the homogeneity of the standard material, the uncertainty introduced by the long-term stability, the combined standard uncertainty of the determination result, and the expanded standard uncertainty of the determination result.

19. An interchangeable active human insulin solution standard substance, characterized in that: Prepared by the preparation method according to any one of claims 1 to 18.

20. Use of the interchangeable active human insulin solution standard substance according to claim 19 as an interchangeable standard substance.

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