Active human insulin solution standard substance with interchangeability as well as preparation method and application thereof

Abstract

The invention discloses an interchangeable active human insulin solution standard substance as well as a preparation method and application thereof, and belongs to the technical field of stoichiometric analysis and detection. The preparation method provided by the invention comprises the steps of purification of human insulin, self-assembly of the human insulin, confirmation of an insulin self-assembly compound structure, purification of active human insulin, determination of immunoaffinity activity concentration and specific activity, and preparation of the active human insulin solution standard substance with interchangeability. The preparation method provided by the invention can be used for calibration of a human insulin conventional in-vitro diagnosis system, development of an in-vitro diagnosis detection reagent, method performance verification and value traceability of a human insulin detection result in serum, so that the accuracy and comparability of a clinical detection result are ensured, and public health and life safety are protected.

Description

Technical Field

[0001] This application belongs to the technical field of stoichiometric analysis and detection, and particularly relates to an active human insulin solution reference material with interchangeability, a preparation method thereof, and an application thereof. Background Art

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

[0003] In vitro diagnosis plays an important role in modern medicine. It is reported in the literature that 60 - 70% of medical decisions rely on the results of in vitro diagnosis. Whether the results of in vitro diagnosis are accurate or not is directly related to the correct diagnosis and effective treatment of diseases. Interchangeability is very important for ensuring the traceability of in vitro diagnosis results and is a necessary condition for effective calibration, accuracy verification, and proficiency testing based on accuracy. The famous standardization expert Greg Miller mentioned that interchangeability is the core of the success of the standardization and coordination work of the entire laboratory medicine.

[0004] To ensure the accuracy and comparability of in vitro diagnosis results, both theory and practice have proven that establishing a metrological traceability transfer chain for in vitro diagnosis results is an effective way to solve this problem. Using a reference material without interchangeability for calibration will cause the interruption of the metrological traceability transfer chain, and thus the expected calibration and standardization purposes cannot be achieved. Reflected in the experimental data, calibrating an in vitro diagnostic system with a reference material without interchangeability may produce unexpected deviations, and these deviations will be transmitted along with the metrological traceability transfer chain to the final in vitro diagnosis results, resulting in inaccurate and incomparable results. For example, after an in vitro diagnostic system is calibrated with a myocardial troponin reference material without interchangeability, significant result variability will still occur, and the purpose of standardization cannot be achieved. At the same time, because using a reference material without interchangeability to calibrate an in vitro diagnostic system may produce unexpected deviations, a reference material without interchangeability cannot be used for accuracy verification and proficiency testing based on accuracy either.

[0005] There are many reasons for non - interchangeability, including differences in matrices, inconsistencies between reference materials and analytes in fresh clinical samples, and inconsistencies in analytes at the microscopic level, etc. Therefore, in order to ensure that the developed reference materials have interchangeability, human - sourced authentic samples are usually used as raw materials for reference materials to develop matrix reference materials, so as to ensure that the developed reference materials 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 six methods for establishing the metrological traceability of in vitro diagnostic results. Among them, the methods that can be completely traced back to SI units are as Figure 1 shown. Among them, the reference materials at the m1 and m2 levels are purity reference materials, which usually do not have interchangeability. Therefore, it is required to transfer the quantity value to the matrix reference material with interchangeability at the m3 level, and then further transfer it to the manufacturer's primary calibrator. Because the quantity - value transfer procedure p4 adopted by the manufacturer is often a routine measurement procedure, only reference materials with interchangeability can be used for the calibration of routine measurement procedures. For the other several traceability methods, it is also required that the reference materials at the m3 level should have interchangeability, otherwise the quantity - value traceability transfer chain will be broken.

[0006] However, there are currently great difficulties in the development and use of matrix reference materials, especially for matrix reference materials of protein-based in vitro diagnostic markers. Protein-based in vitro diagnostic markers account for a significant proportion in in vitro diagnosis. Diagnostic markers for many major diseases such as tumors, diabetes, cardiovascular and cerebrovascular diseases, and neurodegenerative diseases are proteins. The lack of interchangeable matrix reference materials for protein-based in vitro diagnostic markers has become a limiting factor for the standardization of the results of protein-based in vitro diagnostic markers. Since the raw materials of matrix reference materials generally come from real human samples, the raw materials are limited and difficult to obtain. In particular, it is even more difficult to obtain low-value and high-value real human samples in abnormal pathological states. Adding and other methods may lead to non-interchangeability of the reference materials. Therefore, the quantity value level of matrix reference materials is often limited and cannot be used for the calibration of the entire clinically reportable range. Protein-based in vitro diagnostic markers often exist in the form of protein complexes or aggregates in the body, while pure products often exist in the form of monomers. The behavior of directly adding protein pure products to serum to attempt to change the quantity value level is likely to result in non-interchangeability of the added reference materials due to the inconsistency of the analytes. Proteins may have glycosylation structures, and the inconsistency in the microscopic glycosylation state of the same protein between the reference material and real clinical samples may lead to non-interchangeability. The preparation and preservation processes of reference materials such as freeze-drying may also cause the depolymerization of protein aggregates in serum, resulting in non-interchangeability of the reference materials. C-reactive protein is a typical example. At the same time, even when using a metrological benchmark method such as isotope dilution mass spectrometry for the accurate quantification of proteins in the matrix, large differences are still found in international comparisons, making it difficult to meet the requirements of clinical total error. These adverse factors make it difficult to develop interchangeable matrix reference materials for protein-based in vitro diagnostics, and the quantity is very limited, making it difficult to meet the current needs for the standardization of protein-based in vitro diagnostic results.

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

[0008] To overcome the above-mentioned defects in the prior art, this application provides an interchangeable active human insulin solution reference material, its preparation method and application, aiming to fill the gaps in related technologies and ensure the accurate and effective clinical detection results of human insulin. The innovative idea of this application is to find the crux by analyzing the reasons for the non-interchangeability of protein pure solution reference materials, and then regulate and evaluate the interchangeability of the human insulin solution reference material through structural adjustment and confirmation, determination of physical and chemical concentration and immunoaffinity activity concentration, etc., overcome the non-interchangeable factors, and then develop a human insulin pure solution reference material that can be traced to SI units and has interchangeability, so as to achieve wide-range calibration of conventional in vitro diagnostic systems.

[0009] To achieve the above-mentioned invention object, the present application provides the following technical solutions:

[0010] On the one hand, the present application provides a preparation method of a reference material of active human insulin solution with interchangeability, comprising the following steps:

[0011] (1) Purification of human insulin: Select human insulin with a purity of 90-98%, and purify it successively by reverse-phase high-performance liquid chromatography and molecular sieve to obtain a high-purity human insulin raw material;

[0012] (2) Self-assembly of human insulin: Dissolve the high-purity human insulin raw material in a 1×PBS solution containing zinc ions, and let it stand to obtain an insulin self-assembly complex;

[0013] (3) Confirmation of the structure of the insulin self-assembly complex: Perform circular dichroism spectroscopy and mass photometry spectroscopy scans on the insulin self-assembly complex, and at the same time perform circular dichroism spectroscopy and mass photometry spectroscopy scans on a human insulin solution with the same concentration as in step (2) prepared with PBS solution without zinc ions as a comparison. Judge whether the self-assembly complex is formed according to the scan results. If not, repeat step (2);

[0014] (4) Purification of active human insulin: Build a device combining high-performance liquid chromatography and surface plasmon resonance (SPR) spectroscopy, and use a molecular sieve column to perform on-line separation and on-line SPR spectroscopy detection on the insulin self-assembly complex confirmed in step (3). Collect the fractions with a signal slope increase > 30 degrees on the sensorgram and lyophilize them to obtain an active human insulin raw material;

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

[0016] (6) Preparation of a reference material of active human insulin solution with interchangeability: Use a diluent to prepare solutions of the active human insulin raw material in step (4) at multiple concentration levels in the concentration range of 10-1000 pmol / L, dispense and seal them to form candidate reference materials of active human insulin solution with interchangeability, and then perform homogeneity inspection, stability inspection, standard value determination, uncertainty evaluation, and interchangeability evaluation on the candidates in turn to obtain a reference material of active human insulin solution with interchangeability.

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

[0018] Optionally, in step (1), the conditions for 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 at 82:18 and acetonitrile: water at 50:50 as the mobile phase, and separating with a C4, C8, or C18 chromatographic column 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 conditions for the molecular sieve include: using PBS and water as the mobile phase respectively, further purifying with a molecular sieve column, monitoring the elution time by absorbance at 280 nm, separating insulin aggregates and monomeric insulin, collecting the fraction within the elution time period of monomeric insulin, and freeze-drying to obtain the high-purity human insulin raw material.

[0020] Optionally, in step (2), the source of the 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 independently selects any value from 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 the range value between any two of them, and the molar ratio of insulin to zinc ions in the solution independently selects any value from 6:1, 6:5, 6:10, 6:20, 6:30, 6:40, 6:50, 6:60 or the range value between any two of them.

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

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

[0025] Optionally, 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.

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

[0027] Optionally, in step (3), the criterion for judgment is as follows: if the circular dichroism peak moves towards the high wavelength direction and becomes flat, and a peak with a larger molecular weight appears in the mass photometer spectrum, then a self-assembled complex is formed; otherwise, it is not formed.

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

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

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

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

[0032] S1. Prepare an active human insulin solution from the active human insulin raw material with 1×PBST solution, and determine the immunoaflinity activity concentration c of the active human insulin solution raw,active ;

[0033] S2. Take the above-prepared active human insulin solution, weigh its mass and add isotope-labeled amino acids thereto, centrifuge and concentrate or dry it with nitrogen, then add concentrated hydrochloric acid, protect it with nitrogen and seal it, perform hydrolysis, and determine the content of the above amino acids in the hydrolysis solution by isotope dilution mass spectrometry, and calculate the physicochemical concentration C of the active human insulin solution according to the protein sequence of human insulin selected in step (1) raw,chem ;

[0034]

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

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

[0037]

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

[0039] S4. Measure the density of the above-prepared active human insulin solution using a densitometer. According to the measured physicochemical concentration c raw,chem (unit: mg / g) and the immunoaffinity activity concentration c raw,active , then calculate the specific activity of the two according to the following formula:

[0040]

[0041] In the formula, R is the specific activity of the active human insulin raw material, c raw,active is the immunoaffinity activity concentration of the active human insulin solution, c raw,chem is the physicochemical concentration of the active human insulin solution, and ρ is the density of the 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 raw,active of the active human insulin solution is measured by a surface plasmon resonance spectrometer.

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

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

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

[0046] Optionally, in step S2, the molar concentration of the concentrated hydrochloric acid is independently selected from any value of 6 mol / L, 7 mol / L, 8 mol / L or the range value between any two of them, the temperature of the hydrolysis is independently selected from any value of 110 °C, 120 °C, 130 °C, 140 °C, 150 °C or the range value between any two of them, and the time of the hydrolysis is independently selected from any value of 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, 96 h or the range value 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 > 99%, and the bovine serum albumin solution also contains 0 - 500 mmol / L of zinc acetate, and the zinc acetate is of analytical pure or higher grade.

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

[0049]

[0050] In the formula, c CRM represents the certified value of the standard substance of the active human insulin solution, in the unit of pmol / L; m STD represents the mass of the weighed active human insulin raw material, in the unit of g; P raw represents the purity of the active human insulin raw material, in the unit of g / g; M insulin represents the molar mass of human insulin, in the unit of g / mol; V STD represents the volume of the volumetric flask used when preparing the solution, in the unit of L.

[0051] Optionally, in step (6), the uncertainty includes the uncertainty introduced during the certification process, the uncertainty introduced by the homogeneity of the reference material, the uncertainty introduced by the long-term stability, the combined standard uncertainty of the certification result, and the expanded standard uncertainty of the certification result.

[0052] Optionally, in step (6), the solutions at each concentration level are aliquoted and sealed in brown ampoules, with each aliquot being 10 - 5000 μL. The candidate reference material of the active human insulin solution is labeled, stored in a refrigerator at -70 °C, and each aliquot unit is consecutively numbered according to the aliquoting order. The number of aliquot units is P, forming a candidate reference material of the active human insulin solution with interchangeability.

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

[0054] Using the randbetween function in Excel to randomly select and test the consecutively numbered aliquot units. When P ≤ 200, the number of aliquot units m selected is not less than 11; when 200 < P ≤ 500, the number of aliquot units m selected is not less than 15; when 500 < P ≤ 1000, the number of aliquot units m selected is not less than 25; when the number of aliquot units P > 1000, the number of aliquot units m selected is not less than 30. According to the order of selection, the m selected aliquot units are renumbered and arranged in sequence. The insulin concentration of the active human insulin solution reference material in the above selected aliquot units is tested for homogeneity by fluorescence or chemiluminescence immunoassay. During the test, first, the insulin concentration of the active human insulin solution reference material in each selected aliquot unit is tested for the first time in the above order, then the above order is disrupted, and the insulin concentration of the active human insulin solution reference material in each selected aliquot unit is tested for the second time again. The above operation is repeated until the insulin concentration of the active human insulin solution reference material in each selected aliquot unit is tested for the nth time, where n is an integer greater than or equal to 3, so as to obtain n test results, and calculate and statistically analyze according to the following formula: the homogeneity of the insulin concentration of the active human insulin solution reference material in the aliquot unit;

[0055] When m candidate reference materials are selected, according to the above test method, under the condition of repeatability n times, m groups of precision measurement data are obtained as follows:

[0056] Number 1: x 11 , x 12 ...... x 1n , average value

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

[0059] Number m: x m1 , x m2 ...... xmn , average value

[0060] Average value:

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

[0062] Sum of squares of differences between groups:

[0063] Sum of squares of differences within groups:

[0064] Degree of freedom between groups: v 1 = m - 1; Degree of freedom within groups: v 2 = N - m

[0065]

[0066] Statistic F:

[0067] where S 1 2 is the variance between groups, S 2 2 is the variance within groups. According to the degrees of freedom (v 1 , v 2 ) and the given significance level α = 0.05, the critical value α of F is obtained from the table, and then compared with the F value calculated by Formula (9). If F < F α , it is considered that there is no significant difference between within groups and between groups, and the insulin concentration of the active human insulin solution reference material in the dispensing unit is uniform. Otherwise, the insulin concentration of the active human insulin solution reference material in the dispensing unit is non-uniform. α α α

[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 period of the long-term stability test, according to the principle of being dense at the front and sparse at the back, at least 5 time points k are selected. At each time point, at least 2 dispensing units of the active human insulin solution reference material stored at -70°C in step (6) are sampled, and the insulin concentration of the active human insulin solution reference material in each dispensing unit is repeatedly tested 3 times by fluorescence or luminescence immunoassay. Then, the arithmetic mean of these measurement results is calculated to obtain the arithmetic mean Y i of the measurement results at each time point. The arithmetic mean Y i of the above measurement results is fitted with the corresponding measurement time according to the following linear model:

[0070] Y i = b 0 + bX Formula (10)

[0071] In Formula (10), b 0 represents the intercept, b represents the regression coefficient (slope); X represents the test time, unit (month); Y i represents the arithmetic mean of the measurement results of insulin concentration in the active human insulin solution reference substance at each time point.

[0072] Calculate the regression coefficient (slope) and intercept according to Formula (11) and Formula (12):

[0073]

[0074] In Formula (11) and Formula (12), X i represents the test time at the i-th time point, unit in month; Y i represents the arithmetic mean of the measurement results of insulin concentration in the active human insulin solution reference substance in the above-mentioned dispensing unit at the i-th time point; X represents the average value of the test times at all time points, unit in month; Y represents the arithmetic mean of the measurement results of insulin concentration in the active human insulin solution reference substance in the above-mentioned dispensing unit at all time points; k represents the number of time points.

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

[0076]

[0077] The meanings of each symbol 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 to get t 0.95,k-2 value;

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

[0082] The duration of the short-term stability test shall be not less than 7 days. During the short-term stability test period, according to the principle of denser in the front and sparser in the back, select not less than 5 time points k. At each time point, take out at least two sub-packaging units from step (6), place them in the temperature range of -20°C to 40°C for investigation, extract the active human insulin solution reference material in the above sub-packaging units, and use fluorescence or luminescence immunoassay to conduct 3 repeated tests on the insulin concentration of the active human insulin solution reference material in each sub-packaging unit, calculate the arithmetic mean of these measurement results, and 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 =b 0 +bX Formula (15)

[0084] In Formula (15), b 0 represents the intercept; b represents the regression coefficient (slope); X represents the test time, unit (days); Y i represents the arithmetic mean of the measurement results of the insulin concentration in the active human insulin solution reference material at each time point.

[0085] Calculate the regression coefficient (slope) and intercept according to Formula (16) and Formula (17):

[0086]

[0087] In Formula (16) and Formula (17), X i represents the detection time of the i-th time point, unit in days; Y i represents the arithmetic mean of the measurement results of the insulin concentration in the active human insulin solution reference material in the above sub-packaging units at the i-th time point; represents the average value of the detection times of all time points, unit in days; represents the arithmetic mean of the measurement results of the insulin concentration in the active human insulin solution reference material in the above sub-packaging units at all time points; k represents the number of time points.

[0088] Calculate the value of s 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 to obtain the value of t 0.95,k-2 ;

[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 reference material is stable in the short term; otherwise, it indicates that the active human insulin solution reference material is unstable.

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

[0096] According to the uncertainty evaluation mathematical model:

[0097] The uncertainty of the assigned value of the active human insulin solution reference material comes from the uncertainty introduced in the assignment process, the uncertainty introduced by the homogeneity of the reference material, and the uncertainty introduced by the long-term stability.

[0098] ① Uncertainty introduced in the assignment process:

[0099] According to formula (20), considering the influence of the solvent blank detection limit at the same time, the uncertainty of the preparation result of the active human insulin solution reference material can be calculated by formula (21):

[0100]

[0101] In the formula, u r，cCRM represents the relative uncertainty of the preparation result of the active human insulin solution reference material, dimensionless;

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

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

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

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

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

[0107] ② Uncertainty component introduced by homogeneity

[0108] The uncertainty introduced by the homogeneity of the reference material is equal to the standard deviation of the between-bottle homogeneity. When the between-group mean square is greater than the within-group mean square, it is calculated according to formula (22):

[0109]

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

[0111]

[0112] ③ Uncertainty component introduced by stability

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

[0114] u lts =s k，lts ·t formula (24)

[0115] ④ Combined standard uncertainty of the assigned value result

[0116] Calculate the combined standard uncertainty of the assigned value result of the reference material of active human insulin solution according to formula (25):

[0117]

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

[0119] According to formula (26), with the coverage factor k = 2, calculate the expanded uncertainty of the assigned value result of the reference material of active human insulin solution:

[0120] U=ku c formula (26)

[0121] Therefore, the assigned value result of the reference material of active human insulin solution can be expressed as:

[0122] c CRM ±U

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

[0124] Finally, the homogeneity, stability and assigned value result of the human insulin concentration of the reference material of active human insulin solution are obtained.

[0125] In the second aspect, the present application provides a reference material of active human insulin solution with interchangeability, which is prepared by the preparation method described in any one of the 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, this application has the following beneficial effects:

[0128] (1) The insulin solution standard material regulated by the method of the present application is interchangeable between commonly used luminescent immunoassay systems and can be used for calibration of these conventional chemiluminescent immunoassay systems. Therefore, it is no longer necessary to develop complex matrix standard materials to ensure interchangeability, thereby reducing the development cost of interchangeable protein standard materials. 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.

[0129] (2) The active human insulin solution standard material provided by this application replaces the previous matrix standard material, shortening the traceability transfer chain of ISO17511:2020, which helps to reduce the measurement uncertainty of the value transfer terminal. The uncertainty of the interchangeable active human insulin solution standard material determined by the method of this application is much smaller than the uncertainty level of the current isotope dilution mass spectrometry for the determination of human insulin in complex matrices such as serum, and can meet the current total error requirements of human insulin clinical test items, thereby providing new ideas for the development of interchangeable standard materials and contributing to the standardization of protein in vitro diagnostic results. BRIEF DESCRIPTION OF THE DRAWINGS

[0130] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0131] Figure 1 This is a schematic diagram of the traceability transfer of measurement values ​​that can be fully traced to SI units in ISO 17511:2020;

[0132] Figure 2 The cross-linking conditions of disulfide bonds between the two sequences of the applicant's insulin sequence SEQ ID No.1 and SEQ ID No.2, as well as within the sequence (Note: A-Chain represents SEQ ID No.1; B-Chain represents SEQ ID No.2);

[0133] Figure 3 This is a schematic diagram of the uncertainty sources of the active human insulin solution standard material in this application;

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

[0135] Figure 5 For different insulins and Zn in this application 2+ Mass photometer spectra of solutions with different ratios;

[0136] Figure 6 Sensing diagram for determining the immunoaffinity activity concentration of the active insulin raw material in this application;

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

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

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

[0140] Figure 10 Short-term stability results at 4°C of the reference material of the active human insulin solution (level 1) in this application;

[0141] Figure 11 Short-term stability results at 4°C of the reference material of the active human insulin solution (level 2) in this application;

[0142] Figure 12 Short-term stability results at 4°C of the reference material of the active human insulin solution (level 3) in this application;

[0143] Figure 13 Short-term stability results at room temperature of the reference material of the active human insulin solution (level 1) in this application;

[0144] Figure 14 Short-term stability results at room temperature of the reference material of the active human insulin solution (level 2) in this application;

[0145] Figure 15 Short-term stability results at room temperature of the reference material of the active human insulin solution (level 3) in this application;

[0146] Figure 16 Results of the interchangeability evaluation of the reference material of the active human insulin solution according to the CLSI EP14 standard in this application. Detailed implementation manners

[0147] The present application will be further described below in conjunction with specific embodiments. The following descriptions are only several embodiments of the present application and do not impose any form of limitation on the present application. Although the present application is disclosed as preferred embodiments, it is not intended to limit the present application. Any person skilled in the relevant art can make some changes or modifications within the scope of the technical solutions of the present application by using the disclosed technical content, which are equivalent to equivalent implementation cases and all fall within the scope of the technical solutions.

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

[0149] Unless otherwise specified, the analysis methods in the embodiments adopt the conventional settings and conventional analysis methods of instruments or equipment.

[0150] Example 1

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

[0152] I. Purification of human insulin

[0153] The human insulin sequence described in the present application has the protein sequences 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 situation between the sequences of SEQ ID No.1 and SEQ ID No.2 and within the sequences is as Figure 2 shown.

[0157] The raw material is a commercially available human insulin biochemical reagent from Sigm-Aldrich. The HPLC purity of this reagent is 97.5%. Since the purity is relatively low, it cannot be directly used for the development of the reference material. In order to obtain a high-purity candidate for the human insulin reference material, reverse-phase high-performance liquid chromatography and molecular sieve are used to further purify the commercially available human insulin. 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 mobile phase gradient is shown in Table 1.

[0161] Table 1 Mobile Phase Gradient of Reversed-Phase High Performance Liquid Chromatography 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: 220 nm

[0165] Flow rate: 1 mL / min

[0166] Column temperature: 40 °C

[0167] Sample injection volume: 100 μL

[0168] Under the above chromatographic conditions, human insulin and A21-deamino human insulin can be effectively separated, and the chromatographic peak of human insulin is collected to complete the purification. The collected human insulin fractions are further purified using molecular sieve columns with PBS and water as the mobile phases respectively, and the elution time is monitored by absorbance at 280 nm to separate insulin aggregates from monomeric insulin. The specific separation conditions are as follows:

[0169] Sample 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 min

[0174] Detection wavelength: 280 nm

[0175] Sample concentration: 1.0 mg / mL

[0176] Mobile phases: 1×PBS and water respectively

[0177] Fractions within the elution time range of monomeric insulin are collected. The collected fractions are freeze-dried (cold trap temperature -70 °C, vacuum 1 Pa, freeze-drying for 48 h) to obtain high-purity human insulin raw material.

[0178] II. Self-Assembly of Human Insulin

[0179] The high-purity human insulin raw material prepared by purification is dissolved in 1×PBS solution containing zinc acetate, with an insulin concentration of 0.02 mg / mL. The molar ratio of insulin to zinc ions in the solution is 6:60. The solution is placed in a 4 °C refrigerator for self-assembly for 24 h.

[0180] III. Confirmation of the Structure of Insulin Self-Assembly Complex

[0181] The self-assembled complex of insulin in the presence of zinc ions prepared in Step 2 was subjected to circular dichroism spectroscopy and mass photometry spectroscopy scans. For the circular dichroism spectroscopy scan, the optical path length was 1 cm, the scanning wavelength range was 200 - 400 nm, and the scanning speed was 50 nm / min. The mass photometry scan was performed under the default conditions. At the same time, the circular dichroism spectroscopy and mass photometry spectroscopy of a human insulin solution with the same concentration prepared with PBS without zinc ions were scanned as a comparison. The results are as Figure 4 and Figure 5 shown.

[0182] As Figure 4 can be seen, when there is no Zn 2+ in the solution, insulin has a negative peak at 208 nm. When the concentration of Zn 2 + in the solution gradually increases, the circular dichroism spectrum of the insulin solution changes, the intensity of the peak decreases and becomes flatter. These changes indicate that insulin forms aggregates under the action of Zn 2+ . As Figure 5 can be seen, 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 continuously increasing, thus indicating that insulin aggregates are formed in the presence of Zn 2+ .

[0183] IV. Purification of Active Human Insulin

[0184] An efficient liquid chromatography and surface plasmon resonance (SPR) spectroscopy coupled device was set up. 1×PBST was used as the mobile phase and running buffer, and 0.5 mmol / L sodium hydroxide was used as the regeneration solution. The human insulin monoclonal antibody was conjugated to the gold foil surface with carboxymethyl cellulose as the detector for SPR signals. The liquid phase separation conditions are as follows:

[0185] Sample injection volume: 100 μL

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

[0187] Gradient: Isocratic elution

[0188] Flow rate: 0.5 mL / min

[0189] Analysis time: 35 min

[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 the fractions with a signal slope rising by more than 30 degrees on the sensor graph 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 in step 4 into a 50 mL volumetric flask, and use 1×PBST to make an active human insulin solution with a concentration of 0.02 mg / mL. During the preparation, place the volumetric flask on the balance and weigh the added 1×PBST solution to 50096 mg. Use a surface plasmon resonance spectrometer to determine the immunoaffinity activity concentration of the active human insulin solution in step 4. Set the instrument temperature to 25°C and the sample tank temperature to 25°C. Three pre-start cycles were set as background noise references to avoid the influence of baseline drift on the accuracy of the experiment. The flow rate was 30 μL / min. Two blank controls were set to remove the background interference of the sample buffer: the sample buffer was injected at two flow rates of 5 and 100 μL / min, the contact time was 36 s, the dissociation time was 120 s, the regeneration flow rate was 30 μL / min, and the time was 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 own 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 13 C isotope-labeled proline, valine, leucine, isoleucine and phenylalanine (the number of isotope-labeled atoms is not less than 3), centrifuged, concentrated and dried, 500 μL of 8 mol / L concentrated hydrochloric acid was added, nitrogen was passed through and sealed, and hydrolyzed in an oven at 110°C for 48 hours. After hydrolysis, the content of the above amino acids in the hydrolyzate was determined by isotope dilution mass spectrometry using national proline, valine, leucine, isoleucine and phenylalanine standard substances as standards, and the physicochemical concentration of the above 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] In the formula, 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 such amino acids contained in insulin; M insulin is the molecular weight of insulin.

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

[0199]

[0200] Measure the density ρ (unit: g / mL) of the above-prepared active human insulin solution using a densitometer. According to the measured physical and chemical concentration c raw,chem (unit: mg / g) and the result c of the immunoaffinity activity concentration raw,active (unit: mg / mL), calculate the specific activity R of the two according to formula (2), and the result is 0.95.

[0201]

[0202] VI. Preparation of the reference material of the active human insulin solution with interchangeability

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

[0204] Under the condition of ensuring that the environmental temperature is 20 ± 2 °C, accurately weigh 1.0195 mg of the active human insulin raw material in Step 4, dissolve it with the above-prepared solvent, and then transfer it to a 10 mL volumetric flask for volume fixation to obtain an insulin stock solution. According to the weighing mass, the purity of human insulin, and the volume of the volumetric flask, the insulin concentration of the stock solution is 1.504×10 7 pmol / L, and the calculation process is as follows:

[0205]

[0206] In the formula, C hINS，0 represents the concentration of the No. 0 active human insulin solution, m hINS represents the measured mass of the active human insulin raw material, q hINS represents the purity of the active human insulin raw material, V represents the volume of the volumetric flask used for preparing the solution, and M hINS represents the molecular weight of human insulin.

[0207] Continue to pipette 1.000 mL of the stock solution, transfer it to a 100 mL volumetric flask for volume fixation to obtain the 1# solution. According to the pipetted volume, the insulin concentration in the stock solution, and the volume of the volumetric flask, the calculated insulin concentration in the 1# solution is 1.504×10 5pmol / L, and the calculation process is as follows:

[0208]

[0209] In the formula, C hINS，1 represents the concentration of human insulin in Solution 1#, C hINS，0 represents the concentration of the active human insulin solution No. 0, V 0 represents the volume of the active human insulin solution No. 0 taken, V 1 represents the volume of the volumetric flask used when preparing Solution 1#.

[0210] Continue to take 95.5 μL of Solution 1# with a pipette, transfer it to a 100 mL volumetric flask and make up the volume to obtain Solution 2#. According to the taken volume, the concentration of insulin in Solution 1#, and the volume of the volumetric flask, the calculated concentration of insulin in Solution 2# is 143.67 pmol / L, and the calculation process is as follows:

[0211]

[0212] In the formula, C hINS，2 represents the concentration of human insulin in Solution 2#, C hINS，1 represents the concentration of human insulin in Solution 1#, V 1 represents the volume of Solution 1# taken, V 2 represents the volume of the volumetric flask used when preparing Solution 2#.

[0213] Take 174.8 μL of Solution 1# with a pipette, transfer it to a 250 mL volumetric flask and make up the volume to obtain Solution 3#. According to the taken volume, the concentration of insulin in Solution 1#, and the volume of the volumetric flask, the calculated concentration of insulin in Solution 3# is 105.19 pmol / L, and the calculation process is as follows:

[0214]

[0215] In the formula, C hINS，3 represents the concentration of human insulin in Solution 3#, C hINS，1 represents the concentration of human insulin in Solution 1#, V 1 represents the volume of Solution 1# taken, V 3 represents the volume of the volumetric flask used when preparing Solution 3#.

[0216] Take 74.4 μL of Solution 1# with a pipette, transfer it to a 250 mL volumetric flask and make up the volume to obtain Solution 4#. According to the taken volume, the concentration of insulin in Solution 1#, and the volume of the volumetric flask, the calculated concentration of insulin in Solution 4# is 44.77 pmol / L, and the calculation process is as follows:

[0217]

[0218] In the formula, C hINS，4 represents the concentration of human insulin in the 4# solution, and C hINS，1 represents the concentration of human insulin in the 1# solution. V 1 represents the volume of the 1# solution taken, and V 4 represents the volume of the volumetric flask used when preparing the 4# solution.

[0219] The above 2#, 3#, and 4# solutions are the candidate reference materials of active human insulin solution at three levels. The solutions are fully mixed and then dispensed into clean brown ampoules under shaking. Each unit is dispensed with 500 μL, and 200 units are dispensed cumulatively for each level, and stored in a -70 °C refrigerator.

[0220] VII. Homogeneity and Stability Tests of Reference Materials

[0221] 1. Homogeneity Test of Active Human Insulin Solution Reference Material

[0222] Use the randbetween function in Excel to randomly select and test the consecutively numbered dispensed units (P). When P ≤ 200, the number of dispensed units m selected is not less than 11; when 200 < P ≤ 500, the number of dispensed units m selected is not less than 15; when 500 < P ≤ 1000, the number of dispensed units m selected is not less than 25; when the number of dispensed units P > 1000, the number of dispensed units m selected is not less than 30. According to the order of selection, the m selected dispensed units are renumbered and arranged in sequence. Use the Beijing Leadman chemiluminescence immunoassay method to test the homogeneity of the insulin concentration of the active human insulin solution reference material in the above selected dispensed units. During the test, first test the insulin concentration of the active human insulin solution reference material in each selected dispensed unit in the above order for the first time, then disrupt the above order, and repeat the test of the insulin concentration of the active human insulin solution reference material in each selected dispensed unit for the second time. Repeat the above operation until the insulin concentration of the active human insulin solution reference material in each selected dispensed unit is tested for the nth time, where n is an integer greater than or equal to 3, so as to obtain n test results, and calculate and statistically analyze according to the following formula: the homogeneity of the insulin concentration of the active human insulin solution reference material in the dispensed unit;

[0223] When m candidate reference materials are selected and m groups of precision measurement data are obtained under the condition of repeatability n times according to the above 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] Number m: x m1 , x m2 ...... x mn , average value

[0228] Average value:

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

[0230] Sum of squares of differences between groups:

[0231] Sum of squares of differences within groups:

[0232] Degree of freedom between groups: v 1 = m - 1; Degree of freedom within groups: v 2 = N - m

[0233]

[0234] Statistic F:

[0235] where S 1 2 is the variance between groups, S 2 2 is the variance within groups. According to the degrees of freedom (v 1 , v 2 ) and the given significance level α = 0.05, the critical value α of F is obtained from the table, and then compared with the F value calculated by formula (9). If F < F , it is considered that there is no significant difference between within groups and between groups, and the insulin concentration of the active human insulin solution reference material in the dispensing unit is uniform. Otherwise, the insulin concentration of the active human insulin solution reference material in the dispensing unit is non-uniform. α for comparison. If F < F α , it is considered that there is no significant difference between within groups and between groups, and the insulin concentration of the active human insulin solution reference material in the dispensing unit is uniform. Otherwise, the insulin concentration of the active human insulin solution reference material in the dispensing unit is non-uniform.

[0236] Using the chemiluminescence immunoassay system of Beijing Leadman, 11 bottles of candidate reference materials of the active human insulin solution reference material were randomly selected from each level of the dispensed active human insulin solution reference material, and each bottle of sample was analyzed 3 times repeatedly. The results of the homogeneity test are shown in Tables 2 to 4.

[0237] Table 2 Homogeneity Test of the Certified Reference Material of Active Human Insulin Solution (Level 1)

[0238]

[0239] Table 3 Homogeneity Test of the Certified Reference Material of Active Human Insulin Solution (Level 2)

[0240]

[0241]

[0242] Table 4 Homogeneity Test of the Certified Reference Material of Active Human Insulin Solution (Level 3)

[0243]

[0244]

[0245] Therefore, after the homogeneity test, the calculated F values of the certified reference materials of active human insulin solution at the three levels are all less than the F critical value. Therefore, the certified reference materials of active human insulin solution prepared at the three levels are all homogeneous.

[0246] When performing the homogeneity test, the injection volume of the chemiluminescence immunoassay analyzer is 30 μL each time. Therefore, the minimum sampling amount of the certified reference materials of active human insulin solution at the three levels is 30 μL.

[0247] 2. Long-term Stability Test of the Certified Reference Material of Active Human Insulin Solution

[0248] The duration of the long-term stability test shall not be less than 6 months. During the period of the long-term stability test, in accordance with the principle of being dense at the beginning and sparse at the end, at least 5 time points k shall be selected. At each time point, at least 2 aliquot units of the certified reference material of active human insulin solution stored at -70 °C in Step 6 shall be sampled, and the insulin concentration of the certified reference material of active human insulin solution in each aliquot unit shall be repeatedly tested 3 times by fluorescence or luminescence immunoassay method, and then the arithmetic mean of these measurement results shall be calculated to obtain the arithmetic mean Y of the measurement results at each time point i , the arithmetic mean Y of the above measurement results i shall be fitted with the corresponding measurement time according to the following linear model:

[0249] Y i = b 0 + bX Formula (10)

[0250] In Formula (10), b 0 represents the intercept, b represents the regression coefficient (slope); X represents the test time, unit (month); Y iRepresents the arithmetic mean of the determination results of insulin concentration in the active human insulin solution reference material at each time point.

[0251] Calculate the regression coefficient (slope) and intercept according to Formula (11) and Formula (12):

[0252]

[0253]

[0254] In Formula (11) and Formula (12), X i Represents the detection time at the i-th time point, with the unit of month; Y i Represents the arithmetic mean of the determination results of insulin concentration in the active human insulin solution reference material in the above-mentioned sub-packaging unit at the i-th time point; Represents the average value of the detection times at all time points, with the unit of month; Represents the arithmetic mean of the determination results of insulin concentration in the active human insulin solution reference material in the above-mentioned sub-packaging unit at all time points; k represents the number of time points.

[0255] Calculate the value of s 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 to obtain t 0.95,k-2 value;

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

[0262] The stability investigation was carried out by chemiluminescence immunoassay at the 0th, 1st, 2nd, 4th, and 6th months respectively. Each time, 3 packages were sampled, and each package was measured in parallel 2 times. The long-term stability test results of the active human insulin solution reference material at different levels are shown in Tables 5 to 7, and the trend chart is as Figures 7 - 9 shown.

[0263] Table 5 Results of long-term stability study of the reference material of active human insulin solution (Level 1) (pmol / L)

[0264]

[0265]

[0266] Taking the data in Table 5, with x representing time and y representing the characteristic value of the reference material, and fitting them into a straight line, the slope k = 0.161206897 and the intercept b = 143.0008621 are obtained.

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

[0268]

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

[0270]

[0271] The t-distribution factor with degrees of freedom 2 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 candidate reference material of the active human insulin solution (Level 1) is stable at -70°C.

[0274] Table 6 Results of long-term stability study of the reference material of active human insulin solution (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] Taking the data in Table 6, with x representing time and y representing the characteristic value of the reference material, and fitting them into a straight line, the slope k = 0.046551724 and the intercept b = 105.3189655 are obtained.

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

[0278]

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

[0280]

[0281] The t-distribution factor with 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 certified reference material of active human insulin solution (level 2) is stable at -70 °C.

[0284] Table 7 Results of long-term stability study of certified reference material of active human insulin solution (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] Taking the data in Table 7, with x representing time and y representing the characteristic value of the reference material, and fitting them into a straight line, the slope k = -0.094827586 and the intercept b = 45.14655172.

[0287] The standard deviation of the straight line can be calculated by the following formula:

[0288]

[0289] Taking its square root s = 0.44398923, the uncertainty of the slope is calculated by 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 certified reference material of active human insulin solution (level 3) is stable at -70 °C.

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

[0295] The duration of the short-term stability test shall be not less than 7 days. During the short-term stability test period, in accordance with the principle of being dense at the front and sparse at the back, select not less than 5 time points k. At each time point, take out at least two sub-packaging units from the above-mentioned step 2, place them in the temperature range of -20°C to 40°C for investigation, extract the active human insulin solution reference material from the above-mentioned sub-packaging units, and use fluorescence or luminescence immunoassay to conduct 3 repeated tests on the insulin concentration of the active human insulin solution reference material in each sub-packaging unit, calculate the arithmetic mean of these measurement results, and 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 = b 0 + bX Formula (15)

[0297] In Formula (15), b 0 represents the intercept; b represents the regression coefficient (slope); X represents the test time, unit (days); Y i represents the arithmetic mean of the determination results of the insulin concentration in the active human insulin solution reference material at each time point.

[0298] Calculate the regression coefficient (slope) and intercept according to Formula (16) and Formula (17):

[0299]

[0300] In Formula (16) and Formula (17), X i represents the detection time of the i-th time point, unit in days; Y i represents the arithmetic mean of the determination results of the insulin concentration of the active human insulin solution reference material in the above-mentioned sub-packaging units at the i-th time point; represents the average value of the detection times of all time points, unit in days; represents the arithmetic mean of the determination results of the insulin concentration of the active human insulin solution reference material in the above-mentioned sub-packaging units 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 to obtain the value of t 0.95,k-2 ;

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

[0308] The short-term stability of the candidate reference material was examined using a Beijing Leadman chemiluminescence immunoassay analyzer. The temperatures for the short-term stability test were 4°C, 25°C, and 40°C respectively; the investigation time at each temperature was 7 days. The following table lists the average results of the stability investigation of 3 samples taken each time at different intervals.

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

[0310] Table 8 Results of short-term stability investigation of the reference material of active human insulin solution (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] Taking the data in Table 8, with x representing time and y representing the characteristic value of the reference material, fitting them into a straight line, we get the slope k = -0.531097561 and the intercept b = 142.2195122.

[0313] The standard deviation of the straight line can be calculated by the following formula:

[0314]

[0315] Taking the square root, s = 1.346140221. The uncertainty of the slope is calculated by the following formula:

[0316]

[0317] The t-distribution factor with degrees of freedom 2 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 candidate reference material of active human insulin solution (Level 1) is stable at 4°C. Table 9 Results of short-term stability investigation of the reference material of active human insulin solution (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] Taking the data in Table 9, with x representing time and y representing the characteristic value of the reference material, and fitting them into a straight line, the slope k = 0.228658537 and the intercept b = 105.8682927 are obtained.

[0322] The standard deviation of the straight line can be calculated by the following formula:

[0323]

[0324] Taking its square root s = 0.584539983, the uncertainty of the slope is calculated by 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 reference material of insulin (human) solution for calibration of the fully automatic closed-type chemiluminescence immunoassay analyzer (level 2) is stable at 4°C.

[0329] Table 10 Results of short-term stability investigation of the reference material of active human insulin solution (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] Taking the data in Table 10, with x representing time and y representing the characteristic value of the reference material, and fitting them into a straight line, the slope k = -0.17804878 and the intercept b = 45.0097561 are obtained.

[0332] The standard deviation of the straight line can be calculated by the following formula:

[0333]

[0334] Taking its square root s = 0.480345404, the uncertainty of the slope is calculated by 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 not significant, so the certified reference material of active human insulin solution (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 - 15 as follows.

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

[0341] Table 11 Results of the investigation of the room temperature stability of the certified reference material of active human insulin solution (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] Taking the data in Table 11, with x representing time and y representing the characteristic value of the reference material, a straight line was fitted. Then, the slope k = -0.449137931 and the intercept b = 144.6077586.

[0344] The standard deviation of the straight line can be calculated by the following formula:

[0345]

[0346] Taking the square root, s = 1.156428807. The uncertainty of the slope was calculated by the following formula:

[0347]

[0348] The t-distribution factor with degrees of freedom 2 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 not significant, so the certified reference material of active human insulin solution (level 1) is stable for 6 hours at room temperature.

[0351] Table 12 Results of the investigation of the room temperature stability of the certified reference material of active human insulin solution (level 2) (pmol / L)

[0352]

[0353]

[0354] Taking the data in Table 12, with x representing time and y representing the characteristic value of the reference material, and fitting them into a straight line, the slope k = -0.185344828 and the intercept b = 104.2818966 are obtained.

[0355] The standard deviation of the straight line can be calculated by the following formula:

[0356]

[0357] Taking its square root s = 0.966612167, the uncertainty of the slope is calculated by 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 not significant. Thus, the reference material of active human insulin solution (level 2) can be stable for 6 hours at room temperature.

[0362] Table 13 Results of the investigation on the room temperature stability of the reference material of active human insulin solution (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] Taking the data in Table 13, with x representing time and y representing the characteristic value of the reference material, and fitting them into a straight line, the slope k = -0.061206897 and the intercept b = 45.19913793 are obtained.

[0365] The standard deviation of the straight line can be calculated by the following formula:

[0366]

[0367] Taking its square root s = 0.449105113, the uncertainty of the slope is calculated by 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 not significant. Thus, the certified reference material of active human insulin solution (level 3) can be stable for 6 hours at room temperature.

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

[0373] Table 14 Results of the stability investigation of the certified reference material of active human insulin solution (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] Perform a Student's t-test for the means of two groups on the stability test data of day 1 and day 0 in Table 14. The p-value of the mean difference test is 0.0001. Therefore, there is a very significant difference between the short-term stability test data of day 1 and day 0. Thus, the certified reference material of active human insulin solution is unstable at 40 °C.

[0376] Table 15 Results of the stability investigation of the certified reference material of active human insulin solution (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] Perform a Student's t-test for the means of two groups on the stability test data of day 1 and day 0 in Table 15. The p-value of the mean difference test is 0.0003. Therefore, there is a significant difference between the short-term stability test data of day 1 and day 0. Thus, the certified reference material of active human insulin solution (level 2) is unstable at 40 °C.

[0379] Table 16 Results of the stability investigation of the certified reference material of active human insulin solution (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] Perform a Student's t-test for the means of two groups on the stability test data of day 1 and day 0 in Table 16. The p-value of the mean difference test is 0.001. Therefore, there is a significant difference between the short-term stability test data of day 1 and day 0. Thus, the certified reference material of active human insulin solution (level 3) is unstable at 40 °C.

[0382] VIII. Determination of the certified value of the certified reference material of active human insulin solution

[0383] Calculate the certified value of the certified reference material of active human insulin solution at each concentration level according to formula (20).

[0384]

[0385] In formula (20), c CRM represents the certified value of the active human insulin solution reference material, with the unit of pmol / L; m STD represents the mass of the weighed active human insulin raw material, with the unit of g; P raw represents the purity of the active human insulin raw material, with the unit of g / g; M insulin represents the molar mass of human insulin, with the unit of g / mol; V STD represents the volume of the volumetric flask used when preparing the solution, with the unit of L.

[0386] The certified values of the active human insulin solution reference material at 3 levels were calculated and summarized in Table 17.

[0387] Table 17 Certified values of the active human insulin solution reference material at three levels (pmol / L)

[0388] Number Level 1 Level 2 Level 3 Fixed Value Result 143.67 105.19 44.77

[0389] IX. Evaluation of the uncertainty of the certified value of the active human insulin solution reference material

[0390] According to the uncertainty evaluation mathematical model (formula (20)), the uncertainty of the certified value of the active human insulin solution reference material comes from the uncertainty introduced in the certification process, the uncertainty introduced by the homogeneity of the reference material, and the uncertainty introduced by the long-term stability. The specific sources of uncertainty are as Figure 3 shown.

[0391] 1. Uncertainty introduced in the certification process

[0392] (1) When preparing the insulin stock solution, 1.0195 mg of the active human insulin raw material was accurately weighed, dissolved with the prepared solvent, and then transferred to a 10 mL volumetric flask for volume fixation. Its concentration can be calculated by the following formula:

[0393]

[0394] In the formula, c hINS represents the concentration of human insulin in the stock solution, m hINS represents the mass of the weighed active human insulin raw material, P hINS represents the purity of the active human insulin raw material, V represents the volume of the volumetric flask used when preparing the stock solution, and M hINS represents the molecular weight of human insulin.

[0395] According to this model, considering the influence of the detection limit of the solvent blank at the same time, the uncertainty of the prepared result of the insulin stock solution concentration can be calculated by the following formula:

[0396]

[0397] In the formula, represents the relative uncertainty of the preparation result of the insulin stock solution concentration, dimensionless;

[0398] represents the relative uncertainty of the mass of the insulin purity reference material, dimensionless;

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

[0400] u r,P represents the relative uncertainty introduced by the insulin purity reference material, dimensionless;

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

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

[0403] ① For the balance used to weigh the reference material, according to the balance verification certificate, when the weighing value is 1 mg, the indication error is 0.0002 mg, the off-balance error is -0.0014 mg, and the repeatability is 0.0008 mg. All are considered according to the uniform distribution. The uncertainty introduced by one weighing is:

[0404]

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

[0406]

[0407] ② According to the calibration certificate of the volumetric flask, the U r of the 10 mL volumetric flask is 1% (k = 2). Therefore, the standard uncertainty is U r / 2 = 0.5%; the temperature fluctuation range in the laboratory is ±2 °C, and the volume expansion coefficient of the solvent water is 2.1×10 -2 (%·°C -1 ). Using the rectangular distribution, the volume uncertainty caused by the difference between the calibration temperature and the use temperature of the volumetric flask is: For the 10 mL volumetric flask used in this research and development, the uncertainty introduced by the repeatability of 6 volume fixings is 0.00004%. The combined uncertainty introduced by the 10 mL volumetric flask is:

[0408]

[0409] ③ The assigned value of the insulin purity reference material is (0.857 ± 0.024) g / g. Therefore, the relative standard uncertainty introduced by the insulin reference material purity is as follows:

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

[0411] ④ Based on the molecular formula of insulin C 257 H 383 N 65 O 77 S 6 , the uncertainty introduced by calculating the molecular weight of insulin is calculated according to the following formula:

[0412]

[0413] In the formula, represents the relative uncertainty of the insulin molecular weight, dimensionless;

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

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

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

[0417] According to the IUPAC International Atomic Weight Table, the relative atomic masses and uncertainties of the 5 atoms are as follows:

[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 solvent blank detection, the chemiluminescence immunoassay analyzer method is used. The detection limit of this method is 0.09 pmol / L, and the concentration of the prepared stock solution is 1.504 × 10 7pmol / L. Considering a uniform distribution, the uncertainty component introduced by the method detection limit is as follows:

[0426]

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

[0428]

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

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

[0431]

[0432] In the formula, c 1# represents the concentration of the 1# insulin solution, with the unit of pmol / L;

[0433] c 0 represents the molar concentration of the insulin stock solution, with the unit of pmol / L;

[0434] V 0 represents the volume of the insulin stock solution, with the unit of mL;

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

[0436] Therefore, according to the uncertainty evaluation mathematical model and considering the uncertainty introduced by the solvent blank method detection limit at the same time, the uncertainty of the 1# solution concentration result can be calculated by the following formula:

[0437]

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

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

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

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

[0442] represents the uncertainty introduced by the solvent blank method detection limit, dimensionless.

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

[0444]

[0445] ②According to the calibration certificate of the volumetric flask, the correction value at the 100 mL point is -0.03 mL. Considering it in a rectangular distribution, its relative uncertainty is The volume uncertainty caused by the difference between the calibration temperature and the use temperature is 0.024%, and the uncertainty introduced by the repeatability of volume fixing 6 times is 0.0001%. The combined uncertainty introduced by the 100 mL volumetric flask is:

[0446]

[0447] ③When performing the solvent blank test, the chemiluminescence immunoassay analyzer method is used. The detection limit of this method is 0.09 pmol / L, and the concentration of the prepared Solution 1# is 1.504×10 5 pmol / L. Considering it in a uniform distribution, the uncertainty component introduced by the method detection limit is:

[0448]

[0449] Therefore, the uncertainty of Solution 1# is:

[0450]

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

[0452] When preparing the human insulin solution (Level 1), a 200 μL pipette is used to transfer an appropriate volume of Solution 1# into a 100 mL volumetric flask to complete the preparation process. Therefore, the mathematical model for uncertainty evaluation is:

[0453]

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

[0455] c 1 represents the molar concentration of the insulin solution (Level 1), in units of pmol / L;

[0456] V 1# represents the volume of Solution 1# insulin, in units of mL;

[0457] V represents the volume of the insulin solution (Level 1), in mL.

[0458] Therefore, according to the mathematical model for uncertainty evaluation, considering the uncertainty introduced by the method detection limit of the solvent blank, 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 Insulin Solution No. 1, dimensionless;

[0462] represents the relative uncertainty of the volume of Insulin Solution No. 1, dimensionless;

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

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

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

[0466]

[0467] ② According to the calibration certificate of the volumetric flask, the correction value at the 100 mL point is -0.03 mL. Considering it in a rectangular distribution, its relative uncertainty is The volume uncertainty caused by the difference between the calibration temperature and the use temperature is 0.024%, and the uncertainty introduced by the repeatability of volume fixation 6 times is 0.0001%. The combined uncertainty introduced by the 100 mL volumetric flask is:

[0468]

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

[0470]

[0471] Therefore, the uncertainty of the insulin solution (Level 1) is as follows:

[0472]

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

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

[0475]

[0476] In the formula, c 1# represents the concentration of Solution 1 of insulin, with the unit of pmol / L;

[0477] c 2 represents the molar concentration of the insulin solution (Level 2), with the unit of pmol / L;

[0478] V 1# represents the volume of Solution 1 of insulin, with the unit of mL;

[0479] V represents the volume of the insulin solution (Level 2), with the unit of mL.

[0480] Therefore, according to the mathematical model for uncertainty evaluation and considering 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] In the formula, represents the relative uncertainty of the concentration result of the insulin solution (Level 2), dimensionless;

[0483] represents the relative uncertainty of the concentration result of Solution 1 of insulin, dimensionless;

[0484] represents the relative uncertainty of the volume of Solution 1 of insulin, dimensionless;

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

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

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

[0488]

[0489] ②According to the calibration certificate of the volumetric flask, the correction value at the 250 mL point is -0.13 mL. Considering it according to the rectangular distribution, its relative uncertainty is The volume uncertainty caused by the difference between the calibration temperature and the use temperature is 0.024%, and the uncertainty introduced by the repeatability of volume fixing 6 times is 0.0001%. The combined uncertainty introduced by the 250 mL volumetric flask is:

[0490]

[0491] ③When performing the solvent blank detection, the chemiluminescence immunoassay analyzer method is used. The detection limit of this method is 0.09 pmol / L. The prepared insulin solution (level 2) is 105.19 pmol / L. Considering it according to 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 the 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 mathematical model for uncertainty evaluation is:

[0497]

[0498] In the formula, c 1# represents the concentration of solution 1# insulin, in the unit of pmol / L;

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

[0500] V 1# represents the volume of solution 1# insulin, in the unit of mL;

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

[0502] Therefore, according to the mathematical model for uncertainty evaluation, considering the uncertainty introduced by the method detection limit of the solvent blank, the uncertainty of the concentration result of the insulin solution (Level 3) can be calculated by the following formula:

[0503]

[0504] In the formula, represents the relative uncertainty of the concentration result of the insulin solution (Level 3), dimensionless;

[0505] represents the relative uncertainty of the concentration result of Insulin Solution No. 1, dimensionless;

[0506] represents the relative uncertainty of the volume of Insulin Solution No. 1, dimensionless;

[0507] u r,V represents the relative uncertainty of the volume of the insulin solution (Level 3), dimensionless;

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

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

[0510]

[0511] ② According to the calibration certificate of the volumetric flask, the correction value at the 250 mL point is -0.13 mL. Considering it in a rectangular distribution, its relative uncertainty is The volume uncertainty caused by the difference between the calibration temperature and the use temperature is 0.024%. The uncertainty introduced by the 6 - time volume - fixing repeatability is 0.0001%. The uncertainty introduced by the 250 mL volumetric flask after synthesis is:

[0512]

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

[0514]

[0515] Therefore, the uncertainty of the insulin solution (Level 3) is as follows:

[0516]

[0517] Therefore, the uncertainties in the certification process of the active human insulin solution reference materials at each level are shown in Table 18.

[0518] Table 18 Uncertainties introduced in the certification process of the active human insulin solution reference materials

[0519]

[0520] 2. Uncertainty component introduced by homogeneity

[0521] According to JJF1343-2022, the uncertainty introduced by the homogeneity of the reference material is equal to the standard deviation of the between-bottle homogeneity. When the between-group mean square is greater than the within-group mean square, it is calculated according to the following formula:

[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 reference materials of the active human insulin solution at 3 levels are shown in Table 19 as follows.

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

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

[0528] 3. Uncertainty component introduced by stability

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

[0530] u sts = s k,sts ·t

[0531] The uncertainty components introduced by the stability of the reference materials of the active human insulin solution at 3 levels are shown in Table 20 as follows.

[0532] Table 20 Uncertainty components introduced by the stability of the reference materials of the active human insulin solution at three levels pmol / L

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

[0534] 4. Uncertainty of the certified value result

[0535] The uncertainty components introduced by the certification process, homogeneity, and stability are combined to obtain the uncertainty of the certified value result, as shown in Table 21.

[0536] Table 21 Uncertainty of the certified value results of the human insulin solution reference material at three levels pmol / L

[0537]

[0538] Therefore, the certified value results of the human insulin solution reference material with active insulin at three levels are shown in Table 22:

[0539] Table 22 Certified value results of the human insulin solution reference material with active insulin at three levels pmol / L

[0540] Number Level 1 Level 2 Level 3 Fixed Value Result 143.7±6.3 105.2±4.2 44.8±2.0

[0541] X. Evaluation of the commutability of the human insulin solution reference material

[0542] A total of 39 single-person serum samples that can cover the concentration range of the human insulin solution reference material prepared in Step VI are collected. These samples are centrifuged within 24 hours after blood collection and separately placed in a -70°C refrigerator. Together with the human insulin solution reference materials at each level prepared in Step VI, the insulin concentrations of the clinical samples and reference materials are measured on 6 common domestic and foreign chemiluminescence immunoassay systems, including Siemens, Beckman, Roche, Abbott, Mindray, and Antu. Each sample is measured in parallel 2 times. The obtained results are averaged and evaluated according to the CLSI EP14 standard. The results are as Figure 16 shown.

[0543] As can be Figure 16 seen, the three insulin solution reference materials prepared from active insulin (the figure also shows the human insulin solution reference materials not detailed in this example) are all within the 95% prediction interval determined by the patient sample results, indicating that the insulin solution reference materials at these three levels have good commutability.

[0544] In summary, the insulin solution standard material after regulation by 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, it is no longer necessary to develop complex matrix standard materials to ensure interchangeability, thereby reducing the development cost of interchangeable protein standard materials. 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 transfer chain of ISO 17511:2020 is shortened, which helps to reduce the measurement uncertainty of the value transfer 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 are only a few embodiments of the present application and do not constitute any form of limitation to the present application. Although the present application is disclosed as above with preferred embodiments, it is not intended to limit the present application. Any technician familiar with the 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% is selected and purified by reverse phase high performance liquid chromatography and molecular sieves to obtain high-purity human insulin raw material; (2) Self-assembly of human insulin: dissolving the high-purity human insulin raw material in a 1×PBS solution containing zinc ions and allowing the solution 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, and at the same time, a human insulin solution prepared with a PBS solution without zinc ions and having the same concentration as in step (2) is subjected to circular dichroism and mass spectrometry spectrum scanning analysis as a comparison, and 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: construct a high performance liquid chromatography and surface plasmon resonance (SPR) spectroscopy coupling device, use a molecular sieve column to perform online separation and online SPR spectroscopy detection on the insulin self-assembled complex confirmed in step (3), collect the fractions with a signal slope increase of more than 30 degrees on the sensor graph and freeze-dry them to obtain active human insulin raw materials; (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 active human insulin solution standard substance with interchangeability: The active human insulin raw material in step (4) is prepared into solutions with multiple concentration levels ranging from 10 to 1000 pmol / L using a diluent, and the solutions are packaged and sealed to form active human insulin solution standard substance candidates with interchangeability. The candidates are then subjected to homogeneity test, stability test, standard value determination, uncertainty assessment, and interchangeability evaluation in sequence to obtain active human insulin solution standard substances with interchangeability.

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; Preferably, 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, using a C4, C8 or C18 chromatographic column for separation, separating human insulin and A21-deamino human insulin, and collecting the human insulin fraction for the next step of molecular sieve purification; Preferably, 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 a high-purity human insulin raw material.

3. 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 zinc ions includes zinc acetate; Preferably, 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 ion in the solution is 6:1-6:60; Preferably, in step (2), the standing temperature is 4 to 10° C., and the standing time is 4 to 48 hours.

4. 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 to 1 cm, the scanning wavelength range is 200 to 400 nm, and the scanning speed is 10 to 100 nm / min; Preferably, in step (3), the judgment criterion is: if the circular dichroism peak moves toward the high wavelength direction and becomes gentle, and a peak with a larger molecular weight appears on the mass spectrometer spectrum, a self-assembled complex is formed, otherwise it is not formed.

5. 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 and surface plasmon resonance (SPR) spectroscopy coupling 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 the gold foil surface with carboxymethyl cellulose as the detector of the SPR signal; Preferably, the molar concentration of the sodium hydroxide is 0.5-20 mmol / L.

6. 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 comprises: S1. The active human insulin raw material is prepared into an active human insulin solution using 1×PBST solution, and the immunoaffinity activity concentration c of the active human insulin solution is determined. raw,active ; S2. Take the prepared active human insulin solution, weigh its mass and add isotope-labeled amino acids thereto, concentrate by centrifugation or 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 according to the protein sequence of human insulin selected in step (1). raw,chem ; In the formula, 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 this 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 is the mass of 1× PBST solution, C raw,chem is the physicochemical concentration of active human insulin solution; S4. Using a densitometer 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 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.

7. The method for preparing an interchangeable active human insulin solution standard substance according to claim 6, 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 Determined by surface plasmon resonance spectroscopy; Preferably, in step S2, the isotopes include 13 C. 15 At least one of N and D, the amino acid includes at least one of proline, valine, leucine, isoleucine, and phenylalanine, and the number of isotope-labeled atoms is ≥ 3; Preferably, 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.

8. 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 HPLC purity of the bovine serum albumin solution is greater than 99%, and the bovine serum albumin solution further contains 0-500 mmol / L of zinc acetate, and the zinc acetate is of analytical grade or above; Preferably, in step (6), the standard value determination is performed according to the following formula: In the formula, c CRM Indicates the certified value of active human insulin solution standard substance, unit: pmol / L; m STD It indicates the mass of the weighed active human insulin raw material, in g; P raw Indicates the purity of active human insulin raw materials, in 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; Preferably, in step (6), the uncertainty includes the uncertainty introduced in the valuation process, the uncertainty introduced by the uniformity of the standard material, the uncertainty introduced by the long-term stability, the combined standard uncertainty of the valuation result, and the expanded standard uncertainty of the valuation result.

9. An interchangeable active human insulin solution standard substance, characterized in that: The compound is prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the interchangeable active human insulin solution standard substance as claimed in claim 9, or the interchangeable active human insulin solution standard substance prepared by the preparation method as claimed in any one of claims 1 to 8 as an interchangeable standard substance.

Images