Method, system and device for evaluating consistency of heparin drugs

Abstract

The invention relates to a heparin drug consistency evaluation method, system and device, and belongs to the technical field of drug quality control. Comprising the following steps: carrying out disaccharide analysis, oligosaccharide analysis and two-dimensional liquid phase analysis on a selected standard preparation to respectively obtain corresponding peak parameters, and constructing a qualitative database and a quantitative database according to the peak parameters; acquiring the chromatographic data of the reference preparation treated under the same condition as the sample to be detected, and performing consistency evaluation on the chromatographic data and the two databases; wherein the consistency evaluation comprises the following steps: acquiring chromatographic data of a reference preparation, performing automatic integration on the chromatographic data, and performing automatic attribution on components by utilizing a qualitative database to respectively obtain two peak contents; obtaining samples in a quantitative database; according to the sample, carrying out consistency evaluation on the two peak contents; and if yes, acquiring the chromatographic data of a large batch of samples to be detected and carrying out consistency evaluation on the chromatographic data. According to the method, the chromatographic data processing process is simplified, and the efficiency, accuracy and reliability of quality evaluation of the heparin drugs are improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of drug quality control, in particular to a heparin drug consistency evaluation method, system and device. BACKGROUND

[0002] Heparin is a kind of extremely important anticoagulant drug, which plays a key role in clinical treatment. However, the structure of heparin drug is complex, which brings great challenges to quality control. In order to ensure product quality, key quality attributes need to be characterized from multiple dimensions. Specifically, it should cover aspects such as raw material traceability, depolymerization process, physicochemical properties, structural composition, biological activity and immunogenicity, etc. Through systematic verification of these aspects, the consistency of quality is fully ensured.

[0003] In order to ensure the accuracy of the data obtained by each quality evaluation, it is usually necessary to use multiple batches of reference preparations to compare the test samples multiple times. The multi-dimensional structure analysis data processing process involves multiple software, and the automatic integration means of the current liquid- quality platform is not suitable for macromolecular sugar substances, and still needs to be handled manually, which has certain requirements for the use of software and is greatly affected by the experimental personnel's skills, which may interfere with the integration results, at the same time, it also greatly increases the data processing time, affecting the work efficiency. In addition, the attribution of each component in the experimental data still depends on manual work, which greatly increases the risk of errors; in addition, the existing statistical software has scattered functions, is highly professional and has high learning cost, which all make the quality evaluation of heparin drugs face great challenges. Therefore, how to comprehensively, quickly and accurately evaluate the consistency of heparin drugs is an important problem to be solved at present. SUMMARY

[0004] Therefore, the technical problem to be solved by the present application is to overcome the problems of repeated experiments, complex chromatographic data processing process, low efficiency, scattered statistical analysis functions and high learning cost in the prior art.

[0005] In order to solve the above technical problems, the present application provides a heparin drug consistency evaluation method, which comprises: S1, selecting a standard preparation according to the reference preparation and marketed preparation required by the drug to be tested; S2, performing disaccharide analysis, oligosaccharide analysis and two-dimensional liquid chromatography on the standard preparation to obtain first peak parameters, second peak parameters and third peak parameters respectively; constructing a qualitative database and a quantitative database according to the first peak parameters, the second peak parameters and the third peak parameters; S3, obtaining reference preparation chromatographic data processed under the same conditions as the sample to be tested; performing consistency evaluation on the reference preparation chromatographic data and the qualitative database and the quantitative database; wherein the consistency evaluation step is: The reference preparation chromatographic data is automatically integrated, and components are automatically attributed by using the qualitative database, to obtain the key peak content and the peak content of the quantitative database respectively; The sample in the quantitative database is obtained; and the key peak content and the peak content of the qualitative database and the quantitative database are evaluated for consistency according to the sample; S4, when the reference preparation chromatographic data is consistent with the quantitative database, the chromatographic data of a large number of to-be-tested samples are obtained; and the chromatographic data are evaluated for consistency with the qualitative database and the quantitative database.

[0006] In an embodiment of the present application, the reference preparation chromatographic data includes a first chromatographic peak obtained by disaccharide analysis, a second chromatographic peak obtained by oligosaccharide analysis, and a third chromatographic peak obtained by two-dimensional liquid chromatography analysis; and the method for automatically integrating the reference preparation chromatographic data is as follows: the first chromatographic peak is automatically integrated by using template integration; the second chromatographic peak is automatically integrated by using automatic vertical integration; and the third chromatographic peak is automatically integrated by using vertical template integration.

[0007] In an embodiment of the present application, the step of automatically integrating the first chromatographic peak by using template integration is as follows: A plurality of disaccharide standard samples and any set of sample disaccharide analysis data are obtained, and the sample is manually integrated; in the manual integration, baseline start and end peaks and valleys are identified to assist integration, and a first integration result is obtained; The first integration result is set as a first template, the retention time of the remaining sample chromatogram is calibrated according to the plurality of disaccharide standard samples, the chromatographic peak of the preliminary integration is corrected by iteration of cut points, and automatic template integration is completed by adapting to different baseline conditions.

[0008] In an embodiment of the present application, the step of automatically integrating the second chromatographic peak by using automatic vertical integration is as follows: Sample oligosaccharide analysis data are obtained, and a continuous horizontal baseline is generated according to the chromatographic trend; In the horizontal baseline range, a vertical line is drawn downward from each peak valley and shoulder peak to complete automatic integration.

[0009] In an embodiment of the present application, the step of automatically integrating the third chromatographic peak by using vertical template integration is as follows: Any sample two-dimensional liquid chromatography analysis single-polymerization-degree oligosaccharide data are obtained to perform manual integration; According to the manual integration result, a vertical line is automatically drawn downward from each peak valley and shoulder peak to complete integration, and a second integration result is obtained; The second integration result is set as the second template, and integration is performed using the second template to complete the automatic integration of a large number of samples.

[0010] In one embodiment of the present invention, step S2, in the process of constructing a qualitative database and a quantitative database based on the first peak parameter, the second peak parameter, and the third peak parameter, includes performing template integration on the first peak parameter and using the template integration to perform offset calibration on the retention time. The offset calibration step is as follows: Calculate the internal average of the retention time offsets of multiple disaccharides in the sample and template; If the internal average value exceeds the preset time, then during template integration, the retention time of the sample start and end points is corrected based on the template according to the offset standard, and the nearest valley point is found near the corrected retention time to serve as the final sample start and end points.

[0011] In one embodiment of the present invention, in step S3, the method for automatically assigning components using the qualitative database is as follows: for the chromatographic data obtained from the disaccharide analysis and the two-dimensional liquid chromatography analysis, automatic assignment is completed by matching the relative retention time with the components in the qualitative database; for the chromatographic data obtained from the oligosaccharide analysis, automatic assignment is performed by the peak shape and retention time difference.

[0012] In one embodiment of the present invention, in step S3, the method for evaluating the consistency between the critical peak content and the peak content of the quantitative database based on the sample is as follows: the consistency between the critical peak content and the peak content of the quantitative database is evaluated using t-test, mass range analysis, and principal component analysis.

[0013] Secondly, to solve the above-mentioned technical problems, the present invention provides a consistency evaluation system for heparin drugs, comprising: The selection module is used to select a standard formulation based on the reference formulation and marketed formulations required for the drug to be tested. The module performs disaccharide analysis, oligosaccharide analysis, and two-dimensional liquid chromatography analysis on the standard preparation to obtain the first peak parameter, the second peak parameter, and the third peak parameter, respectively; and constructs a qualitative database and a quantitative database based on the first peak parameter, the second peak parameter, and the third peak parameter. The first evaluation module is used to acquire chromatographic data of a reference preparation processed under the same conditions as the sample to be tested; and to evaluate the consistency of the reference preparation chromatographic data with the qualitative database and the quantitative database; wherein, the consistency evaluation includes: The chromatographic data of the reference preparation are automatically integrated, and the components are automatically assigned using the qualitative database to obtain the content of key peaks and the content of peaks in the quantitative database, respectively. Obtain samples from the quantitative database; evaluate the consistency between the key peak content and the peak content in the quantitative database based on the samples; The second evaluation module is used to acquire chromatographic data of a large number of test samples when the chromatographic data of the reference preparation is consistent with the quantitative database; and to evaluate the consistency of the chromatographic data with the qualitative database and the quantitative database.

[0014] Thirdly, in order to solve the above-mentioned technical problems, the present invention provides a consistency evaluation device for heparin drugs, including the above-mentioned consistency evaluation system for heparin drugs.

[0015] Compared with the prior art, the above-described technical solution of the present invention has the following advantages: (1) The consistency evaluation method, system, and apparatus for heparin drugs described in this invention comprehensively evaluate the structural characteristics of heparin drugs through disaccharide analysis, oligosaccharide analysis, and two-dimensional liquid chromatography analysis, more accurately characterizing the key quality attributes of the drugs and ensuring the comprehensiveness and accuracy of quality control. Automatic integration and automatic attribution functions reduce human error, improve data processing efficiency and accuracy, and ensure the consistency and reliability of quality evaluation. By constructing qualitative and quantitative databases and standardized evaluation processes, the quality evaluation work becomes more systematic and standardized, reducing repetitive work and unnecessary steps. The automated consistency evaluation method can quickly process large amounts of data, significantly improving work efficiency and shortening evaluation time. After confirming consistency between the reference preparation and the quantitative database, rapid and accurate consistency evaluation can be performed on a large number of test samples, improving the efficiency and feasibility of large-scale quality control.

[0016] (2) This invention automates the processing of chromatographic data of heparin drugs and performs statistical comparisons with a database to accurately assess their quality consistency. This not only eliminates the cumbersome process of analysts conducting large-scale comparison experiments between reference formulations and sample results, but also fills the gap in existing integrated and automated data processing software. In addition, this method significantly reduces the burden on analysts involved in consistency evaluation in terms of experimental operations and data processing, improving work efficiency and accuracy. Attached Figure Description

[0017] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein: Figure 1 This is a flowchart of a consistency evaluation method for heparin drugs in a preferred embodiment of the present invention; Figure 2 This is a schematic diagram of automatic peak and valley identification in a preferred embodiment of the present invention; Figure 3This is a schematic diagram of the template integration retention time offset before correction in a preferred embodiment of the present invention; Figure 4 This is a schematic diagram of the template integral retention time offset after correction in a preferred embodiment of the present invention; Figure 5 This is a schematic diagram of the template integral tangent point before iterative correction in a preferred embodiment of the present invention; Figure 6 This is a schematic diagram of the template integral tangent point after iterative correction in a preferred embodiment of the present invention; Figure 7 This is a first schematic diagram of the method for obtaining the template integral baseline in a preferred embodiment of the present invention; Figure 8 This is a second schematic diagram of the method for obtaining the template integral baseline in a preferred embodiment of the present invention; Figure 9 This is a third schematic diagram of the method for obtaining the template integral baseline in a preferred embodiment of the present invention; Figure 10 This is a fourth schematic diagram of the method for obtaining the template integral baseline in a preferred embodiment of the present invention; Figure 11 This is a schematic diagram of valley point determination for automatic vertical integration in a preferred embodiment of the present invention; Figure 12 This is a peak curve diagram from the automatic oligosaccharide analysis attribution method in a preferred embodiment of the present invention; Figure 13 This is a flowchart of the automatic oligosaccharide analysis and attribution method in a preferred embodiment of the present invention; Figure 14 This is a schematic diagram illustrating an application of template integration in a preferred embodiment of the present invention; Figure 15 This is a schematic diagram illustrating an application of automatic vertical integration in a preferred embodiment of the present invention; Figure 16 This is a schematic diagram illustrating an application of vertical template integration in a preferred embodiment of the present invention; Figure 17 This is a schematic diagram of a database verification application in a preferred embodiment of the present invention; Figure 18 This is a preferred embodiment of the present invention. Figure 17 The corresponding effect diagram. Detailed Implementation

[0018] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention. Example 1

[0019] Reference Figure 1As shown, this embodiment of the invention provides a method for consistency evaluation of heparin drugs, including but not limited to the following steps: S1. Select the standard formulation based on the reference formulation and marketed formulations required for the drug to be tested; S2. Perform disaccharide analysis, oligosaccharide analysis, and two-dimensional liquid chromatography analysis on the standard preparation to obtain the first peak parameter, second peak parameter, and third peak parameter, respectively; construct a qualitative database and a quantitative database based on the first peak parameter, second peak parameter, and third peak parameter; S3. Obtain chromatographic data of the reference preparation processed under the same conditions as the sample to be tested; evaluate the consistency of the reference preparation chromatographic data with the qualitative and quantitative databases; the consistency evaluation steps are as follows: The chromatographic data of the reference preparation were automatically integrated, and the components were automatically assigned using a qualitative database to obtain the content of key peaks and the content of peaks in the quantitative database, respectively. Obtain samples from the quantitative database; based on the samples, evaluate the consistency between the content of key peaks and the peak content in the quantitative database; S4. When the chromatographic data of the reference preparation is consistent with the quantitative database, obtain the chromatographic data of a large number of test samples; evaluate the consistency of the chromatographic data with the qualitative and quantitative databases.

[0020] This invention provides an automated evaluation method for heparin drugs through multi-dimensional integrated analysis, comprehensively assessing the structural characteristics of heparin drugs through disaccharide analysis, oligosaccharide analysis, and two-dimensional liquid chromatography. This multi-dimensional analysis method can more accurately characterize the key quality attributes of drugs, ensuring the comprehensiveness and accuracy of quality control. Automatic integration and automatic attribution functions reduce errors from manual operation, improve the efficiency and accuracy of data processing, and ensure the consistency and reliability of quality evaluation. By constructing qualitative and quantitative databases and a standardized evaluation process, the quality evaluation work becomes more systematic and standardized, reducing repetitive work and unnecessary steps. The automated consistency evaluation method can quickly process large amounts of data, improving work efficiency and shortening the quality evaluation time. After confirming the consistency between the reference preparation and the quantitative database, rapid and accurate consistency evaluation can be performed on a large number of test samples, improving the efficiency and feasibility of large-scale quality control. This invention also provides a multi-dimensional, automated chromatographic data processing method that accurately determines the statistical differences between the test heparin drugs and the database through automated processing of chromatographic analysis data. Therefore, it eliminates the need for large-scale repetitive experiments and data processing, significantly reducing the burden on analysts. Furthermore, this method overcomes the problems of complex and inefficient chromatographic data processing, fragmented statistical analysis functions, and high learning costs in existing technologies, and significantly improves the efficiency, accuracy, and reliability of heparin drug quality evaluation.

[0021] Specifically, in step S1, qualitative and quantitative databases are established based on reference formulations and marketed formulations required for the drug under test. The drug under test is a heparin-based drug, encompassing low molecular weight heparin (e.g., enoxaparin sodium), standard heparin (e.g., unfractionated heparin), and synthetic heparin (e.g., fondaparinux sodium). Reference formulations are typically validated, high-quality standard samples that provide a reliable benchmark for quality evaluation. Marketed formulations represent the product quality in actual manufacturing processes. By selecting representative reference and marketed formulations, the comprehensiveness and accuracy of the standard database can be ensured.

[0022] Specifically, in step S2, the selected standard preparation is subjected to disaccharide analysis, oligosaccharide analysis, and two-dimensional liquid chromatography analysis to obtain peak parameters (including the first peak parameter, such as peak composition; the second peak parameter, such as peak relative retention time; and the third peak parameter, such as peak area percentage) corresponding to different analytical methods, thereby constructing a qualitative and quantitative database of the standard. The specific steps for obtaining the peak parameters corresponding to different analytical methods are as follows: S210. Obtain the qualitative parameters of the qualitative database. Perform disaccharide analysis, oligosaccharide analysis, and two-dimensional liquid chromatography analysis on the selected preparations in the database using the same chromatographic conditions. Improve the qualitative parameters of the database based on the relative retention time and mass spectrometry data.

[0023] S220. Obtain the quantitative parameters from the quantitative database. Import the raw chromatographic data obtained from the analysis into the software, and refine the quantitative parameters in the database based on the software's automatic integration. The automatic integration steps are as follows: S221. The chromatographic peaks obtained from disaccharide analysis are integrated using a template strategy. Multiple (e.g., 8) disaccharide standards and any set of sample disaccharide analysis data are imported, and the samples are manually integrated. During manual integration, the software automatically identifies the baseline's beginning and end peaks and troughs to assist integration, obtaining the first integration result. After integration, the first integration result is set as the first template. The chromatographic retention times of the remaining samples are calibrated based on multiple (e.g., 8) disaccharide standards. The tangent points of the initially integrated chromatographic peaks are iteratively corrected, adapting to different baseline conditions to complete automatic template integration.

[0024] Furthermore, the disaccharide analysis employs a template integration strategy, including: automatically identifying template integration valleys, calibrating retention time offsets, iteratively correcting integration tangent points, and setting corresponding baseline acquisition methods based on different templates.

[0025] Specifically, the steps to achieve automatic recognition of valley points in the template integration are as follows: Calculate the first derivative of the chromatographic curve using the differentiation formula. The expression for the derivative formula is: (1) in, Indicates in The function value at that location; Indicates in The function value at that location; Indicates the independent variable x The next value. Represent the independent variable x The current value.

[0026] When the first derivative of the chromatographic curve is 0, and the first derivative on the left is less than 0 while the first derivative on the right is greater than 0, this point is considered a valley of the chromatographic peak. After manual integration, when adjusting the results, the nearest valley will be highlighted in red to assist in accurate manual integration. (Refer to...) Figure 2 .

[0027] Specifically, the method for calibrating the template integral against the retention time offset is as follows: Calculate the internal average value of the retention time offset of multiple (e.g., 8) disaccharides in the sample and the first template. If it exceeds the preset time, preferably 0.05 min in this embodiment, the retention time of the sample's start and end points will be corrected according to the offset standard based on the template when the first template is integrated. The nearest valley point is found near the corrected retention time, which is the final sample start and end point.

[0028] For example, taking the samples and templates in Table 1 as examples, the results of their offset calibration are as follows: Figures 3-4 As shown.

[0029] Table 1:

[0030] Specifically, the implementation method of the template integral tangent point iterative correction function is as follows: Calculate the vertical height difference between the chromatographic curve and the baseline within the range from the integration starting point to the nearest vertex. Obtain the number of sites on the chromatographic curve below the baseline and the point of maximum vertical difference between them. If the number of sites below the baseline exceeds 5, modify the integration starting point to the midpoint between the original starting point and the point of maximum vertical difference, using this as the new integration starting point. After the initial calibration, continue comparing and modifying the tangent point, iterating until the number of sites on the chromatographic curve below the baseline is less than 5. This tangent point is then used as the final start and end point of the chromatographic peak. (Refer to...) Figures 5-6 .

[0031] Specifically, Figures 7-10 This demonstrates the baseline acquisition method for template points, which is set according to different template conditions. The specific steps of the method are as follows: When the baseline endpoints of the chromatographic peaks are all on the chromatographic curve, the corresponding start and end valley points of the sample are found based on the template endpoints and the offset standard. If no corresponding start and end valley points are found, the next valley point is searched on the same side (i.e., to the left of the start point and to the right of the end point) as the extended start and end valley point. To prevent errors in the search, the template valley point corresponding to this point is obtained. If it is on a different side compared to the original template valley point (i.e., to the right of the original template start point and to the left of the end point), this point is corrected to a valley point on the same side. Connecting the corrected start and end valley points yields the final sample baseline.

[0032] When the endpoint of the chromatographic peak is not on the chromatographic curve (collinear with the peak and valley in the vertical direction), the starting and ending valley points of the sample are found based on the template extension and the offset standard to form the baseline. If there are other valley points between these baselines and these valley points are below the baseline, the endpoint of the sample baseline is moved to these points to obtain the final extended baseline of the sample. Finally, the final extended baseline is truncated, and the corresponding peak and valley in the sample are found using the offset standard. A perpendicular line is drawn downwards, and the intersection of this line and the final extended baseline is the final starting and ending point of the sample. Connecting the final starting and ending points yields the final sample baseline.

[0033] S222. The chromatographic peaks obtained from oligosaccharide analysis are integrated using an automatic vertical integration strategy. The sample oligosaccharide analysis data is imported, and a continuous horizontal baseline is automatically generated based on the chromatographic trend. This baseline adapts to different types of complex chromatographic peaks and autonomously draws vertical lines downward from each peak valley (including shoulder peaks) within this baseline range to complete the integration.

[0034] Furthermore, the automatic vertical integration strategy includes a valley point determination method. The specific method for the valley point determination method, i.e., the automatic integration of complex UV chromatographic multiplets in automatic vertical integration, is as follows: Calculate the first derivative of the chromatographic curve according to the differentiation formula (1). Peaks and valleys are determined by the first and second derivatives of the chromatographic peaks. Normal peaks have obvious valleys; the first derivative at the valley is 0, and the first derivative on the left is less than 0, while the first derivative on the right is greater than 0. For shoulder peaks, the first derivative changes, resulting in an extreme point. The intersection of a vertical line drawn upwards from this point with the chromatographic curve is the valley of the shoulder peak. For hidden peaks, which are equivalent to shoulder peaks formed on the first derivative, the valley needs to be determined based on the second derivative. Automatic vertical integration is completed by drawing a dividing line downwards from the valley and intersecting the baseline. (Refer to...) Figure 11 As shown.

[0035] S223. The chromatographic peaks obtained from two-dimensional liquid chromatography (2LC) are integrated using a vertical template strategy. Import any sample's 2LC data on oligosaccharides with a single degree of polymerization and perform manual integration. Similar to the integration method in oligosaccharide analysis, a vertical line is automatically drawn downwards from each peak valley (including shoulders) to complete the integration and obtain the second integration result. This integration result is then set as the second template. Through integration using the second template, large batches of samples can be processed.

[0036] Specifically, in step S3, before conducting large-scale sample testing, chromatographic data of a reference formulation processed under the same conditions as the samples are acquired. This reference formulation chromatographic data includes a first chromatographic peak obtained through disaccharide analysis, a second chromatographic peak obtained through oligosaccharide analysis, and a third chromatographic peak obtained through two-dimensional liquid chromatography analysis. The consistency of the reference formulation chromatographic data with the quantitative database is evaluated to verify its usability.

[0037] For example, the steps for obtaining chromatographic data of a reference preparation processed under the same conditions as the sample are as follows: First, select a validated reference preparation; second, prepare the mobile phase and the solution of the test sample according to the standard operating procedure (SOP). Ensure that the concentration, solvent, and processing method are consistent with the test sample. Next, set the chromatographic analysis parameters, including column type, mobile phase composition, flow rate, detection wavelength, column temperature, etc., ensuring that these parameters are completely consistent with the analytical conditions of the test sample. Then, inject the reference preparation solution into the chromatographic system for analysis and record the chromatogram, including key parameters such as retention time (RT), peak area, and peak height. Subsequently, acquire chromatographic data using a chromatographic data system and perform preprocessing, such as baseline correction, noise filtering, and integration, to ensure the accuracy and reliability of the data. After data processing, validate the results, check the integrity and consistency of the data, and resolve any anomalies. Finally, store the validated data and perform repeatability tests to assess the stability of the experiment, providing accurate and reliable reference data for subsequent consistency evaluation and quality control.

[0038] Furthermore, the specific steps for consistency evaluation between the reference preparation chromatographic data and the quantitative database are as follows: S310. Import the raw sample data and perform automatic integration. The specific steps for automatic integration are similar to those in steps S221 to S223. Specifically, the procedure for automatically integrating the first chromatographic peak using template integration can be found in step S221; the procedure for automatically integrating the second chromatographic peak using automatic vertical integration can be found in step S222; and the procedure for automatically integrating the third chromatographic peak using vertical template integration can be found in step S223.

[0039] S320. Automatically assign the raw sample data. Specifically, for disaccharide analysis and two-dimensional liquid chromatography analysis, automatic assignment is completed by matching the relative retention time with the components in the qualitative database; for oligosaccharide analysis, assignment is completed by peak shape and retention time difference.

[0040] Specifically, the different integration strategies in steps S221 to S223 correspond to different automatic attribution methods, including: Disaccharide analysis and two-dimensional liquid chromatography automatically assign components by matching relative retention time with components in a qualitative database; while oligosaccharide analysis assigns components based on peak shape and retention time difference.

[0041] For example, the attribution of peaks in oligosaccharide analysis is based on... Figure 12 As shown, the highest peak is determined to be dp6, and dp8, dp10, dp12, and so on are named sequentially to the left, while dp6, dp4, and so on are named sequentially to the right. After integration, the ΔRT between adjacent peaks is automatically identified. When ΔRT > 0.7, it is considered as if there is no shoulder peak and is named normally; when ΔRT < 0.7, it is considered as a shoulder peak and named dpx-1 / 2 / 3. Refer to Table 2 and... Figure 13 As shown, the specific steps for assigning chromatographic peaks are as follows: Step 1: Locate the highest peak in the chromatogram, such as... Figure 12 As shown, it is assigned to dp6.

[0042] Step 2: Using dp6 as the center, assign it to the left and right.

[0043] Step 3: Compare the retention time (RT) difference (△RT) between the current peak (CP) and the left adjacent peak (LAP) and the right adjacent peak (RAP).

[0044] Step 4: If ΔRT is less than 0.7, then assign LAP / RAP to the current peak (CP).

[0045] Step 5: If ΔRT is greater than or equal to 0.7, then assign LAP / RAP to the peak preceding or following CP (i.e., CP + / - 2).

[0046] Step 6: Check for any unassigned peaks. If unassigned peaks exist, return to Step 3 and continue the assignment process. If no unassigned peaks exist, name peaks with the same degree of aggregation in descending order of their RT (Rapid Regression).

[0047] Table 2:

[0048] S330. Open the organized database sample and evaluate the consistency of the sample to be tested using t-test, quality range analysis, and principal component analysis (PCA).

[0049] Specifically, firstly, a t-test is used to compare whether there are statistically significant differences in key parameters between the tested sample and the standard database samples, thereby assessing their consistency. Secondly, quality range analysis is used to determine whether the component information in the tested sample falls within the preset quality standard range, further verifying its quality consistency. Finally, principal component analysis (PCA) is applied to reduce the dimensionality of the multidimensional data, extract the main variation information, and assess the similarity of the tested sample and the standard database samples in key features, thus comprehensively judging their consistency. These three statistical analysis methods complement each other, providing a comprehensive evaluation of the sample consistency from different perspectives, improving the accuracy and reliability of the evaluation results.

[0050] This invention evaluates the consistency of chromatographic data by automating the processing of chromatographic data. The evaluation process includes semi-automatic integration and assignment of sample chromatograms, t-test with database, quality range analysis, principal component analysis, and statistical evaluation.

[0051] Specifically, in step S4, after successful verification, a large batch of sample chromatographic data can be imported for consistency evaluation with the database to verify its quality consistency. The consistency evaluation in step S4 includes automatic integration, component assignment, and consistency assessment steps, which are similar to the methods in step S3, and therefore will not be repeated here. This process allows for efficient, rapid, and accurate quality consistency evaluation of large batches of samples, ensuring the stability and reliability of product quality during large-scale production.

[0052] This invention provides a method for multi-dimensional automated evaluation of heparin drugs. This method automates the processing of chromatographic data of heparin drugs and performs statistical comparisons with a database to accurately assess their quality consistency. This not only eliminates the cumbersome process of analysts conducting large-scale comparison experiments between reference formulations and samples, but also fills a gap in existing integrated and automated data processing software. Furthermore, this method reduces the burden on analysts involved in consistency evaluation in terms of experimental operations and data processing, improving work efficiency and accuracy.

[0053] To more clearly illustrate the method described in the embodiments of the present invention, exemplary steps are provided below. It should be noted that the implementation conditions can be flexibly adjusted according to the actual situation of the specific manufacturer, and conditions not specifically specified can be performed according to conventional experimental conditions. The specific details are as follows: Experimental objective: To analyze the multidimensional structural characteristics of the drug under test using liquid chromatography-UV and multicenter cut two-dimensional liquid chromatography (MHC 2D LC) to evaluate the consistency of heparin samples from different manufacturers (taking enoxaparin sodium as an example).

[0054] Experimental Methods: The complete enzymatic hydrolysis products were separated using a strong anion exchange column, and the enoxaparin sodium disaccharide units were analyzed using high-resolution mass spectrometry (HMS). Enoxaparin sodium oligosaccharides were also separated using two molecular sieve columns with different pore sizes in series, and analyzed using HMS. Furthermore, enoxaparin sodium oligosaccharides with a single degree of polymerization were separated using a molecular sieve column in the first dimension and a strong anion exchange column in the second dimension, and analyzed using HMS. Because enoxaparin sodium oligosaccharides have double bonds at their non-reducing ends, they exhibit characteristic absorption at 232 nm; therefore, the UV spectrum at 232 nm was analyzed in this experiment.

[0055] For experimental data processing methods, please refer to... Figure 1 As shown, specifically: Step 1: Construct qualitative and quantitative databases. The specific steps are as follows: Step 1-1: Obtain qualitative parameters for the qualitative database. Specifically, identify a substantial number of reference formulations and marketed formulations to be used as database parameters. Perform oligosaccharide analysis, disaccharide analysis, and two-dimensional liquid chromatography analysis on the selected formulations in the database using the same chromatographic conditions. Refine the qualitative analysis parameters of the database based on relative retention times and structural composition.

[0056] Step 1-2: Obtain the quantitative parameters from the quantitative database. The specific steps are as follows: Step 1-2-1: Automatic integration of the chromatographic data obtained from disaccharide analysis is performed using the template integration method. The integration result is as follows: Figure 14 As shown. The specific steps are as follows: Step 1-2-1-1: Export the raw chromatographic data obtained from the experiment as a "csv" file.

[0057] Step 1-2-1-2: Launch the software and open the main interface, which contains three main function icons: "Integration," "Statistical Evaluation," and "Configuration." Click the "Integration" icon, and select the "Disaccharide Integration" option from the drop-down secondary function menu to perform disaccharide integration analysis.

[0058] Steps 1-2-1-3: Click the "Import Standards" function button on the right side of the interface, and select the CSV file containing the chromatographic data of the eight disaccharide standards obtained in the same experiment as the sample. The software will automatically identify and locate the chromatographic peaks of these eight standard disaccharides.

[0059] Step 1-2-1-4: Click the "Import Sample" function button and select the chromatographic data CSV file of the sample to be processed. After manually completing the integration operation, click the "Save Results" button to save the current integration results.

[0060] Step 1-2-1-5: Click the "Template Import" function button and select the template CSV file saved after manual integration. In the pop-up dialog box, select the remaining sample chromatogram CSV files to be integrated.

[0061] Step 1-2-1-6: The software will automatically complete the integration operation of the remaining sample chromatographic data based on the imported template.

[0062] Step 1-2-1-7: Manually review the results of the automatic integration. If necessary, make simple modifications and adjustments to ensure the accuracy of the integration. After completing this step, semi-automatic integration is achieved.

[0063] Step 1-2-1-8: After confirming that the integration results are correct, click the "Save Results" button to save the processed data, and then click the "Export Results" button to export the data in the required format for subsequent analysis and archiving.

[0064] Step 1-2-2: Oligosaccharide integration is performed using the automatic vertical integration method. The integration result is as follows: Figure 15 As shown. The specific steps are as follows: Step 1-2-2-1: Export the raw data as a "csv" file; Step 1-2-2-2: Open the main interface of the software. The main interface includes three icons: "Integration", "Statistical Evaluation" and "Configuration". Click "Integration" and select "Vertical Integration" in the secondary function. Step 1-2-2-3: Click the "Import Sample" function key on the right and select the chromatogram CSV file of the sample to be processed; Step 1-2-2-4: The software completes automatic vertical integration; Step 1-2-2-5: Manually review the scoring results, make minor modifications, and complete the semi-automatic scoring process; Step 1-2-2-6: Click "Save Results" and "Export Results" to save and export the processed data.

[0065] Steps 1-2-3: Perform two-dimensional integration using the vertical template integration method. The integration result is as follows: Figure 16 As shown. The specific steps are as follows: Step 1-2-3-1: Export the raw data as a "csv" file; Step 1-2-3-2: Open the main interface of the software. The main interface includes three icons: "Integration", "Statistical Evaluation" and "Configuration". Click "Integration" and select "Two-dimensional Integration" in the secondary functions. Step 1-2-3-3: Click the "Import Sample" function key on the right, select the chromatogram CSV of the sample to be processed, manually integrate, and then click "Save Results"; Steps 1-2-3-4: Click the "Template Import" function key on the right, select the CSV template after manual integration, and select the chromatogram of the sample to be integrated in the pop-up dialog box; Steps 1-2-3-5: The software completes the vertical template integration; Steps 1-2-3-6: Manually review the scoring results, make minor modifications, and complete the semi-automatic scoring process; Steps 1-2-3-7: Click "Save Results" and "Export Results" to save and export the processed data.

[0066] Based on steps 1-2-1 to 1-2-3, integrate each component and organize the peak area percentages to improve the quantitative analysis parameters in the database.

[0067] Step 2: Verify database availability. The specific steps are as follows: Step 2-1: Automatic integration. Perform oligosaccharide analysis, disaccharide analysis, and two-dimensional liquid chromatography analysis on the reference preparation of the sample in the same experiment, and perform automatic integration according to the method in Step 1-2.

[0068] Step 2-2: Component Assignment. Open the software's main interface, which includes three icons: "Integration," "Statistical Evaluation," and "Configuration." Click "Integration," and in the secondary functions, you can select "Vertical Integration," "Disaccharide Integration," and "Two-Dimensional Integration." Click "Import Samples," and the raw data for disaccharide analysis, oligosaccharide analysis, and two-dimensional liquid chromatography will be automatically integrated. The oligosaccharide analysis results will be directly displayed in the peak information table on the right. For disaccharide analysis and two-dimensional liquid chromatography, click "Standard Assignment" for automatic assignment.

[0069] Steps 2-3: Database verification, with consistency evaluation parameters as follows: Figures 17-18 As shown.

[0070] Step 2-3-1: Export the raw data as a "csv" file; Step 2-3-2: On the main interface of the software, click "Statistical Evaluation". In the secondary functions, you can select "t-test", "quality range" and "PCA". Click "Add Sample" to select the exported result file, click "Add Data" to select the organized database, and perform consistency evaluation.

[0071] Step 3: Consistency evaluation of large batches of samples, the specific steps are as follows: Step 3-1: The steps for automatic integration are similar to those in Step 2-1, and will not be repeated here.

[0072] Step 3-2: The steps for component attribution are similar to those in Step 2-2, and will not be repeated here.

[0073] Step 3-3: The steps for consistency evaluation are similar to those for steps 2-3, and will not be repeated here.

[0074] This invention employs three data processing methods to efficiently process data collected by liquid chromatography-UV (LC-UV) and multicenter split two-dimensional liquid chromatography (MHC 2D LC). This method enables precise qualitative analysis of test samples and comparison with a database to achieve statistical quantitative analysis.

[0075] This invention effectively solves the problems of repetitive experimental procedures, complex chromatographic data processing, and fragmented statistical analysis functions in existing technologies. Through innovative methods and tools, it reduces repetitive experimental operations, simplifies the chromatographic data processing workflow, and integrates disparate statistical analysis functions. This not only significantly reduces the workload of analysts but also improves work efficiency and the accuracy of data processing. Example 2

[0076] Based on the same inventive concept, this embodiment provides a consistency evaluation system for heparin drugs. The principle of solving the problem is similar to the consistency evaluation method for heparin drugs provided in Embodiment 1, and the repeated parts will not be described again.

[0077] This embodiment provides a consistency evaluation system for heparin drugs, including: The selection module is used to select a standard formulation based on the reference formulation and marketed formulations required for the drug to be tested. The module is constructed to perform disaccharide analysis, oligosaccharide analysis, and two-dimensional liquid chromatography analysis on the standard preparation to obtain the first peak parameter, the second peak parameter, and the third peak parameter, respectively; and a qualitative database and a quantitative database are constructed based on the first peak parameter, the second peak parameter, and the third peak parameter. The first evaluation module is used to acquire chromatographic data of the reference preparation processed under the same conditions as the test sample; and to evaluate the consistency of the reference preparation chromatographic data with the qualitative and quantitative databases; wherein, the consistency evaluation includes: The chromatographic data of the reference preparation were automatically integrated, and the components were automatically assigned using a qualitative database to obtain the content of key peaks and the content of peaks in the quantitative database, respectively. Obtain samples from the quantitative database; based on the samples, evaluate the consistency between the content of key peaks and the peak content in the quantitative database; The second evaluation module is used to acquire chromatographic data of a large number of test samples when the chromatographic data of the reference preparation is consistent with the quantitative database; and to evaluate the consistency of the chromatographic data with the qualitative and quantitative databases. Example 3

[0078] This embodiment provides a consistency evaluation device for heparin drugs, including a consistency evaluation system for heparin drugs provided in Embodiment 2.

[0079] Those skilled in the art will understand that embodiments of this application can be provided as methods, systems, or computer program products. Therefore, this application can take the form of a completely hardware embodiment, a completely software embodiment, or an embodiment combining software and hardware aspects. Furthermore, this application can take the form of a computer program product embodied on one or more computer-usable storage media (including but not limited to disk storage, CD-ROM, optical storage, etc.) containing computer-usable program code.

[0080] This application is described with reference to flowchart illustrations and / or block diagrams of methods, apparatus (systems), and computer program products according to embodiments of this application. It will be understood that each block of the flowchart illustrations and / or block diagrams, and combinations of blocks in the flowchart illustrations and / or block diagrams, can be implemented by computer program instructions. These computer program instructions can be provided to a processor of a general-purpose computer, special-purpose computer, embedded processor, or other programmable data processing apparatus to produce a machine, such that the instructions, which execute via the processor of the computer or other programmable data processing apparatus, generate instructions for implementing the flowchart... Figure 1 One or more processes and / or boxes Figure 1 A device that provides the functions specified in one or more boxes.

[0081] These computer program instructions may also be stored in a computer-readable storage medium that can direct a computer or other programmable data processing device to function in a particular manner, such that the instructions stored in the computer-readable storage medium produce an article of manufacture including instruction means, which are implemented in a process Figure 1 One or more processes and / or boxes Figure 1 The function specified in one or more boxes.

[0082] These computer program instructions may also be loaded onto a computer or other programmable data processing equipment to cause a series of operational steps to be performed on the computer or other programmable equipment to produce a computer-implemented process, thereby providing instructions that execute on the computer or other programmable equipment for implementing the process. Figure 1 One or more processes and / or boxes Figure 1 The steps of the function specified in one or more boxes.

[0083] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for consistency evaluation of heparin-type drugs, characterized in that, include: S1. Select the standard formulation based on the reference formulation and marketed formulations required for the drug to be tested; S2. Perform disaccharide analysis, oligosaccharide analysis, and two-dimensional liquid chromatography analysis on the standard preparation to obtain the first peak parameter, the second peak parameter, and the third peak parameter, respectively; construct a qualitative database and a quantitative database based on the first peak parameter, the second peak parameter, and the third peak parameter; S3. Obtain chromatographic data of the reference preparation processed under the same conditions as the sample to be tested; evaluate the consistency of the reference preparation chromatographic data with the qualitative database and the quantitative database; wherein, the consistency evaluation steps are as follows: The chromatographic data of the reference preparation are automatically integrated, and the components are automatically assigned using the qualitative database to obtain the content of key peaks and the content of peaks in the quantitative database, respectively. Obtain samples from the quantitative database; evaluate the consistency between the key peak content and the peak content in the quantitative database based on the samples; S4. When the chromatographic data of the reference preparation is consistent with the quantitative database, obtain the chromatographic data of a large batch of test samples; evaluate the consistency of the chromatographic data with the qualitative database and the quantitative database.

2. The consistency evaluation method for heparin drugs according to claim 1, characterized in that, The reference formulation chromatographic data includes a first chromatographic peak obtained by disaccharide analysis, a second chromatographic peak obtained by oligosaccharide analysis, and a third chromatographic peak obtained by two-dimensional liquid chromatography analysis. The method for automatically integrating the reference formulation chromatographic data is as follows: automatically integrating the first chromatographic peak using template integration; automatically integrating the second chromatographic peak using automatic vertical integration; and automatically integrating the third chromatographic peak using vertical template integration.

3. The consistency evaluation method for heparin drugs according to claim 2, characterized in that, The step of automatically integrating the first chromatographic peak using template integration is as follows: Obtain disaccharide analysis data from multiple disaccharide standards and any set of samples, perform manual integration on the samples, identify the baseline peaks and valleys during manual integration to assist integration, and obtain the first integration result. The first integration result is set as the first template. The chromatographic retention time of the remaining samples is calibrated according to the multiple disaccharide standards. The chromatographic peaks of the initial integration are iteratively corrected for the tangent point. Automatic template integration is completed to adapt to different baseline conditions.

4. The consistency evaluation method for heparin drugs according to claim 2, characterized in that, The step of automatically integrating the second chromatographic peak using automatic vertical integration is as follows: Obtain oligosaccharide analysis data from the sample and generate a continuous horizontal baseline based on the chromatographic trend; Within the horizontal baseline range, draw vertical lines downwards from each peak and valley to complete automatic integration.

5. The consistency evaluation method for heparin drugs according to claim 2, characterized in that, The step of automatically integrating the third chromatographic peak using vertical template integration is as follows: Manual integration was performed on the single degree of polymerization oligosaccharide data obtained from two-dimensional liquid phase analysis of any sample. Based on the manual integration results, vertical lines are automatically drawn downwards from each peak and valley to complete the integration and obtain the second integration result. The second integration result is set as the second template, and integration is performed using the second template to complete the automatic integration of a large number of samples.

6. The consistency evaluation method for heparin drugs according to claim 1, characterized in that, S2, in the process of constructing the qualitative and quantitative databases based on the first peak parameter, the second peak parameter, and the third peak parameter, includes performing template integration on the first peak parameter and using the template integration to perform offset calibration on the retention time. The offset calibration step is as follows: Calculate the internal average of the retention time offsets of multiple disaccharides in the sample and template; If the internal average value exceeds the preset time, then during template integration, the retention time of the sample start and end points is corrected based on the template according to the offset standard, and the nearest valley point is found near the corrected retention time to serve as the final sample start and end points.

7. The consistency evaluation method for heparin drugs according to claim 1, characterized in that, In step S3, the method for automatically assigning components using the qualitative database is as follows: for the chromatographic data obtained from the disaccharide analysis and the two-dimensional liquid chromatography analysis, automatic assignment is completed by matching the relative retention time with the components in the qualitative database; for the chromatographic data obtained from the oligosaccharide analysis, automatic assignment is performed by the peak shape and retention time difference.

8. The consistency evaluation method for heparin drugs according to claim 1, characterized in that, In step S3, the method for evaluating the consistency between the critical peak content and the peak content in the quantitative database based on the sample is as follows: the consistency between the critical peak content and the peak content in the quantitative database is evaluated using t-test, quality range analysis, and principal component analysis.

9. A consistency evaluation system for heparin-type drugs, characterized in that... ,include: The selection module is used to select a standard formulation based on the reference formulation and marketed formulations required for the drug to be tested. The module performs disaccharide analysis, oligosaccharide analysis, and two-dimensional liquid chromatography analysis on the standard preparation to obtain the first peak parameter, the second peak parameter, and the third peak parameter, respectively; and constructs a qualitative database and a quantitative database based on the first peak parameter, the second peak parameter, and the third peak parameter. The first evaluation module is used to acquire chromatographic data of a reference preparation processed under the same conditions as the sample to be tested; and to evaluate the consistency of the reference preparation chromatographic data with the qualitative database and the quantitative database; wherein, the consistency evaluation includes: The chromatographic data of the reference preparation are automatically integrated, and the components are automatically assigned using the qualitative database to obtain the content of key peaks and the content of peaks in the quantitative database, respectively. Obtain samples from the quantitative database; evaluate the consistency between the key peak content and the peak content in the quantitative database based on the samples; The second evaluation module is used to acquire chromatographic data of a large number of test samples when the chromatographic data of the reference preparation is consistent with the quantitative database; and to evaluate the consistency of the chromatographic data with the qualitative database and the quantitative database.

10. A consistency evaluation device for heparin-type drugs, characterized in that, This includes the consistency evaluation system for heparin drugs as described in claim 9.

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