High-field nuclear magnetic resonance-based method and system for evaluating structural consistency of biopharmaceuticals

Abstract

The application discloses a bio-drug structure consistency evaluation method and system based on high-field nuclear magnetic resonance, and belongs to the technical field of nuclear magnetic resonance testing materials. Firstly, the high-field nuclear magnetic resonance 2D 1 H- 1 H TOCSY spectrum and 2D 1 H- 1 H NOESY spectrum of a generic drug and an original research drug are collected, signals with differences are marked, data such as chemical shift difference and spatial distance difference are calculated, then new two-dimensional spectrum diagrams are reconstructed and dimension reduction processing is performed; Pearson fitting of one-dimensional signal peaks is further performed; and the chemical shift difference, the spatial distance difference and the Pearson fitting result are used for consistency evaluation of covalent structure and spatial structure of the bio-drug. The application can be calculated with high efficiency and high accuracy through software, can comprehensively evaluate the consistency of the higher structure which has a greater influence on drug activity, and can evaluate the consistency of the overall structure of the generic drug and the original research drug.

Description

Technical Field

[0001] This invention belongs to the technical field of nuclear magnetic resonance testing materials, and in particular relates to a method and system for evaluating the structural consistency of biopharmaceuticals based on high-field nuclear magnetic resonance technology. Background Technology

[0002] Drug consistency evaluation, also known as drug consistency research, is a high-standard requirement that generic drugs must be consistent with original drugs in terms of "management consistency, intermediate process consistency, quality standard consistency, etc." Drug safety-related plans clearly require that generic drugs that fail to pass the drug quality consistency evaluation will not be re-registered and their drug approval documents will be revoked. Biopharmaceuticals, with their high safety, high tolerability, and high efficiency, have become a new focus for major pharmaceutical companies. Biopharmaceuticals not only include one-dimensional covalent structures, but also three-dimensional spatial higher-order structures, and even three-dimensional molecular aggregation and association to form higher-order structures. Existing studies have shown that some biopharmaceuticals enter the body with incorrect spatial structures, which will activate the human immune system, and the drug will be regarded as an invader and degraded by the immune system (Lundahl MLE, et al, RSC Chem Biol, 2021, 2(4): 1004). Therefore, the higher-order structure of biopharmaceuticals is crucial to the activity of the drug. It is very meaningful and valuable to establish a simple and practical method for a comprehensive evaluation of the structural consistency of biopharmaceuticals.

[0003] Patent CN114878621A discloses a method for quantitatively evaluating the structure of protein drugs based on high-field nuclear magnetic resonance (NMR) technology. This method uses unlabeled generic and original drugs to acquire two-dimensional NMR data, enabling a comprehensive, accurate, and convenient quantitative evaluation of the biopharmaceutical structure. The core of this method is comparing the chemical shift and signal intensity of the acquired two-dimensional NOESY and TOCSY spectra, calculating the chemical shift difference and spatial distance difference. However, this method still has some room for improvement. For two-dimensional spectra, in addition to signal coordinates (chemical shift) and signal magnitude (signal intensity), the signal distribution itself contains much information, which is crucial for the consistency evaluation of biopharmaceutical structures. Therefore, comparing the signal distribution while comparing signal coordinates and magnitudes provides a more comprehensive measure of the consistency of the two-dimensional spectra, enhancing the objectivity of the consistency evaluation of biopharmaceutical structures. Summary of the Invention

[0004] The purpose of this invention is to propose a biopharmaceutical structure consistency evaluation method based on high-field nuclear magnetic resonance technology. This method evaluates the consistency of chemical shifts, signal intensities, and signal distributions of two-dimensional nuclear magnetic resonance spectra that reflect the covalent structure and higher-order spatial structure of generic and original drugs, and assesses the structural similarity between generic and original drugs.

[0005] Another objective of this invention is to propose a biopharmaceutical structure consistency evaluation system based on high-field nuclear magnetic resonance technology. This system can implement the operation steps of the method through software programs to automate the calculation of relevant indicators such as chemical shift difference, spatial distance difference, and correlation coefficient, thereby improving calculation efficiency and accuracy.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A method for evaluating the structural consistency of biopharmaceuticals based on high-field nuclear magnetic resonance includes the following steps:

[0008] 1) High-field 2D nuclear magnetic resonance (NMR) sampling was conducted on two groups of samples: generic and original drugs of peptide and protein biopharmaceuticals. 1 H- 1 HTOCSY spectrum and 2D 1 H- 1 H NOESY spectrum;

[0009] 2) Perform data processing on the spectra, analyze the differences between the NMR spectra of generic drugs and original drugs, and mark the signals with differences;

[0010] 3) Based on the two sets of samples 2D 1 H- 1 Chemical shifts of signals with differences in the H TOCSY spectrum are calculated to determine the chemical shift difference.

[0011] 4) Based on the two sets of samples 2D 1 H- 1 Chemical shifts and relative signal intensities of signals with differences in the H NOESY spectrum are used to calculate the chemical shift difference and spatial distance difference, respectively.

[0012] 5) Based on the 2D of the two groups of samples 1 H- 1 The chemical shifts and intensities of differential signals in the H NOESY spectrum are obtained, and the differential signals are reconstructed into a new two-dimensional spectrum. This spectrum reflects the distribution of the differential signals. Principal component analysis (PCA) is performed on the reconstructed two-dimensional spectrum to obtain eigenvectors. The reconstructed signals of the two-dimensional spectrum are projected onto the eigenvectors according to their positions and intensities to form one-dimensional normally distributed signal peaks, thus achieving dimensionality reduction of the reconstructed two-dimensional spectrum.

[0013] 6) Perform Pearson fitting on the one-dimensional normal distribution signal peaks of the two groups of samples to obtain the Pearson fitting correlation coefficient;

[0014] 7) According to 2D 1 H- 1 Chemical shift differences in H TOCSY spectra are used to evaluate the consistency of covalent structures of biopharmaceuticals.

[0015] 8) According to 2D 1 H- 1 The chemical shift difference, spatial distance difference, and Pearson fitting results in the H NOESY spectrum were used to evaluate the consistency of the spatial structure of the biopharmaceutical.

[0016] 9) The overall structural consistency is determined based on the consistency evaluation results of the covalent structure and spatial structure of the biopharmaceutical. When both the covalent structure and spatial structure consistency evaluations meet the standards, the consistency of the biopharmaceutical structure is determined to meet the standards.

[0017] Further steps in processing the spectrum include: solvent pressing, adding a window function, Fourier transform, baseline phase calibration, and linear prediction.

[0018] Furthermore, 2D 1 H- 1 H TOCSY spectrum and 2D 1 H- 1 H NOESY spectra were acquired using different nuclear magnetic resonance instruments with ultra-low temperature probes and a speed of 500 MHz or higher, based on the actual molecular weight of the sample.

[0019] Furthermore, the chemical shift difference is represented by the root mean square (RMS) value, which is calculated using the following formula: Where Δδ x and Δδ y These are the chemical shift differences in the x and y dimensions, respectively.

[0020] Furthermore, the relative signal intensity is equal to the ratio of the marked differential signal intensity to the reference signal in the spectrum, where the reference signal in the spectra of the two sets of samples is the same signal in both spectra.

[0021] Furthermore, the spatial distance difference refers to the difference in the relative atomic distance between the generic drug and the original drug, calculated using the following formula: Among them I 原 and I 仿 These are the relative NOE signal intensities of the original drug sample and the generic drug sample, respectively.

[0022] Furthermore, when the marked differential signals are reconstructed into a two-dimensional spectrum, the chemical shift of the differential signals is used as the x and y coordinates, the intensity of the differential signals is used as the z-axis coordinate, and the z-axis is represented in the form of contour lines to form a new two-dimensional spectrum.

[0023] Furthermore, the original drug and generic drug 2D 1 H- 1 When the root mean square value of the chemical shift difference between the different signals in the H TOCSY spectrum is less than 0.008 ppm, it indicates that the covalent structures are consistent.

[0024] Furthermore, the original drug and generic drug 2D 1 H- 1 When the root mean square value of the chemical shift difference of the differential signals in the H NOESY spectrum is less than 0.008 ppm, the spatial distance difference is less than 10%, and the Pearson correlation coefficient is between 0.90 and 1.00, it indicates that the generic drug has the same spatial structure as the original drug.

[0025] A biopharmaceutical structure consistency evaluation system based on high-field nuclear magnetic resonance, comprising:

[0026] A high-field nuclear magnetic resonance (NMR) instrument equipped with an ultra-low temperature probe is used to collect high-field 2D NMR data from two groups of samples: generic and original drugs of peptide and protein biopharmaceuticals. 1 H- 1 H TOCSY spectrum and 2D 1 H- 1 H NOESY spectrum;

[0027] Memory, used to store computer programs;

[0028] A processor, used to implement steps 2) to 9) of the above method when executing the above computer program.

[0029] Compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0030] First, in addition to using 2D, this invention... 1 H- 1 H TOCSY spectrum and 2D 1 H- 1 In addition to quantifying the differences in chemical shifts and relative intensities in H NOESY spectra between original and generic drugs, this study also introduced correlation analysis of signal distribution types, utilizing more comprehensive information to assess structural consistency and improving the accuracy of existing methods.

[0031] Secondly, for the analysis of signal distribution types, innovative new data processing and analysis tools such as two-dimensional spectrum reconstruction of nuclear magnetic resonance signals, dimensionality reduction of principal component analysis spectrum, and Pearson correlation analysis were introduced to ensure the accuracy of signal distribution type analysis, thereby enabling a more comprehensive evaluation of the consistency of higher-order structures that have a greater impact on drug activity.

[0032] Furthermore, during the dimensionality reduction of the spectral data in principal component analysis, projecting the reconstructed spectral data onto the eigenvectors yields a one-dimensional signal distribution with maximum resolution, thus improving the accuracy of correlation analysis.

[0033] Finally, this invention uses software to automate the processing of the acquired spectral data, including 2D representations of the structure. 1 H-1 H TOCSY spectrum and 2D 1 H- 1 The differences in chemical shifts and relative intensities in the H NOESY spectra were quantitatively calculated and evaluated, saving manpower and improving computational efficiency and accuracy. Attached Figure Description

[0034] Figure 1 This is a flowchart of the quantitative evaluation process for the structure of generic and original drugs in this invention.

[0035] Figure 2A-2G This refers to the consistency evaluation results between the generic drug and the original drug in the embodiments of this invention. Detailed Implementation

[0036] To make the above features and advantages of the present invention more apparent and understandable, specific embodiments are described below in conjunction with the accompanying drawings.

[0037] This embodiment discloses a method and system for evaluating the structural consistency of biopharmaceuticals based on high-field nuclear magnetic resonance (NMR). The generic drug and the original drug used in this embodiment are both peptide biopharmaceuticals for treating type II diabetes. This embodiment evaluates the structural consistency between the generic and generic drugs by comparing the consistency of their NMR spectra, which reflect the structures of the original and generic drugs. With the development of NMR hardware, especially the increase in instrument field strength and the use of ultra-low temperature probes, the resolution and sensitivity of NMR experiments have been greatly improved, and it has been widely used in the study of various biomolecules. (NMR 2D) 1 H- 1 HTOCSY spectrum and 2D 1 H- 1 H NOESY spectroscopy can reflect the covalent and spatial structure of biopharmaceuticals, and by comparing the spectra, the structural consistency between generic and original drugs can be evaluated. Therefore, this invention uses a high-resolution and high-sensitivity nuclear magnetic resonance method, such as a high-field nuclear magnetic resonance instrument (≥500MHz, depending on the sample molecular weight and other conditions) with an ultra-low temperature probe, to acquire two-dimensional 2D... 1 H- 1 H TOCSY spectrum and 2D 1 H- 1 In addition to intuitive visual comparison, the H NOESY spectrum uses self-developed software to quantitatively evaluate the chemical shift, signal intensity, and signal distribution type of the spectral signal, ultimately achieving consistency evaluation of the structure of generic drugs and original drugs.

[0038] The flowchart of the method in this embodiment is as follows: Figure 1 As shown, the specific operation steps are as follows:

[0039] 1. Sample preparation:

[0040] Dissolve 2.0 mg of the original drug and generic drug powder in 500 μL of water, add 50 μL of heavy water, and add a small amount of NaOH until the sample is completely dissolved.

[0041] 2. Sample data acquisition and data processing:

[0042] High-field 2D NMR data of original and generic drugs were collected on an 800MHz NMR instrument at a temperature of 298K. 1 H- 1 H TOCSY spectrum and 2D 1 H- 1 The H NOESY spectra were prepared with mixing times of 80 ms and 300 ms. The spectra were then processed by solvent suppression, windowing, Fourier transform, baseline phase calibration, and linear prediction to obtain four high-quality, high-resolution NMR spectra for comparison.

[0043] 3. Quantitative evaluation of nuclear magnetic resonance spectra:

[0044] 2D 1 H- 1 The evaluation results of the H TOCSY spectrum are as follows: Figure 2A-2B As shown, Figure 2A This is a direct visual comparison of the spectra, showing that the overall spectra overlap is quite good. Signals with chemical shift changes are marked, and the corresponding chemical shifts are extracted. Software is used to calculate the chemical shift difference, resulting in a bar chart with the signal number as the x-axis and the chemical shift difference as the y-axis, as shown below. Figure 2B As shown in the figure, all chemical shift differences are less than 0.008 ppm, indicating that the generic drug and the original drug have the same covalent structure.

[0045] 2D 1 H- 1 The evaluation results of the H NOESY spectrum are as follows: Figure 2C-2G As shown, Figure 2C The results of the spectral visualization comparison show that, overall, the spectral overlap across different regions is good. Signals with significant changes in chemical shift and signal intensity are marked, and their chemical shifts and intensities are extracted. Self-developed software is used to calculate the chemical shift difference and spatial distance difference, outputting data and charts. Figure 2D The horizontal axis represents the signal number, and the vertical axis represents the chemical shift difference. The results show that the chemical shift difference of the signals is less than 0.008 ppm. Figure 2E The horizontal axis represents the spatial distance difference, and the vertical axis represents the percentage of the spatial distance difference. The results show that the spatial distance difference is less than ±10%.

[0046] Using software and information from the labeled signal, the labeled signal is reconstructed in two dimensions, such as... Figure 2F As shown in the figure, the diagonal lines represent the eigenvectors after principal component analysis. Projecting the signal onto these eigenvectors, the intensity is represented by the intensity of the two-dimensional reconstructed signal, resulting in... Figure 2G ,right Figure 2G Pearson correlation analysis was performed on the data, and the correlation coefficient was 0.958, indicating that the distribution of the two spectral marker signals is highly consistent.

[0047] According to the internationally recognized understanding of Pearson correlation coefficient values ​​(Schober P, et al., AnesthAnalg, 2018, 126(5):1763), and based on the actual situation of nuclear magnetic resonance spectral analysis and comparison, a Pearson correlation coefficient between 0.90 and 1.00 indicates extremely high consistency, meaning that the generic drug and the original drug have extremely high structural consistency; between 0.70 and 0.90 indicates high consistency, meaning that the generic drug and the original drug have high consistency in the main structure, but there may be differences in local structures; between 0.40 and 0.70 indicates moderate consistency, meaning that the generic drug and the original drug have only a certain degree of structural similarity; and less than 0.40 indicates weak consistency, meaning that the generic drug and the original drug have significant structural differences.

[0048] In summary, generic drugs have a very high degree of structural similarity to original drugs.

[0049] The embodiments described above are not intended to limit the present invention. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of this application should be included within the scope of the claims of this application.

Claims

1. A method for evaluating the structural consistency of biopharmaceuticals based on high-field nuclear magnetic resonance, characterized in that, Includes the following steps: 1) High-field 2D nuclear magnetic resonance (NMR) sampling was conducted on two groups of samples: generic and original drugs of peptide and protein biopharmaceuticals. 1 H- 1 HTOCSY spectrum and 2D 1 H- 1 H NOESY spectrum; 2) Perform data processing on the spectra, analyze the differences between the NMR spectra of generic drugs and original drugs, and mark the signals with differences; 3) Based on the two sets of samples 2D 1 H- 1 Chemical shifts of signals with differences in the H TOCSY spectrum are calculated to determine the chemical shift difference. 4) Based on the two sets of samples 2D 1 H- 1 Chemical shifts and relative signal intensities of signals with differences in the H NOESY spectrum are used to calculate the chemical shift difference and spatial distance difference, respectively. 5) Based on the 2D of the two groups of samples 1 H- 1 The chemical shifts and intensities of the differential signals in the H NOESY spectrum are used as x and y coordinates, and the intensity of the differential signals is used as the z-axis coordinate. The z-axis is represented in the form of contour lines to reconstruct a new two-dimensional spectrum. Principal component analysis is performed on the reconstructed two-dimensional spectrum to obtain eigenvectors. The reconstructed signals of the two-dimensional spectrum are projected onto the eigenvectors according to their positions and intensities to form a one-dimensional normally distributed signal peak. 6) Perform Pearson fitting on the one-dimensional normal distribution signal peaks of the two sets of samples; 7) According to 2D 1 H- 1 Chemical shift differences in H TOCSY spectra are used to evaluate the consistency of covalent structures of biopharmaceuticals. 8) According to 2D 1 H- 1 The chemical shift difference, spatial distance difference, and Pearson fitting results in the H NOESY spectrum were used to evaluate the consistency of the spatial structure of the biopharmaceutical. 9) The overall structural consistency is determined based on the consistency evaluation results of the covalent structure and spatial structure of the biopharmaceutical. When both the covalent structure and spatial structure consistency evaluations meet the standards, the consistency of the biopharmaceutical structure is determined to meet the standards. The software program automates steps 3) to 9).

2. The method as described in claim 1, characterized in that, The steps for data processing of the spectrum include: solvent pressing, adding window functions, Fourier transform, baseline phase calibration, and linear prediction.

3. The method as described in claim 1, characterized in that, 2D 1 H- 1 H TOCSY spectrum and 2D 1 H- 1 H NOESY spectra were acquired using different nuclear magnetic resonance instruments with ultra-low temperature probes and a speed of 500 MHz or higher, based on the actual molecular weight of the sample.

4. The method as described in claim 1, characterized in that, The chemical shift difference is expressed as the root mean square (RMS) value, which is calculated using the following formula: Where Δδ x and Δδ y These are the chemical shift differences in the x and y dimensions, respectively.

5. The method as described in claim 1, characterized in that, The relative signal intensity is equal to the ratio of the marked differential signal intensity to the reference signal in the spectrum, where the reference signal in the spectra of the two sets of samples is the same signal in both spectra.

6. The method as described in claim 1, characterized in that, Spatial distance difference refers to the difference in atomic relative distance between a generic drug and the original drug, calculated using the following formula: Among them I 原 and I 仿 These are the relative NOE signal intensities of the original drug sample and the generic drug sample, respectively.

7. The method as described in claim 1, characterized in that, Original drug vs. generic drug 2D 1 H- 1 When the root mean square value of the chemical shift difference between the different signals in the H TOCSY spectrum is less than 0.008 ppm, it indicates that the covalent structures are consistent.

8. The method as described in claim 1, characterized in that, Original drug vs. generic drug 2D 1 H- 1 When the root mean square value of the chemical shift difference of the differential signals in the H NOESY spectrum is less than 0.008 ppm, the spatial distance difference is less than 10%, and the Pearson correlation coefficient is between 0.90 and 1.00, it indicates that the generic drug has the same spatial structure as the original drug.

9. A biopharmaceutical structure consistency evaluation system based on high-field nuclear magnetic resonance, characterized in that, include: A high-field nuclear magnetic resonance (NMR) instrument equipped with an ultra-low temperature probe is used to collect high-field 2D NMR data from two groups of samples: generic and original drugs of peptide and protein biopharmaceuticals. 1 H- 1 H TOCSY spectrum and 2D 1 H- 1 H NOESY spectrum; Memory, used to store computer programs; A processor, configured to implement steps 2) to 9) of the method according to any one of claims 1-8 when executing the computer program described above.

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