Biological medicine structure consistency evaluation method and system based on high-field nuclear magnetic resonance

Abstract

The invention discloses a biological medicine structure consistency evaluation method and system based on high-field nuclear magnetic resonance, and belongs to the technical field of nuclear magnetic resonance test materials. The method comprises the following steps: firstly, collecting a high-field nuclear magnetic resonance 2D 1H-1H TOCSY spectrum and a 2D 1H-1H NOESY spectrum of an imitated drug and an original drug, marking different signals, calculating data such as chemical shift difference and spatial distance difference, then reconstructing into a new two-dimensional spectrogram, and carrying out dimension reduction processing; carrying out Pearson fitting on a one-dimensional signal peak; and carrying out consistency evaluation on the covalent structure and the spatial structure of the biological medicine by utilizing the chemical shift difference, the spatial distance difference and a Pearson fitting result. According to the method, high-efficiency and high-accuracy calculation can be carried out through software, the consistency of a high-level structure which has a greater influence on the drug activity can be comprehensively evaluated, and consistency evaluation is carried out on the overall structure of a generic drug and an original drug.

Description

Technical Field

[0001] The present 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 biological drugs based on high-field nuclear magnetic resonance technology. Background Art

[0002] Drug consistency evaluation, or 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, and other processes". The drug safety-related plan clearly requires that generic drugs that fail to pass the drug quality consistency evaluation will not be re-registered and their drug approval documents will be cancelled. Biological drugs have become a new focus for major pharmaceutical companies with their high safety, high tolerance and high efficiency. Biological drugs not only contain one-dimensional covalent structures, but also three-dimensional spatial advanced structures, and even three-dimensional molecular aggregation and association to form higher-level structures. Studies have shown that some biological drugs enter the body with the wrong spatial structure, which will activate the human immune system. 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 advanced structure of biological drugs is crucial to the activity of the drug. It is very meaningful and valuable to establish a set of simple and practical methods to comprehensively evaluate the structural consistency of biological drugs.

[0003] Patent CN114878621A discloses a method for quantitatively evaluating the structure of protein drugs based on high-field nuclear magnetic resonance technology. The method uses non-labeled generic drugs and original research drugs to collect two-dimensional nuclear magnetic resonance experiments to conduct comprehensive, accurate and convenient quantitative evaluation of the structure of biological drugs. The core of this method is to compare the chemical shift and signal intensity of the collected two-dimensional NOESY and TOCSY spectra, and calculate the chemical shift difference and spatial distance difference. This method still has some aspects that can be improved. For the two-dimensional spectrum, in addition to the signal coordinates (chemical shift) and signal size (signal intensity), the distribution of the signal itself also contains a lot of information, and this information is crucial for the consistency evaluation of the structure of biological drugs. Therefore, while comparing the signal coordinates and size, comparing the distribution of the signal can more comprehensively measure the consistency of the two-dimensional spectrum and enhance the objectivity of the consistency evaluation of the structure of biological drugs. Summary of the invention

[0004] The purpose of the present invention is to propose a method for evaluating the structural consistency of biological drugs based on high-field nuclear magnetic resonance technology. This method evaluates the chemical shift, signal intensity and signal distribution of two-dimensional nuclear magnetic resonance spectra reflecting the covalent structure and high-order spatial structure of generic drugs and original drugs, and evaluates the structural similarity between generic drugs and original drugs.

[0005] Another object of the present invention is to propose a biological drug structure consistency evaluation system based on high-field nuclear magnetic resonance technology, which can implement the operating steps of the method through a software program to realize the automatic 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 purpose, the present invention is achieved through the following technical solutions:

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

[0008] 1) Collect two groups of samples of peptide and protein biopharmaceuticals, generic drugs and original drugs, using high-field NMR 2D 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) According to two groups of samples 2D 1 H- 1 The chemical shifts of the signals with differences in the H TOCSY spectrum were calculated to find the chemical shift differences;

[0011] 4) According to two groups of samples 2D 1 H- 1 The chemical shift and relative intensity of signals with differences in H NOESY spectra are used to calculate the chemical shift difference and spatial distance difference respectively;

[0012] 5) According to the 2D 1 H- 1 The chemical shift and intensity of the differential signal in the H NOESY spectrum are reconstructed into a new two-dimensional spectrum, which reflects the distribution of the differential signal, and the reconstructed two-dimensional spectrum is subjected to principal component analysis (PCA) to obtain a feature vector, and the reconstructed signal of the two-dimensional spectrum is projected onto the feature vector according to its position and intensity to form a signal peak of a one-dimensional normal distribution, thereby realizing dimensionality reduction processing of the reconstructed two-dimensional spectrum;

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

[0014] 7) According to 2D 1 H- 1 The chemical shift difference in H TOCSY spectrum is used to evaluate the consistency of the covalent structure 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 are used to evaluate the consistency of the spatial structure of biopharmaceuticals;

[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 the consistency evaluation of the covalent structure and spatial structure both meet the standards, the consistency of the biopharmaceutical structure is determined to meet the standards.

[0017] Furthermore, the steps of data processing the spectrum include: solvent suppression, adding window function, Fourier transform, baseline phase calibration and linear prediction.

[0018] Furthermore, 2D 1 H- 1 H TOCSY spectra and 2D 1 H- 1 H NOESY spectra were collected using different NMR instruments with ultra-low temperature probes above 500 MHz according to the actual molecular weight of the samples.

[0019] Furthermore, the chemical shift difference is expressed as a root mean square value, and the calculation formula of the root mean square value is: where Δδ x and Δδ y 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 signal intensity of the difference of the marker to the reference signal in the spectrum, and the reference signal of the spectra of the two groups of samples is the same signal in the two spectra.

[0021] Furthermore, the spatial distance difference refers to the relative distance difference between the atoms of the generic drug and the original drug, and the calculation formula is: Among them I 原 and I 仿 are the relative intensities of NOE signals of original drug samples and generic drug samples, respectively.

[0022] Furthermore, when the labeled differential signals are reconstructed into a two-dimensional spectrum, the chemical shifts of the differential signals are used as x, 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, original drugs and generic drugs 2D 1 H- 1 When the root mean square value of the chemical shift difference of the different signals in the H TOCSY spectrum is less than 0.008 ppm, it indicates that the covalent structure is consistent.

[0024] Furthermore, original drugs and generic drugs 2D 1 H- 1 When the root mean square value of the chemical shift difference of the different 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 and the original drug have the same spatial structure.

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

[0026] High-field NMR instrument with ultra-low temperature probe, used to collect high-field NMR 2D samples of generic and original drugs of peptide and protein biological drugs 1 H- 1 H TOCSY spectra and 2D 1 H- 1 H NOESY spectrum;

[0027] Memory for storing computer programs;

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

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] First, in addition to using 2D 1 H- 1 H TOCSY spectra and 2D 1 H- 1 In addition to quantitatively evaluating original and generic drugs based on the differences in chemical shifts and relative intensities of H NOESY spectra, we also introduced correlation analysis of signal distribution types to evaluate structural consistency using more comprehensive information, thereby improving the accuracy of existing methods.

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

[0032] Thirdly, in the process of principal component analysis spectrum dimensionality reduction, projecting the reconstructed spectrum data onto the eigenvector can obtain the one-dimensional signal distribution with the maximum resolution, thus improving the accuracy of correlation analysis.

[0033] Finally, the present invention uses software to automatically process the collected spectral data, including 2D 1 H-1 H TOCSY spectra and 2D 1 H- 1 The chemical shift and relative intensity differences of the H NOESY spectrum were quantitatively evaluated, which saved manpower and improved calculation efficiency and accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is a flow chart of quantitative evaluation of the structure of generic drugs and original research drugs in the embodiments of the present invention.

[0035] Figure 2A-2G It is the consistency evaluation result of the generic drug and the original drug in the embodiment of the present invention. DETAILED DESCRIPTION

[0036] In order to make the above features and advantages of the present invention more obvious and easy to understand, embodiments are given below and described in detail with reference to the accompanying drawings.

[0037] This embodiment discloses a method and system for evaluating the structural consistency of biological drugs based on high-field nuclear magnetic resonance. The generic drug and the original drug used in this embodiment are a polypeptide biological drug for the treatment of type II diabetes. This embodiment completes the structural consistency evaluation of generic drugs and original drugs by comparing the consistency of the nuclear magnetic resonance spectra reflecting the structures of the original drug and the generic drug. With the development of nuclear magnetic resonance hardware, especially the increase in instrument field strength and the use of ultra-low temperature probes, the resolution and sensitivity of nuclear magnetic resonance experiments have been greatly improved and have been widely used in the research of various biological macromolecules. Nuclear magnetic resonance 2D 1 H- 1 HTOCSY spectrum and 2D 1 H- 1 H NOESY spectra can reflect the covalent and spatial structure of biological drugs. By comparing the spectra, the consistency of the structures of generic drugs and original drugs can be evaluated. Therefore, the present 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 molecular weight of the sample, etc.) with an ultra-low temperature probe to collect 2D 1 H- 1 H TOCSY spectra and 2D 1 H- 1 In addition to intuitive visual comparison, H NOESY spectra use self-written software to quantitatively evaluate the chemical shift, signal intensity, and signal distribution type of the spectrum signal, ultimately achieving consistency evaluation of the structures of generic drugs and original drugs.

[0038] The method of this embodiment has a flow chart as follows Figure 1 The specific steps are as follows:

[0039] 1. Sample preparation:

[0040] 2.0 mg of original drug and generic drug powder were dissolved in 500 μL of water, 50 μL of heavy water was added, and a little NaOH was added until the sample was completely dissolved.

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

[0042] High-field NMR 2D images of original and generic drugs were collected at 298K on an 800MHz NMR instrument. 1 H- 1 H TOCSY spectra and 2D 1 H- 1 H NOESY spectra, the mixing time of the experiment was 80ms and 300ms respectively. The spectra were processed by solvent suppression, adding window function, Fourier transform, baseline phase calibration and linear prediction, and four high-quality and high-resolution NMR spectra were obtained for comparison.

[0043] 3. Quantitative evaluation of NMR spectra:

[0044] 2D 1 H- 1 The evaluation results of H TOCSY spectrum are as follows Figure 2A-2B As shown, Figure 2A It is an intuitive visual comparison of the spectra, and it can be seen that the overall spectra overlap well. The signals with chemical shift changes are marked, and the corresponding chemical shifts are extracted. The chemical shift difference is calculated using software to obtain a bar graph with the signal number as the horizontal axis and the chemical shift difference as the vertical axis, such as Figure 2B As shown, all chemical shift differences in the figure are less than 0.008 ppm, indicating that the covalent structures of the generic drug and the original drug are consistent.

[0045] 2D 1 H- 1 The evaluation results of H NOESY spectrum are as follows Figure 2C-2G As shown, Figure 2C The results of the intuitive visualization of the spectra show that the spectra in each region overlap well overall. The signals with large changes in chemical shift and signal intensity are marked, and the chemical shift and intensity of these signals are extracted. The chemical shift difference and spatial distance difference are calculated using self-written software, and the data and charts are output. Figure 2D The horizontal axis of the chemical shift difference is the signal number, and the vertical axis is the chemical shift difference. The results show that the chemical shift difference of the signals is less than 0.008 ppm. Figure 2E is the spatial distance difference, the horizontal axis is the signal number, and the vertical axis is the percentage reflecting the spatial distance difference. The results show that the spatial distance difference is less than ±10%.

[0046] Using software and information about the marker signal, the marker signal is reconstructed in two dimensions, e.g. Figure 2F As shown in the figure, the oblique line represents the eigenvector after principal component analysis. The signal is projected onto the eigenvector, and the intensity is represented by the intensity of the two-dimensional reconstructed signal. Figure 2G ,right Figure 2G The data were subjected to Pearson correlation analysis and the correlation coefficient was 0.958, indicating that the distribution consistency of the marker signals of the two spectra was extremely high.

[0047] According to the internationally recognized understanding of the numerical value of the Pearson correlation coefficient (Schober P, et al, Anesth Analg, 2018, 126(5): 1763), as well as the actual situation of nuclear magnetic resonance spectrum analysis and comparison, a Pearson correlation coefficient between 0.90 and 1.00 indicates extremely high consistency, indicating that the generic drug and the original drug have extremely high structural consistency; a Pearson correlation coefficient between 0.70 and 0.90 indicates high consistency, indicating that the generic drug and the original drug have high structural consistency, but there may be differences in local structure; a Pearson correlation coefficient between 0.40 and 0.70 indicates moderate consistency, indicating that the generic drug and the original drug have only certain similarities in structure; and a Pearson correlation coefficient less than 0.40 indicates weak consistency, indicating that the generic drug and the original drug have large structural differences.

[0048] In summary, the structures of generic drugs and original drugs are highly consistent.

[0049] The above embodiments are not intended to limit the present invention. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present application shall be included in the scope of the claims of the present application.

Claims

1. A method for evaluating the structural consistency of biological drugs based on high-field nuclear magnetic resonance, characterized in that: The following steps are involved: 1) Collect high-field NMR 2D images of two groups of samples of generic and original drugs of peptide and protein biological drugs 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) According to two groups of samples 2D 1 H- 1 The chemical shifts of the signals with differences in the H TOCSY spectrum were calculated to find the chemical shift differences; 4) According to two groups of samples 2D 1 H- 1 The chemical shift and relative intensity of signals with differences in H NOESY spectra are used to calculate the chemical shift difference and spatial distance difference respectively; 5) According to the 2D 1 H- 1 The chemical shift and intensity of the signal with difference in the H NOESY spectrum are reconstructed into a new two-dimensional spectrum, and the principal component analysis is performed on the reconstructed two-dimensional spectrum to obtain a feature vector, and the reconstructed signal of the two-dimensional spectrum is projected onto the feature vector according to its position and intensity to form a signal peak of a one-dimensional normal distribution; 6) Performing Pearson fitting on the signal peaks of the one-dimensional normal distribution of the two groups of samples; 7) According to 2D 1 H- 1 The chemical shift difference in H TOCSY spectrum is used to evaluate the consistency of the covalent structure 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 are used to evaluate the consistency of the spatial structure of biopharmaceuticals; 9) The overall structural consistency is determined based on the consistency evaluation results of the covalent structure and spatial structure of the biopharmaceutical. When the consistency evaluation of the covalent structure and spatial structure both meet the standards, the consistency of the biopharmaceutical structure is determined to meet the standards.

2. The method according to claim 1, characterized in that The steps of data processing for the spectrum include: solvent suppression, adding window function, Fourier transform, baseline phase correction and linear prediction.

3. The method according to claim 1, characterized in that 2D 1 H- 1 H TOCSY spectra and 2D 1 H- 1 H NOESY spectra were collected using different NMR instruments with ultra-low temperature probes above 500 MHz according to the actual molecular weight of the samples.

4. The method according to claim 1, characterized in that The chemical shift difference is expressed as the root mean square value, and the calculation formula of the root mean square value is where Δδ x and Δδ y are the chemical shift differences in the x- and y-dimensions, respectively.

5. The method according to claim 1, characterized in that The relative signal intensity is equal to the ratio of the differential signal intensity of the marker to the reference signal in the spectrum, and the reference signal of the spectra of the two groups of samples is the same signal in the two spectra.

6. The method according to claim 1, characterized in that The spatial distance difference refers to the relative distance difference between the atoms of the generic drug and the original drug, and the calculation formula is: Among them I 原 and I 仿 are the relative intensities of NOE signals of original drug samples and generic drug samples, respectively.

7. The method according to claim 1, characterized in that When the labeled differential signals are reconstructed into a two-dimensional spectrum, the chemical shifts of the differential signals are used as 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.

8. The method according to claim 1, characterized in that Original Drugs and Generic Drugs 2D 1 H- 1 When the root mean square value of the chemical shift difference of the different signals in the H TOCSY spectrum is less than 0.008 ppm, it indicates that the covalent structure is consistent.

9. The method according to claim 1, characterized in that Original Drugs and Generic Drugs 2D 1 H- 1 When the root mean square value of the chemical shift difference of the different 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 and the original drug have the same spatial structure.

10. A biological drug structure consistency evaluation system based on high-field nuclear magnetic resonance, characterized in that: include: High-field NMR instrument with ultra-low temperature probe, used to collect high-field NMR 2D samples of generic and original drugs of peptide and protein biological drugs 1 H- 1 H TOCSY spectra and 2D 1 H- 1 H NOESY spectrum; Memory for storing computer programs; A processor, used to implement steps 2) to 9) of the method according to any one of claims 1 to 9 when executing the above computer program.

Images