Analysis data generation apparatus, analysis data generation method, and analysis data generation program

Abstract

The present invention calculates, on the basis of objective standards, a scaling value that should be reproducible and theoretically-expected, and generates subtraction data as sample solution data for structure analysis. The present invention comprises: a scaling area setting unit 232 which sets a wave number range of a specific scattering vector as a scaling area excluding a data area in which information on a sample emerges; a matching degree evaluation unit 237 which evaluates, on the basis of specific evaluation standards and by using an X-ray scattering profile in the scaling area, the matching degree between an X-ray scattering profile of a sample solution and an X-ray scattering profile of a buffer solution which has been scaled on the basis of a scaling value k; an optimal value identification unit 239 which identifies an optimal value for the scaling value k on the basis of the evaluation of the matching degree; and a difference calculation unit 255 which performs, by using the optimal value, difference calculation in which the X-ray scattering profile of the buffer solution which has been scaled on the basis of the scaling value k is subtracted as a background from the X-ray scattering profile of the sample solution.

Description

Analysis data generation device, analysis data generation method, and analysis data generation program

[0001] The present invention relates to an analytical data generating device, an analytical data generating method, and an analytical data generating program for generating data for structural analysis from scattering image data obtained by irradiating a sample solution containing a biopolymer with X-rays.

[0002] In solution scattering, particularly in small angle X-ray scattering (SAXS) for polymer solutions, in order to extract the signal from the sample molecules, it is necessary to subtract the background X-ray scattering profile of a buffer solution (the X-ray scattering profile of a solution with the same composition as the sample solution but without sample molecules) from the X-ray scattering profile of the sample solution.

[0003] In particular, when measuring buffer solutions of biopolymers, scaling correction between the two X-ray scattering profiles is essential for practical reasons. In this case, the maximum measurable wavenumber q of the scattering vector is limited by the nature of the measurement, so the scaling value is determined empirically and by trial and error.

[0004] In contrast, Non-Patent Document 1 discloses a method in which the solution is replaced using chromatography, and explains that scaling is not necessary in principle. However, in the case of concentrated solutions of biopolymers that are the subject of SAXS measurement, it is unclear how background subtraction is performed, even though scaling is absolutely necessary.

[0005] Furthermore, Non-Patent Document 2 describes the measurement and data processing of solution scattering at a LiX (Life Science X-ray Scattering) beamline, and discloses subtraction of a scaling background as one of the steps. However, fine adjustment of the scaling factor up to 0.5% is permitted, and the scaling is manually adjusted.

[0006] Patent Document 1 discloses that when adjusting the relative scales of the sample solution data and the buffer solution data, the average values ​​of the data are made to match in the wavenumber range on the high-angle side of the scattering vector.

[0007] The scattering angle is equivalent to the wave number of the scattering vector, and conversion between the two is possible. Therefore, the expression "high-angle or low-angle wave number" means "the wave number corresponding to the high-angle or low-angle scattering angle."

[0008] MV Petoukhov, PV Konarev, AG Kikhney and DI Svergun, ATSAS 2.1 - towards automated and web-supported small-angle scattering data analysis, JOURNAL OF APPLIED CRYSTALLOGRAPHY, Volume 40| Part s1| April 2007| Pages s223-s228, https: / / doi.org / 10.1107 / S0021889807002853Lin Yang,* Stephen Antonelli, Shirish Chodankar, James Byrnes, Edwin Lazo and Kun Qian, “Solution scattering at the Life Science

[0009] International Publication No. 2023 / 176330 Pamphlet Japanese Patent Application Laid-Open No. 2023-086675

[0010] As mentioned above, some prior art techniques perform background subtraction by scaling between X-ray scattering profiles. However, for example, to extract physically meaningful data and reconstruct the electron density distribution of biopolymers using X-ray scattering images as described in Patent Document 2, an accuracy of 0.1% or less is required. Low accuracy causes bias in the ensemble, resulting in a decrease in the χ ２It is not possible to identify the electron density distribution at 1. Furthermore, neither patent literature nor non-patent literature can measure sufficiently high angles. Therefore, even in the high-angle wavenumber range, the measurement data includes sample data, making it difficult to separate sample data from background data, and thus making it difficult to achieve the above-mentioned accuracy.

[0011] The method of manually fine-tuning the 0.5% scaling, as described in Non-Patent Document 2, does not allow for the identification of the electron density distribution of the sample. If the high-angle X-ray scattering profile contains sample data, the data between the high-angle X-ray scattering profiles of the sample solution and the buffer solution will not match, requiring subjective fine-tuning. Furthermore, because the measured X-ray scattering profile contains noise, subtracting the X-ray scattering profile of the buffer solution from the X-ray scattering profile of the sample solution may result in negative values. If the X-ray scattering profile contains negative values, analysis becomes difficult because the data cannot be processed.

[0012] Even in the method described in Patent Document 1, measurements are not made sufficiently far up to the high-angle side, so there is no range in the X-ray scattering profile of the sample solution that is not affected by scattering data from the sample. Therefore, even if the X-ray scattering profile of the sample solution is compared with the X-ray scattering profile of the buffer solution and scaling is adjusted so that the average scattering intensity values ​​at the high-angle side match, the sample data is mixed into the X-ray scattering profile, making it impossible to achieve sufficient accuracy. Note that, like the method described in Patent Document 2, the method described in Patent Document 1 also suffers from the problem of negative values ​​being included in the analysis data obtained by subtracting the X-ray scattering profiles of the sample solution and the buffer solution.

[0013] There are fundamental problems with the objectivity and reproducibility of the analytical data subtracted as described above, and these problems are generally considered unavoidable. However, within the resolution range where structure-specific scattering signals disappear, the profiles of the sample solution and the buffer solution should essentially give the same scattering signals. Furthermore, in recent years, highly sensitive detectors have become capable of obtaining highly accurate data even at wavenumbers with high scattering vector angles, which has increased the impact of background subtraction.

[0014] The present invention has been made in consideration of the above circumstances, and aims to provide an analytical data generation device, an analytical data generation method, and an analytical data generation program that can calculate scaling values ​​that are ideal in principle and with good reproducibility based on objective criteria, and generate subtraction data as sample solution data for structural analysis.

[0015] (1) In order to achieve the above object, the analytical data generation device of the present invention is an analytical data generation device that generates data for structural analysis from scattering image data obtained by irradiating a sample solution containing a biopolymer with X-rays, and is characterized by comprising: a scaling area setting unit that sets a wavenumber range of a specific scattering vector as a scaling area excluding a data area in which sample information appears; a coincidence evaluation unit that uses the X-ray scattering profile within the scaling area to evaluate, using specific evaluation criteria, the degree of coincidence between the X-ray scattering profile of the sample solution and the X-ray scattering profile of a buffer solution scaled using a scaling value k; an optimal value identification unit that identifies an optimal value of the scaling value k based on the evaluation of the degree of coincidence; and a difference calculation unit that uses the optimal value to perform a difference calculation by subtracting the X-ray scattering profile of the buffer solution scaled based on the scaling value k as a background from the X-ray scattering profile of the sample solution.

[0016] (2) Furthermore, in the analysis data generation device described in (1) above, the scaling area setting unit is characterized in that it sets the scaling area based on an X-ray scattering profile of the sample solution and an X-ray scattering profile of the buffer solution.

[0017] (3) Furthermore, in the analytical data generation device described in (2) above, the scaling area setting unit is characterized in that it sets the scaling area by providing a certain gap from the wavenumber range of the scattering vector representing the data area in the X-ray scattering profile of the sample solution.

[0018] (4) In the analytical data generating device described in (1) above, the scaling area setting unit sets the scaling area in accordance with the characteristics of the sample.

[0019] (5) In the analysis data generation device described in any one of (1) to (4) above, the coincidence evaluation unit is characterized in that, as the specific evaluation criterion, emphasis is placed on the coincidence at wave numbers of scattering vectors on the high-angle side.

[0020] (6) Furthermore, in the analysis data generation device described in any one of (1) to (5) above, the degree of coincidence evaluation unit is characterized in that, as the specific evaluation criterion, emphasis is placed on the degree of coincidence in data with a small standard deviation σ.

[0021] (7) Furthermore, in the analysis data generation device described in any one of (1) to (6) above, the coincidence evaluation unit is characterized in that it evaluates the coincidence using an evaluation criterion corresponding to the characteristics of the sample as the specific evaluation criterion.

[0022] (8) Furthermore, the analytical data generation device described in any one of (1) to (7) above further includes a compatibility determination unit that determines whether the reliability of the X-ray scattering profile of the sample solution in the scaling area and the X-ray scattering profile of the scaled buffer solution meets a specific acceptance standard, and the difference calculation unit is characterized in that it does not perform the difference calculation if the reliability does not meet the specific acceptance standard.

[0023] (9) In the analytical data generating device described in (8) above, the compatibility determining unit uses, as the specific acceptance criteria, acceptance criteria according to the characteristics of the sample.

[0024] (10) In the analysis data generation device described in (8) or (9) above, the conformity determination unit is characterized in that it determines whether or not the specific acceptance criteria are satisfied at each point of the wave number of the scattering vector for which data exists.

[0025] (11) Furthermore, in the analytical data generation device described in any one of (1) to (10) above, the X-ray scattering profile of the sample solution and the X-ray scattering profile of the buffer solution are based on data acquired in two detection regions having different camera lengths.

[0026] (12) Furthermore, in the analytical data generation device described in any one of (1) to (10) above, the X-ray scattering profile of the sample solution and the X-ray scattering profile of the buffer solution are based on data acquired in two detection regions having the same camera length.

[0027] (13) Furthermore, the analytical data generation method of the present invention is a method for generating analytical data for generating data for structural analysis from scattering image data obtained by irradiating a sample solution containing a biopolymer with X-rays, and is characterized by including the steps of: setting a wavenumber range of a specific scattering vector as a scaling area excluding a data area in which sample information appears; using the X-ray scattering profile within the scaling area, evaluating the degree of agreement between the X-ray scattering profile of the sample solution and the X-ray scattering profile of a buffer solution scaled with a scaling value k using a specific evaluation criterion; specifying an optimal value for the scaling value k based on the evaluation of the degree of agreement; and using the optimal value to perform a difference calculation by subtracting the X-ray scattering profile of the buffer solution scaled with the scaling value k as a background from the X-ray scattering profile of the sample solution.

[0028] (14) Furthermore, the analytical data generation program of the present invention is an analytical data generation program for generating data for structural analysis from scattering image data obtained by irradiating a sample solution containing a biopolymer with X-rays, and is characterized in that it causes a computer to execute the following steps: setting a wavenumber range of a specific scattering vector as a scaling area excluding a data area in which sample information appears; evaluating, using the X-ray scattering profile within the scaling area, the degree of agreement between the X-ray scattering profile of the sample solution and the X-ray scattering profile of a buffer solution scaled using a scaling value k, based on a specific evaluation criterion; specifying an optimal value for the scaling value k based on the evaluation of the degree of agreement; and using the optimal value to perform a difference calculation by subtracting the X-ray scattering profile of the buffer solution scaled using the scaling value k as a background from the X-ray scattering profile of the sample solution.

[0029] FIG. 1 is a schematic diagram showing an analytical data generation system according to the present invention. FIG. 2 is a perspective view showing an X-ray analysis device. FIG. 3 is a block diagram showing the configuration of an analytical data generation system according to the present invention. FIG. 4 is a flowchart showing the process from measurement to analysis. FIG. 5 is a schematic diagram showing the process from acquiring a scattering image to analyzing it. (a) to (c) are a scattering image, a β-direction profile, and a q-direction profile, respectively. FIG. 6 is a flowchart showing the operation of the analytical data generation device. (a) to (c) are graphs showing an example of a graph showing a data area and a scaling area, and graphs showing an example of a correlation between weighting and an example of the relationship of an error function E with respect to a scaling value k. FIG. 7 is a graph showing a conformance example when the acceptance criteria are met in conformance judgment. (a) and (b) are graphs showing non-conformance examples when the acceptance criteria are not met in conformance judgment. Scaling area, weighting function, and tolerance factor t for a type of sample ｔｏｌｒ FIG. 1 is a diagram showing the correspondence relationship between the two detection regions having different camera lengths. FIG. 2 is a schematic diagram showing the configuration of a detector when data is acquired in two detection regions having the same camera length. FIG. 3 is a schematic diagram showing the configuration of a detector when data is acquired in two detection regions having the same camera length. FIG. 4 is an example of a screen displaying a profile of a sample solution and a scaled profile of a buffer solution.

[0030] Next, an embodiment of the present invention will be described with reference to the drawings. To facilitate understanding of the description, the same reference numerals are used to designate the same components in the drawings, and duplicated descriptions will be omitted.

[0031] [Principle] In the present invention, an evaluation criterion (error function) is used to evaluate the degree of agreement between the profile of the sample solution and the profile of the scaled buffer solution, and to objectively determine the scaling value. The scaling area, weighting function, and tolerance factor t are determined according to the characteristics (type, size) of the sample. ｔｏｌｒ is determined, and after scaling with the optimum value, it becomes possible to subtract the profile that should exist in principle.

[0032] The sample characteristics are mainly influenced by the sample molecular size. The scaling area is the wavenumber range of the scattering vector used to evaluate the degree of agreement between the sample solution profile and the scaled buffer solution profile. The weighting function determines whether to increase or decrease the influence of the difference at each wavenumber point of the scattering vector when calculating the error function.

[0033] Scaling may be performed on either profile. For example, in the following description, scaling is performed by multiplying the buffer solution profile by a scaling value k. However, this is equivalent to scaling the sample solution profile by multiplying it by a scaling value 1 / k. Therefore, when scaling is performed on the buffer solution profile, it can also be interpreted as scaling the sample solution profile.

[0034] [First Embodiment] (Analysis Data Generation System) FIG. 1 is a schematic diagram showing an analysis data generation system 10. The analysis data generation system 10 includes an X-ray analysis apparatus 100 and an analysis data generation apparatus 200. The X-ray analysis apparatus 100 irradiates a sample S0 with X-rays and detects small-angle scattered X-rays. The sample S0 is preferably a polymer in solution, particularly a biopolymer. This is particularly effective for pharmaceutical molecules, molecular complexes, or structures in solution that require structural analysis of the sample S0 of 30 Å or less.

[0035] The target solutions include sample solutions and buffer solutions. A sample solution is a solution containing a sample, for example, a solution containing a biopolymer and a special component for retaining the biopolymer. A buffer solution is a solution obtained by removing the sample from the sample solution. For example, the buffer solution in the above example is a solution that does not contain a biopolymer but contains a special component. A buffer solution may be prepared separately with components similar to those of the sample solution, but it is preferable to use a solution obtained by separating the sample from the sample solution.

[0036] The analytical data generating device 200 is composed of a computer 210, an input device 280, and an output device 290, and controls the operation of the X-ray analysis device 100, and also acquires and processes measurement data from the X-ray analysis device 100.

[0037] The X-ray analysis apparatus 100 includes an X-ray generation unit 110, a sample loading mechanism 120, a detector 130, and a control unit 140. The X-ray generation unit 110 has an X-ray source 111 and irradiates X-rays onto a sample S0. The target of the X-ray source 111 is preferably Cu, but Co may also be used. The sample loading mechanism 120 sends a sample solution containing a sample or a buffer solution without a sample, along with the sample holding tube, to the X-ray irradiation position. The detector 130 detects X-rays scattered by the sample S0 and transmits the obtained measurement data to a computer 210.

[0038] In the above configuration, one detector 130 is provided for one X-ray beam emitted from the X-ray source 111, but other configurations may also be employed. For example, the X-ray analysis apparatus 100 may be configured to emit two equal beams in the same direction using a mirror or a slit and detect the scattered rays thereof with one detector. Alternatively, the X-ray analysis apparatus 100 may be configured to emit two equal beams in opposite directions and detect the scattered rays thereof with two detectors, respectively.

[0039] The computer 210 is, for example, a PC, and is configured with a processor that executes processing, and a memory or hard disk that stores programs and data, etc. The computer 210 receives user input from an input device 280 such as a keyboard or mouse, and outputs a profile, etc. to an output device 290 such as a display.

[0040] The computer 210 may be a server device located on the cloud. In addition, in terms of processing load, the function of controlling the operation of the X-ray analysis apparatus 100 and the function of processing the measurement data may be separated, with the control being performed by a PC installed on-site and the data processing being performed by a server device.

[0041] (X-ray analysis apparatus) Fig. 2 is a perspective view showing an X-ray analysis apparatus 100. The X-ray analysis apparatus 100 has an X-ray source 111, an optical system 115, a Kratsky block 117, a sample holding tube 125, and a detector 130. The X-ray source 111 is a line radiation source or a point radiation source, and emits a diverging beam. The optical system 115 is, for example, a KB parallel type or a serial type optical system.

[0042] The pair of Kratky blocks 117 interact with the X-rays through their respective edges to define one side of the X-ray beam, thereby eliminating parasitic scattering from the irradiated X-rays. The sample holding tube 125 delivers and holds 5 to 10 μl of solution.

[0043] The detector 130 detects X-rays scattered by the solution. The X-ray analysis device 100 transmits the detected scattered image to the analysis data generation device 200. The detected scattered image is transmitted as measurement data at predetermined time intervals t.

[0044] (Analysis Data Generation Apparatus) Fig. 3 is a block diagram showing an analysis data generation system. The analysis data generation apparatus 200 generates sample solution data for structural analysis. The functions of the analysis data generation apparatus 200 are mainly realized by a computer 210.

[0045] The computer 210 includes an input / output control unit 211, a measurement control unit 215, a measurement data storage unit 217, a data conversion unit 223, a scaling area setting unit 232, a coincidence evaluation unit 237, an optimum value specification unit 239, a compatibility determination unit 245, and a difference calculation unit 255. Each unit can send and receive information via a control bus L.

[0046] The input / output control unit 211 accepts input from the input device 280 and controls output to the output device 290. The input / output control unit 211 can accept input of measurement conditions, for example. The measurement conditions include the intensity of the generated X-rays, the position of the Kratky block, the blank position where the X-rays are irradiated, the position of the solution, the arrangement of the detector, and the measurement time t when the scattering image is obtained. The input / output control unit 211 can also output the progress of each index and the determination results.

[0047] The measurement control unit 215 controls the operation of the X-ray analysis apparatus 100. The controlled operations include sending out the sample, generating X-rays, and moving the sample position and detector. Control instructions are sent to the control unit 140 in the X-ray analysis apparatus 100, which controls each part of the X-ray analysis apparatus 100.

[0048] The measurement data storage unit 217 stores, as measurement data, the scattering image detected by the X-ray analysis apparatus 100. The stored measurement data is used for conversion into a scattering profile, generation of analysis data, and the like.

[0049] The data conversion unit 223 converts the measurement data into a scattering profile. Specifically, the data conversion unit 223 calculates the scattering profile by integrating the intensity along the circumferential direction (β direction) around the center of the scattering image. The data conversion unit 223 also calculates the standard deviation σ(nq) of the intensity in the β direction for a certain wave number q.

[0050] The scaling area setting unit 232 sets a wavenumber range of a specific scattering vector as a scaling area excluding the data area in which sample information appears. Ideally, the scaling area should be set to a wavenumber range of scattering vectors in which sample data does not appear. For example, when the target of structural analysis is a biopolymer such as a protein or nucleic acid, the side chains are flexible, so the surface fractal dimension is automatically determined to be larger than that size. Therefore, a resolution greater than the resolution determined by the surface degrees of freedom is never achieved. Once the resolution is determined, the wavenumber range of scattering vectors in which sample data does not appear is determined, and this range can be used as the scaling area.

[0051] The area excluding the data area where the sample information appears is, for example, 0.6 Å. －１ The wave number range is preferably 0.8 Å or more. －１ This means a wavenumber range above 8 Å. This is because the contribution of structures of biopolymers below about 8 Å to diffracted X-rays is reduced due to molecular motion in the solution.

[0052] The scaling area setting unit 232 can also set the scaling area according to the characteristics of the sample. For example, if the area where the contribution to the X-ray intensity is almost zero is determined according to the type of sample, the scaling area can be easily set. This makes it possible to set the area where Proud's law holds true and the molecular structure is not reflected as the scaling area.

[0053] However, when actually measuring a sample and performing structural analysis, there are cases where the wavenumber range of the scattering vector that includes the sample data must be used as the scaling area. In such cases, it is preferable for the scaling area setting unit 232 to set the scaling area according to the characteristics of the sample. In this case, the scaling area is basically determined by the type of sample. If the scaling area cannot be determined despite this, the quality of the acquired data is assumed to be low and a loose standard is set.

[0054] The scaling area setting unit 232 can also set a scaling area based on the profile of the sample solution and the profile of the buffer solution. For example, it can specify a data area by calculation from the acquired profile, and then automatically set the scaling area by providing a certain gap from that area. Specific methods will be described later.

[0055] The coincidence evaluation unit 237 evaluates the coincidence between the profile of the sample solution and the profile of the buffer solution scaled by the scaling value k using a specific evaluation criterion. Scaling using the evaluation criterion enables objective background subtraction. The profile within the scaling area is used to evaluate the coincidence. This makes it possible to identify the scaling value k using an area of ​​wave numbers of scattering vectors whose contribution to the X-ray intensity from the sample is extremely small.

[0056] In the present invention, a scaling area can be set separately from the wavenumber range for analyzing scattering from the sample. When the profile of the buffer solution scaled by the scaling value k is subtracted from the profile of the sample solution, negative intensity values ​​occur in the scaling area, but negative intensity values ​​are unlikely to occur in the wavenumber range for analyzing scattering from the sample. Therefore, problems with data processing are unlikely to occur in the wavenumber range for analyzing scattering from the sample.

[0057] The coincidence evaluation unit 237 preferably places emphasis on the degree of coincidence in the wavenumber of the scattering vector on the high-angle side as a specific evaluation criterion. That is, the coincidence of the X-ray scattering profile on the high-angle side is weighted. This allows the degree of coincidence to be evaluated by weighting the side that contributes less to the X-ray intensity. Furthermore, the coincidence evaluation unit 237 preferably places emphasis on the degree of coincidence in data with a small standard deviation as a specific evaluation criterion. This allows the reliability of each point to be reflected in the weighting, and the degree of coincidence to be evaluated by placing emphasis on data with small variations.

[0058] The coincidence evaluation unit 237 can evaluate the coincidence with respect to the scaling value k using an evaluation criterion according to the type of sample as the specific evaluation criterion. Specifically, by substituting each value of the sample solution profile and each value of the buffer solution profile into the evaluation function, the evaluation function is expressed as a function of k. In this case, it is preferable that the evaluation criterion is automatically determined when the type of sample is set. As the specific evaluation criterion, an evaluation function having weighting specific to the type of sample can be used. An example of the evaluation function will be described later.

[0059] The optimal value specifying unit 239 specifies the optimal value of the scaling value k based on the evaluation of the degree of coincidence. For example, when the evaluation function is expressed as a quadratic function of k based on the least squares method, the scaling value k that takes the minimum value may be specified as the optimal value. Note that the optimal value may be determined directly from the results of evaluation using a specific evaluation criterion, or the optimal value may be determined using another criterion based on the evaluation results. For example, the scaling value k that takes the minimum value may be referenced and the optimal value may be specified taking into consideration another criterion.

[0060] The conformity determination unit 245 determines whether the reliability of the sample solution profile in the scaling area and the scaled buffer solution profile meets a specific acceptance standard, where reliability means that the variation at each point is small.

[0061] The tolerance criterion is the tolerance factor t ｔｏｌｒ The tolerance factor t can be used to determine whether the subtraction is meaningful. ｔｏｌｒ is a coefficient obtained by dividing the tolerance by the standard deviation σ. ｔｏｌｒ is the acceptance criterion depending on the type of sample.

[0062] The tolerance factor t ｔｏｌｒThis allows the situation of each type of sample to be reflected. The compatibility determination unit 245 preferably determines whether the reliability satisfies a specific acceptance standard at each point of the wave number of the scattering vector for which data exists. Compared to determining based on the total data at each point, determining at each point allows for more accurate identification of singular values.

[0063] The difference calculation unit 255 subtracts the profile of the buffer solution, which has been scaled as a background, from the profile of the sample solution using the optimal value. The difference calculation unit 255 does not execute processing if the reliability of the profile does not meet a specific tolerance standard. This makes it possible to avoid meaningless subtraction when there is a large variance between the profiles. The profile of the sample solution from which the background has been subtracted is output as data for analysis.

[0064] (Measurement and Analysis Method) A method for performing measurement and analysis using the analysis data generation system 10 configured as described above will be described. Fig. 4 is a flowchart showing the process from measurement to analysis. First, the X-ray analysis device 100 sets measurement conditions based on information input by the user (step S1).

[0065] Upon receiving a command to start measurement input by the user, the X-ray analysis device 100 starts measurement (step S2). The X-ray analysis device 100 sends the buffer solution to a predetermined position, irradiates X-rays, and acquires scattering data using a detector (step S3). Next, scattering data of the sample solution is acquired in the same manner (step S4). The X-ray analysis device transmits the acquired scattering image data to the computer 210 as measurement data.

[0066] The computer 210 stores the received measurement data and converts the measurement data into a scattering profile (step S5). The computer 210 then calculates the difference between the obtained profiles to generate analytical data (step S6), and performs analysis using the data (step S7). However, details of the acquisition of the scattering image, the evaluation of the degree of coincidence using the evaluation criteria, and the suitability determination will be described later. The generation of the analytical data described in step S6 will also be described later in detail.

[0067] (Acquisition and Analysis of Scattered Images) Figure 5 is a schematic diagram showing the process from acquisition to analysis of a scattered image. The buffer solution and the sample solution are alternately irradiated with X-rays, and the detected scattered images are acquired at predetermined time intervals t. The sample loading mechanism switches the sample holding tubes 125, enabling alternate irradiation of each solution.

[0068] The scattering image is converted into a profile. The analytical data is obtained by subtracting the intensity profile obtained by integrating the measurement data of the buffer solution from the intensity profile obtained by integrating the measurement data of the sample solution over the entire measurement time. Details of the data conversion will be described later.

[0069] In this case, in order to appropriately process the data on the high angle side used for structural analysis of 30 Å or less, it is necessary to adjust the relative scale of the sample solution profile and the buffer solution profile, i.e., perform scaling before subtraction. The method of generating analytical data by scaling will be described in detail later.

[0070] The obtained analytical data can be used for structural analysis as a measured X-ray scattering profile. The volume of a cubic particle in real space is represented by cubic voxels discretized into an NxNxN grid, and an electron density map can be calculated by searching for structure factors based on the measured X-ray scattering profile.

[0071] Specifically, multiple structural models are generated from the measured X-ray scattering profile, and a calculated X-ray scattering profile is calculated from each of the multiple structural models. An index representing the degree of agreement between the calculated calculated X-ray scattering profile and the measured X-ray scattering profile is calculated, and a representative structural model is selected from the multiple structural models based on the calculated index. In this way, a structural model of a polymer in solution that has a structure with dynamic fluctuations can be accurately reproduced.

[0072] (Data Conversion) Figures 6(a) to 6(c) show a scattering image, a β-direction profile, and a q-direction profile, respectively. When a solution is irradiated with X-rays, a scattering image such as that shown in Figure 6(a) is obtained. When the intensity I in this scattering image is plotted in the circumferential direction (β-direction) around the center at a given wave number q, a graph such as that shown in Figure 6(b) is obtained. Furthermore, when the intensity I in the β-direction of the scattering image is integrated and the integrated intensity I for each wave number q is plotted, a scattering profile such as that shown in Figure 6(c) is obtained.

[0073] (Method for Generating Analysis Data) A method for generating analysis data will be described (step S6 in the flowchart shown in FIG. 4). FIG. 7 is a flowchart showing the operation of the analysis data generation device 200.

[0074] First, the user's selection of the sample type is accepted (step T1). It is also possible to input not only the sample type but also the size and other characteristics. A scaling area corresponding to the selected sample type is set (step T2). Details of the setting of the scaling area will be described later. Note that an appropriate scaling area may be calculated from the profile regardless of the sample type. Once the scaling area is set, an evaluation function is set (step T3). It is preferable that the evaluation function is set using a weighting function selected according to the sample type.

[0075] The degree of match between the profile of the sample solution and the profile of the buffer solution is evaluated using the set evaluation function (step T4). During the evaluation, the evaluation function is used as a specific evaluation criterion, and a function of k is obtained as the evaluation result. Then, an optimal scaling value k is determined based on the obtained evaluation result (step T5). For example, the optimal scaling value k can be determined by differentiating the error function E and finding the scaling value k that takes the minimum value. The evaluation of the degree of match and the determination of the optimal value will be described in detail below.

[0076] Then, it is determined whether the reliability of the sample solution profile and the buffer solution profile meets the acceptance criteria (step T6), and if the acceptance criteria are not met, an error message is displayed (step T7), and the series of processes is terminated.On the other hand, if the reliability meets the acceptance criteria, the difference is calculated using the optimal value of the scaling value k (step T8), and the series of processes is terminated.

[0077] In the above example, the optimal value of the scaling value k is calculated using the error function E. However, it is also possible to set multiple different tentative scaling values ​​k, repeatedly calculate the error function E for each setting, and identify the scaling value k for which the obtained evaluation value is smallest as the optimal value.

[0078] (Setting the scaling area) Figure 8(a) is an example of a graph showing each profile, a data area, and a scaling area. The data area is the range in which the differential profile is ultimately used as data. In the example shown in Figure 8(a), the difference between the profile of the sample solution and the profile of the buffer solution becomes smaller as q increases, and the two profiles nearly match in the scaling area.

[0079] In the example shown in FIG. 8(a), the measurement range of a normal SAXS is q=0 Å. －１ 0.2 Å or more －１ While the measurement range of MAXS (Middle Angle X-ray Scattering) is q = 0 Å －１ More than 1 Å －１ The range is as follows. MAXS is suitable for analyzing scattering intensity profiles on the high-angle side, and can be used to identify delicate structures of 30 Å or less that cannot be obtained with the SAXS measurement range. In addition to the overall image of biopolymers on the order of 1000 Å, it is possible to observe such fine structures, thereby obtaining structural information that has not been obtained before in the research and development of biopharmaceuticals. The scaling area is set to a range of q where there is no contribution from normal biopolymers.

[0080] In conventional subtraction, the position where there is a contribution from the sample is used as the scaling area. In the present invention, by setting the wavenumber range where there is no contribution from the sample as the scaling area, it is possible to calculate a scaling value that is strictly meaningful and not dependent on the analyst's subjectivity. In order to make the most of the mid-angle data, the present invention uses data on the higher angle side, which would normally be discarded.

[0081] The scaling area is automatically set according to the input sample type. Alternatively, the range in which the sample solution profile and the buffer solution profile are likely to match can be calculated and determined as the scaling area.

[0082] (Evaluation of the Degree of Matching) The degree of match between the profile of the sample solution and the profile of the buffer solution scaled by the scaling value k is determined by a specific evaluation criterion, which may be, for example, an error function as shown in the following formula (1):

[0083] The error function E is calculated by multiplying the square of the difference between the profile Is(nq) of the sample solution and the profile kIb(nq) of the scaled buffer solution by a weighting factor wf(nq) and summing it up for the wave number nq of the scattering vector at each point within the scaling area. ｍｉｎ－ｓｃ is the minimum value in the scaling area, q ｍａｘ－ｓｃ is the maximum value within the scaling area. The weighting wf(nq) is preferably a function of the wave number nq of the scattering vector and the standard deviation σ(nq) of each profile or difference profile.

[0084] FIG. 8(b) is a diagram showing an example of the correlation between the magnitude of the weighting wf(nq) and each characteristic. σ(nq) reflects the reliability of each data point. Data points that give a large σ(nq) have a low reliability, while data points that give a small σ(nq) have a high reliability. In the example shown in FIG. 8(b), the higher the reliability of the data and the smaller the standard deviation σ(nq), the larger the weighting wf(nq). Furthermore, the smaller the contribution to the structure and the smaller the σ(nq), the larger the weighting wf(nq). It is preferable that the weighting wf(nq) have the properties shown in FIG. 8(b).

[0085] (Identifying the Optimal Value) FIG. 8C is a graph showing an example of the relationship between the error function E and the scaling value k. For example, since the error function expressed by equation (1) is a downwardly convex quadratic function as shown in FIG. 8C, the minimum value of the scaling value k can be obtained as the optimal value by calculating the vertex. Note that the error function is not limited to a quadratic function. When the error function is a quadratic function, the minimum value is uniquely determined by its differentiation. However, if an analytical solution cannot be obtained, there is a problem with the initial value (scaling value).

[0086] (Conformity assessment) There may be cases where the measurement data is not suitable for taking the difference between the profiles. Conformity assessment can be performed to assess this. Conformity assessment determines whether the reliability of the sample solution profile and the buffer solution profile within the scaling area meets specific acceptance criteria.

[0087] For example, the tolerance factor t, which is determined depending on the type of sample, ｔｏｌｒ , the standard deviation of the sample solution profile σ Ｉｓ , the standard deviation of the buffer solution profile σ Ｉｂ Using the error function E(a.u.) normalized per point, the suitability can be determined under the conditions shown in the following equation (2).

[0088] Figure 9 is a graph showing an example of a match where the acceptance criteria are met in the match determination. The example in Figure 9 shows a profile of the sample solution within the scaling area and a profile of the scaled buffer solution. The two profiles are nearly identical, with little variance, and the specific acceptance criteria are met with a high degree of confidence.

[0089] 10(a) and 10(b) are graphs showing examples of non-conformance where the acceptance criteria were not met in the conformance assessment. In the example shown in FIG. 10(a), the profile of the buffer solution intersects with the profile of the sample solution at two points. In such cases, it is possible that the buffer solution has a different structure from the remaining portion of the sample solution. For example, it is possible that the composition of the solvent has changed due to mixing of the sample with the solvent.

[0090] In the example shown in Figure 10(b), the profile of the buffer solution intersects with the profile of the sample solution at one point. In such cases, the solvent concentrations of the sample solution and the buffer solution may differ. In either case of incompatibility, an error message is displayed, indicating that there is no need to take a difference between the profiles.

[0091] (Settings according to the type of sample) As described above, in each process for generating analytical data, settings are made according to the type of sample. In setting the scaling area, an area can be set according to the type of sample. In evaluating the degree of coincidence, an error function using weighting wf(nq) according to the type of sample can be used. The tolerance factor t ｔｏｌｒ The scaling area, weighting function, and tolerance factor t can be set according to the type of sample. ｔｏｌｒ Each setting can be made using the correspondence stored in the analysis data generation device 200.

[0092] [Second embodiment] In the above embodiment, the profile of the sample solution and the profile of the buffer solution are converted based on data acquired by a single detector 130, but they may also be converted based on data acquired in two detection regions with different camera lengths.

[0093] FIG. 12 is a schematic diagram showing the configuration of a detector when data is acquired in two detection regions with different camera lengths. The X-ray analysis apparatus 500 includes an X-ray source 111, a detector 130, and a detector 530 dedicated to scaling. Components other than the X-ray source and the detector are not shown in FIG. 12. The detector 530 dedicated to scaling is installed close to the X-ray source 111, and therefore can increase intensity in a range of large scattering vector wavenumbers. In this way, acquiring data on the high-angle side using the detector 530 dedicated to scaling facilitates the acquisition of highly reliable data.

[0094] [Third Embodiment] The profile of the sample solution and the profile of the buffer solution may be converted based on data acquired in two detection regions with the same camera length. Fig. 13 is a schematic diagram showing the configuration of a detector when data is acquired in two detection regions with the same camera length. The X-ray analysis device 600 includes an X-ray source 111 and a detector 630. In Fig. 13, components other than the X-ray source and the detector are not shown.

[0095] The X-ray analysis device 600 has detection areas 631 and 632 with the same camera length. The detection areas 631 and 632 are included in the same detector 630 and are separated by a gap. By providing two detection areas in one detector in this way, it becomes easy to adjust the scaling area. Note that two different detectors with the same camera length may be stacked to provide two detection areas.

[0096] [Example] The above analytical data generation method was applied to a standard solution of humanized IgG1κ monoclonal antibody (NIST mAb). Figure 14 shows an example of a screen displaying the profile of the sample solution and the scaled profile of the buffer solution. In particular, the wavenumber range of the scattering vector on the high-angle side (0.8 to 1 Å) －１) shows that the profile of the sample solution matches the scaled profile of the buffer solution. The example screen in Figure 14 displays the wavenumber range of the scattering vector, sample concentration, and scaling value k of each profile data as numerical values. Measurement conditions such as the beam center position and exposure time are also displayed.

[0097] 10 Analysis data generation system 100 X-ray analysis apparatus 110 X-ray generation unit 111 X-ray source 115 Optical system 117 Kratsky block 120 Sample loading mechanism 125 Sample holding tube 130 Detector 140 Control unit 200 Analysis data generation apparatus 210 Computer 211 Input / output control unit 215 Measurement control unit 217 Measurement data storage unit 223 Data conversion unit 232 Scaling area setting unit 237 Matching evaluation unit 239 Optimum value identification unit 245 Conformity determination unit 255 Difference calculation unit L Control bus 280 Input device 290 Output device 500 X-ray analysis apparatus 530 Detector 600 X-ray analysis apparatus 630 Detector 631, 632 Detection area q Wave number of scattering vector t ｔｏｌｒ Tolerance factor E Error function S0 Sample nq Wave number of scattering vector

Claims

1. An analytical data generation device that generates data for structural analysis from scattering image data obtained by irradiating a sample solution containing biopolymers with X-rays, comprising: a scaling area setting unit that sets a wavenumber range of a specific scattering vector as a scaling area excluding a data area in which sample information appears; a consistency evaluation unit that uses the X-ray scattering profile within the scaling area to evaluate the consistency between the X-ray scattering profile of the sample solution and the X-ray scattering profile of a buffer solution scaled using a scaling value k, using specific evaluation criteria; an optimal value identification unit that identifies an optimal value for the scaling value k based on the evaluation of the consistency; and a difference calculation unit that uses the optimal value to perform a difference calculation by subtracting the X-ray scattering profile of the buffer solution scaled using the scaling value k as a background from the X-ray scattering profile of the sample solution.

2. The analytical data generating device according to claim 1, wherein the scaling area setting unit sets the scaling area based on the X-ray scattering profile of the sample solution and the X-ray scattering profile of the buffer solution.

3. The analytical data generating device according to claim 2, characterized in that the scaling area setting unit sets the scaling area by providing a certain gap from the wavenumber range of the scattering vector representing the data area in the X-ray scattering profile of the sample solution.

4. The analytical data generating device according to claim 1, wherein said scaling area setting section sets said scaling area in accordance with the characteristics of the sample.

5. An analytical data generation device according to any one of claims 1 to 4, characterized in that the degree of coincidence evaluation unit places emphasis on the degree of coincidence at wave numbers of scattering vectors on the high-angle side as the specific evaluation criterion.

6. The analytical data generating device according to any one of claims 1 to 4, characterized in that the coincidence evaluation unit places emphasis on the coincidence in data with a small standard deviation σ as the specific evaluation criterion.

7. An analytical data generation device according to any one of claims 1 to 4, characterized in that the matching evaluation unit evaluates the matching using an evaluation criterion according to the characteristics of the sample as the specific evaluation criterion.

8. An analytical data generation device as described in any one of claims 1 to 4, further comprising a conformity determination unit that determines whether the reliability of the X-ray scattering profile of the sample solution in the scaling area and the X-ray scattering profile of the scaled buffer solution meets a specific acceptance standard, and wherein the difference calculation unit does not perform the difference calculation if the reliability does not meet the specific acceptance standard.

9. The analytical data generating apparatus according to claim 8, wherein said compatibility determining section uses, as said specific acceptance criteria, acceptance criteria according to the characteristics of the sample.

10. The analytical data generating device according to claim 8, wherein said conformity determining unit determines whether or not said specific acceptance criteria are met at each point of wave numbers of scattering vectors for which data exists.

11. An analytical data generation device as described in any one of claims 1 to 4, characterized in that the X-ray scattering profile of the sample solution and the X-ray scattering profile of the buffer solution are based on data acquired in two detection areas with different camera lengths.

12. An analytical data generation device as described in any one of claims 1 to 4, characterized in that the X-ray scattering profile of the sample solution and the X-ray scattering profile of the buffer solution are based on data acquired in two detection areas with the same camera length.

13. A method for generating analytical data for generating data for structural analysis from scattering image data obtained by irradiating a sample solution containing biopolymers with X-rays, comprising the steps of: setting a wavenumber range of a specific scattering vector as a scaling area excluding a data area in which sample information appears; using the X-ray scattering profile within said scaling area to evaluate the degree of agreement between the X-ray scattering profile of the sample solution and the X-ray scattering profile of a buffer solution scaled using a scaling value k, using a specific evaluation criterion; identifying an optimal value for said scaling value k based on said evaluation of the degree of agreement; and using said optimal value to perform a difference calculation by subtracting the X-ray scattering profile of the buffer solution scaled using the scaling value k as a background from the X-ray scattering profile of the sample solution.

14. An analytical data generation program for generating data for structural analysis from scattering image data obtained by irradiating a sample solution containing biopolymers with X-rays, the analytical data generation program causing a computer to execute the following steps: a process for setting a wavenumber range of a specific scattering vector as a scaling area excluding a data area in which sample information appears; a process for evaluating, using an X-ray scattering profile within said scaling area, the degree of agreement between the X-ray scattering profile of the sample solution and the X-ray scattering profile of a buffer solution scaled using a scaling value k, based on a specific evaluation criterion; a process for identifying an optimal value for said scaling value k based on said evaluation of the degree of agreement; and a process for calculating a difference by using said optimal value to subtract the X-ray scattering profile of the buffer solution scaled using the scaling value k as a background from the X-ray scattering profile of the sample solution.

Images