JP7119602B2 - How to calculate chromatogram similarity - Google Patents

Description

The present invention relates to a method of calculating the degree of similarity between a measurement sample and a standard sample based on chromatograms obtained by size exclusion chromatography (SEC).

SEC is used to separate and purify synthetic macromolecules and proteins based on differences in molecular size, but it is also used as a method for measuring the molecular weight distribution of samples, so it can also be used for quality control, etc. in manufacturing. is possible. In the fields of spectroscopic analysis and mass spectrometry, techniques using "similarity" are frequently used for the purpose of specifying/identifying a sample to be measured. This is a method of comparing a spectrum to be compared with a spectrum obtained by measuring an unknown sample and evaluating the similarity numerically.

For example, Patent Document 1 is an example of applying the concept of similarity to analysis of glycohemoglobin by liquid chromatography and applying it to a method of determining whether a chromatogram is normal or abnormal. In this case, the value corresponding to the x value is the elution time, but in the case of liquid chromatography, the elution time may not always be the same depending on the combination of various factors such as the eluent, column lot, equipment operating conditions, and measurement environment. not. If the x value contains an uncertain factor, the reliability of the similarity calculation will be poor.

JP-A-9-251016

An object of the present invention is to provide a method for calculating the degree of similarity between a measurement sample and a standard sample based on a chromatogram obtained by SEC, which reduces uncertainty (fluctuations) due to the effects of equipment, environment, eluent, etc. It is something to do.

The method for calculating the degree of similarity between a measurement sample and a standard sample based on a chromatogram obtained by size exclusion chromatography according to the present invention eliminates drift in the interval in which similarity calculation is performed for the chromatogram of the measurement sample. and correcting the time axis so that the elution time of the reference component peak is the same as the chromatogram of the standard sample, and then recorrecting the time interval so that it is the same as the chromatogram of the standard sample. After performing the step and, the similarity is calculated between the chromatogram of the standard sample from which the drift of the interval for which the similarity calculation is performed is removed.

In one aspect of the calculation method according to the present invention, the similarity is calculated after converting the chromatogram into an integral curve.

Further, in one aspect of the calculation method according to the present invention, there are two or more intervals in the chromatogram for which similarity calculation is performed.

Further, in one aspect of the calculation method according to the present invention, the degree of similarity is calculated by the following formula.

According to the present invention, since the drift of the chromatogram is removed and the time axis is corrected, it is possible to reduce the uncertainty and calculate the similarity between the measurement sample and the standard sample.

FIG. 4 is a diagram explaining a method of correcting the time axis so that the elution time of the component peaks that serve as a reference is the same as that of the chromatogram of the standard sample. FIG. 4 is a diagram explaining a method of recorrection so that the time interval is the same as the chromatogram of the standard sample; The chromatograms before and after time axis correction, time interval recorrection and drift removal are compared. FIG. 10 is a diagram exemplifying target intervals for similarity calculation in the present invention; Figure a is for all polymer components (one section), Figure b is for two sections, and Figure c is for three sections. 1 is a diagram showing a system configuration used in an example; FIG. 4 is a diagram showing the results of similarity calculation in Example 1. FIG. The horizontal axis is the sample #, and the vertical axis is the degree of similarity (maximum 1000). FIG. 10 is a diagram showing chromatograms (after time correction and drift removal processing) of top five samples from the result of similarity calculation in Example 1; For comparison, it is a diagram showing unprocessed chromatograms of the top five samples from the results of similarity calculation in Example 1. FIG. FIG. 10 is a diagram showing the results of similarity calculation in Example 2 to the top five. FIG. 10 is a diagram showing an integral curve used in Example 3 and a target interval for similarity calculation; Figure b shows the corresponding chromatogram. FIG. 10 is a diagram showing the results of similarity calculation in Example 3 to the top five.

The calculation method of the present invention includes a step of removing the drift in the interval in which the similarity calculation is performed on the chromatogram of the measurement sample, and a step of adjusting the elution time of the reference component peak to be the same as that of the chromatogram of the standard sample. After correcting the time axis, the step of recorrecting so that the time interval is the same as the chromatogram of the standard sample, and then the chromatogram of the standard sample from which the drift of the interval for similarity calculation is removed. It calculates the degree of similarity between An embodiment of the present invention will be described below.

In addition, the "chromatogram" in the present invention refers to a chromatogram obtained by size exclusion chromatography.

As an example of the step of removing the drift of the section in which similarity calculation is performed, at least one or more start points (x1, y1) and at least one or more end points (x2, y2) [x: Time, y: output] is determined, a linear equation (y=ax+b) is calculated, and the y value of the linear equation is subtracted from each data point (time, output) of the chromatogram. However, this process is not limited to this method. A method of calculating a polynomial based on a plurality of points at which peaks other than the start point and end point are not eluted, and subtracting the y value of the polynomial from each data point (time, output) of the chromatogram is also useful. .

The starting point may be before the time at which the target component begins to elute, but in order to improve the accuracy of the similarity calculation, it is preferable that the area other than the target component is as small as possible. The end point may be after the time when the target component finishes eluting and before the time when the reference component peak elutes. As little as possible is better.

A method for correcting the time axis so that the elution time of the reference component peak (hereinafter sometimes referred to as the internal standard peak) is the same as that of the chromatogram of the standard sample will be described. First, the value obtained by dividing the elution time (Ts) of the internal standard in the standard sample chromatogram by the elution time (Tu) of the internal standard in the measurement sample chromatogram is defined as the correction factor (Ft), and the x value ( time) by a correction factor (Ft) (see FIG. 1).

A method of recorrecting the chromatogram so that the time interval is the same as the chromatogram of the standard sample will be described. When the time axis is corrected as described above, the time interval (sampling pitch) of the chromatogram deviates from that of the standard sample chromatogram. Therefore, interpolation processing is performed using data points before and after to perform re-correction (see FIG. 2).

The internal standard peak may be a peak derived from a solvent, a peak of a low-molecular component, etc. When a differential refractometer is used as a detector in SEC, the peak derived from the solvent in which the sample is dissolved has the opposite polarity ( negative direction), and it is preferable to use the solvent peak as an internal standard.

"The step of removing drift in the interval for similarity calculation" and "After correcting the time axis so that the elution time of the reference component peak is the same as the chromatogram of the standard sample, the time interval is the same as the standard sample. It does not matter which step is performed first.

After all the steps described above have been completed for the measurement sample chromatogram (see FIG. 3), similarity calculation is then performed between the measurement sample chromatogram and the standard sample chromatogram from which the drift in the similarity calculation interval has been removed. The method of removing the drift of the standard sample chromatogram is the same as the method used for the measurement sample chromatogram, and there is no problem. There is no problem even if the process treatment of is performed.

A typical method for calculating the “similarity” is the method given in Equation 1, but it is not particularly limited as long as it serves as an index of the similarity of waveforms between two chromatograms. The similarity score calculated using Equation 1 is at most 1000, with closer to 1000 indicating greater identity of the chromatograms.

Also, there is no problem in setting a plurality of sections for which the similarity calculation is performed, and weighting each section by taking the average value of the similarity values of each section. For example, in a polymer sample having multiple molecular weight distributions as shown in FIG. 4b, the similarity may be calculated by dividing it into two sections. The calculation may be performed by dividing into three sections such as the molecular side (first section), the low molecular weight side (third section), and the average molecular weight section (second section).

Similarity calculation can also be performed using an integral curve obtained from a chromatogram. The integral curve indicates the ratio of a certain molecular weight to the whole, and is one of the indicators of the properties of the polymer component. When performing similarity calculation using an integral curve, for the chromatogram for which all the processes described above have been completed, the output values are integrated over time within the specified calculation interval, converted to an integral curve, and then the similarity calculation is performed. You can do it.

The degree of similarity calculated according to the present invention can be used to judge quality by whether it exceeds or falls below a preset reference value. Also, when a plurality of sections are provided, it is also possible to determine whether or not conditions are satisfied in a plurality of items.

EXAMPLES The embodiments of the present invention will be described in more detail below with reference to Examples. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects are possible for the details. Furthermore, the present invention is not limited to the above-described embodiments, and can be modified in various ways within the scope of the claims. It is included in the technical scope of the invention. In addition, all documents described in this specification are incorporated by reference.

Actual measurements were performed using the system shown in FIG. The system consists of eluent 1, solvent deaerator 2 (SD-8020), liquid feed pumps 3 and 11 (DP-8020), sample injection device 4 (AS-8020), column oven 6 (CO-8020), differential It consisted of a refractometer 10 (RI-8020), a resistance tube 7, a waste liquid unit 8 and a data processor 9 (GPC-8020II) (all brackets are manufactured by Tosoh Corporation). As the analysis column 5, Tosoh Corporation TSKgel G-Oligo-PW (7.8 mm I.D. × 30 cm × 2) is used, and polyethylene glycol (manufactured by Fujifilm Wako Pure Chemical Co., Ltd.) is separated. rice field. Other conditions are as follows.
Data collection sampling pitch: 200ms
Injection volume: 50 μL
Column temperature: 40°C
Eluent: 200 mM NaNO ₃ +20% acetonitrile Two types of polyethylene glycol were mixed at the ratio shown in Table 1 to prepare an evaluation sample.
Polyethylene glycol 200 (PEG200) 17 mg/mL
Polyethylene glycol 300 (PEG300) 17 mg/mL

As for the flow rate, the measurement was performed twice at 1.000, 1.005, and 1.010 mL/min in order to intentionally vary the measurement results.

(Example 1)
In this example, a chromatogram obtained by measuring Sample_D at a flow rate of 1.000 mL/min (#7) with one similarity calculation interval was used as the chromatogram of the standard sample. The "solvent-derived peak" was used as an internal standard component, and the elution time (Ts) was 20.220 minutes. The interval for similarity calculation was 13.0 to 19.6 minutes, and drift was removed.

For other chromatograms, remove the drift of 13.0 to 19.6 minutes, calculate the correction factor (Ft) from the elution time (Tu) of the internal standard component, correct the time axis, and then the time data Interpolation processing was performed so that the interval was 200 ms.

Next, the degree of similarity between the chromatogram of the standard sample and each chromatogram was calculated based on Equation (1). The results are shown in FIGS. 6 and 7 (dashed lines represent standard sample chromatograms and solid lines represent selected chromatograms). Even though the flow rate was slightly different, the chromatograms of Sample_D, which had the same molecular weight distribution, had a high degree of similarity and could be distinguished.

For comparison, FIG. 8 shows the results of comparison using raw chromatograms without any correction. Chromatograms other than #8 have completely different elution positions of the high-molecular weight components, and it cannot be judged visually that they have a high degree of similarity.

(Example 2)
In this embodiment, an example in which similarity calculation is performed using a plurality of calculation intervals will be shown. Everything is the same as the first embodiment except for the step of calculating the degree of similarity. The similarity calculation interval was divided into three regions as follows.
First half section: 13.00-14.50 minutes Middle section: 14.50-17.50 minutes Second half section: 17.50-19.57 minutes Since components with high molecular weights elute early in molecular exclusion chromatography, the first half section A fraction with a high molecular weight, a fraction with a low molecular weight in the latter half, and a fraction with an average molecular weight in the middle. FIG. 9 is a diagram showing the similarity in all regions and the similarity in each calculation interval obtained in Example 1. In FIG. It was found that the value obtained by averaging the three sections has the same rank as the similarity calculated for one section (Example 1).

(Example 3)
In this example, the similarity calculation based on the integral curve of the chromatogram was verified. Based on the chromatogram obtained in Example 1 after time correction and drift removal, an operation was performed to obtain an integral curve. In addition, normalization was performed with the maximum value set to 100 so that the difference in the integral curve depending on the sample can be visually recognized.

FIG. 10 shows an example of a chromatogram, an integral curve corresponding thereto, and a calculation interval. In this example, the calculation was performed in two intervals of 13.0 to 16.5 minutes when the high molecular weight region of the high molecular weight component elutes and 16.5 to 19.5 minutes when the low molecular weight region elutes. As in the first embodiment, #7 was used as the reference integral curve.

The degree of similarity was calculated in two sections, and the average value was taken as the final degree of similarity. The results are shown in FIG. It was found that even if the similarity calculation (two sections) was performed based on the integral curve, the ranking was the same as the similarity calculated in one section (Example 1).

1. Eluent2. Degassing device3. Liquid transfer pump (sample side)
4. sample injection valve 5 . analytical column6. Column constant temperature bath 7 . resistance tube8. Waste liquid9. system control and data processing unit 10. Differential refractometer 11 . Liquid transfer pump (reference side)

Claims (3)

A method for calculating the degree of similarity between a measurement sample and a standard sample based on a chromatogram obtained by size exclusion chromatography,
For the chromatogram of the measurement sample,
A step of removing the drift of two or more intervals in the chromatogram for similarity calculation;
After correcting the time axis so that the elution time of the reference component peak is the same as the chromatogram of the standard sample, the time interval (data collection sampling pitch) is readjusted to be the same as the chromatogram of the standard sample. After performing the step of correcting and
The above method, wherein the similarity is calculated with the chromatogram of the standard sample from which the drift in two or more intervals in the chromatogram for which the similarity is to be calculated is removed. 2. The method of claim 1, wherein the chromatogram is converted into an integral curve prior to similarity calculation. 3. The method according to claim 1 or 2 , wherein the degree of similarity is calculated by the following formula.

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