A method for quantifying glucose concentration from the Raman spectrum of cell culture medium.

Abstract

To provide a method for easily determining concentration of glucose, from a Raman spectrum of a cell medium.SOLUTION: A cell medium includes glucose and a component (HEPES) affecting analysis of the glucose, and in the invention, a Raman spectrum of the cell medium is acquired. In the invention, a prescribed wave number for the glucose, a calibration curve indicating a relationship between spectrum intensity of the glucose and concentration of the glucose at 1120 cm-1, a calibration curve indicating a relationship between spectrum intensity of HEPES and concentration of the HEPES in a wave number unique to the HEPES which appears in an isolated state and does not overlap other waveform, and a calibration curve indicating a relationship between the spectrum intensity and concentration of the HEPES at 1120 cm-1 are created, for calculating the concentration of the glucose in the cell medium, on the basis of the spectrum intensity at 1120 cm-1 of the cell medium, the spectrum intensity at the wave number unique to the HEPES and the calibration curves.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This invention relates to a method for quantifying glucose concentration from the Raman spectrum of a cell culture medium. [Background technology]

[0002] For measuring glucose, lactic acid, ammonium, and other substances present in aqueous solutions, liquid chromatography, ion chromatography, or specialized measurement kits are commonly used. Depending on the substance being measured, a combination of multiple instruments may be used. When glucose, lactic acid, ammonium, etc., are present in a high-concentration matrix (high concentrations of sodium, chlorine, etc.), such as in cell culture media, separation analysis using liquid chromatography or ion chromatography is a common analytical method.

[0003] On the other hand, in typical Raman spectroscopy, the processing of the obtained measurement spectrum involves comparing the spectrum with a database to perform qualitative analysis and identification of the substance. If the substance can be identified and a standard substance is available, the concentration of only one component is usually quantified.

[0004] Here, the spectra of standard materials are provided by the material manufacturers, public institutions, and database vendors. The spectra of standard materials are databases of spectral patterns that represent the characteristics of each material, and each material has its own individual data. There are no databases that combine multiple matrices, and users must manually perform arithmetic operations on the waveforms and calculate difference spectra from the obtained spectral data, and then qualitatively identify each material by separating the composite spectra.

[0005] Patent Document 1 describes a component analyzer suitable for analyzing components in blood using Raman spectroscopy. Patent Document 2 describes an analyzer suitable for analyzing components contained in blood and interstitial fluid using Raman spectroscopy. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Patent No. 6826463 specification [Patent Document 2] Patent No. 6842322 specification [Overview of the project] [Problems that the invention aims to solve]

[0007] In Raman spectroscopy, the measured spectrum can show multiple spectra depending on the composition of the substance being measured (molecular bonding state, attached functional groups), making it difficult to immediately determine what kind of substance it is. In particular, Raman spectroscopy does not have the function of separating substances like liquid chromatography; qualitative analysis and identification are performed by comparing the molecular structure of the substance and the attached functional groups with a database. Therefore, if overlapping spectra appear, it may become difficult not only to perform qualitative analysis and identification, but also to calculate the concentration.

[0008] In particular, in liquid chromatography, if, for example, a non-target substance is present at a concentration 10 times or more relative to the substance being measured, the target substance may alter the selectivity of the column and affect the separation. Taking cation analysis as an example, if a trace amount (e.g., 0.17 g / l) of the target component is present in a high concentration of sodium (e.g., 7 g / l), the analysis will be concentrated by the column. However, since the sodium is also concentrated instead of the target component, the sodium may exceed the column's exchange capacity, potentially preventing the target component from being trapped.

[0009] The present invention aims to easily quantify the concentration of glucose from the Raman spectrum of a cell culture medium. [Means for solving the problem]

[0010] A method according to one embodiment of the present invention is A method for quantifying glucose concentration from the Raman spectrum of cell culture medium, The cell medium contains glucose and components that affect glucose analysis, and the Raman spectrum of the cell medium is obtained. The spectral intensity at a predetermined wavenumber for the glucose includes the spectral intensity component of glucose derived from the glucose and the spectral intensity component of the component that affects the analysis of glucose derived from the component that affects the analysis of glucose, and the spectral intensity specific to the component that affects the analysis of glucose at a wavenumber specific to the component that affects the analysis of glucose, which is different from the predetermined wavenumber and appears isolated without overlapping with other waveforms. Using a component-specific calibration curve that shows the relationship between the spectral intensity and concentration of the component affecting the analysis of glucose at the aforementioned specific wavenumber, the concentration of the component affecting the analysis of glucose in the cell culture medium is quantified from the component-specific spectral intensity affecting the analysis of glucose at the aforementioned specific wavenumber. Using a predetermined calibration curve for components affecting glucose analysis that shows the relationship between the spectral intensity and concentration of the components affecting glucose analysis at the predetermined wavenumber, the spectral intensity portion of the components affecting glucose analysis at the predetermined wavenumber is calculated from the quantified concentration of the components affecting glucose analysis. From the spectral intensity at the predetermined wavenumber and the spectral intensity of the component that affects the analysis of glucose, the spectral intensity of glucose at the predetermined wavenumber is calculated. This includes quantifying the concentration of glucose in the cell medium from the calculated spectral intensity of glucose at a predetermined wavenumber, using a glucose calibration curve that shows the relationship between the spectral intensity and concentration of glucose at a predetermined wavenumber. The predetermined wave frequency for the glucose is 1120 cm⁻¹. -1 That is the method. [Effects of the Invention]

[0011] According to the present invention, the glucose concentration in the cell culture medium can be easily quantified from the obtained Raman spectral data. [Brief explanation of the drawing]

[0012] [Figure 1] Figure 1 is a conceptual diagram showing an example of the configuration of an analytical mechanism equipped with the analytical device used in the method of this embodiment. [Figure 2] Figure 2 is a conceptual diagram showing an example of the configuration of the analytical apparatus used in the method of this embodiment. [Figure 3] Figure 3 is a calibration curve showing the relationship between the spectral intensity and concentration of glucose at 1120 cm⁻¹. [Figure 4] Figure 4 is a calibration curve showing the relationship between the spectral intensity and concentration of HEPES at 1120 cm⁻¹. [Figure 5] Figure 5 is a calibration curve showing the relationship between the spectral intensity and concentration of HEPES at 1038 cm⁻¹. [Figure 6] Figure 6 shows a comparison of cell culture measurement results (StemFit) and blood gas meter results, illustrating the time course of glucose as measured by Raman spectroscopy. [Figure 7] Figure 7 shows a comparison of glucose levels measured over time using Raman spectroscopy in cell culture media (Essential8) and blood gas analysis. [Figure 8] Figure 8 shows a comparison of cell culture measurement results (CHOM) and blood gas meter results, illustrating the time course of glucose as measured by Raman spectroscopy. [Figure 9] Figure 9 shows a comparison of cell culture measurement results (REGM) and blood gas meter results, illustrating the time course of glucose as measured by Raman spectroscopy. [Figure 10] Figure 10 compares the time-dependent changes in glucose levels measured by Raman spectroscopy and blood gas analysis results for cell culture media from different manufacturers (REGM, StemFit, DMEM / F12, Essential6). [Figure 11]Figure 11 is a calibration curve showing the relationship between lactate concentration and spectral intensity at 1084 cm⁻¹. [Figure 12] Figure 12 is a calibration curve showing the relationship between glucose concentration and spectral intensity at 1084 cm⁻¹. [Figure 13] Figure 13 shows a comparison of cell culture measurement results (StemFit) and blood gas meter results, illustrating the time course of lactate levels using Raman spectroscopy. [Figure 14] Figure 14 shows a comparison of cell culture measurement results (Essential8) and blood gas meter results, illustrating the time course of lactate levels using Raman spectroscopy. [Figure 15] Figure 15 shows a comparison of cell culture measurement results (CHOM) and blood gas meter results, illustrating the time course of lactic acid using Raman spectroscopy. [Figure 16] Figure 16 shows a comparison of cell culture measurement results (REGM) and blood gas meter results, illustrating the time course of lactate levels using Raman spectroscopy. [Figure 17] Figure 17 compares the time-dependent changes in lactic acid levels, measured by Raman spectroscopy, and ion chromatography results for cell culture media from different manufacturers (REGM, StemFit, DMEM / F12, Essential6). [Figure 18] Figure 18 is a calibration curve showing the relationship between ammonium concentration and spectral intensity at 1436 cm⁻¹. [Figure 19] Figure 19 is a calibration curve showing the relationship between glucose concentration and spectral intensity at 1436 cm⁻¹. [Figure 20] Figure 20 is a calibration curve showing the relationship between lactic acid concentration and spectral intensity at 1436 cm⁻¹. [Figure 21] Figure 21 is a calibration curve showing the relationship between lactic acid concentration and spectral intensity at 852 cm⁻¹. [Figure 22] Figure 22 is a calibration curve showing the relationship between acetaldehyde concentration and spectral intensity at 1436 cm⁻¹. [Figure 23]Figure 23 is a calibration curve showing the relationship between acetaldehyde concentration and spectral intensity at 928 cm⁻¹. [Figure 24] Figure 24 is a calibration curve showing the relationship between glutamine concentration and spectral intensity at 1436 cm⁻¹. [Figure 25] Figure 25 is a calibration curve showing the relationship between glutamine concentration and spectral intensity at 1140 cm⁻¹. [Figure 26] Figure 26 is a calibration curve showing the relationship between HEPES concentration and spectral intensity at 1436 cm⁻¹. [Figure 27] Figure 27 is a calibration curve showing the relationship between phenol red concentration and spectral intensity at 1436 cm⁻¹. [Figure 28] Figure 28 is a calibration curve showing the relationship between phenol red concentration and spectral intensity at 1162 cm⁻¹. [Figure 29] Figure 29 shows a comparison of cell culture measurement results (StemFit) and Ammonia Assay Kit results, illustrating the time course of ammonium levels as measured by Raman spectroscopy. [Figure 30] Figure 30 shows a comparison of cell culture measurement results (Essential8) and Ammonia Assay Kit results, illustrating the time course of ammonium levels as measured by Raman spectroscopy. [Figure 31] Figure 31 shows a comparison of cell culture measurement results (CHOM) and Ammonia Assay Kit results, illustrating the time course of ammonium levels as measured by Raman spectroscopy. [Figure 32] Figure 32 shows a comparison of cell culture measurement results (REGM) and Ammonia Assay Kit results, illustrating the time course of ammonium levels as measured by Raman spectroscopy. [Figure 33] Figure 33 compares the time-dependent changes in ammonium from different manufacturers' cell culture media (REGM, StemFit, DMEM / F12, Essential6) using Raman spectroscopy and ion chromatography. [Modes for carrying out the invention]

[0013] Hereinafter, examples of embodiments for carrying out the present invention will be described with reference to the accompanying drawings. The embodiments illustrated below are provided to facilitate understanding of the present invention and are not intended to limit the interpretation of the present invention. The embodiments illustrated below can be modified and improved as appropriate without departing from the spirit of the present invention.

[0014] <Analysis mechanism> Figure 1 is a conceptual diagram showing an example of the configuration of an analytical mechanism 10 equipped with an analytical device 26 used in the method of this embodiment. The analytical mechanism 10 is a mechanism for analyzing the spectrum of Raman scattered light generated in a sample 16 by irradiation with excitation light. The analytical mechanism 10 mainly comprises a light source 12, a reflector 14, a sample 16, an optical system 18, a monochromator 20, and an analytical device 26.

[0015] Light source 12 emits excitation light to irradiate sample 16. A solid-state laser is preferred as light source 12 because a shorter wavelength of excitation light increases the efficiency of Raman scattering and improves spatial resolution. The spectrum of Raman scattered light shows the difference in wavenumber of the Raman scattered light relative to the wavenumber of the excitation light (Raman shift). Therefore, it is preferable that the wavelength of the excitation light be a single wavelength. Specific examples of excitation light wavelengths include, for example, a single wavelength selected from 514 nm, 532 nm, 633 nm, 670 nm, and 785 nm.

[0016] The reflector 14 reflects the excitation light emitted from the light source 12 and guides it to the sample 16. Note that other components may be used instead of or in addition to the reflector 14, as long as they guide the excitation light emitted from the light source 12 to the sample 16.

[0017] Sample 16 consists of a cell culture medium and a container (not shown) for containing the cell culture medium. The container for the cell culture medium is made of inert plastic material, is vertically oriented, has a capacity of 25-50 ml, and is airtight. The measurement cell used is a rectangular, vertically oriented container made of synthetic quartz glass with a capacity of 1-10 ml. In addition to the method of measuring by placing Sample 16 in such a measurement cell, there is also a flow cell measurement method in which Sample 16 is passed through a flow-type measurement cell. In addition to chemically inert inorganic materials, plastic materials, or synthetic resins, the container may be made of a metal with excellent corrosion resistance to acids and alkalis. The container has a structure with an inlet and outlet for Sample 16, and can employ a structure that transmits Raman scattered light, such as a structure with an optical window for receiving Raman scattered light and an optical window for receiving Raman scattered light that has passed through Sample 16, or a reflective structure in which both the incidence and reception of Raman scattered light can be performed by a single optical element. The container is more preferably one that has a structure that allows for the entry and exit of the sample 16, the incidence of Raman scattered light, the irradiation of the sample 16, and the reception of the Raman scattered light after irradiation, although the method is not limited to these.

[0018] <Cell culture medium> Cell culture media typically consist of inorganic substances, amino acids, pH indicators, pH buffers, and nutrients (broadly classified into three types: carbon sources, mineral sources, and nitrogen sources), but are not limited to these. The component concentrations in cell culture media may vary depending on their purpose. In Raman spectroscopy, absorption of inorganic substances is generally minimal, with absorption observed only from organic substances.

[0019] <Metabolic production> When cells begin to proliferate, they consume nutrients such as glucose, and the lactic acid and ammonium produced within the cells, being low-molecular-weight substances, pass through the cell membrane, are released outside the cell, and accumulate in the cell culture medium. The accumulation of waste products such as lactic acid and ammonium causes a decrease in the pH of the cell culture medium and cytotoxicity, hindering cell growth, so monitoring the concentrations of lactic acid and ammonium is necessary.

[0020] <Cell culture medium imitation solution> The cell culture medium simulant can be a mixture of a pH indicator, a pH buffer, glucose, lactic acid, etc. In the cell culture medium simulant, typically, a pH indicator and a pH buffer are formulated. Since the cell culture medium simulant does not show changes over time, it is preferably used for preparing a calibration curve.

[0021] <pH indicator> The pH indicator is for visually and instantaneously determining the pH of the contents when cell culture is performed in a transparent culture vessel. In the present application, phenol red is used as the pH indicator. When cell culture medium contains phenol red as a pH indicator, this is used for visually and instantaneously determining the pH of the contents when performing cell culture in a transparent culture vessel. Phenol red is particularly preferred because the pH to be monitored is 6 - 8.

[0022] <pH buffer> The pH buffer is for suppressing the rapid change caused by lactic acid that increases during cell culture. In the present application, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) is used as the pH buffer. HEPES is particularly preferred because it is excellent at absorbing changes in the carbon dioxide concentration generated by the cultured cells through aerobic respiration and maintaining the physiological pH during cell culture. Also, the phosphate buffer is particularly preferred because it is non-toxic as it is composed of ions commonly found in vivo.

[0023] Before measuring sample 16 with a Raman spectrometer, it is preferable to perform background correction with pure water. It is desirable to use pure water or purified water (distilled water purified with ion exchange resin) with few impurities. Since background measurement greatly affects the integrity of subsequent measurements of sample 16, it is preferable to measure pure water again after background measurement and correction, and repeat until the baseline is as flat as possible. Furthermore, if there are unidentifiable peaks in the cell medium that are difficult to identify, it is preferable to measure the cell medium before cell culture (day 0 of culture) and use the obtained spectrum to perform background correction, in addition to background correction with pure water, assuming that they may appear near the main peaks of lactate or glucose.

[0024] When high concentrations of matrix components are present, an environment with minimal ambient temperature fluctuations is desirable. Here, ambient temperature is, for example, within the range of 20-30°C, with short-term fluctuations of within ±5°C.

[0025] The optical system 18 guides the Raman scattered light generated in the sample 16 to the monochromator 20. When the sample 16 is irradiated with excitation light, the interaction between the excitation light and the sample 16 causes the sample 16 to produce Raman scattered light with a different wavelength than the excitation light, as well as Rayleigh scattered light with the same wavelength as the excitation light. The analysis mechanism 10 has an optical system 18 on the waveguide to remove this Rayleigh scattered light, and guides the Raman scattered light obtained through the optical system 18 to the monochromator 20.

[0026] The monochromator 20 includes a diffraction grating 22 and a CCD (Charge Coupled Device) detector 24. The diffraction grating 22 separates the excitation light supplied from the sample 16 via the optical system 18 into wavelengths and guides them to the light-receiving surface of the CCD detector 24. The CCD detector 24 has multiple photodetectors arranged in one or two dimensions within its light-receiving surface. This CCD detector 24 stores charge proportional to the intensity of Raman scattered light of each wavelength exposed to the photodetectors within its light-receiving surface, and outputs Raman spectral data showing the intensity distribution of Raman scattered light for each wavelength to the analysis instrument 26. Instead of the CCD detector 24, detectors using photodiodes, CMOS (Complementary Metal Oxide Semiconductor), etc., can be used.

[0027] <Analyzer> The analyzer 26 detects components in the cell culture medium of sample 16 based on data provided by the CCD detector 24 of the monochromator 20, and determines the concentration of the detected components. The analyzer 26 also notifies the user, as necessary, of the types and concentrations of components present in sample 16.

[0028] Figure 2 is a conceptual diagram showing an example of the configuration of the analytical apparatus 26 used in the method of this embodiment. The analytical apparatus 26 mainly comprises an input unit 30, a storage unit 32, a control unit 34, and a display unit 36.

[0029] The input unit 30 is a section that can receive various types of information, such as commands and settings, in response to user operations. Specific examples of the input unit 30 include a mouse, keyboard, touch panel, portable semiconductor connector type memory, and communication interfaces that exchange information via network communication or serial communication. Examples of semiconductor connector type memory include USB (Universal Serial Bus) memory and CF (Compact Flash) memory.

[0030] The storage unit 32 is the part that stores data provided by the input unit 30 or the control unit 34 and reads the stored data. Specific examples of the storage unit 32 include semiconductor memory, magnetic disks, and optical disks.

[0031] The memory unit 32 includes a first storage area (not shown) for storing various programs, a second storage area (not shown) for storing various data such as setting data input from the input unit 30, and an expansion area (not shown) for expanding programs and data. The first storage area stores an analysis program for performing the process of analyzing the cell culture medium of sample 16. The specific contents stored in the memory unit 32 will be described in detail later.

[0032] The control unit 34 controls the analysis device 26 based on the program stored in the first storage area of ​​the storage unit 32 and the data stored in the second storage area of ​​the storage unit 32.

[0033] When the control unit 34 reads an analysis program stored in the first storage area of ​​the storage unit 32, it expands the read analysis program into the expansion area of ​​the storage unit 32. In this case, the control unit 34 functions as an acquisition unit 40, an extraction unit 42, a component identification unit 44, a calculation unit 46, and a notification unit 48, and executes the process of analyzing the cell culture medium.

[0034] The display unit 36 ​​is the part that displays information indicated by the data provided by the control unit 34. Specific examples of the display unit 36 ​​include liquid crystal displays, plasma displays, and EL (Electro-Luminescence) displays.

[0035] The acquisition unit 40 acquires a Raman spectrum showing the intensity distribution of Raman scattered light at each wavenumber, which is generated when a sample, which is a cell culture medium containing the desired components, is irradiated with excitation light of a single wavelength. Specifically, the acquisition unit 40 drives the light source 12 by appropriately adjusting the intensity of the excitation light to be emitted from the light source 12, and acquires the Raman spectrum data output from the CCD detector 24 of the monochromator 20 sequentially through the light source 12, reflector 14, sample 16, and optical system 18.

[0036] In this embodiment, the acquisition unit 40 acquires the Raman spectrum of the cell medium, which contains the target components and components that affect the analysis. The components to be analyzed are glucose, lactate, and ammonium.

[0037] It is preferable to measure the glucose concentration in order to adjust the glucose concentration necessary to maintain cell culture. Here, lactate and ammonium are metabolically produced as components that cause toxicity to cells and inhibit cell growth. It is preferable to measure lactate and ammonium in order to monitor and remove such concentrations and maintain an appropriate cell culture medium state.

[0038] When the target component is glucose, the component that affects it is preferably HEPES, a pH buffer. HEPES has a predetermined wavenumber of 1120 cm for glucose. -1 The spectral intensity appears at 1120 cm⁻¹. -1 In this case, there is linearity between the intensity and concentration of the Raman spectrum of HEPES, 1120 cm⁻¹ -1 This is preferable because there are no other overlapping spectral components.

[0039] When the target component is lactic acid, it is preferable that the influencing component is glucose. Glucose has a predetermined wavenumber of 10⁸⁴ cm⁻¹ for lactic acid. -1 The spectral intensity appears at 1084 cm⁻¹. -1There is linearity between the intensity and concentration of the Raman spectrum of glucose, 1084 cm -1 It is preferable because there are no other overlapping spectral components.

[0040] When the target component is ammonium, it is preferable that the components having an influence are at least one of glucose, lactic acid, acetaldehyde, glutamine, HEPES, and phenol red. These components having an influence exhibit spectral intensity at 1436 cm, which is a predetermined wave number for ammonium. -1 At 1436 cm -1 There is linearity between the intensity and concentration of the Raman spectra of these components having an influence, at 1436 cm -1 It is preferable because there are no other overlapping spectral components.

[0041] The extraction unit 42 converts the wavelength of the Raman spectrum acquired by the acquisition unit 40 into a Raman shift amount and extracts the spectral intensity. Specifically, each wavelength in the Raman spectrum is converted into a wave number, which is the number of waves included in a unit length, and each converted wave number is converted into a Raman shift amount, which is the difference from the wave number of the excitation light emitted from the light source 12. As a result, for example, a Raman spectrum is obtained with the spectral intensity on the vertical axis and the wave number on the horizontal axis. It is known that a substance has unique vibration energy according to its molecular structure and crystal structure, and the Raman shift amount, which is the difference between the wave number (frequency) of the Raman scattered light and the wave number (frequency) of the incident light, has a unique waveform reflecting the molecular structure and crystal structure of the substance.

[0042] The component identification unit 44 searches for and identifies the components of the cell culture medium based on the Raman spectrum converted by the extraction unit 42 and the unique wave numbers of the peaks that appear in the Raman spectrum according to the components of the cell culture medium.

[0043] The calculation unit 46 quantifies the concentration of the component corresponding to the peak based on the spectral intensity of the peak detected by the component identification unit 44. Specifically, the calculation unit 46 reads a calibration curve for the component and wavenumber corresponding to the peak detected by the component identification unit 44 from the second storage area, and uses the calibration curve to determine the concentration (mg / dl) of the component corresponding to the detected peak from the spectral intensity of that peak. The specific functions of each component in the calculation unit will be described later.

[0044] The component identification unit 44 recognizes a predetermined wavenumber of a certain component read from the second storage area and a wavenumber specific to that component, and adjusts the frequency by ±6 cm relative to that wavenumber. -1 The system searches for whether or not there is a peak within the specified range. In this case, the range is ±6 cm based on the predetermined wavenumber and the specific wavenumber. -1 If even one peak is detected within this range, it means that the component corresponding to that peak is present in the cell culture medium. Note that, for the sake of simplicity and clarity, only certain wavenumber values ​​are shown as predetermined and characteristic wavenumbers in this application; however, the predetermined and characteristic wavenumbers in this invention are ±6 cm. -1 This means including the width. For example, "852cm -1 " is 852±6cm -1 This means "928cm -1 " is 928±6cm -1 This means "1038cm -1 " is 1038±6cm -1 This means "1084cm" -1 " is 1084±6cm -1 This means "1120cm" -1 " is 1120±6cm -1 This means "1140cm" -1 " is 1140±6cm -1 This means "1162cm -1 " is 1162±6cm -1 This means "1436cm -1 " is 1436±6cm -1 It means...

[0045] The notification unit 48 notifies the type of cell culture medium component detectable by the component identification unit 44, and the concentration of the cell culture medium component detected by the component identification unit 44 and quantified by the calculation unit 46. For example, the type of cell culture medium component and the concentration of the cell culture medium component quantified by the calculation unit 46 are displayed on the display screen of the display unit 36 ​​in a predetermined display format. Alternatively, or in addition to displaying them on the display unit 36, the type of cell culture medium component and the concentration of the cell culture medium component quantified by the calculation unit 46 may be output as audio from the speaker.

[0046] The control unit 34 functions through the acquisition unit 40, extraction unit 42, component identification unit 44, calculation unit 46, and notification unit 48, and performs the process of analyzing the components of the cell culture medium.

[0047] In Raman spectroscopy, sample 16 can be measured directly in the container without any pretreatment. The measurement can be performed as a simultaneous analysis, in which case substances that show absorption in the solution will appear as spectra. In cell culture media where the composition of the solution is known in advance, it is preferable to know which substances are detected as absorption spectra at high concentrations of matrix and which are not. Phenol red is often added as a pH indicator, and HEPES and phosphate saline buffer are often added as pH buffers. Since these can be treated as substances whose concentrations do not change between the start of use and the end of culture, it is preferable to determine their concentrations beforehand.

[0048] Typically, background measurements are performed before starting measurements with a Raman spectrometer. In background measurements, pure water is usually placed in a quartz cell and measured, and the spectra of the quartz cell's absorption, impurities in the pure water, and carbon dioxide are determined. It is desirable to automatically perform background correction after each subsequent measurement. Following the pure water, the next background can be quantified using, for example, phenol red or HEPES of known concentration, or phenol red or HEPES of unknown concentration, and used in background measurements to quantify the component concentrations after spectral measurement. In cell culture media, substances with little concentration change are phenol red and HEPES. Other components that affect spectral absorption due to little concentration change may also be treated in the same way.

[0049] Furthermore, it has been confirmed that linearity is observed in all mixed solutions of glucose (100-300 mg / dl), lactic acid (0-400 mg / dl), ammonium (0-500 mg / dl), and glutamine (30-50 mg / dl) when determining the following calibration curves.

[0050] Furthermore, when actually implementing the present invention, it is preferable to construct a program that performs calculations automatically on a PC and to judge the calculation results. However, depending on the calculation results, the result may be displayed as, for example, zero, undetectable, ND, or ---.

[0051] In the following explanation, an example of the action of each component targeted by each method is provided to facilitate understanding.

[0052] Below, an example of the effect of each target component is explained. When creating a calibration curve, it is preferable to use a cell medium simulation solution containing the specified components instead of actual cell culture medium. The cell medium simulation solution can be prepared, for example, based on Tables 1 to 4 below.

[0053] [Table 1]

[0054] [Table 2]

[0055] [Table 3]

[0056] [Table 4]

[0057] Table 1 shows the preparation table for the base solution of the cell culture medium simulation at pH 6.5. Table 2 shows the preparation table for the base solution of the cell culture medium simulation at pH 6.8. Table 3 shows the preparation table for the base solution of the cell culture medium simulation at pH 7.0. Table 4 shows the preparation table for the base solution of the cell culture medium simulation at pH 7.5.

[0058] For example, a phosphate-buffered saline base (phenol red + HEPES) simulation solution is prepared based on the table above. As a pH buffer, 10×D-PBS(-), Code. No. 048-29805, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., can be used, or it can be prepared based on the table above. Preparation method Based on the SDS of Fujifilm Wako Pure Chemical Industries, prepare the solution with KCl: 2000.0 mg / L, NaCl: 80000.0 mg / L, KH2PO4: 2000.0 mg / L, and Na2HPO4: 11500.0 mg / L. Here, "10×" means that it is a 10-fold concentrated solution and needs to be diluted 10 times before use. Obtain the reagents with the above components, and using ultrapure water that has been distilled, purified, and filtered, prepare 1 L of the above 10-fold concentrated phosphate buffer solution, and dilute it as a stock solution before use. • Adjust the pH by varying the ratio of KH2PO and NaHPO4 before use. The concentrations of NaCl and KCl are kept constant at 137 mM and 2.7 mM, respectively. The pH indicator phenol red should be 15 mg / l. The pH buffer HEPES should be 3000 mg / l. Based on the above solution, glucose is added at concentrations of 50, 75, 150, 200, and 300 mg / dl; lactic acid at concentrations of 9, 45, 90, 180, and 450 mg / dl; and ammonium at concentrations of 26, 53, 160, 267, 500, and 750 mg / dl. Each of these is added to a pH buffer-based solution, and pure water is added to bring the total volume to 100 ml to adjust to the desired concentration.

[0059] Embodiment 1: Analysis of glucose [Calibration curve for glucose analysis] In this embodiment, the component that affects the analysis is HEPES as a pH buffer. In this case, when analyzing glucose contained in the cell culture medium using Raman spectroscopy, the effect of HEPES is taken into consideration. To quantify the glucose concentration, the spectral intensity and concentration of glucose can be calculated by subtracting this effect from the measured spectral intensity.

[0060] <Glucose Calibration Curve> Figure 3 shows 1120 cm -1 This is a glucose calibration curve showing the relationship between the spectral intensity (y) and concentration (x (mg / dl)) of glucose in a given region. The vertical axis represents the spectral intensity y, and the horizontal axis represents the concentration x (the same applies to other calibration curves below).

[0061] 1120cm -1 The glucose calibration curve (Equation 1) in this case is as follows: Glucose: y = 0.809x + 20.211 (@1120cm) -1 )(Formula 1)

[0062] If glucose is present in the cell culture medium, the predetermined wavenumber for glucose in the Raman spectrum is 1120 cm⁻¹. -1 The above 1120cm will be set. -1The glucose calibration curve in [it] is input from the input unit 30 and stored in the second storage area of the storage unit 32.

[0063] <HEPES specific calibration curve> Figure 4 is a HEPES specific calibration curve showing the relationship between the spectral intensity (y) and the concentration (x (mg / dl)) of HEPES at 1120 cm -1 ).

[0064] 1120 cm -1 The (Formula 2) of the HEPES specific calibration curve at [it] is as follows: HEPES: y = 0.08x + 25.516 (@1120 cm -1 )(Formula 2)

[0065] The above-mentioned HEPES specific calibration curve at 1120 cm -1 is input from the input unit 30 and stored in the second storage area of the storage unit 32.

[0066] <HEPES specific calibration curve> In the Raman spectrum of HEPES, there is a main spectrum at 1038 cm -1 and spectra showing concentration dependence were detected at 1448 cm -1 , 1298 cm -1 , 1190 cm -1 ).

[0067] Figure 5 is a HEPES specific calibration curve showing the relationship between the spectral intensity (y) and the concentration (x (mg / dl)) of HEPES at 1038 cm -1 ).

[0068] 1038 cm -1 The (Formula 3) of the HEPES specific calibration curve at [it] is as follows: HEPES: y = 3.1151x + 331.28 (@1038 cm -1 )(Formula 3)

[0069] When HEPES is contained in the cell culture medium, 1038 cm is the wave number specific to HEPES in the Raman spectrum-1 The above 1038cm is set. -1 The HEPES-specific calibration curve is input from the input unit 30 and stored in the second storage area of ​​the memory unit 32.

[0070] Here, the acquisition unit 40 acquires a Raman spectrum showing the intensity distribution of Raman scattered light at each wavenumber, which is generated when a sample, which is a cell culture medium containing glucose, is irradiated with excitation light of a single wavelength.

[0071] Furthermore, the extraction unit 42 uses a predetermined wavenumber for glucose, which is 1120 cm⁻¹. -1 The spectral intensity at 1120 cm², including the spectral intensity component of glucose derived from glucose and the spectral intensity component of HEPES derived from HEPES. -1 The spectral intensity at this point is extracted. Furthermore, the extracted portion is the wavenumber 1038 cm, which is unique to HEPES and appears isolated without overlapping with other waveforms. -1 Extract the spectral intensity specific to HEPES in this context.

[0072] Furthermore, the memory unit 32 is 1120 cm -1 A glucose calibration curve showing the relationship between the spectral intensity and concentration of glucose at 1120 cm², and 1120 cm². -1 The HEPES calibration curve shows the relationship between the spectral intensity and concentration of HEPES at 1038 cm², and the 1038 cm² calibration curve shows the relationship between the spectral intensity and concentration of HEPES at 1038 cm². -1 A HEPES-specific calibration curve showing the relationship between HEPES spectral intensity and concentration is stored.

[0073] The memory unit 32 is 1038 cm -1 Instead of using the HEPES-specific calibration curve that shows the relationship between the spectral intensity and concentration of HEPES at a given wavenumber, one may store the calibration curves at other wavenumbers that show the concentration dependence of HEPES as described above.

[0074] The calculation unit 46 quantifies the concentration of HEPES using a HEPES-specific calibration curve based on the spectral intensity specific to HEPES, and then calculates the concentration of HEPES using a predetermined HEPES calibration curve at 1120 cm⁻¹. -1The spectral intensity component of HEPES at 1120 cm was calculated, and -1 From the spectral intensity of [location] and the spectral intensity of HEPES, 1120 cm⁻¹ -1 The spectral intensity of glucose is calculated, and the glucose concentration is quantified using a glucose calibration curve based on the spectral intensity of glucose.

[0075] [Example 1: Glucose Analysis] The following describes an example in which glucose concentration was quantified from the peaks of the Raman spectrum. However, the present invention is not limited to this example. In this example, a cell medium containing glucose was used. Table 5 shows the type and main components of the cell medium.

[0076] [Table 5]

[0077] <About Cell Culture Sample 1> As cell medium sample 1, StemFit AK02N manufactured by Ajinomoto Co., Inc., as shown in Table 5 above, was used. This StemFit AK02N was used to culture NPCs (Nephron progenitor cells) isolated from mouse fetuses, and cell medium sample 1 containing glucose, as in Example 1, was used as the cell medium sample on day 0 of culture. Cell medium sample 1 was irradiated with a single wavelength laser light of 785 nm, and the Raman spectrum of cell medium sample 1 was obtained. A Raman analyzer manufactured by TSI, model number ProRaman-L-785-B1S, was used. A blood gas analyzer (blood gas meter) manufactured by Radiometer, Inc., model ABL800 BASIC analyzer was used. For ammonium analysis, the Ammonia Assay Kit (AA0100) from Sigma-Aldrich was used.

[0078] <Regarding cell culture sample 2> Cell medium sample 2 was prepared using the cell medium sample 1 after 7 days of culture.

[0079] In cell culture sample 1, the wavenumber of the main spectrum of HEPES is 1038 cm⁻¹. -1 The spectral intensity at (the specific wavenumber of HEPES) was 650.0925. Therefore, from (Equation 3), 3.031x = 650.0925 - 363.51 x=(650.0925-363.51) / 3.031=94.55 Therefore, the concentration of HEPES was determined to be 94.55 mg / dl.

[0080] In cell culture sample 1, the wavenumber for glucose analysis was 1120 cm⁻¹. -1 The spectral intensity at a given wavenumber for glucose was 284.4756.

[0081] The glucose concentration is given by (Equation 1) and (Equation 2), x=(284.4756-(((0.08×94.55)+25.516)+20.211)) / 0.809=285.8mg / dl Therefore, the glucose concentration was found to be 285.8 mg / dl.

[0082] Figures 6-9 show the tracking performance of the blood gas meter when measuring glucose in several different cell culture media from different manufacturers listed in Table 5. Each figure shows the results of a total of four experiments conducted at different times. Figure 10 compares the time-dependent changes in glucose measured by Raman spectroscopy in different cell culture media from different manufacturers (REGM, StemFit, DMEM / F12, Essential6) with the results from the blood gas meter. Here, the blood gas meter measurement results are used as a comparison point for relative evaluation with the concentration detected by the Raman spectrometer and calculated; therefore, tracking performance indicates relative responsiveness. From the figures, it was found that measurements could be performed with good reproducibility even when the time of day was changed, and the indication tendency and responsiveness with the blood gas meter were also good.

[0083] The wavenumber at which the newly discovered glucose spectrum is detected remains unchanged regardless of the presence or absence of phenol red and HEPES.

[0084] Through diligent investigation by the inventors, it was discovered that the peak of HEPES, a pH buffering agent, was causing an augmentation of the peak of glucose, the target of measurement, at a certain wavenumber, and that the overlapping and influencing of these peaks was the cause.

[0085] The above configuration is based on the following findings discovered by the inventors: [1] Between the intensity and concentration of the glucose spectrum in the cell medium, there is a difference of 1120 cm⁻¹. -1 Linearity was found in the following case: that is, the 1120 cm³ of glucose in the cell culture medium -1 It was found that the intensity and concentration of the spectrum can be used to calculate the other. [2] Between the spectral intensity and concentration of HEPES in the cell medium, 1120 cm⁻¹ -1 and 1038cm -1 Linearity was found in the following case. That is, the 1120 cm³ of HEPES in cell culture medium -1 and 1038cm -1 It was found that the intensity and concentration of the spectrum can be used to calculate the other. [3] 1120 cm in cell culture medium -1 The intensity of the Raman spectrum at 1120 cm⁻¹ was found to be essentially the sum of the spectral intensities of glucose and HEPES. That is, in cell culture at 1120 cm⁻¹ -1 Subtracting the spectral intensity of HEPES from the spectral intensity of [location] gives 1120 cm⁻¹. -1 It was found that the spectral intensity of glucose could be practically calculated using this method.

[0086] Embodiment 2: Analysis of lactic acid [Calibration curve for lactic acid analysis] In this embodiment, the influencing component is glucose. In this case, when analyzing lactate contained in the cell culture medium using Raman spectroscopy, the bulking effect of glucose is taken into consideration. To quantify the concentration of lactate, the spectral intensity and concentration of lactate can be calculated by subtracting this influencing factor from the measured spectral intensity.

[0087] <Lactate Calibration Curve> Figure 11 shows 1084 cm. -1 This is a lactate calibration curve showing the relationship between the spectral intensity (y) and concentration (x (mg / dl)) of lactate in a given location.

[0088] 1084cm -1 The lactate calibration curve in (Equation 4) is as follows: Lactic acid:y=0.2626x+17.054(@1084cm -1 )(Formula 4)

[0089] If lactate is present in the cell culture medium, the predetermined wavenumber for lactate in the Raman spectrum is 1084 cm⁻¹. -1 The above 1084cm is set. -1 The lactate calibration curve is input from the input unit 30 and stored in the second storage area of ​​the memory unit 32.

[0090] <Glucose Calibration Curve> Figure 12 shows 1084 cm. -1 This is a glucose calibration curve showing the relationship between the spectral intensity (y) and concentration (x (mg / dl)) of glucose in a given location.

[0091] 1084cm -1 The predetermined calibration curve for glucose in (Equation 5) is as follows: Glucose: y = 0.2959x + 18.003 (@1084cm) -1 )(Formula 5)

[0092] The above 1084cm -1 The predetermined glucose calibration curve is input from the input unit 30 and stored in the second storage area of ​​the memory unit 32.

[0093] <Glucose-specific calibration curve> In the Raman spectrum of glucose, 1120 cm⁻¹ -1 The main spectrum is located at 1120 cm⁻¹, which includes this spectrum. -1 , 1058cm -1 , 904cm -1 A spectrum showing concentration dependence was detected.

[0094] 1120cm -1 The glucose-specific calibration curve in this case is the same as the one shown in Figure 3, so its explanation is omitted.

[0095] If glucose is present in the cell culture medium, the glucose-specific wavenumber in the Raman spectrum will be 1120 cm⁻¹. -1 The above 1120cm will be set. -1 The glucose-specific calibration curve is input from the input unit 30 and stored in the second storage area of ​​the memory unit 32.

[0096] Here, the acquisition unit 40 acquires a Raman spectrum showing the intensity distribution of Raman scattered light at each wavenumber, which is generated when a sample, which is a cell culture medium containing lactic acid, is irradiated with excitation light of a single wavelength.

[0097] Furthermore, the extraction unit 42 has a predetermined wavenumber for lactic acid, which is 1084 cm⁻¹. -1 The spectral intensity at 1084 cm², which includes the spectral intensity component of lactic acid derived from lactic acid and the spectral intensity component of glucose derived from glucose. -1 The spectral intensity at [location] is extracted. Furthermore, the extraction unit 42 extracts the 1120 cm⁻¹ wavenumber, which is specific to glucose and appears isolated without overlapping with other waveforms. -1 Extract the glucose-specific spectral intensity in the given region.

[0098] Furthermore, the memory unit 32 is 1084 cm -1 A lactate calibration curve showing the relationship between the spectral intensity and concentration of lactate at 1084 cm², and 1084 cm². -1A glucose specific calibration curve showing the relationship between the spectral intensity and concentration of glucose at 1120 cm -1 and a glucose specific calibration curve showing the relationship between the spectral intensity and concentration of glucose at -1 are stored.

[0099] Note that the storage unit 32 may store a calibration curve at another wavenumber showing the concentration dependency of glucose described above instead of the glucose specific calibration curve showing the relationship between the spectral intensity and concentration of glucose at 1084 cm -1 The calculation unit quantifies the concentration of glucose from the spectral intensity specific to glucose using the glucose specific calibration curve, and calculates the spectral intensity of glucose at 1084 cm from the concentration of glucose using the glucose specific calibration curve.

[0100] From the spectral intensity specific to glucose, the concentration of glucose is quantified using the glucose specific calibration curve, and from the concentration of glucose, the spectral intensity of glucose at 1084 cm is calculated using the glucose specific calibration curve. -1 From the spectral intensity at 1084 cm and the spectral intensity of glucose, the spectral intensity of lactic acid at 1084 cm is calculated, and from the spectral intensity of lactic acid, the concentration of lactic acid is quantified using the lactic acid calibration curve. -1 From the spectral intensity at 1084 cm and the spectral intensity of glucose, the spectral intensity of lactic acid at 1084 cm is calculated, and from the spectral intensity of lactic acid, the concentration of lactic acid is quantified using the lactic acid calibration curve. -1 From the spectral intensity at 1084 cm and the spectral intensity of glucose, the spectral intensity of lactic acid at 1084 cm is calculated, and from the spectral intensity of lactic acid, the concentration of lactic acid is quantified using the lactic acid calibration curve.

[0101] [Example 2: Analysis of Lactic Acid] Hereinafter, an example of quantifying the lactic acid concentration from the peak of the Raman spectrum will be described. However, the present invention is not limited to this example. Note that since the cell culture medium sample 1 is the same as the sample simultaneously analyzed with the analysis of glucose, the description thereof will be omitted.

[0102] Cell culture medium samples 1 and 2 containing lactic acid were irradiated with laser light of a single wavelength of 785 nm, and the Raman spectra of cell culture medium samples 1 and 2 were obtained. The analyzer used was ProRaman-L-785-B1S manufactured by TSI. The blood gas analyzer (blood gas meter) used was the ABL800 BASIC analyzer manufactured by Radiometer Co., Ltd. The ion chromatograph was a 7320 portable ion chromatograph device manufactured by Nikkiso Co., Ltd., and lactic acid was measured under predetermined measurement conditions.

[0103] <Lactic acid concentration of cell culture medium sample 1> From the results of the above glucose analysis examples, the glucose concentration of cell culture medium sample 1 was 285.8 mg / dl. Therefore, from (Equation 5), 0.2959×285.8 + 18.003 = 102.57122 Therefore, the spectral intensity of glucose at 1084 cm -1 was calculated to be 102.57122.

[0104] In cell culture medium sample 1, the spectral intensity at 1084 cm -1 (predetermined wave number of lactic acid) was 120.5383. Therefore, 120.5383 - 102.57122 = 17.96708 Therefore, the spectral intensity of lactic acid at 1084 cm -1 was calculated to be 17.96708.

[0105] The lactic acid concentration was obtained from (Equation 4): x = ((17.96708 - 17.054) / 0.2626 = 3.477 mg / dl Assuming the molar mass of lactic acid = 178 g / mol, 3.477 (mg / dl) = 3.477×10 / 178 (mM) = 0.2 (mM) Therefore, it was found that the lactic acid concentration of cell culture medium sample 1 was 0.2 mM.

[0106] <Lactic acid concentration of cell culture medium sample 2> The glucose concentration of cell culture medium sample 2 was 137 mg / dl. Therefore, from (Equation 5), 0.2959×137 + 18.003 = 58.5413 Therefore, the spectral intensity of glucose at 1084 cm -1 was calculated to be 58.5413.

[0107] In cell culture medium sample 2, at 1084 cm which is the wave number of the spectrum for lactic acid analysis -1The spectral intensity at a given wavenumber for lactic acid was 155.772. Therefore, 155.772 - 58.5413 = 97.2307 Therefore, 1084cm -1 The spectral intensity of lactic acid in this region was calculated to be 97.2307.

[0108] The lactic acid concentration is given by (Equation 4), x=(97.2307-17.054) / 0.2626=305.32mg / dl Assuming the molar mass of lactate is 178 g / mol, 305.32(mg / dl)=305.32×10 / 178(mM)=17.2(mM) Therefore, the lactate concentration in cell culture sample 2 was found to be 17.2 mM.

[0109] Figures 13-16 show the trackability of blood gas meters when measuring lactate using several different cell culture media from different manufacturers listed in Table 5. Each figure shows the results of four experiments conducted at different times. Figure 17 compares the time-dependent changes in lactate measured by Raman spectroscopy and ion chromatography for different cell culture media from different manufacturers (REGM, StemFit, DMEM / F12, Essential6). The figure shows that measurements could be performed with good reproducibility even when the time of day was changed, and the indication tendencies with blood gas meters and ion chromatography were also good.

[0110] Furthermore, it has been confirmed that the wavenumber for spectral detection of lactic acid, newly discovered by this invention, remains unchanged regardless of the presence or absence of phenol red and HEPES.

[0111] The above configuration is based on the following findings discovered by the inventors: [1] Between the spectral intensity and concentration of lactate in cell culture medium, 1084 cm⁻¹ -1 Linearity was found in this case. Specifically, the 1084 cm³ of lactate in cell culture medium. -1It was found that the intensity and concentration of a Raman spectrum can be used to calculate the other. [2] Between the spectral intensity and concentration of glucose in the cell medium, there is a difference of 1084 cm⁻¹. -1 and 1120cm -1 Linearity was found in the following case: specifically, the 1084 cm³ of glucose in the cell culture medium. -1 and 1120cm -1 It was found that the spectral intensity and concentration could be calculated from one of them. [3] 1084 cm in cell culture medium -1 The spectral intensity at 1084 cm⁻¹ was found to be essentially the sum of the spectral intensities of lactate and glucose. That is, 1084 cm⁻¹ in cell culture medium. -1 Subtracting the spectral intensity of glucose from the spectral intensity at 1084 cm⁻¹ gives 1084 cm⁻¹. -1 It was found that the spectral intensity of lactic acid could be practically calculated.

[0112] Embodiment 3: Analysis of ammonium [Calibration curve for ammonium analysis] In this embodiment, the component that affects the analysis is at least one of glucose, lactic acid, acetaldehyde, glutamine, HEPES, and phenol red. When analyzing ammonium (NH4) contained in the cell culture medium using Raman spectroscopy, the influence of the component that affects the analysis is taken into consideration. To quantify the concentration of ammonium, the spectral intensity and concentration of ammonium can be quantified by subtracting the influence of the component that affects the analysis from the measured spectral intensity.

[0113] <Ammonium Calibration Curve> Figure 18 shows 1436 cm. -1 This is a calibration curve showing the relationship between the spectral intensity (y) and concentration (x (mg / dl)) of ammonium in a given location.

[0114] 1436cm -1 The ammonium calibration curve (Equation 6) in this case is as follows: Ammonium: y = 0.0961x + 70.752 (@1436cm) -1 )(Formula 6)

[0115] If ammonium is present in the cell culture medium, the predetermined wavenumber for ammonium in the Raman spectrum is 1436 cm⁻¹. -1 The above 1436cm is set. -1 The ammonium calibration curve is input from the input unit 30 and stored in the second storage area of ​​the memory unit 32.

[0116] <Glucose Calibration Curve> Figure 19 shows 1436 cm. -1 This is a calibration curve showing the relationship between the spectral intensity (y) and concentration (x (mg / dl)) of glucose in a given location.

[0117] 1436cm -1 The predetermined calibration curve for glucose in (Equation 7) is as follows: Glucose: y = 0.1234x + 71.878 (@1436cm) -1 )(Formula 7)

[0118] The above 1436cm -1 The predetermined glucose calibration curve is input from the input unit 30 and stored in the second storage area of ​​the memory unit 32.

[0119] <Glucose-specific calibration curve> Regarding the glucose-specific calibration curve, the same glucose-specific calibration curve used for the analysis of lactate can be used, so its explanation will be omitted.

[0120] <Lactate Calibration Curve> Figure 20 shows 1436 cm. -1 This is a calibration curve showing the relationship between the spectral intensity (y) and concentration (x (mg / dl)) of lactate in [location].

[0121] 1436cm -1 The predetermined calibration curve for lactate in (Equation 8) is as follows: Lactic acid:y=0.2572x+73.409(@1436cm-1 )(Formula 8)

[0122] The above 1436cm -1 The predetermined calibration curve for lactate is input from the input unit 30 and stored in the second storage area of ​​the memory unit 32.

[0123] <Lactate-specific calibration curve> In the Raman spectrum of lactate, 852 cm⁻¹ -1 The main spectrum is located at 852 cm⁻¹, which includes this spectrum. -1 , 1038cm -1 , 1084cm -1 A spectrum showing concentration dependence was detected.

[0124] Figure 21 shows 852 cm -1 This is a calibration curve showing the relationship between the spectral intensity (y) and concentration (x (mg / dl)) of lactate in [location].

[0125] The main spectrum is 852 cm⁻¹. -1 The lactate-specific calibration curve in (Equation 9) is as follows: Lactic acid:y=0.7626x+5.12(@852cm -1 )(Formula 9)

[0126] If lactic acid is present in the cell culture medium, the specific wavenumber for lactic acid in the Raman spectrum will be 852 cm⁻¹. -1 The above 852cm will be set. -1 The lactic acid-specific calibration curve is input from the input unit 30 and stored in the second storage area of ​​the memory unit 32.

[0127] <Acetaldehyde Calibration Curve> Figure 22 shows 1436 cm. -1 This is a calibration curve showing the relationship between the spectral intensity (y) and concentration (x (mg / dl)) of acetaldehyde in a given location.

[0128] 1436cm -1 The prescribed calibration curve for acetaldehyde in (Equation 10) is as follows: Acetaldehyde: y = 0.4955x + 33.837 (@1436cm) -1 )(Equation 10)

[0129] The above 1436cm -1 The predetermined calibration curve for acetaldehyde is input from the input unit 30 and stored in the second storage area of ​​the memory unit 32.

[0130] <Acetaldehyde-specific calibration curve> In the Raman spectrum of acetaldehyde, at 928 cm⁻¹, -1 The main spectrum is located at 858 cm⁻¹, which includes this spectrum. -1 , 928cm -1 , 1436cm -1 A spectrum showing concentration dependence was detected.

[0131] Figure 23 shows 928 cm -1 This is a calibration curve showing the relationship between the spectral intensity (y) and concentration (x (mg / dl)) of acetaldehyde in a given location.

[0132] The main spectrum is 928 cm⁻¹. -1 The acetaldehyde-specific calibration curve (Equation 11) in this context is as follows: Acetaldehyde: y = 0.3071x + 21.035 (@928cm) -1 )(Equation 11)

[0133] If acetaldehyde is present in the cell culture medium, the wavenumber specific to acetaldehyde in the Raman spectrum will be 928 cm⁻¹. -1 The above 928cm will be set. -1 The acetaldehyde-specific calibration curve is input from the input unit 30 and stored in the second storage area of ​​the memory unit 32.

[0134] <Glutamine Calibration Curve> Figure 24 shows 1436 cm. -1 This is a calibration curve showing the relationship between the spectral intensity (y) and concentration (x (mg / dl)) of glutamine in [location].

[0135] 1436 cm -1 The glutamine calibration curve (Equation 12) at -1 is as follows: Glutamine: y = 0.0947x + 124.78 (@ 1436 cm -1 (Equation 12)

[0136] The above-mentioned glutamine calibration curve at 1436 cm -1 is input from the input unit 30 and stored in the second storage area of the storage unit 32.

[0137] <Glutamine-specific calibration curve> In the Raman spectrum of glutamine, there is a main spectrum at 1140 cm -1 , and spectra showing concentration dependence at 900 cm -1 including this, 1140 cm -1 , and 1436 cm -1 were detected.

[0138] Figure 25 is a calibration curve showing the relationship between the spectral intensity (y) of glutamine at 1140 cm -1 and the concentration.

[0139] The glutamine calibration curve (Equation 13) at 1140 cm -1 is as follows: Glutamine: y = 0.2839x + 23.074 (@ 1140 cm -1 (Equation 13)

[0140] When glutamine is contained in the cell culture medium, 1140 cm -1 is set as the wave number specific to glutamine in the Raman spectrum. The glutamine-specific calibration curve at the above-mentioned 1140 cm -1 s input from the input unit 30 and stored in the second storage area of the storage unit 32.

[0141] <HEPES calibration curve> Figure 26 is a calibration curve showing the relationship between the spectral intensity (y) of HEPES at 1436 cm -1 and the concentration (x (mg / dl)).

[0142] 1436 cm -1 The (Equation 14) of the HEPES calibration curve at 1436 cm is as follows: HEPES: y = 1.0737x + 118.78 (@ 1436 cm -1 (Equation 14)

[0143] The above 1436 cm -1 The HEPES calibration curve at 1436 cm is input from the input unit 30 and stored in the second storage area of the storage unit 32.

[0144] <HEPES-specific calibration curve> Regarding the HEPES-specific calibration curve, since the HEPES-specific calibration curve used in the analysis of glucose can be used, the description is omitted.

[0145] <Phenol red calibration curve> Figure 27 is a calibration curve showing the relationship between the spectral intensity (y) and the concentration (x (mg / dl)) of phenol red at 1436 cm -1 .

[0146] 1436 cm -1 The (Equation 15) of the phenol red calibration curve at 1436 cm is as follows: Phenol red: y = 5.9107x + 34.724 (@ 1436 cm -1 (Equation 15)

[0147] The above 1436 cm -1 The phenol red calibration curve at 1436 cm is input from the input unit 30 and stored in the second storage area of the storage unit 32.

[0148] <Phenol red-specific calibration curve> Figure 28 is a calibration curve showing the relationship between the spectral intensity (y) and the concentration (x (mg / dl)) of phenol red at 1162 cm -1 .

[0149] A1162 cm -1The phenol red specific calibration curve (Equation 16) in this case is as follows: Phenol Red: y = 10.304x + 33.441 (@1162cm) -1 )(Equation 16)

[0150] The above 1162cm -1 The phenol red-specific calibration curve is input from the input unit 30 and stored in the second storage area of ​​the memory unit 32.

[0151] In this embodiment, the acquisition unit 40 acquires a Raman spectrum showing the intensity distribution of Raman scattered light at each wavenumber, which is generated when a sample, which is a cell culture medium containing ammonium, is irradiated with excitation light of a single wavelength.

[0152] Furthermore, in this embodiment, the extraction unit 42 has a predetermined wavenumber for ammonium, which is 1436 cm⁻¹. -1 The spectral intensity at 1436 cm² includes at least one of the following: the spectral intensity component of ammonium derived from ammonium, the spectral intensity component of glucose derived from glucose, the spectral intensity component of lactic acid derived from lactic acid, the spectral intensity component of acetaldehyde derived from acetaldehyde, the spectral intensity component of glutamine derived from glutamine, the spectral intensity component of HEPES derived from HEPES, and the spectral intensity component of phenol red derived from phenol red. -1 The spectral intensity at this point, and the 1120 cm⁻¹ wavenumber, which is unique to glucose and appears isolated without overlapping with other waveforms. -1 The spectral intensity specific to glucose and the wavenumber specific to lactate, 852 cm⁻¹. -1 The spectral intensity characteristic of lactic acid, and the wavenumber characteristic of acetaldehyde, 928 cm⁻¹. -1 The spectral intensity specific to acetaldehyde and the wavenumber specific to glutamine, 1140 cm⁻¹. -1 The spectral intensity specific to glutamine in this region, and the wavenumber specific to HEPES, 1038 cm⁻¹. -1 The spectral intensity specific to HEPES, and the wavenumber specific to phenol red, 1162 cm⁻¹. -1Extract at least one of the spectral intensities specific to phenol red in the sample.

[0153] Furthermore, in this embodiment, the storage unit 32 is 1436 cm². -1 Ammonium calibration curve showing the relationship between spectral intensity and concentration of ammonium at 1436 cm⁻¹ -1 A glucose calibration curve showing the relationship between the spectral intensity and concentration of glucose at 1120 cm². -1 A glucose-specific calibration curve showing the relationship between the spectral intensity and concentration of glucose at 1436 cm⁻¹. -1 A predetermined calibration curve for lactate, showing the relationship between spectral intensity and concentration of lactate in 852 cm². -1 A lactic acid-specific calibration curve showing the relationship between the spectral intensity and concentration of lactic acid at 1436 cm⁻¹. -1 The acetaldehyde calibration curve, 928 cm², shows the relationship between the spectral intensity and concentration of acetaldehyde. -1 Acetaldehyde-specific calibration curve showing the relationship between spectral intensity and concentration of acetaldehyde at 1436 cm⁻¹ -1 A predefined calibration curve for glutamine, showing the relationship between spectral intensity and concentration, at 1140 cm⁻¹. -1 A glutamine-specific calibration curve showing the relationship between the spectral intensity and concentration of glutamine in 1436 cm⁻¹. -1 HEPES calibration curve showing the relationship between spectral intensity and concentration of HEPES at 1038 cm⁻¹ -1 A HEPES-specific calibration curve showing the relationship between HEPES spectral intensity and concentration at 1436 cm⁻¹. -1 A phenol red calibration curve showing the relationship between spectral intensity and concentration of phenol red at 1162 cm⁻¹. -1 Store at least one of the phenol red-specific calibration curves that show the relationship between the spectral intensity and concentration of phenol red in a given location.

[0154] Furthermore, in this embodiment, the calculation unit 46 calculates at least one of the following: the glucose concentration using a glucose-specific calibration curve based on the spectral intensity specific to glucose; the lactic acid concentration using a lactic acid-specific calibration curve based on the spectral intensity specific to lactic acid; the acetaldehyde concentration using an acetaldehyde-specific calibration curve based on the spectral intensity specific to acetaldehyde; the glutamine concentration using a glutamine-specific calibration curve based on the spectral intensity specific to glutamine; the HEPES concentration using a HEPES-specific calibration curve based on the spectral intensity specific to HEPES; and the phenol red concentration using a phenol red-specific calibration curve based on the spectral intensity specific to phenol red. From the glucose concentration, it calculates 1436 cm⁻¹ using a predetermined glucose calibration curve. -1 The spectral intensity of glucose and the concentration of lactate were used to determine the lactate calibration curve at 1436 cm⁻¹. -1 The spectral intensity of lactic acid and the concentration of acetaldehyde were used to determine the acetaldehyde calibration curve at 1436 cm⁻¹. -1 The spectral intensity of acetaldehyde and the concentration of glutamine were used to determine the glutamine concentration at 1436 cm⁻¹. -1 The spectral intensity of glutamine and the concentration of HEPES were used to determine the HEPES calibration curve at 1436 cm⁻¹. -1 Using the spectral intensity of HEPES and the concentration of phenol red, a predetermined calibration curve for phenol red was used at 1436 cm⁻¹. -1 Calculate at least one spectral intensity component of phenol red at 1436 cm⁻¹. -1 From the spectral intensity at and at least one of the spectral intensity components of glucose, lactate, acetaldehyde, glutamine, HEPES, and phenol red, 1436 cm⁻¹ -1 The spectral intensity of ammonium is calculated, and the concentration of ammonium is quantified from the spectral intensity using an ammonium calibration curve.

[0155] [Example 3: Analysis of Ammonium] The following describes an example in which the concentration of ammonium was quantified from the peaks of the Raman spectrum. However, the present invention is not limited to this experimental example. Note that cell culture medium sample 1 is the same as the sample analyzed simultaneously with glucose analysis, so its description is omitted.

[0156] A Raman spectrum was obtained from cell culture sample 1 containing ammonium, which was irradiated with a single-wavelength laser beam of 785 nm. The analyzer used was a TSI ProRaman-L-785-B1S. The ammonium was analyzed using the Sigma-Aldrich Ammonia Assay Kit (AA0100). Ion chromatography was performed using a Nikkiso 7320 portable ion chromatograph, and ammonium was measured under specified measurement conditions.

[0157] <Ammonium concentration in cell culture sample 1> From the results of the glucose analysis example described above, the glucose concentration of cell culture medium sample 1 was 285.8 mg / dl. Therefore, from (Equation 8), 1436cm -1 The spectral intensity of glucose at this point = 0.1234 × 285.8 + 71.878 = 107.14572

[0158] In the cell culture medium, lactic acid is not present in the cell culture medium before the cells are cultured. Cell culture medium sample 1 is a sample with 0 days of culture, and since lactic acid is a metabolite, the lactic acid concentration in cell culture medium sample 1 was set to 0 mg / dl. Therefore, from (Equation 9), 1436cm -1 The spectral intensity of lactate in this region = 0.2572 × 0 + 73.409 = 73.409

[0159] In cell culture media, acetaldehyde is not present in the cell culture medium before the cells are cultured. Cell culture medium sample 1 is a sample with 0 days of culture, and since acetaldehyde is a metabolite, the acetaldehyde concentration in cell culture medium sample 1 was set to 0 mg / dl. Therefore, from (Equation 11), 1436cm-1 The spectral intensity of acetaldehyde at this point = 0.4955 × 0 + 33.837 = 33.837

[0160] The glutamine concentration in cell culture sample 1 was 29.1 mg / dl. Therefore, from (Equation 12), 1436cm -1 The spectral intensity of glutamine in this region = 0.0947 × 29.1 + 124.78 = 127.527

[0161] The HEPES concentration in cell culture sample 1 was 94.55 mg / dl. Therefore, from (Equation 14), 1436cm -1 The spectral intensity of HEPES at this location = 1.0737 × 94.55 + 118.78 = 220.298355

[0162] The phenol red concentration in cell culture sample 1 was 10 mg / dl. Therefore, from (Equation 15), 1436cm -1 The spectral intensity of phenol red in this case = 5.9107 × 10 + 34.724 = 93.831

[0163] In cell culture sample 1, the wavenumber for ammonium analysis was 1436 cm⁻¹. -1 The spectral intensity at [location] was 688.2881. Therefore, the ammonium concentration, with ammonium molar mass = 18.03 g / mol, can be calculated from (Equation 7): x=(688.2881-107.145-73.409-33.837-127.527-220.298355-93.831-70.752) / (0.0961×10×18.03)=-2.2mM Therefore, the ammonium concentration in cell culture sample 1 was found to be -2.2 mM.

[0164] <Ammonium concentration in cell culture sample 2> The glucose concentration in cell culture medium sample 2 was 137 mg / dl. Therefore, from (Equation 8), 1436cm-1 Spectral intensity of glucose at -1 = 0.1234×137 + 71.878 = 88.7838

[0165] The lactic acid concentration of cell culture medium sample 2 was 29.7 mg / dl. Therefore, from (Equation 9), 1436 cm -1 Spectral intensity of lactic acid at -1 = 0.2572×29.7 + 73.409 = 81.04784

[0166] The acetaldehyde concentration of cell culture medium sample 2 was 24.5 mg / dl. Therefore, from (Equation 11), 1436 cm -1 Spectral intensity of acetaldehyde at -1 = 0.4955×24.5 + 33.837 = 45.97675

[0167] The glutamine concentration of cell culture medium sample 2 was 17.6 mg / dl. Therefore, from (Equation 12), 1436 cm -1 Spectral intensity of glutamine at -1 = 0.0947×17.6 + 124.78 = 126.44672

[0168] The HEPES concentration of cell culture medium sample 2 was 94.55 mg / dl. Therefore, from (Equation 14), 1436 cm -1 Spectral intensity of HEPES at -1 = 1.0737×94.55 + 118.78 = 220.298

[0169] The phenol red concentration of cell culture medium sample 2 was 10 mg / dl. Therefore, from (Equation 15), 1436 cm -1 Spectral intensity of phenol red at -1 = 5.9107×10 + 34.724 = 93.831

[0170] At 1436 cm, which is the wavenumber of the spectrum for ammonium analysis in cell culture medium sample 2 -1The spectral intensity at [location] was 829.5327. Therefore, the ammonium concentration, with ammonium molar mass = 18.03 g / mol, can be calculated from (Equation 7): x=(829.5327-88.7838-81.04784-45.97675-126.44672-220.298-93.831-70.752) / (0.0961×10×18.03)=5.9mM Therefore, the ammonium concentration in cell culture sample 2 was found to be 5.9 mM.

[0171] Figures 29-32 show the trackability of ammonium assays when measured in several different cell media from different manufacturers listed in Table 5. Each figure shows the results of four experiments conducted at different times. Figure 33 compares the time-dependent changes in ammonium measured by Raman spectroscopy and ion chromatography for different cell media from different manufacturers (REGM, StemFit, DMEM / F12, Essential6). The figure shows that measurements were reproducible with good accuracy even when the time of day was changed, and the indication tendencies with ion chromatography were also good.

[0172] Experiments based on the present invention have shown that the ammonium peak is detected in an augmented form compared to other peaks (glucose, lactic acid, acetaldehyde, glutamine, HEPES, and phenol red).

[0173] The above configuration is based on the following findings discovered by the inventors: [1] Between the intensity and concentration of the Raman spectrum of ammonium in cell culture medium, there is a difference of 1436 cm⁻¹. -1 Linearity was found in the following case: 1436 cm -1 It was found that the intensity and concentration of the Raman spectrum of ammonium in a given location can be used to calculate the other. [2] Between the intensity and concentration of the Raman spectrum of glucose in cell culture medium, there is a difference of 1436 cm⁻¹. -1 and 1120cm -1Linearity was found in the following case: specifically, the 1436 cm³ of glucose in the cell culture medium. -1 and 1120cm -1 It was found that the intensity and concentration of a Raman spectrum can be used to calculate the other. [3] Between the intensity and concentration of the Raman spectrum of lactate in cell culture medium, 1436 cm⁻¹ -1 and 852cm -1 Linearity was found in the following case: 1436 cm³ of lactate in cell culture medium. -1 and 852cm -1 It was found that the intensity and concentration of a Raman spectrum can be used to calculate the other. [4] Between the intensity and concentration of the Raman spectrum of acetaldehyde in cell culture medium, there is a difference of 1436 cm⁻¹. -1 and 928cm -1 Linearity was found in the following case: acetaldehyde in cell culture medium at 1436 cm³. -1 and 928cm -1 It was found that the intensity and concentration of a Raman spectrum can be used to calculate the other. [5] Between the intensity and concentration of the Raman spectrum of glutamine in cell culture medium, there is a difference of 1436 cm⁻¹. -1 and 1140cm -1 Linearity was found in the following case: that is, glutamine in cell culture medium at 1436 cm⁻¹. -1 and 1140cm -1 It was found that the intensity and concentration of a Raman spectrum can be used to calculate the other. [6] Between the intensity and concentration of the Raman spectrum of HEPES in cell culture medium, there is a difference of 1436 cm⁻¹. -1 and 1038cm -1 Linearity was found in the following case: HEPES in cell culture medium at 1436 cm². -1 and 1038cm -1 It was found that the intensity and concentration of a Raman spectrum can be used to calculate the other. [7] Between the intensity and concentration of the Raman spectrum of phenol red in cell culture medium, there is a difference of 1436 cm⁻¹. -1 and 1162cm-1 Linearity was found in the following case: 1436 cm³ of phenol red in cell culture medium. -1 and 1162cm -1 It was found that the intensity and concentration of a Raman spectrum can be used to calculate the other.

[0174] A first embodiment of the present invention is: A method for quantifying glucose concentration from the Raman spectrum of cell culture medium, The cell medium contains glucose and components that affect glucose analysis, and the Raman spectrum of the cell medium is obtained. The spectral intensity at a predetermined wavenumber for the glucose includes the spectral intensity component of glucose derived from the glucose and the spectral intensity component of the component that affects the analysis of glucose derived from the component that affects the analysis of glucose, and the spectral intensity specific to the component that affects the analysis of glucose at a wavenumber specific to the component that affects the analysis of glucose, which is different from the predetermined wavenumber and appears isolated without overlapping with other waveforms. Using a component-specific calibration curve that shows the relationship between the spectral intensity and concentration of the component affecting the analysis of glucose at the aforementioned specific wavenumber, the concentration of the component affecting the analysis of glucose in the cell culture medium is quantified from the component-specific spectral intensity affecting the analysis of glucose at the aforementioned specific wavenumber. Using a predetermined calibration curve for components affecting glucose analysis that shows the relationship between the spectral intensity and concentration of the components affecting glucose analysis at the predetermined wavenumber, the spectral intensity portion of the components affecting glucose analysis at the predetermined wavenumber is calculated from the quantified concentration of the components affecting glucose analysis. From the spectral intensity at the predetermined wavenumber and the spectral intensity of the component that affects the analysis of glucose, the spectral intensity of glucose at the predetermined wavenumber is calculated. This includes quantifying the concentration of glucose in the cell medium from the calculated spectral intensity of glucose at a predetermined wavenumber, using a glucose calibration curve that shows the relationship between the spectral intensity and concentration of glucose at a predetermined wavenumber. The predetermined wave frequency for the glucose is 1120 cm⁻¹. -1 That is the method.

[0175] This allows for the quantitative determination of glucose concentration in cell culture media from the obtained Raman spectral data. Furthermore, the glucose concentration in cell culture media can be determined quickly from the obtained Raman spectral data. Since no reagents are used during measurement, consumables are not generated, leading to reduced running costs. Solutions containing multiple matrix concentrations can be measured directly without pretreatment, and quantitative analysis of glucose in cell culture media can be performed from the detected spectrum. In Raman spectroscopy, unlike liquid chromatography, the target substance is generally not separated; instead, qualitative analysis and identification are performed by comparing the molecular structure and functional groups of the substance with a database of standard substance spectra. This allows for the quantitative determination of glucose concentration in cell culture media by subtracting the influence of other substances present.

[0176] A second embodiment of the present invention is, in the first embodiment, the component affecting the analysis of glucose is HEPES, and the wavenumber specific to HEPES is 10³⁸ cm⁻¹. -1 That is the case.

[0177] Therefore, if the HEPES component is present in the cell culture medium, the Raman spectrum will show 10³⁸ cm⁻¹. -1 Experiments have confirmed the appearance of a specific peak and its reproducibility. This allows for the confirmation of the presence or absence of HEPES components in the cell culture medium and for the accurate quantification of HEPES in the cell culture medium.

[0178] A third embodiment of the present invention is: In the first or second embodiment described above, a method for quantifying the concentration of lactic acid in addition to the concentration of glucose, wherein the method for quantifying the concentration of lactic acid is: The cell medium contains lactic acid and components that affect the analysis of lactic acid, and the Raman spectrum of the cell medium is obtained. The spectral intensity at a predetermined wavenumber for lactic acid, including the spectral intensity component of lactic acid derived from the lactic acid and the spectral intensity component of the component that affects the analysis of lactic acid derived from the component that affects the analysis of lactic acid, and the spectral intensity specific to the component that affects the analysis of lactic acid at a wavenumber specific to the component that affects the analysis of lactic acid, which is different from the predetermined wavenumber for lactic acid and appears isolated without overlapping with other waveforms, are extracted. Using a calibration curve specific to the components affecting the analysis of lactic acid, which shows the relationship between the spectral intensity and concentration of the components affecting the analysis of lactic acid at wavenumbers specific to those components, the concentration of the components affecting the analysis of lactic acid is quantified from the spectral intensity specific to those components. Using a predetermined calibration curve for components affecting the analysis of lactic acid, which shows the relationship between the spectral intensity and concentration of components affecting the analysis of lactic acid at a predetermined wavenumber for lactic acid, the spectral intensity portion of the components affecting the analysis of lactic acid at a predetermined wavenumber for lactic acid is calculated from the concentration of the components affecting the analysis of lactic acid. From the spectral intensity of lactic acid at a predetermined wavenumber and the spectral intensity of components that affect the analysis of lactic acid, the spectral intensity of lactic acid at a predetermined wavenumber is calculated. This includes quantifying the concentration of lactic acid from the spectral intensity of lactic acid using a lactic acid calibration curve that shows the relationship between the spectral intensity and concentration of lactic acid at a predetermined wavenumber for the lactic acid, The predetermined wave frequency for lactic acid is 1084 cm². -1 That is the method.

[0179] This allows for the quantitative determination of the lactic acid concentration in the cell medium from the obtained Raman spectral data. Furthermore, the lactic acid concentration in the cell medium can be determined quickly from the obtained Raman spectral data. Since no reagents are used during measurement, no consumables are generated, leading to reduced running costs. Solutions containing multiple concentrations of matrix can be measured directly without pretreatment, and quantitative analysis of lactic acid in the cell medium can be performed from the detected spectrum. In Raman spectroscopy, unlike liquid chromatography, the target substance is generally not separated; instead, qualitative analysis and identification are performed by comparing the molecular structure and functional groups of the substance with a database of standard substance spectra. This allows for the quantitative determination of the lactic acid concentration in the cell medium by subtracting the influence of other substances present.

[0180] A fourth embodiment of the present invention is, in the third embodiment, the component that affects the analysis of lactic acid is glucose, and the characteristic wavenumber of glucose is 1120 cm⁻¹. -1 That is the case.

[0181] Therefore, if glucose components are present in the cell culture medium, the Raman spectrum at 1120 cm⁻¹ will be... -1 Experiments have confirmed the appearance of a specific peak and its reproducibility. This allows for the confirmation of the presence or absence of glucose components in the cell culture medium and for accurate quantification of glucose in the cell culture medium.

[0182] A fifth embodiment of the present invention is: In any one of the embodiments described above (1 to 3), a method for determining glucose concentration, or a method for determining glucose concentration and lactate concentration, in addition to determining ammonium concentration, wherein the method for determining ammonium concentration is: The cell medium contains ammonium and components that affect the analysis of ammonium, and the Raman spectrum of the cell medium is obtained. The spectral intensity for the ammonium at a predetermined wavenumber is extracted, including the spectral intensity component of ammonium derived from the ammonium and the spectral intensity component of the component that affects the analysis of ammonium derived from the component that affects the analysis of ammonium, and the spectral intensity specific to the component that affects the analysis of ammonium at a wavenumber specific to the component that affects the analysis of ammonium, which is different from the predetermined wavenumber and appears isolated without overlapping with other waveforms. Using a component-specific calibration curve that shows the relationship between the spectral intensity and concentration of the component affecting the analysis of ammonium at a component-specific wavenumber, the concentration of the component affecting the analysis of ammonium is quantified from the component-specific spectral intensity affecting the analysis of ammonium. Using a predetermined calibration curve for components affecting the analysis of ammonium, which shows the relationship between the spectral intensity and concentration of components affecting the analysis of ammonium at a predetermined wavenumber for the ammonium, the spectral intensity of the components affecting the analysis of ammonium at a predetermined wavenumber for the ammonium is calculated from the concentration of the components affecting the analysis of ammonium. From the spectral intensity of the ammonium at a predetermined wavenumber and the spectral intensity of components that affect the analysis of the ammonium, the spectral intensity component of the ammonium at the predetermined wavenumber is calculated. This includes quantifying the concentration of the ammonium from the spectral intensity of the ammonium using an ammonium calibration curve that shows the relationship between the spectral intensity and concentration of the ammonium at a predetermined wavenumber for the ammonium. The predetermined wave frequency for the ammonium is 1436 cm⁻¹ -1 That is the method.

[0183] This allows for the quantitative determination of the ammonium component concentration in the cell medium from the obtained Raman spectral data. Furthermore, the ammonium concentration in the cell medium can be determined quickly from the obtained Raman spectral data. Since no reagents are used during measurement, no consumables are generated, leading to reduced running costs. Solutions containing multiple concentrations of matrix can be measured directly without pretreatment, and quantitative analysis of ammonium in the cell medium can be performed from the detected spectrum. In Raman spectroscopy, unlike liquid chromatography, the target substance is generally not separated; instead, qualitative analysis and identification are performed by comparing the molecular structure and functional groups of the substance with a database of standard substance spectra. This allows for the quantitative determination of the ammonium concentration in the cell medium by subtracting the influence of other substances present.

[0184] A sixth embodiment of the present invention is, in the fifth embodiment, wherein the component affecting the analysis of the ammonium is glucose, and the characteristic wavenumber of the glucose is 1120 cm⁻¹. -1 That is the case.

[0185] Therefore, if glucose components are present in the cell culture medium, the Raman spectrum at 1120 cm⁻¹ will be... -1 Experiments have confirmed the appearance of a specific peak and its reproducibility. This allows for the confirmation of the presence or absence of glucose components in the cell culture medium and for accurate quantification of glucose in the cell culture medium.

[0186] A seventh embodiment of the present invention is, in the fifth or sixth embodiment, a component that affects the analysis of ammonium is lactic acid, and the characteristic wavenumber of lactic acid is 852 cm⁻¹. -1 That is the case.

[0187] Therefore, if lactic acid components are present in the cell culture medium, the Raman spectrum will show 852 cm⁻¹. -1Experiments have confirmed the appearance of a specific peak and its reproducibility. This allows for the confirmation of the presence or absence of lactic acid components in the cell culture medium and for accurate quantification of lactic acid in the cell culture medium.

[0188] An eighth embodiment of the present invention is, in the fifth to seventh embodiments, wherein the component affecting the analysis of ammonium is acetaldehyde, and the characteristic wavenumber of acetaldehyde is 928 cm⁻¹. -1 That is the case.

[0189] Therefore, if acetaldehyde is present in the cell culture medium, the Raman spectrum at 928 cm⁻¹ will be affected. -1 Experiments have confirmed the appearance of a specific peak and its reproducibility. This allows for the confirmation of the presence or absence of acetaldehyde in the cell culture medium and for accurate quantification of acetaldehyde in the cell culture medium.

[0190] A ninth embodiment of the present invention is, in the fifth to eighth embodiments, wherein the component affecting the analysis of the ammonium is glutamine, and the characteristic wavenumber of the glutamine is 1140 cm⁻¹. -1 That is the case.

[0191] Therefore, if glutamine is present in the cell culture medium, the Raman spectrum at 1140 cm⁻¹ will be... -1 Experiments have confirmed the appearance of a specific peak and its reproducibility. This allows for the confirmation of the presence or absence of glutamine components in the cell culture medium and for the accurate quantification of glutamine in the cell culture medium.

[0192] A tenth embodiment of the present invention is, in any one of the fifth to ninth embodiments, the component affecting the analysis of the ammonium is HEPES, wherein the characteristic wavenumber of the HEPES is 10³⁸ cm⁻¹. -1 That is the case.

[0193] Therefore, if the HEPES component is present in the cell culture medium, the Raman spectrum will show 10³⁸ cm⁻¹. -1Experiments have confirmed the appearance of a specific peak and its reproducibility. This allows for the confirmation of the presence or absence of HEPES components in the cell culture medium and for the accurate quantification of HEPES in the cell culture medium.

[0194] An eleventh embodiment of the present invention is, in any one of the fifth to ten embodiments, wherein the component affecting the analysis of the ammonium is phenol red, and the characteristic wavenumber of the phenol red is 1162 cm⁻¹. -1 That is the case.

[0195] Therefore, if the phenol red component is present in the cell culture medium, the Raman spectrum will show 1162 cm⁻¹. -1 Experiments have confirmed the appearance of a specific peak and its reproducibility. This allows for the confirmation of the presence or absence of phenol red components in the cell culture medium and for accurate quantification of phenol red in the cell culture medium.

[0196] A twelfth embodiment of the present invention is to perform the determination of glucose concentration and lactic acid concentration in a single analytical instrument, in accordance with the third or fourth embodiment described above.

[0197] This allows for the simultaneous analysis and detection of two components, glucose and lactate, and their differentiation and quantitative determination of concentrations. It overcomes the conventional challenge of processing spectra in typical Raman spectroscopy, where the obtained spectral intensity must be compared with a database to perform qualitative analysis of substances or calculate the concentration of each component individually. When analyzing the components of cell culture media containing high-concentration matrix, simultaneous analysis enables the detection and concentration calculation of both glucose and lactate. Furthermore, since glucose and lactate concentrations can be determined in a single measurement, quantitative analysis of glucose and lactate concentrations can be performed with a single analyzer without the need for ion chromatography or blood gas meters, thus eliminating consumables and reducing running costs.

[0198] A thirteenth embodiment of the present invention is to perform the determination of ammonium concentration in addition to the determination of glucose concentration or glucose concentration and lactate concentration in any one of the fifth to eleventh embodiments described above, using a single analytical instrument.

[0199] This allows for the simultaneous analysis and detection of glucose and ammonium, or glucose, lactate, and ammonium, enabling differentiation and quantitative determination of their concentrations. It overcomes the conventional challenge of processing spectra in typical Raman spectroscopy, where obtained spectral intensities must be compared with a database to qualitatively identify substances or calculate the concentration of each component individually. When analyzing the components of cell culture media containing high-concentration matrix, simultaneous analysis enables the detection and concentration calculation of glucose and ammonium, or glucose, lactate, and ammonium. Furthermore, since glucose, lactate, and ammonium concentrations can be determined in a single measurement, it eliminates the need for liquid chromatography, ion chromatography, or ammonia assay kits, thus reducing consumables and allowing for quick determination of glucose, lactate, and ammonium concentrations with a single analyzer, leading to expected cost reductions. [Explanation of Symbols]

[0200] 10 Analytical mechanism 12 light source 14 Reflectors 16 samples 18 Optical system 20 Monochromemeter 22 Diffraction gratings 24 CCD detectors 26 Analyzer 30 Input section 32 Storage section 34 Control Unit 36 Display section 40 Acquisition Department 42 Extraction part 44 Component identification unit 46 Calculation Section 48 Notification Department

Claims

1. A method for quantifying glucose concentration from the Raman spectrum of cell culture medium, The cell medium contains glucose and components that affect glucose analysis, and the Raman spectrum of the cell medium is obtained. The spectral intensity at a predetermined wavenumber for the glucose includes the spectral intensity component of glucose derived from the glucose and the spectral intensity component of the component that affects the analysis of glucose derived from the component that affects the analysis of glucose, and the spectral intensity specific to the component that affects the analysis of glucose at a wavenumber specific to the component that affects the analysis of glucose, which is different from the predetermined wavenumber and appears isolated without overlapping with other waveforms. Using a component-specific calibration curve that shows the relationship between the spectral intensity and concentration of the component affecting the analysis of glucose at the aforementioned specific wavenumber, the concentration of the component affecting the analysis of glucose in the cell culture medium is quantified from the component-specific spectral intensity affecting the analysis of glucose at the aforementioned specific wavenumber. Using a predetermined calibration curve for components affecting glucose analysis that shows the relationship between the spectral intensity and concentration of the components affecting glucose analysis at the predetermined wavenumber, the spectral intensity portion of the components affecting glucose analysis at the predetermined wavenumber is calculated from the quantified concentration of the components affecting glucose analysis. From the spectral intensity at the predetermined wavenumber and the spectral intensity of the component that affects the analysis of glucose, the spectral intensity of glucose at the predetermined wavenumber is calculated. This includes quantifying the concentration of glucose in the cell medium from the calculated spectral intensity of glucose at a predetermined wavenumber, using a glucose calibration curve that shows the relationship between the spectral intensity and concentration of glucose at a predetermined wavenumber. The predetermined wavenumber for glucose is 1120 ± 6 cm. -1 And, The component that affects the analysis of glucose is HEPES, and the wavenumber specific to HEPES is 1038 ± 6 cm⁻¹. -1 The method.

2. The method according to claim 1, wherein in addition to determining the glucose concentration, the concentration of lactic acid is also determined, and the method for determining the concentration of lactic acid is: The cell medium contains lactic acid and components that affect the analysis of lactic acid, and the Raman spectrum of the cell medium is obtained. The spectral intensity at a predetermined wavenumber for lactic acid, including the spectral intensity component of lactic acid derived from the lactic acid and the spectral intensity component of the component that affects the analysis of lactic acid derived from the component that affects the analysis of lactic acid, and the spectral intensity specific to the component that affects the analysis of lactic acid at a wavenumber specific to the component that affects the analysis of lactic acid, which is different from the predetermined wavenumber for lactic acid and appears isolated without overlapping with other waveforms, are extracted. Using a calibration curve specific to the components affecting the analysis of lactic acid, which shows the relationship between the spectral intensity and concentration of the components affecting the analysis of lactic acid at wavenumbers specific to those components, the concentration of the components affecting the analysis of lactic acid is quantified from the spectral intensity specific to those components. Using a predetermined calibration curve for components affecting the analysis of lactic acid, which shows the relationship between the spectral intensity and concentration of components affecting the analysis of lactic acid at a predetermined wavenumber for lactic acid, the spectral intensity portion of the components affecting the analysis of lactic acid at a predetermined wavenumber for lactic acid is calculated from the concentration of the components affecting the analysis of lactic acid. From the spectral intensity of lactic acid at a predetermined wavenumber and the spectral intensity of components that affect the analysis of lactic acid, the spectral intensity of lactic acid at a predetermined wavenumber is calculated. This includes quantifying the concentration of lactic acid from the spectral intensity of lactic acid using a lactic acid calibration curve that shows the relationship between the spectral intensity and concentration of lactic acid at a predetermined wavenumber for the lactic acid, The predetermined wavenumber for lactic acid is 1084 ± 6 cm -1 The method.

3. The component that affects the analysis of lactic acid is glucose, and the characteristic wavenumber of glucose is 1120 ± 6 cm⁻¹. -1 The method according to claim 2.

4. A method according to any one of claims 1 to 3, wherein a method for determining glucose concentration, or a method for determining glucose concentration and lactate concentration, in addition to determining ammonium concentration, wherein the method for determining ammonium concentration is: The cell medium contains ammonium and components that affect the analysis of ammonium, and the Raman spectrum of the cell medium is obtained. The spectral intensity for the ammonium at a predetermined wavenumber is extracted, including the spectral intensity component of ammonium derived from the ammonium and the spectral intensity component of the component that affects the analysis of ammonium derived from the component that affects the analysis of ammonium, and the spectral intensity specific to the component that affects the analysis of ammonium at a wavenumber specific to the component that affects the analysis of ammonium, which is different from the predetermined wavenumber and appears isolated without overlapping with other waveforms. Using a component-specific calibration curve that shows the relationship between the spectral intensity and concentration of the component affecting the analysis of ammonium at a component-specific wavenumber, the concentration of the component affecting the analysis of ammonium is quantified from the component-specific spectral intensity affecting the analysis of ammonium. Using a predetermined calibration curve for components affecting the analysis of ammonium, which shows the relationship between the spectral intensity and concentration of components affecting the analysis of ammonium at a predetermined wavenumber for the ammonium, the spectral intensity of the components affecting the analysis of ammonium at a predetermined wavenumber for the ammonium is calculated from the concentration of the components affecting the analysis of ammonium. From the spectral intensity of the ammonium at a predetermined wavenumber and the spectral intensity of components that affect the analysis of the ammonium, the spectral intensity component of the ammonium at the predetermined wavenumber is calculated. This includes quantifying the concentration of the ammonium from the spectral intensity of the ammonium using an ammonium calibration curve that shows the relationship between the spectral intensity and concentration of the ammonium at a predetermined wavenumber for the ammonium. The predetermined wavenumber for the ammonium is 1436 ± 6 cm -1 The method.

5. The component affecting the analysis of the ammonium is glucose, and the characteristic wavenumber of the glucose is 1120 ± 6 cm². -1 The method according to claim 4.

6. The component affecting the analysis of the ammonium is lactic acid, and the characteristic wavenumber of the lactic acid is 852 ± 6 cm. -1 The method according to claim 4 or 5.

7. A method for quantifying glucose concentration from the Raman spectrum of cell culture medium, The cell medium contains glucose and components that affect glucose analysis, and the Raman spectrum of the cell medium is obtained. The spectral intensity at a predetermined wavenumber for the glucose includes the spectral intensity component of glucose derived from the glucose and the spectral intensity component of the component that affects the analysis of glucose derived from the component that affects the analysis of glucose, and the spectral intensity specific to the component that affects the analysis of glucose at a wavenumber specific to the component that affects the analysis of glucose, which is different from the predetermined wavenumber and appears isolated without overlapping with other waveforms. Using a component-specific calibration curve that shows the relationship between the spectral intensity and concentration of the component affecting the analysis of glucose at the aforementioned specific wavenumber, the concentration of the component affecting the analysis of glucose in the cell culture medium is quantified from the component-specific spectral intensity affecting the analysis of glucose at the aforementioned specific wavenumber. Using a predetermined calibration curve for components affecting glucose analysis that shows the relationship between the spectral intensity and concentration of the components affecting glucose analysis at the predetermined wavenumber, the spectral intensity portion of the components affecting glucose analysis at the predetermined wavenumber is calculated from the quantified concentration of the components affecting glucose analysis. From the spectral intensity at the predetermined wavenumber and the spectral intensity of the component that affects the analysis of glucose, the spectral intensity of glucose at the predetermined wavenumber is calculated. This includes quantifying the concentration of glucose in the cell medium from the calculated spectral intensity of glucose at a predetermined wavenumber, using a glucose calibration curve that shows the relationship between the spectral intensity and concentration of glucose at a predetermined wavenumber. The component that affects the analysis of glucose is HEPES, and the predetermined wavenumber is 1120 ± 6 cm. -1 In this method, A method for determining the concentration of glucose, in addition to determining the concentration of ammonium, wherein the method for determining the concentration of ammonium is: The cell medium contains ammonium and components that affect the analysis of ammonium, and the Raman spectrum of the cell medium is obtained. The spectral intensity for the ammonium at a predetermined wavenumber is extracted, including the spectral intensity component of ammonium derived from the ammonium and the spectral intensity component of the component that affects the analysis of ammonium derived from the component that affects the analysis of ammonium, and the spectral intensity specific to the component that affects the analysis of ammonium at a wavenumber specific to the component that affects the analysis of ammonium, which is different from the predetermined wavenumber and appears isolated without overlapping with other waveforms. Using a component-specific calibration curve that shows the relationship between the spectral intensity and concentration of the component affecting the analysis of ammonium at a component-specific wavenumber, the concentration of the component affecting the analysis of ammonium is quantified from the component-specific spectral intensity affecting the analysis of ammonium. Using a predetermined calibration curve for components affecting the analysis of ammonium, which shows the relationship between the spectral intensity and concentration of components affecting the analysis of ammonium at a predetermined wavenumber for the ammonium, the spectral intensity of the components affecting the analysis of ammonium at a predetermined wavenumber for the ammonium is calculated from the concentration of the components affecting the analysis of ammonium. From the spectral intensity of the ammonium at a predetermined wavenumber and the spectral intensity of components that affect the analysis of the ammonium, the spectral intensity component of the ammonium at the predetermined wavenumber is calculated. This includes quantifying the concentration of the ammonium from the spectral intensity of the ammonium using an ammonium calibration curve that shows the relationship between the spectral intensity and concentration of the ammonium at a predetermined wavenumber for the ammonium. The predetermined wave number for the ammonium is 1436 ± 6 cm -1 and The component affecting the analysis of the ammonium is acetaldehyde, and the characteristic wavenumber of the acetaldehyde is 928 ± 6 cm⁻¹. -1 The method.

8. A method for quantifying glucose concentration from the Raman spectrum of cell culture medium, The cell medium contains glucose and components that affect glucose analysis, and the Raman spectrum of the cell medium is obtained. The spectral intensity at a predetermined wavenumber for the glucose includes the spectral intensity component of glucose derived from the glucose and the spectral intensity component of the component that affects the analysis of glucose derived from the component that affects the analysis of glucose, and the spectral intensity specific to the component that affects the analysis of glucose at a wavenumber specific to the component that affects the analysis of glucose, which is different from the predetermined wavenumber and appears isolated without overlapping with other waveforms. Using a component-specific calibration curve that shows the relationship between the spectral intensity and concentration of the component affecting the analysis of glucose at the aforementioned specific wavenumber, the concentration of the component affecting the analysis of glucose in the cell culture medium is quantified from the component-specific spectral intensity affecting the analysis of glucose at the aforementioned specific wavenumber. Using a predetermined calibration curve for components affecting glucose analysis that shows the relationship between the spectral intensity and concentration of the components affecting glucose analysis at the predetermined wavenumber, the spectral intensity portion of the components affecting glucose analysis at the predetermined wavenumber is calculated from the quantified concentration of the components affecting glucose analysis. From the spectral intensity at the predetermined wavenumber and the spectral intensity of the component that affects the analysis of glucose, the spectral intensity of glucose at the predetermined wavenumber is calculated. This includes quantifying the concentration of glucose in the cell medium from the calculated spectral intensity of glucose at a predetermined wavenumber, using a glucose calibration curve that shows the relationship between the spectral intensity and concentration of glucose at a predetermined wavenumber. The component that affects the analysis of glucose is HEPES, and the predetermined wavenumber is 1120 ± 6 cm. -1 In this method, A method for determining the concentration of glucose, in addition to determining the concentration of ammonium, wherein the method for determining the concentration of ammonium is: The cell medium contains ammonium and components that affect the analysis of ammonium, and the Raman spectrum of the cell medium is obtained. The spectral intensity for the ammonium at a predetermined wavenumber is extracted, including the spectral intensity component of ammonium derived from the ammonium and the spectral intensity component of the component that affects the analysis of ammonium derived from the component that affects the analysis of ammonium, and the spectral intensity specific to the component that affects the analysis of ammonium at a wavenumber specific to the component that affects the analysis of ammonium, which is different from the predetermined wavenumber and appears isolated without overlapping with other waveforms. Using a component-specific calibration curve that shows the relationship between the spectral intensity and concentration of the component affecting the analysis of ammonium at a component-specific wavenumber, the concentration of the component affecting the analysis of ammonium is quantified from the component-specific spectral intensity affecting the analysis of ammonium. Using a predetermined calibration curve for components affecting the analysis of ammonium, which shows the relationship between the spectral intensity and concentration of components affecting the analysis of ammonium at a predetermined wavenumber for the ammonium, the spectral intensity of the components affecting the analysis of ammonium at a predetermined wavenumber for the ammonium is calculated from the concentration of the components affecting the analysis of ammonium. From the spectral intensity of the ammonium at a predetermined wavenumber and the spectral intensity of components that affect the analysis of the ammonium, the spectral intensity component of the ammonium at the predetermined wavenumber is calculated. This includes quantifying the concentration of the ammonium from the spectral intensity of the ammonium using an ammonium calibration curve that shows the relationship between the spectral intensity and concentration of the ammonium at a predetermined wavenumber for the ammonium. The predetermined wavenumber for the ammonium is 1436 ± 6 cm -1 And, The component affecting the analysis of the ammonium is glutamine, and the characteristic wavenumber of the glutamine is 1140 ± 6 cm². -1 The method.

9. A method for quantifying glucose concentration from the Raman spectrum of cell culture medium, The cell medium contains glucose and components that affect glucose analysis, and the Raman spectrum of the cell medium is obtained. The spectral intensity at a predetermined wavenumber for the glucose includes the spectral intensity component of glucose derived from the glucose and the spectral intensity component of the component that affects the analysis of glucose derived from the component that affects the analysis of glucose, and the spectral intensity specific to the component that affects the analysis of glucose at a wavenumber specific to the component that affects the analysis of glucose, which is different from the predetermined wavenumber and appears isolated without overlapping with other waveforms. Using a component-specific calibration curve that shows the relationship between the spectral intensity and concentration of the component affecting the analysis of glucose at the aforementioned specific wavenumber, the concentration of the component affecting the analysis of glucose in the cell culture medium is quantified from the component-specific spectral intensity affecting the analysis of glucose at the aforementioned specific wavenumber. Using a predetermined calibration curve for components affecting glucose analysis that shows the relationship between the spectral intensity and concentration of the components affecting glucose analysis at the predetermined wavenumber, the spectral intensity portion of the components affecting glucose analysis at the predetermined wavenumber is calculated from the quantified concentration of the components affecting glucose analysis. From the spectral intensity at the predetermined wavenumber and the spectral intensity of the component that affects the analysis of glucose, the spectral intensity of glucose at the predetermined wavenumber is calculated. This includes quantifying the concentration of glucose in the cell medium from the calculated spectral intensity of glucose at a predetermined wavenumber, using a glucose calibration curve that shows the relationship between the spectral intensity and concentration of glucose at a predetermined wavenumber. The component that affects the analysis of glucose is HEPES, and the predetermined wavenumber is 1120 ± 6 cm. -1 In this method, A method for determining the concentration of glucose, in addition to determining the concentration of ammonium, wherein the method for determining the concentration of ammonium is: The cell medium contains ammonium and components that affect the analysis of ammonium, and the Raman spectrum of the cell medium is obtained. The spectral intensity for the ammonium at a predetermined wavenumber is extracted, including the spectral intensity component of ammonium derived from the ammonium and the spectral intensity component of the component that affects the analysis of ammonium derived from the component that affects the analysis of ammonium, and the spectral intensity specific to the component that affects the analysis of ammonium at a wavenumber specific to the component that affects the analysis of ammonium, which is different from the predetermined wavenumber and appears isolated without overlapping with other waveforms. Using a component-specific calibration curve that shows the relationship between the spectral intensity and concentration of the component affecting the analysis of ammonium at a component-specific wavenumber, the concentration of the component affecting the analysis of ammonium is quantified from the component-specific spectral intensity affecting the analysis of ammonium. Using a predetermined calibration curve for components affecting the analysis of ammonium, which shows the relationship between the spectral intensity and concentration of components affecting the analysis of ammonium at a predetermined wavenumber for the ammonium, the spectral intensity of the components affecting the analysis of ammonium at a predetermined wavenumber for the ammonium is calculated from the concentration of the components affecting the analysis of ammonium. From the spectral intensity of the ammonium at a predetermined wavenumber and the spectral intensity of components that affect the analysis of the ammonium, the spectral intensity component of the ammonium at the predetermined wavenumber is calculated. This includes quantifying the concentration of the ammonium from the spectral intensity of the ammonium using an ammonium calibration curve that shows the relationship between the spectral intensity and concentration of the ammonium at a predetermined wavenumber for the ammonium. The predetermined wavenumber for the ammonium is 1436 ± 6 cm -1 And, The component affecting the analysis of the ammonium includes HEPES, and the characteristic wavenumber of HEPES is 1038 ± 6 cm⁻¹. -1 The method.

10. A method for quantifying glucose concentration from the Raman spectrum of cell culture medium, The cell medium contains glucose and components that affect glucose analysis, and the Raman spectrum of the cell medium is obtained. The spectral intensity at a predetermined wavenumber for the glucose includes the spectral intensity component of glucose derived from the glucose and the spectral intensity component of the component that affects the analysis of glucose derived from the component that affects the analysis of glucose, and the spectral intensity specific to the component that affects the analysis of glucose at a wavenumber specific to the component that affects the analysis of glucose, which is different from the predetermined wavenumber and appears isolated without overlapping with other waveforms. Using a component-specific calibration curve that shows the relationship between the spectral intensity and concentration of the component affecting the analysis of glucose at the aforementioned specific wavenumber, the concentration of the component affecting the analysis of glucose in the cell culture medium is quantified from the component-specific spectral intensity affecting the analysis of glucose at the aforementioned specific wavenumber. Using a predetermined calibration curve for components affecting glucose analysis that shows the relationship between the spectral intensity and concentration of the components affecting glucose analysis at the predetermined wavenumber, the spectral intensity portion of the components affecting glucose analysis at the predetermined wavenumber is calculated from the quantified concentration of the components affecting glucose analysis. From the spectral intensity at the predetermined wavenumber and the spectral intensity of the component that affects the analysis of glucose, the spectral intensity of glucose at the predetermined wavenumber is calculated. This includes quantifying the concentration of glucose in the cell medium from the calculated spectral intensity of glucose at a predetermined wavenumber, using a glucose calibration curve that shows the relationship between the spectral intensity and concentration of glucose at a predetermined wavenumber. The component that affects the analysis of glucose is HEPES, and the predetermined wavenumber is 1120 ± 6 cm. -1 In this method, A method for determining the concentration of glucose, in addition to determining the concentration of ammonium, wherein the method for determining the concentration of ammonium is: The cell medium contains ammonium and components that affect the analysis of ammonium, and the Raman spectrum of the cell medium is obtained. The spectral intensity for the ammonium at a predetermined wavenumber is extracted, including the spectral intensity component of ammonium derived from the ammonium and the spectral intensity component of the component that affects the analysis of ammonium derived from the component that affects the analysis of ammonium, and the spectral intensity specific to the component that affects the analysis of ammonium at a wavenumber specific to the component that affects the analysis of ammonium, which is different from the predetermined wavenumber and appears isolated without overlapping with other waveforms. Using a component-specific calibration curve that shows the relationship between the spectral intensity and concentration of the component affecting the analysis of ammonium at a component-specific wavenumber, the concentration of the component affecting the analysis of ammonium is quantified from the component-specific spectral intensity affecting the analysis of ammonium. Using a predetermined calibration curve for components affecting the analysis of ammonium, which shows the relationship between the spectral intensity and concentration of components affecting the analysis of ammonium at a predetermined wavenumber for the ammonium, the spectral intensity of the components affecting the analysis of ammonium at a predetermined wavenumber for the ammonium is calculated from the concentration of the components affecting the analysis of ammonium. From the spectral intensity of the ammonium at a predetermined wavenumber and the spectral intensity of components that affect the analysis of the ammonium, the spectral intensity component of the ammonium at the predetermined wavenumber is calculated. This includes quantifying the concentration of the ammonium from the spectral intensity of the ammonium using an ammonium calibration curve that shows the relationship between the spectral intensity and concentration of the ammonium at a predetermined wavenumber for the ammonium. The predetermined wavenumber for the ammonium is 1436 ± 6 cm -1 And, The component that affects the analysis of the ammonium is phenol red, and the characteristic wavenumber of the phenol red is 1162 ± 6 cm⁻¹. -1 The method.

11. The method according to claim 2 or 3, wherein the determination of glucose concentration and the determination of lactate concentration are performed in a single analytical instrument.

12. The method according to any one of claims 4 to 6, wherein the determination of glucose concentration or the determination of glucose concentration and lactate concentration, in addition to the determination of ammonium concentration, is performed in a single analytical instrument.

13. An analytical apparatus for performing the method according to any one of claims 1 to 12, comprising a control unit for controlling the analytical apparatus, wherein the control unit comprises an acquisition unit for acquiring the Raman spectrum, an extraction unit for extracting the spectral intensity, a component identification unit for searching for and identifying components of the cell culture medium, a calculation unit for quantifying the concentration of the components, and a notification unit for notifying the type and concentration of the components.

Images