Fluorescence analysis method, fluorescence analysis device, and operation program for fluorescence analysis device

Abstract

A fluorescence analysis method for irradiating, with excitation light, a suspension in which a target substance is dispersed and analyzing measured intensity of fluorescence, the method comprising: acquiring a first intensity of fluorescence having a reference emission wavelength corresponding to the target substance and a second intensity of fluorescence having the reference emission wavelength or having an emission wavelength different from the reference emission wavelength, as measured by irradiating the suspension with excitation light having a reference excitation wavelength corresponding to the target substance; calculating representative values of the first intensity and the second intensity; and estimating the concentration of the target substance in the suspension on the basis of the representative values.

Description

Fluorescence analysis method, fluorescence analysis device, and operation program for fluorescence analysis device

[0001] The technology of the present disclosure relates to a fluorescence analysis method, a fluorescence analysis device, and an operating program for the fluorescence analysis device.

[0002] For example, as described in Japanese Patent Laid-Open No. 2022-142057, a technique is known in which a suspension containing a dispersed target substance is irradiated with excitation light, the intensity of the fluorescence generated by the irradiation is measured, and the concentration of the target substance is estimated from the fluorescence intensity. One method of measuring fluorescence is to use a plate reader or the like to irradiate the target substance with excitation light of a reference excitation wavelength corresponding to the target substance and measure only the intensity of the fluorescence of the reference emission wavelength corresponding to the target substance.

[0003] Here, particularly when the suspension contains only a small amount of the target substance, i.e., when the concentration of the target substance is relatively low, the influence of noise can reduce the S / N ratio of the fluorescence intensity, resulting in a decrease in the accuracy of estimating the concentration of the target substance. For this reason, conventional methods have involved irradiating the sample with excitation light of a reference excitation wavelength and measuring the fluorescence intensity of a reference emission wavelength multiple times, and then using the arithmetic mean of the multiple intensities obtained to estimate the concentration. However, this conventional method has the problem of requiring a very long measurement time.

[0004] One embodiment of the technique of the present disclosure provides a fluorescence analysis method, a fluorescence analysis device, and an operating program for the fluorescence analysis device that can shorten the time required to measure the fluorescence intensity of a target substance in a suspension.

[0005] The fluorescence analysis method disclosed herein is a fluorescence analysis method that analyzes the intensity of fluorescence measured by irradiating excitation light onto a suspension in which a target substance is dispersed, and includes acquiring a first intensity of fluorescence at a reference emission wavelength corresponding to the target substance and a second intensity of fluorescence at the reference emission wavelength or an emission wavelength different from the reference emission wavelength, measured by irradiating at least excitation light at a reference excitation wavelength corresponding to the target substance, calculating representative values ​​of the first intensity and the second intensity, and estimating the concentration of the target substance in the suspension based on the representative values.

[0006] Preferably, there are multiple second intensities.

[0007] It is preferable that the first intensity and the second intensity are obtained by irradiating the suspension with excitation light in an excitation wavelength range consisting of a plurality of excitation wavelengths including a reference excitation wavelength, and measuring the intensity of fluorescence in an emission wavelength range consisting of a plurality of emission wavelengths including a reference emission wavelength for each of the plurality of excitation wavelengths.

[0008] Preferably, the plurality of excitation wavelengths are spaced apart by a first wavelength interval, and the plurality of emission wavelengths are spaced apart by a second wavelength interval.

[0009] The representative value is preferably a weighted average value of the first intensity and the second intensity.

[0010] The weighting coefficients used to calculate the weighted average value are preferably derived using a function.

[0011] Preferably, the function is a Gaussian function.

[0012] The suspension is a liquid obtained during the manufacturing process of a biopharmaceutical, and the target substance is preferably an impurity that is unnecessary for the biopharmaceutical.

[0013] The fluorescence analyzing device disclosed herein is a fluorescence analyzing device that analyzes the intensity of fluorescence measured by irradiating excitation light onto a suspension in which a target substance is dispersed, and includes a processor. The processor acquires a first intensity of fluorescence at a reference emission wavelength corresponding to the target substance and a second intensity of fluorescence at the reference emission wavelength or an emission wavelength different from the reference emission wavelength, measured by irradiating the suspension with at least excitation light at a reference excitation wavelength corresponding to the target substance, calculates representative values ​​of the first intensity and the second intensity, and estimates the concentration of the target substance in the suspension based on the representative values.

[0014] The operating program for a fluorescence analyzer disclosed herein is an operating program for a fluorescence analyzer that analyzes the intensity of fluorescence measured by irradiating excitation light onto a suspension in which a target substance is dispersed, and includes acquiring a first intensity of fluorescence at a reference emission wavelength corresponding to the target substance and a second intensity of fluorescence at the reference emission wavelength or an emission wavelength different from the reference emission wavelength, measured by irradiating at least excitation light at a reference excitation wavelength corresponding to the target substance, calculating representative values ​​of the first intensity and the second intensity, and estimating the concentration of the target substance in the suspension based on the representative values.

[0015] According to the technology of the present disclosure, it is possible to provide a fluorescence analysis method, a fluorescence analysis device, and an operating program for a fluorescence analysis device that can shorten the time required to measure the fluorescence intensity of a target substance in a suspension.

[0016] 1 is a diagram showing a fluorescence analysis system; 2 is a diagram showing the internal configuration of a fluorescence measuring device; 3 is a diagram showing fluorescence intensity data; 4 is a block diagram showing a computer constituting a fluorescence analyzer; 5 is a block diagram showing a processing unit of a CPU of a fluorescence analyzer; 6 is a diagram showing weighting coefficient data; 7 is a diagram showing calibration curve data; 8 is a diagram showing processing by a calculation unit; 9 is a diagram showing processing by an estimation unit; 10 is a diagram showing a target substance selection screen; 11 is a diagram showing an estimation result display screen; 12 is a flowchart showing processing procedures of a fluorescence analyzer; 13 is a graph showing the relationship between concentration and intensity in Comparative Example 1; 14 is a graph showing the relationship between actual concentration and estimated concentration in Comparative Example 2; 15 is a graph showing the contribution of intensity to concentration estimation in Comparative Example 2; 16 is a graph showing the relationship between concentration and intensity in Example 1; 17 is a graph showing the relationship between concentration and intensity in Example 2;

[0017] 1, a fluorescence analysis system 10 includes a fluorescence measurement device 12 and a fluorescence analysis device 13. The fluorescence measurement device 12 and the fluorescence analysis device 13 are connected via a network such as a LAN (Local Area Network).

[0018] The fluorescence measuring device 12 measures the intensity IN (see FIG. 3, etc.) of autofluorescence emitted from impurities 16 in the purified solution 15 or fluorescence emitted from a fluorescent reagent added to the impurities 16. The purified solution 15 is obtained during the manufacturing process of a biopharmaceutical 17. The purified solution 15 is an example of a "suspension" according to the technology of the present disclosure. The impurities 16 are an example of a "target substance" according to the technology of the present disclosure. Note that, hereinafter, the autofluorescence emitted from the impurities 16 and the fluorescence emitted from the fluorescent reagent are collectively referred to as fluorescence FL (see FIG. 2).

[0019] The biopharmaceutical 17 is produced by culturing host cells 19 in a culture medium 18. The host cells 19 are, for example, Chinese hamster ovary cells into which an antibody gene has been introduced. The culture medium 18 contains the host cells 19, an antibody 20 produced by the host cells 19, impurities 16, and the like. The process of producing the biopharmaceutical 17 begins with removing the host cells 19 from the culture medium 18 to produce a culture supernatant 21. The culture supernatant 21 is then purified by passing it through various chromatography devices, leaving behind the antibody 20 that serves as the active ingredient of the biopharmaceutical 17 and removing the impurities 16 that are not necessary for the biopharmaceutical 17. The liquid obtained during this purification process is the purified liquid 15. In addition to the antibody 20, the purified liquid 15 contains trace amounts of impurities 16 that were not completely removed by the chromatography device. Here, DNA (deoxyribonucleic acid), a type of nucleic acid, is used as an example of the impurity 16. In this case, for example, Quant-iT (registered trademark) or PicoGreen (registered trademark) can be used as the fluorescent reagent. The impurities 16 may be vitamins, amino acids, peptides, proteins, cellular metabolites, etc. In particular, examples of amino acids include tryptophan and phenylalanine, which are essential amino acids, and tyrosine, which is produced from phenylalanine and is a raw material for thyroxine, adrenaline, melanin, etc.

[0020] The purified liquid 15 is caused to flow at a set flow rate through a dedicated measuring device such as a flow cell. The fluorescence measuring device 12 measures the intensity IN of the fluorescence FL of the impurities 16 in the purified liquid 15 flowing through the measuring device. The fluorescence measuring device 12 transmits fluorescence intensity data 22, which is the measurement result of the intensity IN of the fluorescence FL, to the fluorescence analyzing device 13 via the network.

[0021] The fluorescence analyzer 13 is, for example, a desktop personal computer. The fluorescence analyzer 13 is operated by an operator OP who is involved in practical work at a manufacturing facility for the biopharmaceutical 17. The fluorescence analyzer 13 estimates the concentration EC (see FIG. 9 ) of the impurities 16 in the purified solution 15 based on the fluorescence intensity data 22. The fluorescence analyzer 13 makes the estimated concentration EC available for viewing by the operator OP. The fluorescence analyzer 13 may be a laptop personal computer or a tablet terminal.

[0022] As shown in FIG. 2 , the fluorescence measuring device 12 includes an excitation light source 30, a spectroscope 31, and a measuring device 32. The excitation light source 30 is, for example, a xenon lamp, and emits white light WL. The spectroscope 31 includes a prism or a diffraction grating and a slit. The spectroscope 31 converts the white light WL into monochromatic excitation light EL and irradiates the purified liquid 15 with the excitation light EL. The spectroscope 31 switches the excitation wavelength of the excitation light EL at set intervals. The set interval is, for example, a value in microseconds to milliseconds. The excitation wavelength range of the excitation light EL handled by the spectroscope 31 is 250 nm to 610 nm. The wavelength interval of the excitation light EL dispersed by the spectroscope 31 is 5 nm. This 5 nm is an example of a “first wavelength interval” according to the technology disclosed herein. The reference excitation wavelength of the excitation light EL corresponding to the impurities 16 is 500 nm. That is, the fluorescence measuring device 12 switches the excitation wavelength of the excitation light EL at set intervals using the spectrometer 31, thereby sequentially irradiating (scanning) the purified liquid 15 with excitation light EL in an excitation wavelength range consisting of multiple excitation wavelengths including the reference excitation wavelength.

[0023] In this way, the purified liquid 15 is irradiated with the excitation light EL, thereby generating fluorescence FL. The fluorescence FL is captured by the measuring instrument 32. The emission wavelength range of the fluorescence FL handled by the measuring instrument 32 is 250 nm to 620 nm. The wavelength interval of the fluorescence FL measured by the measuring instrument 32 is 5 nm. This 5 nm is an example of the "second wavelength interval" according to the technique of the present disclosure. The reference emission wavelength of the fluorescence FL corresponding to the impurities 16 is 535 nm.

[0024] The measuring instrument 32 has a fluorescence detecting element prepared for each of the multiple emission wavelengths that make up the emission wavelength range. The fluorescence detecting element is, for example, a CCD (Charge-Coupled Device) sensor covered with a filter for each emission wavelength. The measuring instrument 32 measures the intensity IN of the fluorescence FL of each emission wavelength and outputs the measurement results as fluorescence intensity data 22.

[0025] 3, the fluorescence intensity data 22 is a collection of intensities IN of fluorescence FL in an emission wavelength range consisting of multiple emission wavelengths, for each of excitation light EL with multiple excitation wavelengths, such as 250 nm, 255 nm, ..., 500 nm, ..., 605 nm, and 610 nm, i.e., a collection of fluorescence spectra FLS. Such fluorescence intensity data 22 is called an excitation-emission matrix (EEM). In other words, the fluorescence measuring device 12 is a device capable of measuring EEM.

[0026] The intensity IN (500, 535) at a reference emission wavelength of 535 nm of the fluorescence spectrum FLS at a reference excitation wavelength of 500 nm is important for estimating the concentration EC of the impurity 16 in the purified solution 15. This intensity IN (500, 535) is an example of a "first intensity" according to the technology of the present disclosure. The intensity IN at an emission wavelength other than the reference emission wavelength of 535 nm of the fluorescence spectrum FLS at a reference excitation wavelength of 500 nm, for example, the intensity IN (500, 540) at an emission wavelength of 540 nm (see FIG. 8 ), is an example of a "second intensity" according to the technology of the present disclosure. Furthermore, the intensity IN at an emission wavelength of 535 nm of the fluorescence spectrum FLS at an excitation wavelength other than the reference excitation wavelength of 500 nm, for example, the intensity IN (490, 535) at an excitation wavelength of 490 nm (see FIG. 8 ), is also an example of a "second intensity" according to the technology of the present disclosure. Furthermore, the intensity IN at an emission wavelength other than the reference emission wavelength of 535 nm of the fluorescence spectrum FLS at an excitation wavelength other than the reference excitation wavelength of 500 nm, for example, the intensity IN (490, 525) at an emission wavelength of 525 nm of the fluorescence spectrum FLS at an excitation wavelength of 490 nm (see FIG. 8 ), is also an example of the "second intensity" according to the technology of the present disclosure. In any case, all of the intensities IN other than the intensity IN (500, 535) are examples of the "second intensity" according to the technology of the present disclosure.

[0027] 4 , the computer constituting the fluorescence analyzer 13 includes a storage 35, a memory 36, a CPU (Central Processing Unit) 37, a communication unit 38, a display 39, and an input device 40. These components are interconnected via a bus line 41.

[0028] The storage 35 is a hard disk drive built into the computer constituting the fluorescence analyzer 13 or connected via a cable or network. Alternatively, the storage 35 is a disk array consisting of multiple hard disk drives. The storage 35 stores control programs such as an operating system, various application programs (hereinafter referred to as APs (Application Programs)), and various data associated with these programs. Note that a solid state drive may be used instead of a hard disk drive.

[0029] The memory 36 is a work memory for the CPU 37 to execute processing. The CPU 37 loads programs stored in the storage 35 into the memory 36 and executes processing in accordance with the programs. In this way, the CPU 37 comprehensively controls each part of the computer. The CPU 37 is an example of a "processor" according to the technology of the present disclosure. The memory 36 may be built into the CPU 37.

[0030] The communication unit 38 is a network interface that controls the transmission of various information via a network or the like. The display 39 displays various screens. Each screen is provided with an operation function using a GUI (Graphical User Interface). The computer that constitutes the fluorescence analyzer 13 accepts input of operation instructions from an input device 40 via each screen. The input device 40 is a keyboard, mouse, touch panel, microphone for voice input, etc.

[0031] 5, an operating program 45 is stored in the storage 35 of the fluorescence analyzer 13. The operating program 45 is an AP for causing a computer to function as the fluorescence analyzer 13. In other words, the operating program 45 is an example of an "operating program for a fluorescence analyzer" according to the technology of the present disclosure. The storage 35 also stores weighting coefficient data 46, calibration curve data 47, and the like.

[0032] When the operating program 45 is started, the CPU 37 of the computer constituting the fluorescence analyzer 13 works in cooperation with the memory 36 and the like to function as an instruction receiving unit 50, a measurement control unit 51, an acquisition unit 52, a read / write (hereinafter abbreviated as RW (Read Write)) control unit 53, a calculation unit 54, an estimation unit 55, and a display control unit 56.

[0033] The instruction receiving unit 50 receives various instructions from the operator OP via the input device 40. The various instructions include an instruction to estimate the concentration EC of the impurities 16 in the purified liquid 15. When an estimation instruction is received, the instruction receiving unit 50 outputs a message to the measurement control unit 51 indicating that the estimation instruction has been received. The instruction receiving unit 50 also outputs the content of the estimation instruction to the calculation unit 54.

[0034] The measurement control unit 51 controls the operation of the fluorometer 12. Specifically, when the measurement control unit 51 receives an input from the instruction receiving unit 50 indicating that an estimation instruction has been received, the measurement control unit 51 transmits a measurement control signal 60 to the fluorometer 12. The measurement control signal 60 is a signal for causing the fluorometer 12 to measure the intensity IN of the fluorescence FL. The measurement control signal 60 includes measurement conditions for the intensity IN of the fluorescence FL. The measurement conditions include the set interval for switching the excitation light EL in the spectroscope 31, the excitation wavelength range of the excitation light EL handled by the spectroscope 31, and the emission wavelength range of the fluorescence FL handled by the measurement instrument 32. When the measurement control signal 60 is received, the fluorometer 12 operates the excitation light source 30, the spectroscope 31, and the measurement instrument 32 in accordance with the measurement conditions included in the measurement control signal 60, and measures the intensity IN of the fluorescence FL.

[0035] The acquisition unit 52 acquires fluorescence intensity data 22 from the fluorescence measuring device 12. As described above, the fluorescence intensity data 22 includes intensity IN (500, 535), which is the first intensity of fluorescence FL at a reference emission wavelength measured by irradiating excitation light EL at a reference excitation wavelength. The fluorescence intensity data 22 also includes intensity IN (500, 540), intensity IN (490, 535), intensity IN (490, 525), or the like, which is the second intensity of fluorescence FL at the reference emission wavelength or an emission wavelength different from the reference emission wavelength. Therefore, by acquiring the fluorescence intensity data 22, the acquisition unit 52 acquires the first intensity and the second intensity. The acquisition unit 52 outputs the fluorescence intensity data 22 to the RW control unit 53.

[0036] The RW control unit 53 controls the storage of various data in the storage 35 and the reading of various data from the storage 35. For example, the RW control unit 53 stores the fluorescence intensity data 22 from the acquisition unit 52 in the storage 35. The RW control unit 53 also reads the fluorescence intensity data 22 from the storage 35 and outputs the read fluorescence intensity data 22 to the calculation unit 54.

[0037] The RW control unit 53 reads the weighting coefficient data 46 from the storage 35 and outputs the read weighting coefficient data 46 to the calculation unit 54. The RW control unit 53 also reads the calibration curve data 47 from the storage 35 and outputs the read calibration curve data 47 to the estimation unit 55.

[0038] The calculation unit 54 calculates a weighted average value IN_WA (see FIG. 8 ) as a representative value of the intensity IN based on the fluorescence intensity data 22 and the weighting coefficient data 46. The calculation unit 54 outputs a calculation result 61 including the weighted average value IN_WA to the estimation unit 55.

[0039] The estimation unit 55 estimates the concentration EC of the impurity 16 in the purified solution 15 based on the calibration curve data 47 and the calculation result 61. The estimation unit 55 outputs an estimation result 62 including the estimated concentration EC to the display control unit 56.

[0040] The display control unit 56 controls the display of various screens on the display 39. The various screens include a target substance selection screen 70 (see FIG. 10 ) for selecting a target substance for which the concentration EC is to be estimated, an estimated concentration display screen 80 (see FIG. 11 ) for displaying the estimated concentration EC, and the like.

[0041] 6, the weighting coefficient data 46 is a set of 5 × 5 = 25 weighting coefficients derived using a Gaussian function GF shown in the following formula (1). That is, the weighting coefficient data 46 is a 5 × 5 Gaussian filter. In formula (1), x is the excitation wavelength of the excitation light EL, and y is the emission wavelength of the fluorescence FL. σ is a variable for determining the width of the Gaussian function GF, and here σ = 1.

[0042] The weighting coefficient for the central box is 36 / 256, for the boxes above, below, left, and right of the central box, 24 / 256, and for the boxes diagonally up, down, left, and right of the central box, 16 / 256. The weighting coefficient for the boxes above, below, left, and right one box away from the central box is 6 / 256, for the boxes on either side of those, 4 / 256, and for the boxes diagonally up, down, right, and left one box away from the central box, 1 / 256. In this way, the weighting coefficient for the central box is the largest, and the value gradually decreases as you move away from the central box. The sum of all these weighting coefficients is 1.

[0043] 7, the calibration curve data 47 is literally data on the calibration curve CL. Specifically, the calibration curve data 47 is a linear function that expresses the calibration curve CL using the intensity IN, the concentration EC, and a coefficient. The calibration curve CL is created by measuring the intensity IN of the fluorescence FL of multiple standard samples with known concentrations EC.

[0044] 8 , the calculation unit 54 applies the weighting coefficient data 46 to 5×5=25 intensities IN centered on the shaded intensity IN (500, 535) at the reference emission wavelength of 535 nm in the fluorescence spectrum FLS with a reference excitation wavelength of 500 nm, and calculates a weighted average value IN_WA of the intensities IN. In other words, the calculation unit 54 multiplies the 25 intensities IN centered on the intensity IN (500, 535) by the coefficients corresponding to the positional relationships of the 5×5 Gaussian filters in the weighting coefficient data 46, and then adds them together to calculate the weighted average value IN_WA of the intensities IN. The weighted average value IN_WA is an example of a "representative value" according to the technology of the present disclosure.

[0045] Specifically, the weighting coefficient data 46 is applied to the intensities at each combination of a reference excitation wavelength of 500 nm, 495 nm and 505 nm (which are ±5 nm from the reference excitation wavelength 500 nm), 490 nm and 510 nm (which are ±10 nm from the reference excitation wavelength 500 nm), a reference emission wavelength of 535 nm, 530 nm and 540 nm (which are ±5 nm from the reference emission wavelength 535 nm), and 525 nm and 545 nm (which are ±10 nm from the reference emission wavelength 535 nm). Note that, in this example, the weighting coefficient data 46 is applied only to 5 × 5 = 25 intensities IN centered on intensity IN (500, 535) to calculate the weighted average value IN_WA of the intensities IN, but this is not limited to this. The weighting coefficient data 46 may also be applied to intensities IN other than intensity IN (500, 535) to calculate the weighted average value IN_WA of the intensities IN in a similar manner. However, the concentration EC of the impurities 16 in the purified liquid 15 is estimated using the weighted average value IN_WA calculated by applying the weighting coefficient data 46 to 5 x 5 = 25 intensities IN centered around the intensity IN (500, 535).

[0046] As an example, as shown in FIG. 9, the estimation unit 55 derives the concentration EC by substituting the weighted average value IN_WA into a linear function that represents the calibration curve CL of the calibration curve data 47.

[0047] The display control unit 56 displays a target substance selection screen 70, as shown in FIG. 10 , on the display 39 in response to an operation instruction from the operator OP via the input device 40. The target substance selection screen 70 is provided with a pull-down menu 71 for alternatively selecting a target substance for which the concentration EC is to be estimated. The target substance selection screen 70 also has a measurement condition setting button 72 for setting the measurement conditions for the intensity IN of the fluorescence FL. When the measurement condition setting button 72 is selected, the display control unit 56 displays a measurement condition setting screen (not shown) on the display 39.

[0048] An estimate button 73 and an end button 74 are provided at the bottom of the target substance selection screen 70. When the estimate button 73 is selected, the instruction receiving unit 50 receives an estimate instruction. The measurement control unit 51 then transmits a measurement control signal 60 including the target substance and measurement conditions selected in the pull-down menu 71 at that time to the fluorometer 12. This causes the fluorometer 12 to measure the intensity IN of the fluorescence FL. On the other hand, when the end button 74 is selected, the display of the target substance selection screen 70 is cleared and the fluorescence analysis is terminated.

[0049] The reference excitation wavelength and reference emission wavelength vary depending on the target substance. For this reason, the operator OP selects the target substance on the target substance selection screen 70. Information on the reference excitation wavelength and reference emission wavelength corresponding to the target substance is stored in the storage 35. The calculation unit 54 reads out from the storage 35 the reference excitation wavelength and reference emission wavelength corresponding to the target substance selected on the target substance selection screen 70, and applies weighting coefficient data 46 to 5 × 5 = 25 intensities IN centered around the intensity IN of the fluorescence FL at the read reference excitation wavelength and reference emission wavelength to calculate a weighted average value IN_WA of the intensities IN.

[0050] When the estimation result 62 is input from the estimation unit 55, the display control unit 56 displays an estimated concentration display screen 80, as shown in Fig. 11 as an example, on the display 39. The concentration EC included in the estimation result 62 is displayed on the estimated concentration display screen 80. The estimated concentration display screen 80 also has a back button 81. When the back button 81 is selected, the display returns to the target substance selection screen 70, making it possible to select a target substance again.

[0051] The estimated concentration display screen 80 has an estimate button 73 and an end button 74 at the bottom, similar to the target substance selection screen 70. This allows the concentration EC to be repeatedly estimated.

[0052] Next, the operation of the above configuration will be described with reference to the flowchart shown in Fig. 12. When the operating program 45 is started in the fluorescence analyzer 13, the CPU 37 functions as an instruction receiving unit 50, a measurement control unit 51, an acquisition unit 52, a RW control unit 53, a calculation unit 54, an estimation unit 55, and a display control unit 56, as shown in Fig. 5.

[0053] Under the control of the display control unit 56, the display 39 displays the target substance selection screen 70 shown in Fig. 10. On the target substance selection screen 70, the operator OP operates a pull-down menu 71 to select a desired target substance, and in some cases selects a measurement condition setting button 72 to set the measurement conditions for the fluorescence FL intensity IN, and then selects an estimation button 73. When the estimation button 73 is selected, an estimation instruction is accepted by the instruction accepting unit 50 (YES in step ST100). Then, a measurement control signal 60 including the target substance and measurement conditions selected in the pull-down menu 71 at that time is transmitted from the measurement control unit 51 to the fluorescence measurement device 12 (step ST110).

[0054] In the fluorescence measuring device 12 that has received the measurement control signal 60, the excitation light source 30, the spectroscope 31, and the measuring device 32 are operated in accordance with the measurement conditions included in the measurement control signal 60, and the intensity IN of the fluorescence FL is measured. Fluorescence intensity data 22, which is the measurement result of the intensity IN, is transmitted from the fluorescence measuring device 12 to the fluorescence analyzing device 13.

[0055] In the fluorescence analyzer 13, the acquisition unit 52 acquires the fluorescence intensity data 22 from the fluorescence measurement device 12 (step ST120). The fluorescence intensity data 22 is output from the acquisition unit 52 to the RW control unit 53. Then, the fluorescence intensity data 22 is stored in the storage 35 under the control of the RW control unit 53 (step ST130).

[0056] The RW control unit 53 reads out the fluorescence intensity data 22 from the storage 35 (step ST140), and outputs the read fluorescence intensity data 22 to the calculation unit 54. In addition, the RW control unit 53 reads out the weighting coefficient data 46 from the storage 35, and outputs the read weighting coefficient data 46 to the calculation unit 54.

[0057] 8 , the calculation unit 54 applies the weighting coefficient data 46 to 5×5=25 intensities IN centered around the first intensity IN (500, 535), and calculates a weighted average value IN_WA of the intensities IN (step ST150). The calculation result 61 including the weighted average value IN_WA is output from the calculation unit 54 to the estimation unit 55.

[0058] The RW control unit 53 reads the calibration curve data 47 from the storage 35 and outputs the read calibration curve data 47 to the estimation unit 55. As shown in Fig. 9 , the estimation unit 55 estimates the concentration EC of the impurity 16 based on the weighted average value IN_WA and the calibration curve data 47 (step ST160). An estimation result 62 including the estimated concentration EC is output from the estimation unit 55 to the display control unit 56.

[0059] 11 is displayed on the display 39 under the control of the display control unit 56 (step ST170). The operator OP checks the concentration EC on the estimated concentration display screen 80, and determines whether the purification step has been completed or whether maintenance of the chromatography apparatus should be performed.

[0060] As described above, the CPU 37 of the fluorescence analyzer 13 includes an acquisition unit 52, a calculation unit 54, and an estimation unit 55. The acquisition unit 52 acquires the first intensity IN (500, 535) and the second intensity IN (500, 540). The intensity IN (500, 535) is the intensity of the fluorescence FL at a reference emission wavelength corresponding to the impurity 16, which is the target substance, measured by irradiating the impurity 16 with excitation light EL at a reference excitation wavelength corresponding to the impurity 16. The intensity IN (500, 540) and the like are the intensities of the fluorescence FL at the reference emission wavelength or an emission wavelength different from the reference emission wavelength, which are measured by irradiating the impurity 16 with at least excitation light EL at a reference excitation wavelength corresponding to the impurity 16. The calculation unit 54 calculates a weighted average value IN_WA, which is a representative value of these intensities IN. The estimation unit 55 estimates the concentration EC of the impurity 16 in the purified solution 15 based on the weighted average value IN_WA.

[0061] Conventionally, excitation light EL of a reference excitation wavelength was irradiated, and the intensity IN of fluorescence FL of a reference emission wavelength was measured multiple times, and the arithmetic mean value of the multiple intensities IN obtained thereby was used to estimate the concentration EC. Therefore, in order to measure multiple intensities IN, excitation light EL of the reference excitation wavelength had to be irradiated multiple times, which took a very long measurement time. In contrast, with the technology disclosed herein, in order to measure multiple intensities IN, it is only necessary to irradiate excitation light EL of a reference excitation wavelength corresponding to the impurity 16 at least once. Therefore, it is possible to shorten the measurement time of the intensity IN of fluorescence FL of the impurity 16 in the purified solution 15.

[0062] When the amount of impurities 16 contained in the purified solution 15 is small, the S / N ratio of the intensity IN of the fluorescence FL is reduced due to the influence of noise, which may reduce the accuracy of estimating the concentration EC of the impurities 16. However, in the technology disclosed herein, the concentration EC of the impurities 16 in the purified solution 15 is estimated based on the weighted average value IN_WA. Therefore, even when the amount of impurities 16 contained in the purified solution 15 is small, the concentration EC of the impurities 16 in the purified solution 15 can be estimated with high accuracy.

[0063] 3 and 5, there are multiple second intensities, so that the intensities IN for estimating the concentration EC of the impurities 16 in the purified liquid 15 can be measured more frequently in a short period of time.

[0064] As shown in Figures 2 and 3, the multiple intensities IN (first intensity and second intensity) are obtained by irradiating the purified liquid 15 with excitation light EL in an excitation wavelength range consisting of multiple excitation wavelengths including a reference excitation wavelength, and measuring the intensity IN of fluorescence FL in an emission wavelength range consisting of multiple emission wavelengths including a reference emission wavelength for each of the multiple excitation wavelengths.

[0065] For example, if the 25 intensities IN shown in Figure 8 were to be measured using conventional methods, the process of irradiating excitation light EL at each excitation wavelength and measuring the intensities IN of fluorescence FL at each emission wavelength would have to be repeated 25 times. In contrast, with the technology disclosed herein, it is sufficient to change the excitation wavelength and irradiate excitation light EL only five times. This makes it possible to measure more intensities IN in a shorter time.

[0066] 2, the multiple excitation wavelengths are spaced apart at 5 nm wavelength intervals, and the multiple emission wavelengths are also spaced apart at 5 nm wavelength intervals, so there is no bias in the density of the fluorescence intensity data 22, making it easy to handle the fluorescence intensity data 22.

[0067] As shown in FIG. 8 , the representative value is the weighted average IN_WA of multiple intensities IN (first intensity and second intensity). Considering the accuracy of estimating the concentration EC, it would be best to use the arithmetic average of multiple first intensities to estimate the concentration EC, as in conventional methods. However, this would result in a long measurement time, as described above. Therefore, in the technology disclosed herein, the first intensity and the second intensity, which have characteristics similar to the first intensity, are measured simultaneously or approximately simultaneously over a period of several milliseconds to several seconds. Then, to reflect the difference in importance of the first intensity and the second intensity in estimating the concentration EC (naturally, the first intensity is more important than the second intensity), the weighted average IN_WA of these intensities is calculated as the representative value. Therefore, the impact of using the second intensity in addition to the first intensity on the accuracy of estimating the concentration EC can be mitigated. Note that instead of the weighted average IN_WA of multiple intensities IN (first intensity and second intensity), the arithmetic average of multiple intensities IN (first intensity and second intensity) may be used to estimate the concentration EC.

[0068] As shown in Fig. 6, the weighting coefficients used to calculate the weighted average value are derived using a function, which makes it possible to set the weighting coefficients easily.

[0069] As shown in FIG. 6 , the function is a Gaussian function GF. The Gaussian function GF is used, for example, in the field of image processing for the purpose of smoothing (noise reduction). The weighted average value IN_WA calculated using the weighting coefficients derived from such a Gaussian function GF is a harmonious value. Therefore, the influence of using the second intensity in addition to the first intensity on the estimation accuracy of the concentration EC can be further mitigated.

[0070] 1 , the suspension is a purified liquid 15 obtained in the manufacturing process of a biopharmaceutical 17, and the target substance is an impurity 16 that is unnecessary for the biopharmaceutical 17. The amount of impurities 16 in the purified liquid 15 is usually very small. Therefore, even when the suspension contains only a small amount of the target substance, the effect of being able to estimate the concentration of the target substance in the suspension with high estimation accuracy can be fully exerted.

[0071] [Example] Using λDNA as the impurity 16 (substance of interest), the concentration EC of λDNA was estimated using the fluorescent reagent Quant-iT PicoGreen (manufactured by Invitrogen). First, λDNA was dissolved in TE (Tris-EDTA (Ethylenediaminetetraacetic Acid)) buffer (hydrogen ion exponent 8.0) instead of the purified solution 15, and after adding PicoGreen, the solution was left at room temperature for approximately 30 minutes. During this process, multiple solutions with different λDNA concentrations EC (the concentrations EC were known) were prepared. The fluorescence intensity IN of each solution was then measured. For the measurement, an Aqualog (trade name, manufactured by HORIBA Scientific) was used, with an exposure time of 5 seconds and wavelength intervals of the excitation light EL and the fluorescence FL, respectively, of 5 nm.

[0072] As an example, the graph shown in Figure 13 is a graph showing the relationship between concentration EC and intensity IN in Comparative Example 1. Comparative Example 1 is a case of a conventional method in which the arithmetic mean value of the fluorescence FL intensity IN (500, 535) at a reference excitation wavelength of 500 nm and a reference emission wavelength of 535 nm obtained from multiple measurements is used to estimate the concentration EC. In this case, when the concentration was higher than 0.05 ng / mL, a relationship between concentration EC and intensity IN that roughly resembled the calibration curve CL was obtained. However, as shown by the two-dot chain circle, at low concentrations of 0.05 ng / mL or less, the relationship between concentration EC and intensity IN collapsed, making it difficult to estimate the concentration EC. The coefficient of determination R in this Comparative Example 1 ２ was 0.969.

[0073] As an example, the graph shown in FIG. 14 is a graph showing the relationship between the actual concentration and the estimated concentration EC in Comparative Example 2. Comparative Example 2 is a case where the concentration EC was estimated using a regression model. Specifically, a Lasso regression model was generated based on the Lasso function in Python's scikit-learn library. Then, the intensities IN at 21 x 41 = 861 points in the excitation wavelength range of 450 nm to 550 nm and the emission wavelength range of 450 nm to 650 nm (both in 5 nm increments) of the fluorescence intensity data 22 were used as input data for the Lasso regression model, and the estimated concentration EC was output from the Lasso regression model.

[0074] In Figure 14, the actual concentration and the estimated concentration EC are nearly consistent not only when the concentration is higher than 0.05 ng / mL, but also when the concentration is low, such as 0.05 ng / mL or less. Therefore, at first glance, the estimation of concentration EC using a regression model appears to be successful. However, as shown in Figure 15, when the contribution of 861 intensity IN input data to the estimation of concentration EC was examined, it was found that the contribution of intensity IN different from the intensity IN (500, 535) at the reference excitation wavelength of 500 nm and the reference emission wavelength of 535 nm, which should be considered important, was high. This generally occurs when statistical methods are applied to a large number of input data (explanatory variables), as input data that have a strong correlation with concentration EC are selected as important variables regardless of the characteristics of the input data. This phenomenon is called chance correlation. If input data with a different noise profile than the input data used to generate the regression model is input, the estimated concentration EC may be far from the actual concentration. In other words, the estimation of concentration EC using a regression model can be said to have poor generalization performance.

[0075] As an example, the graph shown in FIG. 16 is a graph showing the relationship between concentration and intensity in Example 1. Example 1 is a case where a weighted average IN_WA of the fluorescence FL intensity IN (500, 535) at a reference excitation wavelength of 500 nm and a reference emission wavelength of 535 nm and multiple surrounding intensities IN (5 x 5 = 25 intensities IN) using weighting coefficient data 46 derived from a Gaussian function GF is used to estimate the concentration EC. The Gaussian_filter function in the Python Scipy package was used as the Gaussian function GF. In this case, a relationship between concentration EC and intensity IN that roughly mimics the calibration curve CL was obtained not only when the concentration was higher than 0.05 ng / mL, but also at low concentrations of 0.05 ng / mL or less. Therefore, the concentration EC could be estimated with high accuracy even at 0.01 ng / mL. The coefficient of determination R in Example 1 ２ was 0.996, which was improved compared to 0.969 in Comparative Example 1.

[0076] As an example, the graph shown in Figure 17 is a graph showing the relationship between concentration and intensity in Example 2. In Example 2, the arithmetic mean value of the fluorescence FL intensity IN (500, 535) at a reference excitation wavelength of 500 nm and a reference emission wavelength of 535 nm and the surrounding multiple intensities IN (5 x 5 = 25 intensities IN) was used to estimate the concentration EC. In this case, as in Example 1, a relationship between concentration EC and intensity IN that roughly mimicked the calibration curve CL was obtained not only when the concentration was higher than 0.05 ng / mL, but also at low concentrations of 0.05 ng / mL or less. Therefore, the concentration EC could be estimated with high accuracy even at 0.01 ng / mL. The coefficient of determination R in Example 2 ２ was 0.993, which is slightly inferior to 0.996 in Example 1, but is better than 0.969 in Comparative Example 1. From the above, it was confirmed that the technology disclosed herein can shorten the measurement time for the intensity IN of the fluorescence FL and can estimate the concentration EC with high estimation accuracy.

[0077] In the above embodiment, the excitation light EL within an excitation wavelength range consisting of multiple excitation wavelengths is sequentially irradiated onto the purified liquid 15 to obtain the intensities IN of the fluorescence FL at multiple emission wavelengths. However, this is not limited to this. For example, multiple excitation light sources 30 (e.g., laser light sources) emitting excitation light EL at multiple excitation wavelengths and multiple measuring devices 32 corresponding to each excitation light source 30 may be prepared. Then, the excitation light EL may be simultaneously irradiated onto different locations of the purified liquid 15 from the multiple excitation light sources 30, and the fluorescence spectrum FLS may be measured by each measuring device 32. This further shortens the measurement time for the intensity IN of the fluorescence FL. However, since multiple excitation light sources 30 and measuring devices 32 must be prepared, the cost of the device increases. As can be seen from the above description, the phrase "irradiating the suspension with excitation light" in claim 2 includes both sequential irradiation with the excitation light EL and simultaneous irradiation with the excitation light EL.

[0078] The excitation wavelength range, emission wavelength range, first wavelength interval, second wavelength interval, etc. described in the above embodiment are merely examples. For example, the excitation wavelength range may be 250 nm to 800 nm, and the first wavelength interval and second wavelength interval may be 1 nm.

[0079] Although a 5×5 Gaussian filter is used as an example of the weighting coefficient data 46, the present invention is not limited to this. A 3×3 Gaussian filter may also be used. Furthermore, although a Gaussian function GF is used as an example of the function for deriving the weighting coefficient, the present invention is not limited to this. Weighting coefficients may also be derived from an exponential function other than the Gaussian function GF, a polynomial function, or the like.

[0080] The second intensity may be the intensity of fluorescence FL having an emission wavelength other than the reference emission wavelength of 500 nm measured by irradiating excitation light EL having a reference excitation wavelength of 500 nm, specifically, the intensity IN(500, 525), the intensity IN(500, 530), the intensity IN(500, 540), and the intensity IN(500, 545). Alternatively, the second intensity may be the intensity of fluorescence FL having an emission wavelength of 535 nm measured by irradiating excitation light EL having a reference excitation wavelength other than 500 nm, specifically, the intensity IN(490, 535), the intensity IN(495, 535), the intensity IN(505, 535), and the intensity IN(510, 535).

[0081] The suspension is not limited to the exemplified purified liquid 15. It may be a culture medium 18. It may also be water collected from a river, lake, sea, etc. Therefore, the target substance is not limited to the exemplified impurities 16. It may also be a contaminant mixed into water collected from a river, lake, sea, etc.

[0082] The hardware configuration of the computer constituting the fluorometric analyzer 13 according to the technology of the present disclosure can be modified in various ways. For example, the fluorometric analyzer 13 can be configured with multiple computers separated as hardware in order to improve processing power and reliability. For example, the functions of the instruction receiving unit 50, measurement control unit 51, and display control unit 56, and the functions of the calculation unit 54 and estimation unit 55 can be distributed and performed by two computers. In this case, the fluorometric analyzer 13 is configured with two computers. Furthermore, some or all of the functions of the fluorometric analyzer 13 may be performed by the fluorometric measurement device 12.

[0083] In this way, the hardware configuration of the computer of the fluorescence analyzer 13 can be changed as appropriate depending on the required performance, such as processing power, safety, and reliability. Furthermore, not only the hardware, but also APs such as the operating program 45 can be duplicated or stored in multiple storage devices in order to ensure safety and reliability.

[0084] A plurality of fluorescence measurement devices 12 in a plurality of different facilities may be connected to the fluorescence analyzer 13. In other words, the fluorescence analyzer 13 may be configured to operate as a server computer, acquire fluorescence intensity data 22 from a plurality of fluorescence measurement devices 12 in a plurality of different facilities, and distribute the estimated concentration EC to the computer of each facility.

[0085] In the above embodiment, the following various processors can be used as the hardware structure of processing units that perform various processes, such as the instruction receiving unit 50, the measurement control unit 51, the acquisition unit 52, the RW control unit 53, the calculation unit 54, the estimation unit 55, and the display control unit 56. The various processors include the CPU 37, which is a general-purpose processor that executes software (operation program 45) to function as various processing units, as described above, as well as dedicated electrical circuits that are processors having a circuit configuration specifically designed to perform specific processes, such as a programmable logic device (PLD) that is a processor whose circuit configuration can be changed after manufacture, such as an FPGA (Field Programmable Gate Array), and an ASIC (Application Specific Integrated Circuit).

[0086] A single processing unit may be configured with one of these various processors, or may be configured with a combination of two or more processors of the same or different types (e.g., a combination of multiple FPGAs and / or a combination of a CPU and an FPGA).Furthermore, multiple processing units may be configured with a single processor.

[0087] Examples of configuring multiple processing units with a single processor include, first, a form in which one processor is configured with a combination of one or more CPUs and software, as typified by computers such as client and server, and this processor functions as multiple processing units. Second, a form in which a processor is used to realize the functions of the entire system including multiple processing units with a single IC (Integrated Circuit) chip, as typified by systems on chips (SoCs). In this way, various processing units are configured using one or more of the above-mentioned various processors as a hardware structure.

[0088] Furthermore, more specifically, the hardware structure of these various processors can be an electric circuit (circuitry) that combines circuit elements such as semiconductor elements.

[0089] From the above description, the technology described in the following supplementary paragraphs can be understood.

[0090] [Supplementary Item 1] A fluorescence analysis method for analyzing fluorescence intensity measured by irradiating a suspension containing a dispersed target substance with excitation light, the method comprising: acquiring a first intensity of fluorescence at a reference emission wavelength corresponding to the target substance and a second intensity of fluorescence at the reference emission wavelength or an emission wavelength different from the reference emission wavelength, the first intensity and the second intensity being measured by irradiating the suspension with at least excitation light at a reference excitation wavelength corresponding to the target substance; calculating representative values ​​of the first intensity and the second intensity; and estimating the concentration of the target substance in the suspension based on the representative value. [Supplementary Item 2] The fluorescence analysis method according to Supplementary Item 1, in which the second intensity is plural. [Supplementary Item 3] The fluorescence analysis method according to Supplementary Item 1 or Supplementary Item 2, in which the second intensity is plural. [Supplementary Item 3] The fluorescence analysis method according to Supplementary Item 1 or Supplementary Item 2, in which the first intensity and the second intensity are obtained by irradiating the suspension with excitation light in an excitation wavelength range consisting of plural excitation wavelengths including the reference excitation wavelength, and measuring, for each of the plural excitation wavelengths, the intensity of fluorescence in an emission wavelength range consisting of plural emission wavelengths including the reference emission wavelength. [Supplementary Item 4] The fluorescence analysis method according to Supplementary Item 3, wherein the plurality of excitation wavelengths are spaced apart by a first wavelength interval, and the plurality of emission wavelengths are spaced apart by a second wavelength interval. [Supplementary Item 5] The fluorescence analysis method according to any one of Supplementary Item 1 to Supplementary Item 4, wherein the representative value is a weighted average of the first intensity and the second intensity. [Supplementary Item 6] The fluorescence analysis method according to Supplementary Item 5, wherein a weight coefficient used to calculate the weighted average is derived using a function. [Supplementary Item 7] The fluorescence analysis method according to Supplementary Item 6, wherein the function is a Gaussian function. [Supplementary Item 8] The fluorescence analysis method according to any one of Supplementary Item 1 to Supplementary Item 7, wherein the suspension is a liquid obtained in the production process of a biopharmaceutical, and the target substance is an impurity unnecessary for the biopharmaceutical.

[0091] The technology of the present disclosure can be appropriately combined with the various embodiments and / or various modified examples described above. Furthermore, it is not limited to the above embodiments, and various configurations can be adopted without departing from the spirit of the present disclosure. Furthermore, the technology of the present disclosure extends not only to programs, but also to storage media that non-temporarily store programs, and computer program products that include programs.

[0092] The above-described description and illustrations are a detailed explanation of the parts related to the technology of the present disclosure and are merely an example of the technology of the present disclosure. For example, the above description of the configuration, functions, actions, and effects is an explanation of an example of the configuration, functions, actions, and effects of the parts related to the technology of the present disclosure. Therefore, it goes without saying that unnecessary parts may be deleted, new elements may be added, or replacements may be made to the above-described description and illustrations within the scope of the gist of the technology of the present disclosure. Furthermore, to avoid confusion and facilitate understanding of the parts related to the technology of the present disclosure, the above-described description and illustrations omit explanations of common technical knowledge that do not require particular explanation to enable the implementation of the technology of the present disclosure.

[0093] In this specification, "A and / or B" is synonymous with "at least one of A and B." In other words, "A and / or B" means that it may be only A, only B, or a combination of A and B. Furthermore, in this specification, the same concept as "A and / or B" is also applied when three or more things are expressed by connecting them with "and / or."

[0094] All publications, patent applications, and technical standards mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent application, or technical standard was specifically and individually indicated to be incorporated by reference.

Claims

1. A fluorescence analysis method for analyzing the intensity of fluorescence measured by irradiating excitation light onto a suspension in which a target substance is dispersed, the method comprising: acquiring a first intensity of fluorescence at a reference emission wavelength corresponding to the target substance, and a second intensity of fluorescence at the reference emission wavelength or an emission wavelength different from the reference emission wavelength, measured by irradiating at least excitation light at a reference excitation wavelength corresponding to the target substance; calculating representative values ​​of the first intensity and the second intensity; and estimating the concentration of the target substance in the suspension based on the representative values.

2. The fluorescence analysis method according to claim 1, wherein the second intensity is plural.

3. The fluorescence analysis method according to claim 1, wherein the first intensity and the second intensity are obtained by irradiating the suspension with excitation light in an excitation wavelength range consisting of a plurality of excitation wavelengths including the reference excitation wavelength, and measuring the intensity of fluorescence in an emission wavelength range consisting of a plurality of emission wavelengths including the reference emission wavelength for each of the plurality of excitation wavelengths.

4. A method for fluorescence analysis according to claim 3, wherein said plurality of excitation wavelengths are spaced apart by a first wavelength interval and said plurality of emission wavelengths are spaced apart by a second wavelength interval.

5. The fluorescence analysis method according to claim 1, wherein the representative value is a weighted average value of the first intensity and the second intensity.

6. The fluorescence analysis method according to claim 5, wherein the weighting coefficients used to calculate the weighted average value are derived using a function.

7. The fluorescence analysis method according to claim 6, wherein said function is a Gaussian function.

8. The fluorescence analysis method according to claim 1, wherein the suspension is a liquid obtained in the manufacturing process of a biopharmaceutical, and the target substance is an impurity that is unnecessary for the biopharmaceutical.

9. A fluorescence analyzer that analyzes the intensity of fluorescence measured by irradiating a suspension in which a target substance is dispersed with excitation light, comprising a processor, which acquires a first intensity of fluorescence at a reference emission wavelength corresponding to the target substance and a second intensity of fluorescence at the reference emission wavelength or an emission wavelength different from the reference emission wavelength, measured by irradiating at least excitation light at a reference excitation wavelength corresponding to the target substance, calculates representative values ​​of the first intensity and the second intensity, and estimates the concentration of the target substance in the suspension based on the representative values.

10. An operating program for a fluorescence analyzer that analyzes the intensity of fluorescence measured by irradiating excitation light onto a suspension in which a target substance is dispersed, the operating program including: acquiring a first intensity of fluorescence at a reference emission wavelength corresponding to the target substance, and a second intensity of fluorescence at the reference emission wavelength or an emission wavelength different from the reference emission wavelength, measured by irradiating at least excitation light at a reference excitation wavelength corresponding to the target substance; calculating representative values ​​of the first intensity and the second intensity; and estimating the concentration of the target substance in the suspension based on the representative values.

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