Method for analysis by electrophoresis

Abstract

To provide an electrophoretic analysis method using a standard substance as an internal standard.SOLUTION: In one aspect, the present disclosure provides a method for measuring an analyte by electrophoresis, the method including the steps of preparing a mixture by mixing a sample including the analyte with a standard substance; subjecting the mixture to electrophoresis; detecting optical signals derived from the analyte and the standard substance; and measuring the analyte on the basis of the optical signals. In one embodiment, the electrophoresis is capillary electrophoresis.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to methods for analyzing an analyte of interest by electrophoresis. [Background technology]

[0002] There are a great many types of biological molecules such as proteins, including those modified by post-translational modifications, and various analytical methods have been developed due to the complexity of their molecular structures. Electrophoretic analytical methods such as capillary electrophoresis are often used to analyze biological molecules such as proteins. Biological molecules, such as antibody drugs, are often used as pharmaceutical ingredients, and electrophoretic analytical methods can be simple and convenient, making them useful not only for research but also for verifying the quality of products produced during the manufacture of pharmaceutical ingredients.

[0003] Although electrophoretic analysis allows for robust analysis, the reproducibility of absolute detection times between experiments (runs) is not necessarily high. Therefore, it is beneficial to increase the versatility of the information obtained by electrophoretic analysis. Summary of the Invention [Means for solving the problem]

[0004] As a result of extensive research, the present inventors have developed a method for improving electrophoretic analysis by using a standard (marker). Based on this, the present disclosure provides an electrophoretic analysis method using a standard, a standard therefor, an apparatus therefor, and the like.

[0005] Thus, the present disclosure provides: (Item 1) 1. A method for measuring an analyte by electrophoresis, comprising: mixing a sample containing the analyte with a standard to prepare a mixture; subjecting the mixture to electrophoresis; detecting optical signals from the analyte and the standard; determining the analyte based on the optical signal; A method comprising: (Item 2) 2. The method of claim 1 for determining the isoelectric point of an analyte, wherein the standard has a known isoelectric point. (Item 3) The method of any of the preceding items, wherein the standards include a plurality of standards having different known isoelectric points in the range of 3 to 11. (Item 4) The method of any of the preceding items, wherein the plurality of standards comprises pairs of first and second standards having a difference in isoelectric point of less than two. (Item 5) 2. The method of any of the preceding items for determining the molecular weight of an analyte, wherein the standard has a known molecular weight. (Item 6) The method of any of the preceding items, wherein the standard comprises a plurality of standard substances having different known molecular weights in the range of 5 kDa to 1000 kDa. (Item 7) 2. The method of any of the preceding items, wherein the plurality of standards comprises pairs of first and second standards that differ in molecular weight by no more than three-fold. (Item 8) Any of the methods described above, deriving an equation expressing the correlation between the isoelectric point and the detection time or position, or between the molecular weight and the detection time or position, based on the optical signals derived from the plurality of standard substances. (Item 9) The method of any of the preceding items, comprising converting the detection time or detection position into a pH value or molecular weight based on the formula. (Item 10) The method of any of the preceding items, wherein the standard is a tryptophan-free peptide or protein. (Item 11) The method of any of the preceding items, wherein the standard has a dye label. (Item 12) The method of any preceding item, wherein the step of detecting the optical signal comprises irradiating with light having two wavelengths: a wavelength of about 280 nm and a wavelength suitable for detecting the dye label. (Item 13) The method of any of the preceding items, wherein the standard is labeled with rhodamine. (Item 14) 10. The method of any preceding claim, wherein the step of detecting an optical signal comprises irradiating with light having a single wavelength. (Item 15) 2. The method of any of the preceding items, wherein the single wavelength is about 280 nm. (Item 16) 10. The method of any preceding claim, wherein the step of detecting an optical signal comprises detecting light of a different wavelength than the irradiating light. (Item 17) 10. The method of any preceding claim, wherein the step of detecting an optical signal includes detecting light at two wavelengths. (Item 18) The method of any of the preceding items, wherein the standards include at least five standards. (Item 19) The method of any of the preceding items, wherein a plurality of standards are mixed with the sample at different concentrations. (Item 20) The method of any of the preceding items, wherein the electrophoresis is capillary electrophoresis. (Item 21) The method of any of the preceding items, wherein the analyte comprises a peptide and / or a protein. (Item 22) 1. An apparatus for measuring an analyte by electrophoresis, comprising: a channel and electrodes for electrophoresis; a light source for illuminating at least a portion of the flow path; a photodetector that receives light from the flow path; Equipped with the flow path includes an inlet for receiving a sample containing the analyte; Device. (Item 23) 10. The apparatus of any preceding claim, wherein the light source is configured to illuminate the flow path with light of a single wavelength. (Item 24) 10. The device of any preceding claim, wherein the light source is configured to illuminate the flow path with light having a wavelength of about 280 nm. (Item 25) 10. The apparatus of any preceding claim, wherein the photodetector is configured to detect light of a different wavelength than the illumination light of the light source. (Item 26) 10. The apparatus of any preceding item, wherein the photodetector is configured to detect light at two wavelengths. (Item 27) 10. The apparatus of any preceding item, wherein the photodetector comprises a dichroic mirror, a dichroic filter, or a beam splitter. (Item 28) 10. The device of any preceding item, wherein the photodetector is configured to detect light having a wavelength of about 340 nm and light having a wavelength of about 575 nm. (Item 29) 1. A composition for determining the isoelectric point of an analyte, comprising a plurality of standards, the composition is for mixing with a sample containing an analyte and then subjecting it to isoelectric focusing; The plurality of standards have known isoelectric points that are different from each other. composition. (Item 30) 1. A composition for determining the molecular weight of an analyte, comprising a plurality of standards, the composition is for mixing with a sample containing an analyte and then subjecting it to electrophoresis; The plurality of standards have known molecular weights that are different from each other. composition. (Item 31) 1. A method for producing a standard for determining the molecular weight of an analyte, comprising: Identifying a gene encoding a protein of known molecular weight; Identifying a portion encoding all tryptophans in the base sequence encoding the protein in the gene; obtaining a modified gene by replacing all of the tryptophan-encoding portions with base sequences encoding amino acids other than tryptophan; Obtaining the protein produced using the modified gene; and A step of binding a labeling dye to the protein to form a conjugate and obtaining the conjugate as a standard substance. A method comprising:

[0006] It is contemplated that the present disclosure may provide one or more of the above-described features in combinations other than those explicitly stated. Still further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary. [Effects of the Invention]

[0007] The present disclosure provides a convenient electrophoretic analysis using a calibrator as an internal standard. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 is a schematic diagram of an exemplary capillary electrophoresis fluorescence detector. [Figure 2] The results of CIEF separation of cetuximab and both end markers and both flanking markers (Table 1) are shown. 33 ng of cetuximab and 0.3 pmol each of four tryptophan-containing peptide pI markers (EM3.38, FM7.00, FM9.50, EM10.17) were separated by CIEF using a carrier amphoteric generating a pH gradient from pH 3 to 10. The detection results are shown for the eighth scan after the start of focusing at 10 kV. The solid line indicates the 340 nm fluorescence from tryptophan residues. EM: both end markers, FM: both flanking markers, C: cetuximab. The horizontal axis indicates detection time, which is proportional to the axial position of the capillary, and the vertical axis indicates the fluorescence signal value, which is proportional to the sample amount. [Figure 3]The results of CIEF separation of cetuximab and 12 ubiquitous markers (Table 1) are shown. 33 ng of cetuximab and 8 fmol each of 12 tetramethylrhodamine-labeled peptide pI markers (UM7.58, UM8.21, UM8.77, UM9.56, etc.) were separated by CIEF using a carrier amphoteric generating a pH gradient from pH 3 to 10. The results are from the eighth scan after focusing at 10 kV. The solid line indicates the 340 nm fluorescence from tryptophan residues, and the dotted line indicates the 575 nm fluorescence of the tetramethylrhodamine label. UM: ubiquitous marker; C: cetuximab. The horizontal axis indicates detection time, and the vertical axis indicates the fluorescence signal value. [Figure 4] The isoelectric points of the cetuximab peaks determined using both-end markers, both-side markers, and ubiquitous markers are shown, along with their changes with the number of scans. A) Both-end markers (pI 3.38 and pI 10.17) were used, B) Both-side markers (pI 7.00 and pI 9.50) were used, and C) Ubiquitous markers (pI 7.58, pI 8.21, pI 8.77, and pI 9.56) were used. The correspondence between the symbols and the cetuximab peaks is shown at the bottom. [Figure 5] The results of CIEF separation of pI markers are shown. The horizontal axis indicates detection time, and the vertical axis indicates fluorescence signal value. [Figure 6] The graph shows an approximation of the pH value versus detection time using a third-order polynomial for the separation results in Figure 5. The horizontal axis is detection time (min), and the vertical axis is the pH value of the pH gradient. DETAILED DESCRIPTION OF THE INVENTION

[0009] The present disclosure will now be described, illustrating the best mode thereof. Throughout this specification, singular expressions should be understood to include the plural concept unless otherwise specified. Therefore, articles used in the singular (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise specified. Furthermore, terms used in this specification should be understood to have the meaning commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the event of conflict, the present specification (including definitions) will control.

[0010] The following provides definitions of terms particularly used in this specification and / or explains basic technical content as appropriate.

[0011] (Definition, etc.) As used herein, "electrophoresis" refers to the process of generating a potential difference between two distant points in a fluid-filled channel, causing a charged substance present between the two points to move. When electrophoresis is performed using a capillary tube (typically a hollow tube with a small inner diameter of about 0.01 to about 1 mm), it can be referred to as capillary electrophoresis.

[0012] As used herein, "isoelectric focusing" refers to electrophoresis that performs separation by utilizing the phenomenon in which the net charge of an ampholyte becomes zero at a certain pH value (isoelectric point) and no longer migrates.

[0013] As used herein, "analyte" refers to a substance to be separated and analyzed. Typically, the nature of the analyte to be analyzed is unknown. Since it may be unknown whether the analyte is present in a sample, a sample containing the analyte described herein also refers to a sample that is expected to contain the analyte, and may be determined to not contain the analyte as a result of measurement. Typically, the analytes described herein are biological molecules (proteins, nucleic acids, sugars, lipids, etc.), particularly proteins (or peptides).

[0014] As used herein, proteins encompass peptides, but generally, the term "protein" refers to a molecule having a relatively large molecular weight, and the term "peptide" refers to a molecule having a relatively small molecular weight (e.g., 30 amino acids or less, 20 amino acids or less, 15 amino acids or less). Biological molecules described herein also include molecules containing modifications such as phosphorylation and disulfide bonds in proteins and pseudouridine in nucleic acids, as well as molecules conjugated with labels such as fluorescent labels.

[0015] As used herein, the term "standard" (sometimes referred to as "marker") refers to a substance whose property to be measured (such as isoelectric point or molecular weight) is known. Typically, the standard described herein is an internal standard that is mixed with the analyte and detected in the same measurement (run).

[0016] As used herein, the term "kit" refers to a unit in which the components to be provided are provided, usually separated into two or more compartments. The kit preferably advantageously includes instructions or manuals describing how to use or operate the components provided.

[0017] As used herein, the term "about" refers to a range of plus or minus 10% of the indicated value unless otherwise specified. When "about" is used in reference to temperature, it refers to a range of plus or minus 5°C of the indicated temperature.

[0018] (Preferred embodiment) Preferred embodiments of the present disclosure will be described below. The embodiments provided below are provided for a better understanding of the present disclosure, and it is understood that the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that those skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description herein. It is also understood that the following embodiments of the present disclosure can be used alone or in combination.

[0019] In one aspect, the present disclosure provides a method for measuring an analyte by electrophoresis, comprising the steps of: mixing a sample containing the analyte with a standard to prepare a mixture; subjecting the mixture to electrophoresis; detecting optical signals derived from the analyte and the standard; and measuring the analyte based on the optical signals. In one embodiment, the electrophoresis is capillary electrophoresis or microfluidic chip electrophoresis.

[0020] In one embodiment, the isoelectric point of the analyte is determined by electrophoresis. In this embodiment, the standards have known isoelectric points and the electrophoresis is isoelectric focusing, etc. In one embodiment, the molecular weight of the analyte is determined by electrophoresis. In this embodiment, the standards have known molecular weights and the electrophoresis is gel electrophoresis, polymer solution electrophoresis, etc.

[0021] (standard material) The standard substance is any substance that can be electrophoresed. In electrophoretic analysis, substances are easily detected by light, so the standard substance preferably has an optical group (fluorescent group, light-absorbing group, chromogenic group) that allows optical detection. In one embodiment, the standard substance is a protein (peptide) or a nucleic acid, preferably a protein (peptide).

[0022] In many embodiments of the present disclosure, the analyte is a protein (peptide), and the standard is preferably detectable in a manner that distinguishes it from the analyte. In particular, when the standard is detected at a detection time close to that of the analyte during electrophoresis, it is preferable to be able to distinguish the standard from the analyte based on differences in optical properties. In one embodiment, the standard is a protein (peptide) that does not contain tryptophan. Because proteins (peptides) typically contain tryptophan, the optical properties of tryptophan, namely, an excitation (absorption) spectrum with a peak at approximately 280 nm and a fluorescence (emission) spectrum with a peak at approximately 340 nm, can be utilized to optically detect the protein (peptide). Therefore, by designing the standard to include an optical group (e.g., a fluorescent dye label) that can be detected at a wavelength different from that used to detect tryptophan, the protein (peptide) analyte can be detected in a manner that distinguishes it from the standard. For example, examples of optical groups having fluorescence at a wavelength different from that of tryptophan include rhodamine, fluorescein, cyanine, indocyanine, indocarbocyanine, pyronine, Lucifer Yellow, quinacrine, squaric acid, coumarin, and fluoroanthenylmaleimide. For example, optical groups having an absorption peak at a wavelength different from that of tryptophan can be the same as the optical groups having fluorescence at a wavelength different from that of tryptophan described above, but are not limited to these. Those skilled in the art will be able to appropriately perform electrophoretic analysis, taking into account the influence of electrophoretic conditions (e.g., mobile phase conditions) on optical properties.

[0023] Similarly, when the analyte is a nucleic acid, the standard can be designed to contain an optical group that is detectable at a wavelength different from adenine, guanine, thymine, cytosine, and uracil, thereby making it possible to distinguish the analyte from the standard.

[0024] In one embodiment, the standard comprises a plurality of standards (e.g., two, three, four, five, seven, ten, fifteen, twenty, or more). In a preferred embodiment, the plurality of standards comprises a common optical group (or different optical groups with a common emission, fluorescence, or absorption wavelength) so that they can be detected together. In a preferred embodiment, the plurality of standards has a known isoelectric point or known molecular weight that is different from one another.

[0025] The standard can be selected appropriately depending on the purpose of the analysis. For example, when the analyte is completely unknown, multiple standard substances with a wide range of isoelectric points or molecular weights can be used. For example, when the analyte is approximately identified, such as a pharmaceutical ingredient, two or more standard substances with isoelectric points or molecular weights close to the isoelectric point or molecular weight of interest can be used.

[0026] The amount of standard to be used can be appropriately determined by one skilled in the art so that the standard can be detected. In one embodiment, the concentration of the standard in the mixture obtained by mixing with the sample can be about 0.1 nM to 100 μM, for example, about 1 nM to 100 μM, about 10 nM to 100 μM, about 100 nM to 100 μM, about 1 μM to 100 μM, about 0.1 nM to 10 μM, about 1 nM to 10 μM, about 10 nM to 10 μM, about 100 nM to 10 μM, about 1 μM to 10 μM, about 0.1 nM to 1 μM, about 1 nM to 1 μM, about 10 nM to 1 μM, about 100 nM to 1 μM, about 0.1 nM to 100 nM, about 1 nM to 100 nM, about 10 nM to 100 nM, about 0.1 nM to 10 nM, or about 1 nM to 10 nM. Even when irradiating the standard with a wavelength (e.g., about 280 nm) that is not the optimal excitation wavelength for the standard (e.g., about 520 to 550 nm for rhodamine), a standard at a concentration of about 10 nM may be detectable. In one embodiment, multiple standards are mixed with the sample at different concentrations, which allows the detected peaks to vary in magnitude for each standard, making it easier to associate the standards with the peaks.

[0027] In one embodiment, the standards include multiple standards having known different isoelectric points within the range of about 1 to 13, about 2 to 12, or about 3 to 11. In one embodiment, the difference in isoelectric points between adjacent standards for isoelectric point measurement is less than about 4, less than about 3.5, less than about 3, less than about 2.5, less than about 2, less than about 1.5, or less than about 1.

[0028] In one embodiment, the standard has a molecular weight of about 1 kDa to 100 MDa, for example, about 1 kDa to 10 MDa, about 1 kDa to 5 MDa, about 1 kDa to 2 MDa, about 1 kDa to 1 MDa, about 1 kDa to 500 kDa, about 1 kDa to 200 kDa, about 2 kDa to 100 MDa, about 2 kDa to 10 MDa, about 2 kDa to 5 MDa, about 2 kDa to 2 MDa, about 2 kDa to 1 MDa, about 2 kDa to 500 kDa, about 2 kDa to 200 kDa, about 5 kDa to 100 MDa, or about 5 kDa to The sample contains a plurality of standards having different known molecular weights in the ranges of 10 MDa, about 5 kDa to 5 MDa, about 5 kDa to 2 MDa, about 5 kDa to 1 MDa, about 5 kDa to 500 kDa, about 10 kDa to 100 MDa, about 10 kDa to 10 MDa, about 10 kDa to 5 MDa, about 10 kDa to 2 MDa, about 10 kDa to 1 MDa, about 20 kDa to 100 MDa, about 20 kDa to 10 MDa, about 20 kDa to 5 MDa, about 20 kDa to 2 MDa, or about 20 kDa to 1 MDa. In one embodiment, the difference in molecular weight between adjacent standards for the quantitative sample measurement is about 2 to 1000 times, for example, about 2 to 500 times, about 2 to 200 times, about 2 to 100 times, about 2 to 50 times, about 2 to 20 times, about 2 to 10 times, about 5 to 1000 times, about 5 to 500 times, about 5 to 200 times, about 5 to 100 times, about 5 to 50 times, about 5 to 20 times, about 10 to 1000 times, about 10 to 500 times, about 10 to 200 times, about 10 to 100 times, about 10 to 50 times, about 20 to 1000 times, about 20 to 500 times, about 20 to 200 times, about 20 to 100 times, about 50 to 1000 times, about 50 to 500 times, or about 50 to 200 times.

[0029] Those skilled in the art can appropriately prepare standards having specific isoelectric points or molecular weights. Protein or peptide standards having specific isoelectric points or molecular weights can be obtained using any known recombinant gene expression technology, peptide synthesis technology, and peptide modification technology.

[0030] The isoelectric point of a peptide can also be predicted to design a peptide standard with a specific isoelectric point. j The amino acid releases a proton and becomes negatively charged (acid dissociation constant K j ), and n i The amino acid accepts a proton and becomes positively charged (acid dissociation constant K i ), the amino acid has the following formula: Z=Σ i (n i / (1+K i / [H + ]))-Σ j (n j / (1+[H + ] / K j )) The charge Z of the peptide as a whole can be estimated by (Chemical Areas, 36, 470-486, 1982). In this formula, -log([H + The pI of a peptide is determined by the pH (pI = pH ≈ 0.01), which allows for the prediction of its isoelectric point (pI). For example, the pKa of amino acid functional groups can be used to estimate the pI of a peptide, such as α-carboxyl (C-terminus) (3.6), β-carboxyl (Asp) (3.95), γ-carboxyl (Glu) (4.45), imidazole (His) (6.45), α-amino (N-terminus) (7.6), thiol (Cys) (8.5), phenolic hydroxyl (Tyr) (9.8), ε-amino (Lys) (10.2), and guanidinium group (Arg) (12.5). The designed polypeptides can then be synthesized and their isoelectric points confirmed experimentally.

[0031] Protein standards with specific molecular weights can be easily obtained by purifying proteins (e.g., albumin) that are produced in large quantities in living organisms. However, they can also be synthesized in vitro or modified by cleavage (e.g., cleavage with an enzyme such as trypsin) or fusion. Tryptophan-free proteins can be easily produced by those skilled in the art by identifying the gene encoding the protein of interest, modifying the gene to remove the tryptophan-encoding nucleotide sequence, cloning the modified gene, and introducing it into protein-producing cells. Assuming that the frequency of tryptophan among the amino acids constituting a protein (or peptide) is 1.3% and the average molecular weight of one amino acid residue is 100 Da, the probability that a 50 kDa protein (or peptide) does not contain any tryptophans is approximately 0.14%, and the probability that a 100 kDa protein (or peptide) does not contain any tryptophans is approximately 0.00021%. The absence of tryptophan can be a novel feature of a protein.

[0032] In one embodiment, the present disclosure provides a method for producing a standard for determining the molecular weight of an analyte, the method comprising the steps of identifying a gene encoding a protein having a known molecular weight, identifying a portion of the gene encoding all tryptophans in a base sequence encoding the protein, replacing the portion encoding all tryptophans with a base sequence encoding amino acids other than tryptophan to obtain a modified gene, and obtaining a protein produced using the modified gene as a standard.

[0033] Methods for introducing labels (e.g., fluorescent dye labels) into proteins (or peptides) are well known to those skilled in the art. Examples include using labeled amino acids as starting amino acid materials in in vitro peptide synthesis, and chemical conjugation of proteins (or peptides) with labels bearing reactive groups (e.g., NHS-modified carboxyl groups). For the purposes of this disclosure, the location of label introduction does not significantly affect analytical results. Standards with specific isoelectric points can be purified after label introduction to obtain standards with uniform isoelectric points. For standards with specific molecular weights, the rate of change in molecular weight due to label introduction can be negligible in analysis. Furthermore, labeling can be performed by specifying the number and location of labels. This can be achieved by first introducing free (non-cystine-forming) cysteine ​​residues into the protein or peptide and then labeling with a labeling dye bearing an iodoacetyl or maleimide group that specifically reacts with SH groups. In this case, the change in molecular weight due to label introduction can be calculated.

[0034] (light detection) In one embodiment, in the step of subjecting the sample to electrophoresis and / or the step of detecting an optical signal according to the present disclosure, light may be irradiated onto a channel (such as a capillary) through which the sample and standard substance flow. The irradiated light may be absorbed by the sample and / or standard substance, and this light absorption may be detected, or the irradiated light may excite chemical groups contained in the sample and / or standard substance, resulting in the emission of fluorescence.

[0035] In one embodiment, the irradiating light may be limited to a specific wavelength or may include light of a specific wavelength. The wavelength of the light can be adjusted using an optical filter or the like, for example, to generate light with a wavelength range of ±5 nm, 10 nm, 20 nm, or 30 nm from a specific wavelength. Unless otherwise specified, a specific wavelength refers to light having a maximum wavelength peak within ±30 nm of that value. Because proteins (peptides) typically contain tryptophan, a protein (peptide) analyte can be measured by irradiating it with light of approximately 280 nm, which is suitable for exciting tryptophan, and detecting the emitted light of approximately 340 nm. Because the fluorescence yield of tryptophan may be lower than that of groups designed as fluorophores, the irradiating wavelength preferably includes approximately 280 nm, which is suitable for exciting tryptophan. In one embodiment, light of a second wavelength suitable for exciting a fluorescent dye label incorporated into the standard is further irradiated. In one embodiment, a single type of light having a maximum wavelength peak within ±30 nm of the excitation maximum wavelength of the analyte is irradiated. It is preferable to detect the analyte with high sensitivity using a suitable excitation wavelength, but the standard can still be detected even if a wavelength outside the range of suitable excitation wavelengths is used by, for example, adjusting the concentration of the standard added, so the benefits of reducing the number of excitation light sources may outweigh the benefits. It is preferable that the analyte and the standard can be detected using different types of light, so the standard can have a fluorescent dye label that emits fluorescence with a maximum wavelength that is about 50 nm or more away from the maximum wavelength of fluorescence from the analyte. Since the excitation wavelength and the fluorescence wavelength are usually different, in one embodiment, light of a wavelength different from the irradiating light is detected.

[0036] The light source is not particularly limited, but a laser that has high wavelength purity and enables stable light irradiation is preferred, such as an argon laser, a semiconductor-pumped YAG laser, or a helium-neon laser.

[0037] (Determination of isoelectric point) The method of the present disclosure can accurately determine the isoelectric point by using a standard substance as an internal standard. In one embodiment, an equation expressing the correlation between isoelectric point and detection time is derived based on optical signals from multiple standards, and the detection time is converted to a pH value based on this equation, thereby accurately determining the isoelectric point. The equation may be a linear approximation using a first-order approximation, but accuracy can be improved by approximating using a higher-order polynomial, etc. It is also effective to convert the detection position or time to pH by assuming a proportional relationship between the isoelectric points (pH) of two adjacent standards and the positions or times at which the peaks of those two standards are detected. In this case, a pH gradient for n standards can be depicted by n-1 straight lines connected in series.

[0038] Using multiple standards allows for the provision of standards with similar isoelectric points for many types of analytes. Providing reference isoelectric points based on multiple standards improves the accuracy of the approximation formula, resulting in accurate isoelectric point determination. Highly accurate isoelectric points provide information that can be shared among multiple practitioners working with the same substance, independent of laboratory conditions. They can also be effectively used to identify peaks found in different samples. Similarly, for molecular weights, an equation expressing the correlation between molecular weight and detection time can be derived based on the optical signals derived from multiple standards, and the detection time can be converted to molecular weight based on this equation, allowing for accurate molecular weight determination.

[0039] (Composition, kit) In one aspect, the present disclosure provides a composition or kit for measuring an analyte by electrophoresis, comprising a standard. The composition or kit can be used in the methods described herein. In one embodiment, the composition or kit comprises a plurality of standards having different known isoelectric points, and the compositions or standards are mixed with a sample containing the analyte and then subjected to isoelectric focusing. In one embodiment, the composition or kit comprises a plurality of standards having different known molecular weights, and the compositions or standards are mixed with a sample containing the analyte and then subjected to electrophoresis.

[0040] (Device) In one aspect, the present disclosure provides an apparatus for measuring an analyte by electrophoresis, the apparatus comprising a flow path and electrodes for electrophoresis, a light source for illuminating at least a portion of the flow path, and a photodetector for receiving light from the flow path, the flow path comprising an inlet for receiving a sample containing the analyte.

[0041] In one embodiment, the light source is configured to irradiate the flow path with light of a single wavelength. In one embodiment, the light source is configured to detect light of two wavelengths. In one embodiment, the light source is configured to irradiate the flow path with light of a wavelength of about 280 nm. For example, the light source can include an optical filter to adjust the wavelength of the light. The device may also include a light collector such as a collimator.

[0042] In one embodiment, the photodetector is configured to detect light of a wavelength different from the illumination light of the light source. In one embodiment, the photodetector is configured to detect light of a wavelength of about 340 nm and / or light of a wavelength of about 575 nm. In one embodiment, the photodetector comprises a dichroic mirror, a dichroic filter, or a beam splitter.

[0043] In one embodiment, the flow path is a capillary. Commercially available capillaries can be used, for example, commercially available tubing for capillary electrophoresis or HPLC. The capillary can have an outer diameter of, for example, about 0.05 to 5 mm, about 0.05 mm, about 0.1 mm, about 0.18 mm, about 0.2 mm, about 0.36 mm, about 0.5 mm, about 1 mm, about 2 mm, or about 5 mm. The capillary can have an inner diameter of, for example, about 0.01 to 1 mm, about 0.01 mm, about 0.02 mm, about 0.05 mm, about 0.1 mm, about 0.2 mm, about 0.5 mm, or about 1 mm. The capillary can be made of, for example, fused silica or glass. The capillary may be coated on the outside with polyimide or the like.

[0044] (Note) In this specification, "or" is used when "at least one or more" of the items listed in the sentence can be employed. The same applies to "alternative." In this specification, when it is specified that "within a range" of "two values," the range includes the two values ​​themselves.

[0045] The present disclosure will be described below based on examples, but the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present invention. Therefore, the scope of the present invention is not limited to the embodiments or examples specifically described herein, but is limited only by the claims. [Example]

[0046] Example 1: Determination of isoelectric point using an internal standard marker We investigated a method for determining the isoelectric point with high accuracy and ease of use using pI markers as internal standards.

[0047] method: (1) Capillary isoelectric focusing (CIEF) The antibody drug cetuximab was separated by CIEF using a scanning detection capillary isoelectric focusing system (Nichiei Kogyo, Fukushima City). The capillary was a fused silica capillary with an inner diameter of 50 μm, an outer diameter of 375 μm, and a length of 420 mm, and the inner wall was coated with a hydrophilic polymer. The polyimide coating on the outer wall of the capillary was removed in the scanning detection region to enable fluorescence scanning detection. A contactless conductivity detector was installed 156 mm from the cathode end of the capillary. The amphoteric carrier solution used for separation, mixed with the sample, consisted of 2.5% (v / v) Pharmalyte 3-10, 0.1% (v / v) acetic acid, and 0.35% (v / v) tetramethylethylenediamine. The anolyte was 0.1 M phosphoric acid, and the catholyte was 1 M sodium hydroxide. The protein sample solution and a solution of a known isoelectric point standard (pI marker) were added to the amphoteric carrier solution to form the CIEF separation solution. A capillary filled with physiological phosphate buffer solution (PBS) was injected with the CIEF separation solution from the anode side at 50 kPa for 1 minute, followed by the anolyte solution at 50 kPa from the anode side until an increase in electrical conductivity due to the anolyte was detected. The capillary end was then immersed in the anolyte and catholyte solutions, and the current was applied at 5 kV for 1.5 minutes, then at 7 kV for 1.5 minutes, after which focusing was performed at 10 kV. During focusing at 10 kV, the anode side was scanned at a speed of 1 mm / s over a range of 115 mm to 35 mm from the cathode end, once every 1.5 minutes.

[0048] A schematic diagram of the fluorescence detection system is shown in Figure 1. A 280 nm LED was used as the excitation light source for fluorescence detection, and the light was irradiated onto the capillary through a 280 nm bandpass filter. The collected fluorescence was then split into a beam splitter, with 70% of the light passing through a 340 nm bandpass filter and detected by a photomultiplier tube, and 30% of that passing through a 575 nm bandpass filter and detected by another photomultiplier tube. The 340 nm fluorescence was derived from tryptophan (Trp) contained in the sample protein and the pI marker, and the 575 nm fluorescence was derived from tetramethylrhodamine, a fluorescent dye contained in the pI marker.

[0049] (2) Types of pI markers and preparation of CIEF separation solution The pI markers used were four tryptophan-containing peptidic pI markers and 12 tetramethylrhodamine-labeled tryptophan-free peptidic pI markers. These pI markers are shown in Table 1. [Table 1] Note 1) All amino acids except glycine are L-amino acids. Note 2) Cys * indicates a cysteine ​​residue whose SH group is labeled with tetramethylrhodamine. Note 3) Lys(H-Gly) indicates a lysine residue in which the ε-amino group is glycylated.

[0050] Of the four tryptophan-containing pI markers, two are located at almost both ends of the pH gradient from pH 3 to 10, with pIs of 3.38 and 10.17. These are called edge markers (EM). The remaining two tryptophan-containing pI markers are located on either side of the isoelectric point region in which the protein sample cetuximab is distributed, with pIs of 7.00 and 9.50. These are called flanking markers (FM). The edge marker and flanking marker solutions are 1 mM HCl solutions containing two markers at 0.1 mM concentrations, and the final concentrations in the separation solution are 1 μM. The tetramethylrhodamine-labeled pI marker solution contained 12 markers with pIs of 3.64, 3.99, 4.50, 4.99, 5.53, 6.18, 6.86, 7.58, 8.21, 8.77, 9.56, and 10.12, each at a concentration of 2–3 μM. The final concentration in the separation solution was 20–30 nM. The 12 tetramethylrhodamine-labeled pI markers were dispersed throughout the pH gradient and therefore referred to as ubiquitous markers (UM). The CIEF separation solution was prepared by adding 0.2 μL of cetuximab (5 mg / mL), 0.1 μL each of the end marker solution and the side marker solution, or 0.1 μL of the ubiquitous marker solution to 9 μL of amphoteric carrier solution.

[0051] CIEF of cetuximab and pI markers Figure 2 shows the separation results of cetuximab and both end markers and both flanking markers by CIEF. Because these markers contain tryptophan, they were simultaneously detected with cetuximab at a fluorescence wavelength of 340 nm. When determining the isoelectric point of a sample with an unknown isoelectric point using CIEF, it is often assumed that there is a proportional relationship between the detection time (or position) and the isoelectric point of the two isoelectric point markers present on either side of the unknown sample. The isoelectric point of the unknown sample is then determined from the detection time of the peak. In the experiment shown in Figure 2, the cetuximab peaks (C3–C9) and the peaks of the two end markers and the two flanking markers are separated. The advantage of using both end markers is that they can be used in most cases without overlapping with the sample peaks, even when the isoelectric point of the sample is unknown. On the other hand, when the isoelectric point of the sample is roughly known, both flanking markers can be selected to avoid overlap with the sample and separate closer to the sample, which is expected to improve the accuracy of isoelectric point determination.

[0052] Alternatively, peptides labeled with dyes that fluoresce in the visible range can also be used as pI markers. In this case, visible fluorescence detection is required to detect the pI marker, but if the pI marker does not contain tryptophan, it is possible to detect the sample cetuximab and the pI marker independently. In other words, the marker does not affect the separation results of the sample, and the sample does not affect the separation results of the marker. As a result, even if multiple pI markers are added to a sample, the signals from the sample and the markers do not become contaminated. Therefore, even for samples with unknown isoelectric points, it is possible to determine the isoelectric point of the sample by appropriately selecting the most desirable pI marker that is closest to the sample peak.

[0053] Figure 3 shows the results of adding 12 pI markers, which are tryptophan-free peptides labeled with rhodamine, to cetuximab and separating them using CIEF. After irradiating with 280 nm UV light, cetuximab was detected by fluorescence at 340 nm (solid line), and the rhodamine-labeled pI markers by fluorescence at 575 nm (dotted line). As is clear from the figure, there is no mutual interference between the sample signal and the marker signal.

[0054] Next, the isoelectric points of the seven major cetuximab peaks, C3–C9, were determined for each separation run, from the third to eleventh scans after focusing at 10 kV, using both-end markers, both-side markers, and ubiquitous markers. The isoelectric points were determined based on the assumption that there is a proportional relationship between the isoelectric point and detection time between the two markers on either side of the sample peak. For both-end markers and both-side markers, all cetuximab peaks were determined based on the detection times of the two markers. For ubiquitous markers, peak C3 was determined based on the detection times of UM7.58 and UM8.21, peaks C4–C7 were determined based on the detection times of UM8.21 and UM8.77, and peaks C8 and C9 were determined based on the detection times of UM8.77 and UM9.56. The isoelectric point (pI) values ​​of each peak, grouped by marker type, are listed in Table 1. Figure 4 plots the isoelectric points of each peak for each scan run. [Table 2]

[0055] The determined isoelectric points for both the end markers and the side markers gradually decreased with increasing number of scans, i.e., with increasing focusing time. For the C9 peak, the difference in isoelectric point between the fourth and eleventh scans was 0.24 pH units smaller for both the end markers and the side markers. Even for the most acidic C3 peak, where the change was smaller, decreases of 0.15 and 0.11 were observed for the respective markers. This change in the determined isoelectric point with focusing time is likely due to the deviation from the proportional relationship between position in the capillary and pH increasing with increasing focusing time. Because the focusing time required varies depending on the protein, the change in the determined isoelectric point due to differences in focusing time is problematic. Furthermore, uniformly long focusing times reduce analytical efficiency.

[0056] On the other hand, the change in the ubiquitous marker with increasing focusing time was significantly smaller, less than ±0.01 for C3–C8, and even C9, which showed a relatively large change, showed a decrease of 0.05. The small change in the isoelectric point values ​​determined using the ubiquitous marker is also evident from the average CV values ​​for seven peaks, which represent the variation in the isoelectric point values ​​determined for each peak every eight scans. That is, the average CV values ​​were 0.81% for the end marker, 0.83% for the side marker, and 0.08% for the ubiquitous marker. This indicates that the use of the ubiquitous marker allows for highly accurate isoelectric point determination regardless of the focusing time.

[0057] These results demonstrate that using multiple markers distributed throughout the pH gradient allows for more accurate isoelectric point determination, regardless of focusing time, compared with determining the isoelectric point of a sample using two markers at either end of the pH gradient or on both sides of the sample. Tryptophan-containing markers have the advantage of being able to detect both the sample and the marker at a single fluorescence wavelength. However, when used as a ubiquitous marker, the sample and marker peaks may overlap, making peak identification difficult. Furthermore, for samples with multiple peaks with different isoelectric points, such as cetuximab in this example, a separate separation of the marker-free sample alone is required to determine the composition ratio of each peak.

[0058] In contrast, in the case of a marker that does not contain tryptophan, the marker must be detected at a different fluorescence wavelength from the sample. However, because the sample and marker can be detected independently, overlap between the sample peak and the marker peak is not a problem. Therefore, by simply adding a ubiquitous marker to the sample and performing a single separation, it is possible to simultaneously determine both the isoelectric point and the peak composition ratio with high accuracy. Furthermore, the method disclosed herein can determine the isoelectric point with high accuracy by using a pI marker as an internal standard as described above. This allows for easy determination of the isoelectric point of an analyte, not only as a separation pattern under specific analytical conditions, but also as a physical property value, i.e., the isoelectric point information provided in this way, which can be commonly used by multiple practitioners working with the same substance as the analyte, regardless of laboratory conditions, and is therefore highly valuable.

[0059] Furthermore, tetramethylrhodamine, the ubiquitous marker used in this experiment, has an excitation maximum at approximately 500 nm, but in this example, excitation was performed with light at 280 nm. Although 280 nm illumination light is suitable for the fluorescent detection of tryptophan, it is not suitable for the fluorescent detection of tetramethylrhodamine. However, the pI marker could be detected without any problems. The ability to easily measure using a single light source is advantageous.

[0060] Example 2: Approximate formula for determining pI values In Example 1, a linear approximation (linear approximation) was performed for adjacent pI markers, but other approximations may also be used. Because the pH gradient in isoelectric focusing may not actually be linear, curve approximation can be used to determine the isoelectric point more accurately.

[0061] Capillary isoelectric focusing was performed by mixing 12 types of pI markers with the sample in the same manner as in Example 1. The results of detecting the pI markers using fluorescence at 575 nm are shown in FIG.

[0062] Based on this result, we performed a third-order approximation using the least squares method. pH = -2.7227 × t 3 + 137.38×t2 - 2303.9×t + 12850 The following equation was obtained. The graph reflecting this is shown in Figure 6.

[0063] For such a more sophisticated approximation formula, it is preferable to measure the distribution of many types of pI markers. Here, an example of a third-order approximation is shown, but it will be understood by those skilled in the art that an approximation of a different order or an approximation other than a polynomial may also be used.

[0064] (Note) While the present disclosure has been illustrated by preferred embodiments thereof, it is understood that the scope of the present invention is to be construed solely in terms of the claims that follow. It is understood that the patents, patent applications, and other documents cited herein are incorporated by reference into this specification in their entirety as if the contents themselves were specifically set forth herein. [Industrial Applicability]

[0065] The present disclosure provides an electrophoretic analysis method using an internal standard, which can be used in the analysis of various biomolecules, such as proteins. [Explanation of symbols]

[0066] 1: Capillary 2: Excitation light 3: Fluorescence 4: Lens 5: Beam splitter 6: 280nm bandpass filter 7: 340nm bandpass filter 8: 575nm bandpass filter 9:Light source 10: Photodetector

Claims

1. 1. A method for measuring an analyte by electrophoresis, comprising: mixing a sample containing the analyte with a standard to prepare a mixture; subjecting the mixture to electrophoresis; detecting optical signals from the analyte and the standard; determining the analyte based on the optical signal; A method comprising:

2. 10. The method of claim 1 for determining the isoelectric point of an analyte, wherein the standard has a known isoelectric point.

3. 3. The method of claim 2, wherein the standards comprise a plurality of standards having known different isoelectric points ranging from 3 to 11.

4. 4. The method of claim 3, wherein the plurality of standards comprises pairs of first and second standards having an isoelectric point difference of less than two.

5. 10. The method of claim 1 for determining the molecular weight of an analyte, wherein the standard has a known molecular weight.

6. 6. The method of claim 5, wherein the standards comprise a plurality of standards having different known molecular weights ranging from 5 kDa to 1000 kDa.

7. 7. The method of claim 6, wherein the plurality of standards comprises pairs of first and second standards that differ in molecular weight by no more than three-fold.

8. The method according to claim 1, further comprising deriving an equation expressing the correlation between the isoelectric point and the detection time or position, or between the molecular weight and the detection time or position, based on the optical signals derived from the plurality of standard substances.

9. The method of claim 8, further comprising converting the detection time or detection position into a pH value or a molecular weight based on the formula.

10. The method of claim 1 , wherein the standard is a tryptophan-free peptide or protein.

11. The method of claim 1 , wherein the standard has a dye label.

12. 2. The method of claim 1, wherein the step of detecting an optical signal comprises irradiating with light having two wavelengths, a wavelength of about 280 nm and a wavelength suitable for detecting the dye label.

13. The method of claim 1 , wherein the standard is labeled with rhodamine.

14. The method of claim 1 , wherein the step of detecting the optical signal comprises irradiating with light having a single wavelength.

15. 15. The method of claim 14, wherein the single wavelength is about 280 nm.

16. The method of claim 1 , wherein detecting an optical signal comprises detecting light of a different wavelength than the illuminating light.

17. The method of claim 1 , wherein the step of detecting an optical signal comprises detecting light at two wavelengths.

18. The method of claim 1 , wherein the standards include at least five standards.

19. The method of claim 1 , wherein a plurality of standards are mixed with the sample at different concentrations.

20. The method of claim 1 , wherein the electrophoresis is capillary electrophoresis.

21. The method of claim 1 , wherein the analyte comprises a peptide and / or a protein.

22. 1. An apparatus for measuring an analyte by electrophoresis, comprising: a channel and electrodes for electrophoresis; a light source for illuminating at least a portion of the flow path; a photodetector that receives light from the flow path; Equipped with the flow path includes an inlet for receiving a sample containing the analyte; Device.

23. 23. The apparatus of claim 22, wherein the light source is configured to illuminate the flow path with light of a single wavelength.

24. 23. The apparatus of claim 22, wherein the light source is configured to illuminate the flow path with light having a wavelength of about 280 nm.

25. 23. The apparatus of claim 22, wherein the photodetector is configured to detect light of a wavelength different from the illumination light of the light source.

26. 23. The apparatus of claim 22, wherein the photodetector is configured to detect light at two wavelengths.

27. 27. The apparatus of claim 26, wherein the photodetector comprises a dichroic mirror, a dichroic filter, or a beam splitter.

28. 27. The apparatus of claim 26, wherein the photodetector is configured to detect light at a wavelength of about 340 nm and light at a wavelength of about 575 nm.

29. 1. A composition for determining the isoelectric point of an analyte, comprising a plurality of standards, the composition is for mixing with a sample containing an analyte and then subjecting it to isoelectric focusing; The plurality of standards have known isoelectric points that are different from each other. composition.

30. 1. A composition for determining the molecular weight of an analyte, comprising a plurality of standards, the composition is for mixing with a sample containing an analyte and then subjecting it to electrophoresis; The plurality of standards have known molecular weights that are different from each other. composition.

31. 1. A method for producing a standard for determining the molecular weight of an analyte, comprising: Identifying a gene encoding a protein of known molecular weight; Identifying a portion encoding all tryptophans in the base sequence encoding the protein in the gene; a step of replacing all of the tryptophan-encoding portions with base sequences encoding amino acids other than tryptophan to obtain a modified gene; Obtaining the protein produced using the modified gene; and A step of binding a labeling dye to the protein to form a conjugate and obtaining the conjugate as a standard substance. A method comprising:

Images