JP5973677B2 - Protein quantification method using mass spectrometry - Google Patents

Description

  The present invention relates to a method for quantifying proteins in a sample, and specifically, a high performance liquid chromatograph combining high performance liquid chromatography (hereinafter, HPLC) and mass spectrometry (hereinafter, MS). The present invention relates to a protein quantification method by mass spectrometry (HPLC-MS).

  The present invention also relates to a sample pretreatment method for high performance liquid chromatograph mass spectrometry.

  In recent years, in certain diseases, it has become clear that the increase or decrease in the expression of specific proteins is associated with the disease. For example, in cancer, overexpression of a specific oncogene causes the disease. There are known cases. In neurodegenerative diseases such as Alzheimer's disease, overexpression / accumulation of specific proteins is a cause of onset. One option for the treatment of these diseases is the administration of antibodies that can specifically bind to the protein. For example, in breast cancer, overexpression of HER2 protein, which is an expression product of HER2 / neu oncogene, may be observed. As a specific treatment for this, trastuzumab (trastuzumab, trade name Herceptin ( Herceptin®) has been developed and applied clinically.

  On the other hand, since the efficacy of a drug can be related to its blood concentration in a patient, setting of an optimal dose from the blood concentration is expected. Conventionally, ligand binding assays (hereinafter referred to as LBS) and enzyme-linked immunosorbent assays (hereinafter referred to as ELISA) have been widely used to measure the concentration of protein drugs in the blood. The above trastuzumab Quantification by ELISA is also possible.

  In recent years, it has become possible to measure the concentration of protein pharmaceuticals in blood by using a combination of high performance liquid chromatography (HPLC) and mass spectrometry (MS). In this method, sample information is first separated by high performance liquid chromatography, and then specific sequence fragments derived from protein drugs are analyzed by mass spectrometry, so that structural information and quantitative analysis can be performed simultaneously. It is possible (Patent Document 1, Non-Patent Document 1-2). However, this method has not yet been put into practical use in clinical practice.

  Sample preparation is very important in order to generate specific sequence fragments from the protein drug to be analyzed. Fragmentation is necessary to analyze protein drugs that are macromolecules. In order to efficiently perform fragmentation using protease treatment, prior to protease treatment, heat, pH, pressure, and denaturation are used as conventional methods. Modification using chemicals, dithiothreitol (DTT), 2-mercaptoethanol (2-ME) and tris (2-carboxyethyl) phosphine hydrochloride for cleaving disulfide bonds Reduction with salt (Tris (2-carboxyethyl) phosphine hydrochloride: TCEP), protection of thiol group, alkylation with 2-iodoacetamide (IAA) to maintain irreversible dissociation Has been done. After that, protease treatment is performed, purification and elution are performed using a desalting column, etc., and then subjected to HPLC, and the separated sample is analyzed in Multiple Reaction Monitoring (MRM) mode. Quantitative analysis is possible. In mass spectrometry, a stable isotope-labeled peptide or the like is generally added to a sample as an internal standard.

Special Table 2010-515020

Journal of pharmaceutical and biomedical analysis.2000 Jan; 21 (6): 1099-128. Journal of pharmaceutical and biomedical analysis. 2008 Feb 13; 46 (3): 449-55.

  The dose of a pharmaceutical is generally calculated based on the patient's body weight, but the pharmacokinetics in the body of each individual patient may vary depending on the drug metabolic ability, health condition, etc. of the patient. Therefore, even if the same amount of protein pharmaceutical is administered, the blood concentration differs depending on the patient, and the therapeutic effect obtained for this may vary. If the blood concentration of the protein drug is lower than the therapeutic concentration range (effective blood concentration), the desired effect cannot be obtained, while if it is higher than the therapeutic concentration range, an adverse event may occur. Therefore, in order to achieve the optimal therapeutic effect for each individual patient, it is important to grasp the pharmacokinetics thereof. If the blood concentration can be quickly and accurately quantified, it is possible to avoid adverse events due to overdose of protein drugs and to adjust the dosage according to patient characteristics. In particular, antibody drugs are expensive, and optimizing the dosing schedule is important from a cost standpoint.

  In LBS and ELISA, which are currently widely used measurement methods, it is essential to identify antigens and produce antibodies for each protein drug. However, since fluctuations in antibody quality lead to analysis errors, high-quality antibodies are required. However, it takes an enormous amount of time to produce the antibodies, and the structure of the protein drug itself cannot be confirmed. Furthermore, it is known that the standard curve is a sigmoid curve, which makes it difficult to accurately measure the concentration near the detection range limit, and in principle, it also has an abnormal value, making verification difficult. .

  Even in the quantitative method combining HPLC and MS, there are many pre-processing steps and a long time is required, so the processing time for measuring a large amount of sample becomes enormous, while the analysis result of blood concentration is reflected in the treatment. It was impossible to respond quickly. Furthermore, since there is an inhibitory effect on a large amount of protein present in the blood, taking into account the loss during the pretreatment of the protein drug, the amount of blood collected at one time becomes large, and the invasiveness can be increased.

  In addition, as with the preparation of antibodies, it takes time and money to prepare an internal standard according to the target. However, in some cases, it is preferable to use an internal standard in order to correct errors during operation and obtain highly reproducible measurement results.

  The problem to be solved by the present invention is to provide a method for detecting a fragment of a specific sequence derived from a protein drug in order to easily and rapidly measure the concentration of the protein drug in the blood.

  The present inventors first examined a sample pretreatment method for analysis by HPLC-MS, and found that some steps could be further shortened. Further, when performing analysis in the MRM mode, it has been found that a highly accurate analysis can be performed without using a conventional internal standard, and the present invention has been completed.

  That is, the aspects of the present invention are as follows.

1. A method for quantifying a protein in a biological sample by high performance liquid chromatography mass spectrometry, wherein the protein has an amino acid sequence specific for the protein, and other endogenous that is different from the protein to be measured The peak area value for the peptide having the specific amino acid sequence is corrected by using the peak area value obtained by mass spectrometry for the peptide having both the amino acid sequence and the protein and the common amino acid sequence. The above method, comprising:

2. 2. The method according to 1 above, wherein the protein in the biological sample is a protein administered to a subject.

3. 3. The method according to 1 or 2 above, wherein the protein is an antibody.

4). 4. The method according to 3 above, wherein the antibody is a monoclonal antibody.

5. 5. The method according to 3 or 4 above, wherein the antibody is a humanized antibody.

6). It is characterized by simultaneously analyzing a peptide having an amino acid sequence specific to a protein in a biological sample and a peptide having an amino acid sequence common to other endogenous proteins in a multiple reaction monitoring (MRM) mode. The method according to any one of 1 to 5 above.

7). A biological sample pretreatment method for use in the method according to any one of 1 to 6 above, wherein the protein in the sample is subjected to denaturation / reduction treatment for 15 minutes or more and protease treatment for 3 to 6 hours. Method.

8). A high-performance liquid that simultaneously quantifies two or more peptides in a sample, with high-performance liquid chromatography to separate peptides derived from proteins in the sample and a mass spectrometer to quantify the separated peptides A chromatographic mass spectrometry system, wherein a peak area value of a fragment generated from a signal derived from a peptide having an amino acid sequence specific to a protein in a sample is shared with another endogenous protein different from the protein A system further comprising a calculation unit that corrects a peak area value of a fragment generated from a signal derived from a peptide consisting of the amino acid sequence.

9. A computer having an input device, a storage device, and a computing device is used to calculate the peak area value of a fragment generated from a signal derived from a peptide having an amino acid sequence specific to a protein in a sample, from other endogenous proteins different from the protein. A program for high-performance liquid chromatograph mass spectrometry, which executes a process of correcting with a peak area value of a fragment generated from a signal derived from a peptide consisting of a common amino acid sequence of the protein.

  Using the method of the present invention, the peak area value of a fragment generated from a signal derived from a peptide of a protein drug-specific sequence obtained by analyzing a protein drug in the MRM mode is derived from a peptide of a sequence common to the endogenous molecule. By correcting with the peak area value of the fragment generated from this signal, a more accurate calibration curve can be created, and it can be detected accurately in a wide concentration range from low concentration to high concentration.

  The method of the present invention can perform the pretreatment in a shorter time than the conventional method. This pretreatment method can be used as a standardized protocol, and can be a basic technology for developing pretreatment equipment that can be installed in the front part of LC-MS.

  In addition, the method of the present invention enables analysis of all protein drugs such as antibody drugs and can be widely applied to clinical applications. By quickly determining the appropriate dose of a protein drug, it is possible to reduce medical costs and to apply to other new protein drugs.

The flowchart of the conventional processing method of the blood concentration measurement of the protein pharmaceutical using mass spectrometry is shown. The result of examining the time required for the reduction treatment is shown as a result of subjecting the sample after the reduction treatment to SDS-PAGE. The comparison of the detection peak intensity by the presence or absence of the alkylation process is shown. It was clear that alkylation treatment was necessary for peptide fragments containing cysteine residues, whereas alkylation treatment was not necessary for peptide fragments not containing cysteine residues. The result of having examined the time required for protease (Trypsin / Lys-c) treatment is shown. The detection intensity was about the same as the treatment for 12 hours in the treatment for 3 to 6 hours. The figure explaining the method of antibody concentration dilution is shown. The calibration curve created by plotting the peak area values of the peptide LLIYSASFLYSGVPSR (SEQ ID NO: 15) (m / z 887.0, 2+> m / z 359.2) obtained at a sample injection amount of 0.0001 to 1 μg is shown. The lower row shows an enlarged view of the low volume region. The peak area value of peptide LLIYSASFLYSGVPSR (SEQ ID NO: 15) (m / z 887.0,2+> m / z 359.2) obtained at a sample injection amount of 0.0001 to 1 μg was expressed as peptide SGTASVVCLLNNFYPR (SEQ ID NO: 18) (m / z 899.5,2 A calibration curve created by plotting the value divided by the peak area value of +> m / z 272.2) is shown. The lower row shows an enlarged view of the low volume region.

  The method of the present invention is a method for quantifying proteins in a biological sample by mass spectrometry using high performance liquid chromatography mass spectrometry. The method of the present invention can also quantify a specific protein present in the body of a subject, but in many cases the protein is a protein administered to the subject.

  A biological sample is a sample taken from a subject in which a specific protein, in particular an administered protein drug, such as an antibody drug, is assumed to be present, such as blood (whole blood or plasma), saliva, It may be a body fluid such as pleural effusion or ascites, a sample derived from a target region tissue, or the like.

  The biological sample is first subjected to pretreatment necessary for removing other impurities unnecessary for quantifying the protein to be analyzed. For the pretreatment, any method known in the art can be used. Examples thereof include centrifugation, various chromatographies, separation by dialysis, denaturation / reduction treatment, alkylation treatment, protease treatment, and the like. At this stage, the sample contains protein administered as a pharmaceutical and protein inherently possessed by the subject, and pretreatment is performed at the same time.

  In the method of the present invention, the subject is a mammal, particularly a human.

  The protein quantified by the method of the present invention is not particularly limited, but has an amino acid sequence specific to the protein and an amino acid sequence common to other endogenous proteins different from the protein to be measured. It must be a thing. Prior to the analysis, a peptide having a specific amino acid sequence and a peptide having a common amino acid sequence are obtained by protein protease treatment. Therefore, the protein itself to be quantified may be a protein consisting of only amino acids, but may be any protein as long as an appropriate peptide can be obtained by pretreatment, and modifications necessary for formulation, for example, modification with a protecting group. It may be one that has been prepared or may have a sugar chain attached.

  All peptides used for analysis in the present invention are composed of amino acid sequences contained in proteins in biological samples. The peptide for quantification of the protein contains an amino acid sequence specific to the protein.

  In the method of the present invention, a preferred protein is an antibody. Since an antibody has a variable region for binding to a specific antigen and a constant region, the method of the present invention can be performed using these regions. In the variable region, it is preferable to quantify the peptide comprising the amino acid sequence of the complementarity determining region (CDR), which is preferably a hypervariable region in direct contact with the antigen. The antibody is preferably an immunoglobulin (IgG) monoclonal antibody. As is well known in the art, an IgG antibody is composed of two heavy chains and two light chains, each of which has a variable region for specifically binding an antigen. .

  When the protein to be measured is an antibody, the specific amino acid sequence is an amino acid sequence derived from the variable region of the antibody. In particular, the amino acid sequence in CDR is preferable. In that case, it is sufficient that the peptide fragment to be analyzed contains all or part of the CDR. For high-accuracy analysis by high performance liquid chromatography / mass spectrometry, one or more, two or more, 3 As described above, 4 or more, 5 or more, 6 or more, 7 or more, or 8 or more residues may be included in the peptide. On the other hand, the amino acid sequence common to the endogenous protein can be an amino acid sequence derived from the constant region. The specific amino acid sequence and the common amino acid sequence may be included in the amino acid sequence of the antibody to be measured, and may be derived from either a heavy chain or a light chain, respectively.

  In addition, human antibodies, non-human antibodies, chimeric antibodies, and humanized antibodies can be used as antibodies. When the subject is a human, the antibodies are human antibodies, chimeric antibodies, or humanized antibodies that are administered. It is particularly preferred to use humanized antibodies in order to avoid subject rejection of the subject. The chimeric antibody has a human-derived constant region and a non-human-derived variable region. The humanized antibody has a human-derived antibody framework and a non-human-derived CDR.

  In the method of the present invention, the peptide to be subjected to mass spectrometry is not particularly limited, but has an amino acid length of about 5 to 50, preferably about 6 to 40 in consideration of analysis sensitivity and accuracy. is there. Therefore, proteins that are pharmaceuticals are made to have an appropriate length by protease treatment before mass spectrometry. Examples of the protease used for the treatment include trypsin, Lysc, GluC, AspN, chymotrypsin and the like.

  The case where quantification of HER2-specific monoclonal antibody trastuzumab (trade name Herceptin (registered trademark)) is carried out by the method of the present invention will be specifically described.

  Trastuzumab is a monoclonal IgG antibody whose heavy chain consists of 449 amino acids (SEQ ID NO: 1) and whose light chain consists of 214 amino acids (SEQ ID NO: 2). In the heavy chain amino acid sequence shown in SEQ ID NO: 1, the amino acid sequences corresponding to CDR1-3 are residues 26-35 (SEQ ID NO: 3), residues 50-66 (SEQ ID NO: 4), and residues 99-116, respectively. This is a sequence consisting of (SEQ ID NO: 5). On the other hand, in the light chain amino acid sequence shown in SEQ ID NO: 2, the amino acid sequences corresponding to CDR1-3 are residues 24-34 (SEQ ID NO: 6), residues 50-56 (SEQ ID NO: 7), residues 89, respectively. This is a sequence consisting of -97 (SEQ ID NO: 8). Therefore, when quantifying trastuzumab by the method of the present invention, a target peptide may be prepared so as to include a part or all of these CDR sequences.

Table 1 below shows examples of target peptides contained in heavy chains and light chains obtained by protease (Trypsin / Lys-C Mix) treatment, respectively. It is considered that peptides H1 to H5 (SEQ ID NOs: 9-13) can be used for heavy chain CDR1-3 and peptides L1-L3 (SEQ ID NOs: 14-16) can be used for light chain CDR1-3. In the table, the underlined sequence parts in the heavy chain and light chain amino acid sequences correspond to the CDR sequences, and the sequence parts shown in bold are the sequences corresponding to the target peptides (candidates). Those skilled in the art can understand that different peptides can be obtained when different proteases are used, and in that case, a suitable target peptide can be envisaged for the method of the present invention. Similarly, when the purpose is to quantify other antibodies, a target peptide can be obtained in the same manner.

  The method of the present invention is particularly characterized by using high performance liquid chromatograph mass spectrometry.

  High performance liquid chromatography is a technique known in the art, and those skilled in the art can appropriately use an apparatus that can be used in the method of the present invention. HPLC can be performed using, for example, a Nexera UHPLC / HPLC system (Shimadzu Corporation).

  In the method of the present invention, a peptide having the specific amino acid sequence and a peptide having the common amino acid sequence can be simultaneously analyzed in a multiple reaction monitoring (MRM) mode from a sample separated by HPLC. . Mass spectrometry methods by MRM mode are known in the art. In the method of the present invention, mass spectrometry can be performed using, for example, LCMS-8080 (Shimadzu Corporation) in combination with the above HPLC.

  After the analysis, a peak area value derived from a peptide having an amino acid sequence specific to the target protein is corrected with a peak area value derived from a peptide having a sequence in common with another endogenous protein different from the target protein. Specifically, a numerical value obtained by dividing a peak area value derived from a peptide having an amino acid sequence specific to the target protein by a peak area value derived from a peptide having a sequence common to another endogenous protein different from the target protein To create a calibration curve.

By this correction, a multiple correlation coefficient R ² of 0.9 or more can be ensured when the amount of the target protein in the sample is in the range of 0.0001 to 1 μg. In particular, when the amount of protein is very small, for example, even when the injection amount is in the range of 0.0001 to 0.001 μg, it can be quantified with very high accuracy.

  The inventors have also found that the time required for pretreatment prior to mass spectrometry can be shortened.

  FIG. 1 shows the procedure of a conventional HPLC-MS method for quantifying an IgG antibody drug using plasma as a sample.

  Plasma is separated from blood collected from the subject, and then added to a Melon gel (Thermo Fisher Scientific Co., Ltd.) that adsorbs other than antibodies (immunoglobulin G, IgG), for example. Minutes are taken as an IgG pool. This IgG pool is subjected to denaturation / reduction / alkylation treatment, and the IgG cleaved into the heavy chain and the light chain is decomposed into a peptide by protease treatment with a protease treatment. After desalting, it is subjected to HPLC, separated, and analyzed in MRM mode (LC-MS / MS) (FIG. 1).

The present inventors have found that the time can be shortened for each step of the above-described pretreatment as compared with the conventional method. For example, protein denaturation / reduction treatment that previously required about 90 minutes can give good results if it takes 15 minutes or longer, and protease treatment, which was performed in about 12 hours, is performed within 6 hours. Confirmed that it can. This treatment can be performed for a very short time of about several minutes, for example, 1-2 minutes, but is preferably 15 minutes or longer, 30 minutes or longer, 1 hour or longer, 2 hours or longer, or 3 hours or longer. It was also found that the alkylation treatment that conventionally required about 40 minutes can be omitted by appropriately selecting the peptide to be analyzed. As a result, when the alkylation treatment was unnecessary, a reduction of more than 8 hours was achieved as compared with the conventional pretreatment method (Table 2). This is very advantageous when a large amount of specimens must be analyzed at the same time, or when it is desired to immediately feed back the analysis results of a sample derived from a patient to be used for subsequent treatment.

  Therefore, the method of the present invention further varies the amount of protein to be subsequently administered, for example, increases the amount of protein to be administered, based on the measured blood concentration of the protein drug in the subject obtained by the above method. Or a reducing step can be included.

  The present invention also provides two or more peptides in a sample, comprising high performance liquid chromatography for separating peptides derived from proteins in the sample, and a mass spectrometer for quantifying the separated peptides. A high-performance liquid chromatograph mass spectrometry system for simultaneous quantification, wherein a peak area value of a fragment generated from a signal derived from a peptide having an amino acid sequence specific to a protein in a sample is different from that of the other endogenous protein. And a calculation unit that corrects with a peak area value of a fragment generated from a signal derived from a peptide consisting of the amino acid sequence of the protein in common. Specifically, the mass spectrometer includes a sample introduction unit, an ionization chamber, an analysis unit, a detection unit, a recording unit, and the like. Examples of ionization methods include chemical ionization (CI), field desorption (FD), fast atom bombardment (FAB), matrix-assisted laser desorption ionization (MALDI), and electrospray ionization (ESI). That's fine As the analysis unit, a double-focusing mass spectrometer, a quadrupole mass spectrometer, a time-of-flight mass spectrometer, a Fourier transform ion sacrotron resonance mass spectrometer, or the like is used.

  The present invention further provides a computer equipped with an input device, a storage device, and an arithmetic device in which a peak area value of a fragment generated from a signal derived from a peptide having an amino acid sequence specific to a protein in a sample is different from that of the protein. There is provided a program for high performance liquid chromatography mass spectrometry which executes a process of correcting with a peak area value of a fragment generated from a signal derived from a peptide consisting of an amino acid sequence of the protein in common with the endogenous protein of.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

[Example 1]
Examination of pretreatment method for high performance liquid chromatography mass spectrometry In order to perform high performance liquid chromatography mass spectrometry of HER2-specific monoclonal antibody trastuzumab in the blood of subjects, the following pretreatment method was examined. .

1. Acquisition of antibody-containing sample After collecting blood from a subject, plasma is separated and acquired, and then plasma is added to Melon gel (Thermo Fisher Scientific Co., Ltd.) that adsorbs other than antibodies (Immunoglobulin G and IgG) and centrifuged. Fractions containing IgG were obtained by treatment and used as an IgG pool.

2. Denaturation / reduction treatment The obtained antibody-containing sample was subjected to denaturation / reduction treatment. Comparative evaluation was carried out using sodium lauryl sulfate (hereinafter referred to as SDS) -polyacrylamide gel electrophoresis (hereinafter referred to as PAGE). Treatment time of trastuzumab (20mg / ml) with 8M Urea, 25mM tris (2-carboxyethyl) phosphine hydrochloride (TCEP) was examined (90min, 60min, 30min, 15min, 5min). By treatment at room temperature (25 ° C.) for 15 minutes, the appearance of bands was confirmed at the positions of the heavy chain and light chain sizes generated by the cleavage of SS bonds (FIG. 2). As a result, it was confirmed that the reduction treatment was sufficient for 15 minutes or longer.

3. Alkylation A comparative evaluation was conducted on the necessity of alkylation treatment in the pretreatment. After denaturation and reduction treatment of trastuzumab (20 mg / ml), alkylation treatment (+) and alkylation treatment (-), and fragment treatment with protease (Trypsin / Lys-C Mix, Mass Spec Grade, Promega) And performed mass spectrometry using MRM mode using peptides derived from trastuzumab (AEDTAVYYCSR (SEQ ID NO: 17) and LLIYSASFLYSGVPSR (SEQ ID NO: 15)), and compared and evaluated the area values of the peaks generated from the respective fragments. went. The results are shown in FIG. The peptide used here was used only for the study on the necessity of alkylation treatment and the like, and is independent of whether it is specific to trastuzumab in the present invention.

  As a result, AEDTVYYCSR (SEQ ID NO: 17) (m / z 1278.6) containing a cysteine residue was detected as a divalent ion (m / z 667.8) of +57 Da (m / z 1335.6), and the product peak was m / z 847.4. 748.3, 585.2. On the other hand, LLIYSASFLYSGVPSR (SEQ ID NO: 15) (m / z 1773.0) was detected as a divalent ion (m / z 887.0), and the generated peaks were m / z 1183.6, 1112.6, 1025.5, and 359.2. When fragments containing cysteine residues in the sequence were used, the detection intensity was higher when alkylation was performed, and better results were obtained when alkylation was performed (left figure). When using a fragment that does not contain a cysteine residue in the sequence, the detection intensity due to the presence or absence of the alkylation treatment was at the same level (right figure), and it was confirmed that the alkylation treatment was unnecessary.

4). Protease treatment The digestion time for protease treatment was determined by mixing Trypsin / Lys-C at a ratio of protein: protease = 25: 1 (w / w) to the sample after the conventional pretreatment, and various times at 37 ° C ( 12 hours, 6 hours, 3 hours, 1 hour, 0.5 hours, and 0.25 hours), and mass spectrometry results by peptide degradation were compared. As a result, the detection intensity equivalent to the treatment for 12 hours was obtained with the protease treatment for 3 to 6 hours (FIG. 4).

[Example 2]
1. Quantification of trastuzumab in sample Using trastuzumab (Herceptin (registered trademark) 20 mg / ml) and human IgG (PURIFIED HUMAN IGG, Cappel) (20 mg / ml), trastuzumab was diluted 2-fold with human IgG, 10-fold, A 20-fold dilution, a 100-fold dilution, a 200-fold dilution, a 1000-fold dilution, a 2000-fold dilution, and a 10000-fold dilution and additional dilution series were prepared and used as samples (see FIG. 5).

2. High performance liquid chromatography (HPLC)
5 μl of each diluted sample was subjected to denaturation / reduction treatment at room temperature (25 ° C.) for 15 minutes in an 8M urea 43.75 μl, 25 mM TCEP (final concentration) environment, and then 80 mM iodoacetamide (IAA, final concentration) was added. The alkylation was carried out in the dark at room temperature (25 ° C.) for 40 minutes. Next, dilute the final concentration of urea to 1 M with 50 mM Tris-HCl (pH 8.0) and mix trypsin / Lys-C at a ratio of protein: protease = 25: 1 (w / w). Protease treatment at 37 ° C. for 6 hours was performed. Thereafter, desalting was performed, and elution was repeated twice with 50 μl of 70% acetonitrile / 0.1% formic acid (Monospin-C18, GL Sciences Inc.), and the eluted sample was diluted 2-fold with 100 μl of 0.1% formic acid. 4 μl of the pretreated sample was applied to the LC section. Mobile phase A: 0.1% formic acid, mobile phase B: 0.1% formic acid / 70% acetonitrile, 0.01 min 2% B, 0.01 min to 10.00 min 95% B, 10.00 min to 12.50 min 95% B, 12.50 min 2% B, 12.5 min Separation was carried out under the conditions of 2% B for 15.00 minutes, a column temperature of 50 ° C., and a flow rate of 0.4 ml / min. HPLC was performed with a Nexera UHPLC / HPLC system (Shimadzu Corporation).

3. Mass spectrometry (MS)
Samples separated by HPLC are continuously introduced into the MS part, and in MRM mode, the peak area value of the fragment generated from the signal derived from the peptide of the trastuzumab-specific amino acid sequence (LLIYSASFLYSGVPSR (SEQ ID NO: 15)), and Quantitative analysis was performed based on the peak area value of the fragment generated from the peptide-derived signal of the common amino acid sequence (SGTASVVCLLNNFYPR (SEQ ID NO: 18)) with endogenous human IgG. MS was performed in MRM mode with QTRAP 4500 (AB SCIEX).

When a calibration curve is created from the acquired data, even when correction by the method of the present invention is not performed, the multiple correlation coefficient R ² = 0.9877 and the injection amount in the range of 0.0001 to 0.001 μg in the range of trastuzumab injection amount of 0.0001 to 1 μg. Even so, a calibration curve could be created with a high accuracy of R ² = 0.9983. Furthermore, correction is performed by dividing the area value obtained from the trastuzumab-specific amino acid sequence (LLIYSASFLYSGVPSR (SEQ ID NO: 15)) by the area value obtained from the amino acid sequence common to the endogenous antibody (SGTASVVCLLNNFYPR (SEQ ID NO: 18)). When implemented, the calibration curve of LLIYSASFLYSGVPSR (SEQ ID NO: 15) is a wide variety of sample injection amount 0.0001～1Myug, next R ² = 0.993 than R ² = 0.9877, linearity is further improved (Figs. 6 and 7).

[Example 3]
Various peptides derived from trastuzumab Table 3 shows examples of peptide fragments that ensure linearity (R ² > 0.9) in preparing a calibration curve using peptides having various specific amino acid sequences derived from trastuzumab. For both peptides, the accuracy of R ² > 0.9 was achieved by the method of the present invention.

  Since the blood concentration of a protein drug can be easily and rapidly quantified, it is possible to avoid an adverse event caused by overdose of the protein drug and to adjust the dose according to the characteristics of the patient. Previously, the dose was converted based on the patient's body weight, but information on the appropriate dose to each patient can be easily obtained, reducing medical costs and other newly emerging proteins Application to pharmaceuticals is possible, and a ripple effect can be expected.

  All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

Claims (9)

  A method for quantifying a protein in a biological sample by high performance liquid chromatography mass spectrometry, wherein the protein has an amino acid sequence specific for the protein, and other endogenous that is different from the protein to be measured The peak area value for the peptide having the specific amino acid sequence is corrected by using the peak area value obtained by mass spectrometry for the peptide having both the amino acid sequence and the protein and the common amino acid sequence. The above method, comprising:   The method of claim 1, wherein the protein in the biological sample is a protein administered to a subject.   The method according to claim 1 or 2, wherein the protein is an antibody.   4. The method of claim 3, wherein the antibody is a monoclonal antibody.   The method according to claim 3 or 4, wherein the antibody is a humanized antibody.   It is characterized by simultaneously analyzing a peptide having an amino acid sequence specific to a protein in a biological sample and a peptide having an amino acid sequence common to other endogenous proteins in a multiple reaction monitoring (MRM) mode. The method of any one of Claims 1-5.   A biological sample pretreatment method used in the method according to any one of claims 1 to 6, wherein the protein in the sample is subjected to denaturation / reduction treatment for 15 minutes or more and protease treatment for 3 to 6 hours. , Pre-processing method.   A high-performance liquid that simultaneously quantifies two or more peptides in a sample, with high-performance liquid chromatography to separate peptides derived from proteins in the sample and a mass spectrometer to quantify the separated peptides A chromatographic mass spectrometry system, wherein a peak area value of a fragment generated from a signal derived from a peptide having an amino acid sequence specific to a protein in a sample is shared with another endogenous protein different from the protein A system further comprising a calculation unit that corrects a peak area value of a fragment generated from a signal derived from a peptide consisting of the amino acid sequence.   A computer having an input device, a storage device, and an arithmetic device has a peak area value derived from a peptide having an amino acid sequence specific to the protein in the sample, and the amino acid of the protein in common with another endogenous protein different from the protein. A program for high performance liquid chromatograph mass spectrometry that executes a process of correcting with a peak area value derived from a peptide consisting of a sequence.

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