Method for measuring concentration of monoclonal protein in body fluid

Abstract

The present invention relates to a method for measuring the concentration of a monoclonal protein in a biological sample, and a method for diagnosing plasma cell disorders using same. The present invention can provide a quantification result with significantly improved accuracy and reproducibility compared to the prior art quantifying M-protein by mass spectrometry, by estimating an M-protein peak ratio on the basis of a region connecting a monoclonal peak on a polyclonal light chain spectrum and tangential skim points located on respective sides thereof. Therefore, even a trace amount of monoclonal protein can be detected with high reliability by non-invasive mass spectrometry using a body fluid sample, and thus early plasma cell disorders that have not yet progressed to multiple myeloma can also be diagnosed, whereby the present invention can be usefully employed to early establish a treatment strategy and improve the survival rate of patients through same.

Description

Method for measuring the concentration of monoclonal proteins in body fluids

[0001] The present invention relates to a method for accurately quantifying monoclonal proteins (M-proteins) in biological samples using mass spectrometry, specifically MALDI-TOF mass spectrometry.

[0002]

[0003] Multiple myeloma is a type of blood cancer caused by the abnormal proliferation of plasma cells within the bone marrow. Plasma cells are a type of white blood cell that produces antibodies; normally, they play a vital role in the immune system by generating antibodies to protect the body from infection. However, abnormally proliferating plasma cells overproduce monoclonal antibodies (M-proteins), disrupting the protein balance in the blood. Furthermore, this inhibits the production of normal blood cells and leads to problems in various organs, such as bone destruction, weakened immunity, and kidney damage.

[0004] The detection and analysis of M-proteins are critical for the diagnosis, disease monitoring, and evaluation of treatment responsiveness of multiple myeloma. To this end, various tests are utilized, including serum and urine protein electrophoresis (SPEP and UPEP), serum and urine immunofixation electrophoresis (IFE), serum free light chain (FLC) testing, immunoglobulin quantification, and Hevylite™ testing, among other combinations used to measure M-proteins. Among these, IFE is a key technology for identifying and analyzing the types of M-proteins, and it can distinguish between the types of immunoglobulins (IgG, IgA, etc.) and light chains (κ, λ) of the M-protein. However, IFE has several disadvantages, such as difficulty in accurate interpretation due to low resolution, a high likelihood of monoclonal antibody interference that distorts test results because therapeutic monoclonal antibodies have characteristics similar to IgG / κ-type M-proteins, inability to provide quantitative results despite being able to distinguish M-protein types, and the necessity of invasive testing for bone marrow-based minimal residual disease (MRD) analysis due to low detection sensitivity.

[0005] Testing methods utilizing MALDI-TOF mass spectrometry are attracting attention as a more precise and efficient diagnostic approach capable of overcoming the shortcomings of IFE. This method can accurately confirm the presence and type of M-protein through MALDI-TOF spectral patterns and can distinguish therapeutic antibodies identical to M-protein based on mass values, thereby resolving the interference issues associated with monoclonal antibody therapies. Furthermore, by quantifying M-protein, it not only enables the analysis of the Minimum Residual Disease (MRD) status but also offers the advantage of non-invasive diagnosis and monitoring using only blood samples, replacing bone marrow biopsy.

[0006] Accordingly, the inventors intended to develop an efficient analytical method for quantifying patient-specific M-proteins in multiple myeloma patients by utilizing MALDI-TOF, which has various advantages.

[0007]

[0008] Throughout this specification, numerous papers and patent documents are referenced and cited. The disclosures of the cited papers and patent documents are incorporated by reference into this specification in their entirety to more clearly explain the state of the art to which the present invention pertains and the content of the present invention.

[0009]

[0010] The inventors have made diligent research efforts to develop an efficient diagnostic system capable of more accurately predicting the concentration of monoclonal antibodies (M-proteins), which are diagnostic markers for various plasma cell diseases, within a sample. As a result, the present invention was completed by discovering that M-proteins can be detected, identified, and quantified with higher sensitivity and accuracy by calculating the ratio of the area of ​​the monoclonal peak to the area of ​​the light chain spectrum based on the area of ​​the region formed by connecting the tangential skim points and the monoclonal peaks on the polyclonal light chain spectrum obtained through mass spectrometry.

[0011] Therefore, the objective of the present invention is to provide a method for measuring the concentration of a monoclonal protein in a biological sample.

[0012] Another objective of the present invention is to provide a method for diagnosing plasma cell disorders.

[0013]

[0014] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims, and drawings.

[0015]

[0016] According to one aspect of the present invention, the present invention provides a method for measuring the concentration of a monoclonal protein in a biological sample separated from a subject, comprising the following steps:

[0017] (a) a step of obtaining a mass spectrum for a biological sample separated from a subject; and

[0018] (b) A step of measuring the ratio of the area of ​​the monoclonal peak to the area of ​​the polyclonal light chain spectrum in the above mass spectrum.

[0019] The inventors have made diligent research efforts to develop an efficient diagnostic system capable of more accurately predicting the concentration of monoclonal antibodies in the blood, which are important markers for plasma cell hyperplasia-related diseases, including multiple myeloma. As a result, they discovered that by deriving the ratio of the area of ​​the monoclonal peak to the area of ​​the polyclonal light chain spectrum based on the area of ​​the region formed by connecting the tangential skim point and the monoclonal peak on the polyclonal light chain spectrum obtained through mass spectrometry, reliable quantitative information regarding M-protein can be provided with higher sensitivity and improved reproducibility, even in samples containing trace amounts of M-protein.

[0020] In this specification, the term “subject” refers to an individual that provides a sample for measuring the concentration of a monoclonal protein (M-protein) and is ultimately the subject of analysis regarding the onset of plasma cell diseases such as multiple myeloma. The subject includes, without limitation, humans, mice, rats, guinea pigs, dogs, cats, horses, cattle, pigs, monkeys, chimpanzees, baboons, or rhesus monkeys, and specifically, humans. Since the method of the present invention provides information for predicting not only the current onset of plasma cell disease but also the metabolic and genetic risk of future onset of plasma cell disease, the subject of the present invention may be a patient with plasma cell disease or a healthy individual that has not yet developed plasma cell disease.

[0021] In this specification, the term “biological specimen” refers to any specimen containing a variable-region immunoglobulin obtained from mammals, including humans, and includes, but is not limited to, tissues, organs, cells, or cell culture media. More specifically, the biological specimen of the present invention is a liquid specimen. A liquid specimen includes, without limitation, various body fluids that are in a liquid state at the time of separation from the human body, as well as all biological specimens that are in a solid state at the time of separation but become liquid as a whole after or during separation by adding a solvent or a culture medium.

[0022] According to a specific embodiment of the present invention, the biological sample of the present invention is a body fluid. More specifically, the body fluid is one or more body fluids selected from the group consisting of urine, saliva, semen, tears, amniotic fluid, cerebrospinal fluid, synovial fluid, pericardial fluid, peritoneal fluid, and blood. More specifically, the biological sample of the present invention is urine or blood, and most specifically, it may be urine, whole blood, plasma, or serum.

[0023] In this specification, the term “whole blood” generally refers to blood composed of unclotted plasma and cellular components. Plasma accounts for about 50 to 60 percent of the volume of whole blood, and cellular components (e.g., red blood cells, white blood cells, or platelets) may account for about 40 to 50 percent.

[0024] In this specification, the term “plasma” refers to the liquid component of blood and functions as a transport medium for supplying nutrients to the cells and organs of the body.

[0025] In this specification, the term “serum” refers to a pale yellow liquid collected from blood. Specifically, it refers to the pale yellow body fluid component remaining after removing the red coagulated blood, which forms as the fluidity of the blood decreases when the blood is left unattended after collection.

[0026] In this specification, the term “monoclonal protein” refers to an abnormal immunoglobulin that is overproduced in malignant plasma cells and accumulates in the blood or is excreted in the urine, and is also referred to as “M-protein.” M-protein consists of an immunoglobulin heavy chain including IgG, IgA, and IgM, and a type of immunoglobulin light chain composed of kappa and lambda. Distinguishing M-protein from normal immunoglobulin is very important in the diagnosis of various diseases caused by the overactivation and overproliferation of plasma cells. In this specification, the term “monoclonal peak” refers to a peak corresponding to the monoclonal protein on the mass spectrum.

[0027] In this specification, the term “mass spectrometry” refers to a procedure for determining the presence and quantity of a substance to be detected based on its quantitative mass value. Mass spectrometry is performed by filtering, detecting, and measuring ions using the mass-to-charge ratio (m / z value). Generally, mass spectrometry includes (1) a step of ionizing and charging a compound and (2) a step of measuring the molecular weight of the charged compound and calculating the m / z value. The calculated m / z value is used as a reference to identify and quantify a target compound in a complex mixture. Mass spectrometry in this specification can be performed through any type of mass spectrometry method utilizing the above-described principles.

[0028] According to a specific embodiment of the present invention, the acquisition of the mass spectrum is performed by a mass spectrometry method selected from the group consisting of MALDI-TOF (Matrix-Assisted Laser Desorption / Ionization Time of Flight) mass spectrometry, SELDI-TOF (Surface Enhanced Laser Desorption / Ionization Time of Flight) mass spectrometry, ESI-TOF (Electrospray ionization time-of-flight) mass spectrometry, liquid chromatography-mass spectrometry (LC-MS), and LC-MS / MS (liquid chromatography-mass spectrometry / mass spectrometry). More specifically, it is performed by the MALDI-TOF mass spectrometry method.

[0029] MALDI-TOF mass spectrometry is a method that analyzes the molecular weight of ions by irradiating a matrix-supported sample with a laser to desorb and ionize it, and then measuring the time-of-flight required for the generated ions to reach the detector. Because the target substance does not undergo fragmentation, the mass of large biomolecules, such as proteins, can be measured rapidly and accurately. When ionized molecules are accelerated by an electric field and their time-of-flight is measured, a mass-to-charge ratio (m / z) is generated, and the molecular weight of the target substance can be determined using this m / z value.

[0030] According to a specific embodiment of the present invention, the method of the present invention additionally includes a step of selecting a peak of a monoclonal protein on the mass spectrum obtained in step (a) between steps (a) and (b). The selection of the peak can be performed by general methods known in the art, for example, by selecting peak candidate points through differentiation and removing noise peaks using a half-window size method based on a window of a defined size, and then selecting the peak based on its prominence and intensity. Among the selected candidate peaks, a peak with a sharp end can be finally selected by reflecting the curvature. The peak prominence (P) and curvature (κ) applied in the selection process can be derived, for example, through Equations 1 to 3 described below.

[0031] According to a specific embodiment of the present invention, the polyclonal light chain spectral region is 22000 m / z to 25000 m / z (z = +1) or 11000 m / z to 12500 m / z (z = +2).

[0032]

[0033] According to a specific embodiment of the present invention, the ratio of the area of ​​the monoclonal (M-protein) peak to the area of ​​the polyclonal light chain spectrum is derived by the following Equation 4-1 or Equation 4-2:

[0034] [Equation 4-1] (z = +1)

[0035] [Equation 4-2] (z = +2)

[0036] In the above formulas 4-1 and 4-2, ∫M-protein is the area of ​​the monoclonal peak, and and is the area of ​​the polyclonal light chain spectrum when z = +1 and z = +2, respectively, and the ∫M-protein is obtained by calculating the area of ​​the region formed by connecting the tangent scheme point and the monoclonal peak on the polyclonal light chain spectrum.

[0037] In this specification, the term “tangential skim point” refers to a point where a tangent line is drawn from the valley of a peak to the beginning or end of a peak to separate the peak from the background in the mass spectrum, or to separate the shoulder peak from a larger peak. In the prior art, to derive the area of ​​a monoclonal peak in the mass spectrum, a vertical line drawn from the tangent skim point was used to determine the point where it intersects the X-axis (i.e., the m / z value at the tangent skim point), and the area of ​​the region formed by connecting the tangent skim point and five points of the monoclonal peak was calculated (drop perpendicular method). However, the inventors defined only the region formed by connecting the tangent skim point and three points of the monoclonal peak as the area of ​​the monoclonal peak to exclude the overlapping portion included in the background area of ​​the polyclonal light chain spectrum, and confirmed that this allows even trace amounts of M-protein in the sample to be detected / quantified with significantly improved sensitivity and reproducibility (Fig. 7).

[0038] An exemplary process for deriving the area of ​​a monoclonal peak by connecting three points of the monoclonal peak and the tangent scheme point is summarized in FIG. 3b. In addition to the monoclonal peak (x_mz, y_Int), the tangent scheme point (x_1, y_1) located to its left, and the tangent scheme point (x_2, y_2) located to its right, a fourth virtual point (x_mz, max(y_1, y_2)) is derived that has the same y value (intensity) (= y_3) as the higher value between the x value (m / z) of the monoclonal peak and y_1 and y_2. Then, the area of ​​the region formed by connecting the four points (x_1, y_1), (x_mz, max(y_1, y_2)), (x_2, y_2), and (x_mz, y_Int) is calculated. To this end, by calculating the value A integrated from x_1 to x_2 and substituting y_3 = max(y_1, y_2)), the area of ​​the monoclonal peak can be derived through the final formula below:

[0039] (x_mz - x_1) * (y_1 + y_3) / 2 + (x_2 - x_mz) * (y_2 + y_3) / 2

[0040]

[0041] According to a specific embodiment of the present invention, the method further comprises the step of measuring the concentration of gamma globulin in the sample.

[0042] The concentration of gamma globulin in a sample can be measured using various methods known in the art; for example, serum protein electrophoresis can be performed to analyze the concentration of gamma globulin in serum. This method measures the concentrations of various protein fractions in the blood by separating them according to electrical charge, and separates the proteins in the serum into a total of five groups: albumin and four types of globulins (α1, α2, β, γ). The normal range for gamma globulin is generally 0.7 to 1.6 g / dL.

[0043] According to a specific embodiment of the present invention, the concentration of the monoclonal protein is derived by the following formulas 6 to 8:

[0044] [Equation 6]

[0045] [Equation 7]

[0046] [Equation 8]

[0047] In the above Equations 6 to 8 for deriving the calibration curve, [M] represents the concentration of M-protein, and represents the concentration of gamma globulin, and Ratio represents the ratio of the area of ​​the monoclonal (M-protein) peak to the area of ​​the polyclonal light chain spectrum.

[0048] In this specification, the term “calibration curve” refers to a function or mathematical relationship representing a quantitative correlation between a signal value and the actual concentration of a standard substance, based on a signal of one or more standard substances having a known concentration, e.g., a peak signal acquired by a mass spectrometer (e.g., peak intensity, peak area, integrated intensity, or signal-to-internal standard ratio, etc.). Such a calibration curve is typically derived by regression analysis (linear or non-linear regression) and is used to convert a mass spectrometric signal obtained from an unknown sample into an actual concentration.

[0049] V among the component functions of Equation 7 for calculating the Ratio max ( ) represents the maximum value of the Hill function and It is inversely proportional to and can be expressed by Equation 7-1:

[0050] [Equation 7-1] .

[0051] K d ( ) is Ratio It refers to the concentration of M-protein and can be expressed by the following Equation 7-2:

[0052] [Equation 7-2] .

[0053] C( ) is the y-intercept term of the Hill function, has a value less than or equal to 0, and can be expressed by Equation 7-3 below:

[0054] [Equation 7-3] .

[0055] N( ) is the Hill coefficient representing the direction and degree of convexity of the Hill function curve, and can be expressed by Equation 7-4 below:

[0056] [Equation 7-4] .

[0057] N( ) is all real numbers greater than 0, and N( When )< 1, the curve is in the shape of an up-concave log function; N( If )≥ 1, it takes the form of a sigmoid function.

[0058] The coefficients having the relationships in Equations 7-1 to 7-4 above are called parameters, and if defined in the form of a vector (an ordered array of numbers representing multiple variables as a single unit), they are as follows: θ=(θ1,θ2,θ3,θ4,θ5,θ6,θ7,θ8).

[0059]

[0060] In this specification, the term “Hill function” refers to a non-linear saturation model that mathematically represents the phenomenon in which binding, activity, reaction signals, etc., increase with changes in the concentration of a specific substance until they saturate at a certain level. Such a Hill function typically refers to the maximum response and the semi-maximum effective concentration (EC). 50It is expressed as an equation including the value corresponding to Kd), and the Hill coefficient, and can reflect the cooperativity of the reaction.

[0061] Each parameter θ i θ is a dependent factor that acts interdependently within the model rather than independently, and all θ values ​​are greater than 0. The meaning of each parameter is as follows:

[0062] (θ1,θ2): V max Parameters related to (maximum value of the correction curve);

[0063] (θ3,θ4): K d (Ratio= When [M] value) related parameter;

[0064] (θ5,θ6): Parameter related to N (degree of bending);

[0065] (θ7,θ8): Parameters related to C (y-intercept).

[0066] In addition, for each calibration curve data point, the actual measured value (yi) and the predicted value calculated by the calibration curve ( The Mean Squared Error (MSE), calculated by squaring and averaging the differences between ), is the objective function ( If set to ), the parameter vector that minimizes the objective function is the optimal parameter( It can be defined as ).

[0067] [Equation 7-5]

[0068] [Essence 7-6]

[0069] In this specification, the term “objective function” refers to a mathematical function to be minimized or maximized for the estimation or optimization of model parameters, and is generally represented by J(θ). For example, in the case of maximizing the likelihood in statistical estimation, the objective function is the likelihood function Alternatively, it may be defined as a log-likelihood function in its logarithmic form (Maximum Likelihood Estimation, MLE), but is not limited thereto. The objective function may take various forms, such as a Mean Squared Error (MSE), a sum of squared errors, a negative log-likelihood, or a function including a regularization term, and may be applied to linear or non-linear optimization.

[0070] In this specification, the term “optimization” means the process of searching, calculating, and determining the value of a parameter θ through mathematical or numerical methods so that the function value becomes minimum or maximum for a predefined objective function.

[0071] After applying the inverse function to the calibration curve with the 8 parameter values ​​optimized through the above process, the M-protein concentration [M] can be obtained by applying the Ratio of the measured values.

[0072] [Equation 8-1]

[0073]

[0074] According to another aspect of the present invention, the present invention provides a method for providing information necessary for the diagnosis of a plasma cell disorder, comprising the step of measuring the concentration of a monoclonal antibody in a biological sample separated from a subject using the method of the present invention described above.

[0075] In this specification, the term “diagnosis” includes the determination of an individual’s susceptibility to a specific disease, the determination of whether an individual currently possesses a specific disease, and the determination of the prognosis of an individual afflicted with a specific disease.

[0076] In this specification, the term “plasma cell disorder” refers to a series of diseases caused by the excessive production of abnormal plasma cells derived from the final differentiation of B lymphocytes, and is also referred to as monoclonal gammopathy, plasma cell dyscrasia, dysproteinemias, and paraproteinemias. Malignant plasma cell disorders, such as multiple myeloma, amyloidosis, and monoclonal gammopathy of unknown significance, are always accompanied by the accumulation of high concentrations of M-protein in serum and / or urine. Since small amounts of M-protein are also detected in asymptomatic precancerous diseases such as monoclonal gammopathy of unknown significance (MGUS) and subclinical multiple myeloma (SMM), the detection of trace amounts of M-protein in these early-stage plasma cell disorders can be of great help in predicting the likelihood of progression to multiple myeloma at an early stage and thereby establishing treatment strategies early.

[0077] According to a specific embodiment of the present invention, the plasma cell disease for which information regarding diagnosis is to be provided in the present invention is selected from the group consisting of multiple myeloma, monoclonal gammopathy of unknown significance, asymptomatic myeloma, non-secretory myeloma, plasma cell leukemia, plasmacytoma, solitary plasmacytoma of bone, extramedullary plasmacytoma, immunoglobulin deposition diseases, primary amyloidosis, and osteosclerotic myeloma (POEMS syndrome), and more specifically, multiple myeloma.

[0078] If the concentration of monoclonal antibodies in a biological sample isolated from a subject by the method of the present invention is higher than that of a normal control group, it can be determined that the risk of plasma cell disease has increased. A higher concentration compared to a normal control group means that the concentration of monoclonal immunoglobulin, which serves as a direct marker of abnormal proliferation of plasma cells, has increased to a measurable level. Specifically, it may mean a state in which the concentration has increased by 20% or more compared to the control group, more specifically, a state in which it has increased by 40% or more, and even more specifically, a state in which it has increased by 60%.

[0079] According to another aspect of the present invention, the present invention provides a method for deriving the ratio of the area of ​​a monoclonal (M-protein) peak to the area of ​​a polyclonal light chain spectrum using the following Equation 4-1 or Equation 4-2 in a mass spectrum obtained by performing mass spectrometry on a biological sample separated from a subject:

[0080] [Equation 4-1] (z = +1)

[0081] [Equation 4-2] (z = +2)

[0082] In the above formula, ∫M-protein is the area of ​​the monoclonal peak, and and is the area of ​​the polyclonal light chain spectrum when z = +1 and z = +2, respectively, and the ∫M-protein is obtained by calculating the area of ​​the region formed by connecting the tangent scheme point and the monoclonal peak on the polyclonal light chain spectrum.

[0083] As the subject, biological sample, monoclonal peak, tangent scheme point, and mass spectrometry used in the present invention have already been described above, their description is omitted to avoid excessive duplication.

[0084]

[0085] The features and advantages of the present invention are summarized as follows:

[0086] (a) The present invention provides a method for measuring the concentration of a monoclonal protein in a biological sample and a method for diagnosing a plasma cell disorder using the same.

[0087] (b) The present invention can provide quantification results with significantly improved accuracy and reproducibility compared to conventional techniques for quantifying M-proteins by mass spectrometry by estimating the M-protein peak ratio based on the region connecting the monoclonal peak on the polyclonal light chain spectrum and the tangent skim points located on both sides thereof.

[0088] (c) The present invention, in particular, can detect trace amounts of monoclonal proteins with high reliability using non-invasive mass spectrometry with body fluid samples, and can be usefully utilized to diagnose early plasma cell diseases that have not yet progressed to multiple myeloma, thereby facilitating the early establishment of treatment strategies and improving patient survival rates.

[0089]

[0090] Figure 1 is a schematic diagram of the algorithm for M-protein identification and M-protein peak ratio estimation.

[0091] Figure 2 is a diagram illustrating the process of M-protein quantification through calibration curve estimation.

[0092] Figure 3a is a figure showing the process of measuring the area of ​​a monoclonal peak relative to the polyclonal light chain spectrum region of immunoglobulin for M-protein identification among the first sub-algorithms. Figure 3b is a figure showing an exemplary process of deriving the area of ​​the monoclonal peak region formed by connecting the tangent scheme point, the monoclonal peak, and the fourth virtual point (x_mz, y_3).

[0093] Figure 4 shows the actual calibration curve created through curve fitting. Each marked point represents the ratio of monoclonal peaks measured by the tangent scheme method relative to spiked MAb, and the curve represents the fitted curve. The spectra indicated by dashed arrows at each point show how the ratio was measured, and the area enclosed in red on each spectrum corresponds to the area of ​​the monoclonal peak.

[0094] Figure 5 is a figure showing the change in the calibration curve according to the concentration of gamma globulin.

[0095] Figure 6 shows the derived K dThis figure shows examples of the Hill equation according to various gamma values ​​using the values ​​of and n.

[0096] Figure 7 is a figure comparing the results of measuring M-protein concentration using the conventional Mayo Clinic method (drop perpendicular method), which derives the area of ​​the monoclonal peak by drawing a vertical line within the spectrum, and the method of the present invention, which derives a calibration curve based on gamma globulin concentration values ​​after calculating the area ratio of the monoclonal peak by applying the tangent scheme method.

[0097]

[0098] The present invention will be described in more detail below through examples. These examples are intended solely to explain the invention more specifically, and it will be obvious to those skilled in the art that the scope of the invention is not limited by these examples according to the gist of the invention.

[0099]

[0100] Examples

[0101] Pretreatment for M-protein elution

[0102] 50 μL of Thermo Scientific™’s CaptureSelect™ FcXP Affinity Matrix was added to 10 μL of patient serum at room temperature, and the CaptureSelect™ FcXP Affinity Matrix was diluted 10-fold for use in the experiment. The adsorption reaction was carried out using a Thermomixer for 15 minutes, with vortexing performed at 15-minute intervals. Afterward, the supernatant was removed, and the remaining Affinity Matrix was washed three times with 150 μL of PBS and three times with 150 μL of distilled water. The captured Ig was eluted and reduced with 30 μL of 5% acetic acid and 20 mM TCEP.

[0103]

[0104] Preprocessing for deriving a calibration curve

[0105] Three serum samples (IRB no. 2410-034-1575) lacking M-protein and having gamma globulin concentrations of 0.478 g / dL (Hypo), 1.043 g / dL (Normo), and 1.369 g / dL (Hyper), respectively, were used as a matrix for deriving the calibration curve. The gamma globulin concentrations mentioned above were measured via SPEP (serum protein electrophoresis), and [γ] Original It was defined as follows. For each serum matrix, a human monoclonal antibody (MAb) at a concentration of 20 mg / mL was serially diluted to adjust the final M-protein concentrations to 0.03 g / dL, 0.125 g / dL, and 0.5 g / dL. Accordingly, a total of nine calibration points were constructed using combinations of three types of matrices and three types of M-protein concentrations.

[0106] 9 calibration points for obtaining the calibration curve No. γ-globulin level [γ] Original [γ] Corrected [M]R1Hypo-γ0.4780.2700.0310.0602Hypo-γ0.4780.3640.1250.1353Hypo-γ0.4780.7390.50.2594Normo-γ1.0860.5740.0310.0405Normo-γ1.08 60.6680.1250.1246Normo-γ1.0861.0430.50.2327Hyper-γ1.7380.9000. 0310.0148Hyper-γ1.7380.9940.1250.0679Hyper-γ1.7381.3690.50.206

[0107]

[0108] Serum Samples by Gamma Globulin Concentration (Matrix) and Calibration Curve Points Using Human MAb Serum Sample (Matrix) Serum γ-Globulin Concentration (g / dL) Final Calibration Curve Points (g / dL) Cal series 10.47 80.03, 0.125, 0.5 Cal series 21.086 0.03, 0.125, 0.5 Cal series 31.738 0.03, 0.125, 0.5

[0109] Five calibration curve series according to gamma globulin concentration were derived by performing a pretreatment process for M-protein elution on the fabricated calibration series.

[0110] When M-protein is spiked into serum, the monoclonal antibody itself corresponds to the gamma globulin region, so the gamma globulin concentration of the final mixture is simply [γ] Original It is not the same as the value. That is, the total amount of gamma globulin increases with the addition of M-protein, and since serum and M-protein solution were mixed in a 1:1 volume ratio, the gamma globulin concentration [γ] in the final mixture Corrected It can be corrected as follows:

[0111]

[0112] Corrected gamma globulin concentration [γ] Corrected was used as the gamma variable in subsequent analyses. As a result of the actual analysis, [γ] Original [γ] rather than when using the value Corrected It was confirmed that the fit of the correction curve and quantitative accuracy were improved when using the value, and accordingly, the corrected gamma concentration was applied in the present invention.

[0113] Finally, when calculating the calibration curve using 27 values ​​(bold in Table 1) with 9 calibrator data points, the initial parameters were set to θ0 = (1.4, 0.5, 0.3, 1.7, 0.4, 1.9, 0.8, 5.5), and accordingly, the optimized parameters are = (0.884, 0.212, 2.176, 2.469, 0.905, 3.957, 1.129, 8.730). The optimization process of the parameters is described below.

[0114]

[0115] Identification of M-protein in calibrator and sample using mass spectrometry

[0116] Mass spectrometry spectra of polyclonal light chains or monoclonal light chains of patient serum and a calibrator constructed with human monoclonal antibodies were obtained using a Bruker Biotyper SmartLT MALDI-TOF MS instrument. To perform sequential spotting, 1 μl of a tag-labeled protein (KPC-2_6xhis-tag), in which 6 x his is conjugated to KPC-2 protein, was first spotted onto a MALDI plate as an internal standard. Immediately thereafter, 1 μl of a sample in which the polyclonal or monoclonal light chain of the M-protein had eluted was spotted onto the internal standard solution. Subsequently, 1 μl of the substrate SA (sinapinic acid, 20 mg / mL in 0.1% TFA / 50% acetonitrile) was spotted and covered, and then mixed by three aspirations and dispensings using a pipette. The plate was completely dried in the air, and MALDI-TOF analysis was performed.

[0117] Mass spectrum data was acquired using Bruker’s Flex Control version 4.1 software. Random location acquisition was performed to irradiate a total of 2,000 laser shots at 40-shot intervals, and the spectral data was accumulated. Mass spectrometry spectra were acquired in the cation and linear mode with a laser frequency of 200 Hz for the 9,000 to 32,000 m / z range, and +1 and +2 ions were detected simultaneously.

[0118]

[0119] Derivation of a Mass Spectrum-Based M-Protein Measurement Algorithm

[0120] All mass spectrum data were functionalized using Gaussian Kernel Density Estimation (Gaussian KDE). Spectral preprocessing involved smoothing using a Savitzky-Golay filter, followed by baseline correction using the Statistics-sensitive Non-linear Iterative Peak-clipping algorithm (SNIP algorithm). Additionally, peak picking was performed using differentiation, and mass correction via hKPC-2 was applied. The M-protein measurement algorithm was divided into the following two sub-algorithms: 1) M-protein identification and M-protein peak ratio estimation; and 2) M-protein quantification through calibration curve estimation. Through these two algorithms, various clinical information, such as the diagnosis of multiple myeloma, identification and quantification of M-protein isotypes, and confirmation of drug administration, was provided as objective indicators. The pipelines for the two sub-algorithms are summarized in Figures 1 and 2, respectively.

[0121]

[0122] Sub-algorithm 1: M-protein identification and M-protein peak ratio estimation

[0123] Among the first sub-algorithms, M-protein identification was performed by comparing the distributions of κ and λ light chains in the polyclonal light chain spectral region of immunoglobulin (22,000 m / z to 25,000 m / z). For the quantification of M-protein, the area ratio of monoclonal peaks relative to the spectrum between 22,000 m / z and 25,000 m / z was estimated (Fig. 3). First, peak picking was performed through differentiation, and then noise peaks were removed using a filtering algorithm based on a specific window size (half-window size method). Major peaks were selected considering their prominence and intensity, and this process was performed for all polyclonal light chains. Subsequently, only sharp peaks were retained through curvature, and the M-protein was identified by applying the aforementioned method. The definitions of the derivative f'(χ), peak prominence P, and curvature κ used here are given by Equations 1, 2, and 3 below:

[0124] [Equation 1]

[0125] [Equation 2]

[0126] [Equation 3]

[0127] In Peak Prominence, H represents the height. Also, κ is the curvature of the one-dimensional curve y = f(χ).

[0128] A “peak” refers to a point in a spectrum that has a local maximum, and a specific peak i The peak intensity of ) I i It can be defined as. The above Peak i Moving to the left and right directions regarding Peak i As a result of searching for the section where a higher peak exists, the peak is located in the left direction. iIf a higher adjacent peak exists, the minimum intensity value in the interval between those two peaks is Valley Left Defined as, Peak from the right direction i If a higher adjacent peak exists, the minimum intensity value in the interval between those two peaks is Valley Right It is defined as. Afterwards, the peak reference valley (V i ) can be defined as follows: V i = max(Valley Left , Valley Right This is to select the higher baseline of the two directions.

[0129] “Peak Prominence” refers to a specific peak (Peak i It refers to a value indicating how independently ) protrudes compared to surrounding peaks. Peak i Prominence of (P i ) can be defined as follows: P i = I i - V i The prominence value reflects how much a specific peak protrudes from a higher baseline and can be used as a criterion for peak filtering and quantitative signal selection.

[0130] The M-protein region, indicated in red in Figure 1, was derived using a tangential skim method that processes data in a tangential direction. In this case, the tangential skim points were determined by considering peak tailing and adductor peaks. An adductor peak refers to the detection of a small peak corresponding to the mass value of the matrix bound to the protein within a specific range to the right of the main peak during MALDI-TOF mass spectrometry. Based on the information obtained in this way, the ratio of the monoclonal peak portion presumed to be the M-protein in the entire polyclonal light chain spectrum was calculated, and this value was subsequently used for quantification. The ratio is derived through Equation 4-1 or Equation 4-2 below, applying charge values ​​of +1 and +2, respectively.

[0131] [Equation 4-1] (z = +1)

[0132] [Equation 4-2] (z = +2)

[0133]

[0134] Sub-algorithm 2: Calibration curve estimation

[0135] M-protein quantification through calibration curve estimation, the second sub-algorithm, begins with estimating calibration curves for different gamma globulin concentrations. At this time, the calibration curve is estimated based on the Hill equation rather than a straight line, which can be expressed by Equation 5 below.

[0136] [Equation 5]

[0137] Here, V max is the maximum reach constant, K d is V maxIt refers to the χ value at / 2. n represents the direction and degree of convexity of the equation (n<1: convex upward; n=1: linear; n>1: convex downward). In the case of gamma globulin, it is typically named 'hypo-' when the concentration is ~0.6 g / dL, 'normo-' when the concentration is 0.6 to 1.6 g / dL, and 'hyper-' when the concentration is 1.6 to 1.6 g / dL.

[0138] In this algorithm, the calibration curve series consists of the ratio of monoclonal peaks according to different MAb concentrations and gamma globulin concentration. and is determined by the standard concentration [M] of the monoclonal protein applied to derive the calibration curve, which can be obtained by the following formulas 6 to 8.

[0139] [Equation 6]

[0140] [Equation 7]

[0141] [Equation 8]

[0142] In the above equations 6 to 8, [M] represents the concentration of M-protein, and represents the concentration of gamma globulin, and Ratio represents the ratio of the area of ​​the monoclonal (M-protein) peak to the area of ​​the polyclonal light chain spectrum.

[0143] The meanings of the component functions in Equation 7 for calculating the ratio are as follows:

[0144] V max ( ) represents the maximum value of the Hill function and It is inversely proportional to and, specifically, can be expressed by the following Equation 7-1:

[0145] [Equation 7-1] .

[0146] K d ( ) is Ratio It refers to the concentration of M-protein, and specifically, can be expressed by the following Equation 7-2:

[0147] [Equation 7-2] .

[0148] C( ) is the y-intercept term of the Hill function, has a value less than or equal to 0, and specifically can be expressed by Equation 7-3 below:

[0149] [Equation 7-3] .

[0150] N( ) is the Hill coefficient representing the direction and degree of convexity of the Hill function curve, and can be expressed by Equation 7-4 below:

[0151] [Equation 7-4] .

[0152] N( ) is all real numbers greater than 0, and N( When )< 1, the curve is in the shape of an up-concave log function; N( If )≥ 1, it takes the form of a sigmoid function.

[0153] The coefficients having the relationships in Equations 7-1 to 7-4 above are called parameters, and if defined in the form of a vector (an ordered array of numbers representing multiple variables as a single unit), they are as follows: θ=(θ1,θ2,θ3,θ4,θ5,θ6,θ7,θ8).

[0154] Each parameter θ i θ is a dependent factor that acts interdependently within the model rather than independently, and all θ values ​​are greater than 0. The meaning of each parameter is as follows:

[0155] (θ1,θ2): V max Parameters related to (maximum value of the correction curve);

[0156] (θ3,θ4): K d (Ratio= When [M] value) related parameter;

[0157] (θ5,θ6): Parameter related to N (degree of bending);

[0158] (θ7,θ8): Parameters related to C (y-intercept).

[0159] According to the present invention, when a calibration curve is derived by Equation 7, the inverse function of the calibration curve is calculated through Equation 8. Subsequently, the ratio value measured from the spectrum of the sample to be analyzed and the gamma globulin concentration The concentration of monoclonal protein [M] in the sample corresponding to can be derived.

[0160] Specifically, for each calibration curve data point, the actual measured value (yi) and the predicted value calculated by the calibration curve ( Since the difference between ) (residual) can be positive or negative and can be canceled out by simple summation, the Mean Squared Error (MSE), calculated by squaring and averaging the residuals, is used as the objective function ( It was set to ).

[0161] [Equation 7-5]

[0162] The parameter vector that minimizes the objective function represented by Equation 7-5 above is the optimal parameter ( It was defined as ).

[0163] [Essence 7-6]

[0164] Once the optimization process is complete Eight optimal parameter values ​​corresponding to [M] were determined, and after applying the inverse function to the calibration curve, the M-protein concentration [M] was obtained by applying the measured value Ratio.

[0165] [Equation 8-1]

[0166]

[0167] Relationship between calibration curve and Hill equation

[0168] Using serum as a matrix, the monoclonal peak area ratios of calibrators (Table 1) with different MAb concentrations are estimated, and calibration curves are derived through the ratio values ​​for each concentration. Generally, a linear function y = aχ + b is adopted as the curve fitting function to obtain the calibration curve; however, since the calibration points measured in this algorithm exhibit a non-linear pattern that appears to be saturated, curve fitting was performed using the Hill equation, a curvilinear function (Fig. 4).

[0169]

[0170] Calibration curve pattern according to gamma globulin concentration

[0171] A high concentration of gamma globulin implies a high concentration of polyclonal light chains, which means that the spectral area of ​​polyclonal light chains is proportional to the concentration of gamma globulin. In other words, even if the same amount of M-protein is present, the proportion of the monoclonal peak decreases, and as shown in Fig. 5, the maximum value of the ratio on the calibration curve decreases as the concentration of gamma globulin increases. In the Hill function, V max Wow K d Since , and n are not independent factors, they are also affected by gamma globulin concentration, and the same applies to the y-intercept term c. Experimentally, K d and V maxIn the case of , they are inversely proportional to each other, and c always has a negative value. In the case of n, it is proportional to c. Among the multiple functions reflecting these relationships, the optimal combination is Equations 7-1 to 7-4 mentioned above. Accordingly, in Equation 6, the concentration of gamma globulin g exists as an independent variable of the calibration curve series along with the ratio r of the monoclonal peak. However, when the concentration of gamma globulin exceeds the limit of quantification (LOQ) indicated by a star in the graph of Fig. 6, most of the gamma globulin concentration was considered to be equal to the M-protein concentration.

[0172]

[0173] Comparison of the effects of the algorithm of the prior art and the present invention

[0174] One of the biggest differences between the Mayo Clinic assay and this algorithm is that the ratio of monoclonal peaks is calculated, and the quantitative value of M-protein is determined using an empirical virtual calibration curve. The Mayo Clinic method uses the drop perpendicular method, which calculates the area under a vertical line, but does not use a calibration curve; in contrast, this algorithm calculates the ratio using the tangent scheme method and then quantifies the result by applying a calibration curve based on gamma values. Although the Mayo Clinic method does not use a calibration curve, the same Hill equation was applied to each dataset to compare reproducibility. Figure 7 compares the results of applying the Mayo Clinic method and the algorithm of this invention, respectively, to samples mixed with MAb at concentrations of 0–0.5 g / dL. The Hill equation was used for curve fitting in both cases, but the coefficient of determination (R-squared) was checked, and the algorithm of this invention showed a higher value. In particular, unlike the Mayo Clinic method, the algorithm of the present invention shows a stable standard deviation even at very low concentrations of 0.125 g / dL or less (vertical line in the graph of Fig. 7). This means that the algorithm of the present invention shows higher reproducibility compared to the prior art.

[0175]

[0176] Algorithm verification using actual clinical specimens

[0177] The algorithm of the present invention was applied to input the gamma values ​​and M-protein concentration values ​​of actual clinical samples, and the calculated values ​​were compared and verified. The four clinical samples used showed estimated M-protein concentrations of 0.50, 0.70, 1.53, and 0.75 g / dL, respectively. Regardless of the gamma concentration (Normo or Hyper), the relative error with respect to the actual measured values ​​was <11% (Table 3).

[0178] M-Protein Measurement Results in 4 Clinical Samples No. Subtype γ-Globulin Levels Measured (g / dL) Estimated (g / dL) Bias %1 IgG / kappaNormo 0.50 0.50 0.02 IgG / kappaHyper 0.66 0.70 6.13 IgG / lambdaHyper 1.76 1.53 -13.14 IgG / lambdaHyper 0.68 0.75 10.3

[0179]

[0180] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for measuring the concentration of a monoclonal protein in a biological sample isolated from a subject, comprising the following steps: (a) a step of obtaining a mass spectrum for a biological sample separated from a subject; and (b) A step of measuring the ratio of the area of ​​the monoclonal peak to the area of ​​the polyclonal light chain spectrum in the above mass spectrum.

2. The method according to claim 1, wherein the acquisition of the mass spectrum is performed by a mass spectrometry method selected from the group consisting of MALDI-TOF (Matrix-Assisted Laser Desorption / Ionization Time of Flight) mass spectrometry, SELDI-TOF (Surface Enhanced Laser Desorption / Ionization Time of Flight) mass spectrometry, ESI-TOF (Electrospray ionization time-of-flight) mass spectrometry, liquid chromatography-mass spectrometry (LC-MS), and LC-MS / MS (liquid chromatography-mass spectrometry / mass spectrometry).

3. A method according to claim 2, characterized in that the acquisition of the mass spectrum is performed by the MALDI-TOF mass spectrometry method.

4. The method according to claim 1, characterized in that the polyclonal light chain spectral region is 22000 m / z to 25000 m / z (z = +1) or 11000 m / z to 12500 m / z (z = +2).

5. A method according to claim 1, characterized in that the ratio of the area of ​​the monoclonal (M-protein) peak to the area of ​​the polyclonal light chain spectrum is derived by the following Equation 4-1 or Equation 4-2: [Equation 4-1] 　 (z = +1) [Equation 4-2] 　(z = +2)　　 In the above formula, ∫M-protein is the area of ​​the monoclonal peak, and and is the area of ​​the polyclonal light chain spectrum when z = +1 and z = +2, respectively, and the ∫M-protein is obtained by calculating the area of ​​the region formed by connecting the tangent scheme point and the monoclonal peak on the polyclonal light chain spectrum.

6. The method of claim 1, characterized in that the method additionally includes the step of measuring the concentration of gamma globulin in the sample.

7. A method according to claim 6, characterized in that the concentration of the monoclonal protein is derived by the following formulas 6 to 8: [Equation 6] [Equation 7] [Equation 8] In the above formulas 6 to 8, [M] is the concentration of the monoclonal protein; is the concentration of gamma globulin; Ratio is the ratio of the area of ​​the monoclonal peak to the area of ​​the polyclonal light chain spectrum; and V max ( ) is the maximum value of the Hill function; K d ( )silver When it is the concentration of monoclonal protein; C( ) is the y-intercept term of the Hill function; N( ) is the Hill coefficient, which indicates the direction and degree of convexity of the function curve.

8. The method according to claim 1, characterized in that the biological sample is urine, whole blood, plasma, or serum.

9. A method for providing information necessary for the diagnosis of a plasma cell disorder, comprising the step of measuring the concentration of a monoclonal antibody in a biological sample separated from a subject using the method of any one of claims 1 to 8.

10. A method according to claim 9, wherein the plasma cell disease is selected from the group consisting of multiple myeloma, monoclonal gammopathy of unknown significance, asymptomatic myeloma, non-secretory myeloma, plasma cell leukemia, plasmacytoma, solitary plasmacytoma of bone, extramedullary plasmacytoma, immunoglobulin deposition diseases, primary amyloidosis, and osteosclerotic myeloma (POEMS syndrome).

11. A method according to claim 10, characterized in that the plasma cell disease is multiple myeloma.

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