Mass Spectrometry Control

Abstract

The present invention provides a method for immunopurifying and characterizing an analyte from a sample, the method comprising the steps of: (i) providing a predetermined amount of a control substance bound to a substrate via a linkage cleavable by acidic pH and / or a reducing agent, and an optional further analyte-specific antibody or fragment thereof bound to the substrate, wherein the control substance is specific for the analyte or is not specific for the analyte; (ii) allowing the analyte, if present in the sample, to bind to the control substance or said optional further analyte-specific antibody or fragment, wherein the substrate-bound control substance (i) is capable of binding the analyte to any desired target. (iii) washing unbound material from the substrate; (iv) acid eluting the bound analyte from at least one substrate; (v) performing mass spectrometry to identify two or more peaks, at least one peak associated with the presence of the analyte and at least a second peak associated with at least a portion of the control material; and (vi) comparing the size or intensity of the second peak to a predetermined calibration value to allow for calibrating the first peak associated with the analyte.

Description

TECHNICAL FIELD

[0001] The present invention relates to methods for immunopurifying and characterizing analytes, as well as kits for use in such methods.

[0002] The use of mass spectrometry in in vitro diagnostics for the quantification of analytes such as proteins including immunoglobulins is generally known in the art. For example, WO2015 / 154052, which is entirely incorporated herein, discloses a method for detecting immunoglobulin light chains, immunoglobulin heavy chains, or mixtures thereof using mass spectrometry (MS). A sample containing immunoglobulin light chains, heavy chains or mixtures is immunopurified and subjected to mass spectrometry to obtain a mass spectrum of the sample. This can be used, for example, to detect monoclonal proteins in samples derived from patients. It can also be used to fingerprint, isotype and identify disulfide bonds in monoclonal antibodies.

[0003] MS is used, for example, to separate lambda chains and kappa chains in a sample by mass and charge. It can also be used, for example, to detect the heavy chain component of immunoglobulins after reducing disulfide bonds between heavy and light chains using a reducing agent. MS is also described, for example, in WO2015 / 131169, which is entirely incorporated herein.

[0004] Purification of analytes in samples typically uses anti-analyte specific antibodies. These are typically bound to a substrate such as nanobeads or microbeads by techniques generally known in the art. A sample containing the analyte is mixed or incubated with the antibodies attached to the beads, allowing the analyte to bind to the antibodies, and unbound substances are washed away. The analyte is then washed or separated or eluted from the beads and analyzed by mass spectrometry. For example, one problem associated with mass spectrometry in vitro diagnostics for the quantification of serum proteins is that it presents challenges with respect to the accuracy and precision of the reported results.

[0005] Solutions to date include, for example, the use of controls at individual specific steps in the procedure. For instance, in MALDI-TOF, controls can be included in the matrix at the time of target preparation, thus allowing control over the laser crystallization process and subsequent ionization. However, this does not account for variability introduced upstream. In many pre-acquisition purification techniques used in mass spectrometry, the analyte is purified by immunoprecipitation. This requires the use of controls at each step of the purification process, along with a known amount of target protein, to estimate the amount of analyte lost at each step of the purification process. However, this does not control for exemplary variability in the precipitation and subsequent preparation steps of the sample, for example, due to pipetting errors. [Overview of the project]

[0006] The applicant has confirmed that a releaseable control substance, such as a protein or peptide, heteropolymer, or antibody, can be used as a control in the purification process up to the detection step by mass spectrometry by binding the releaseable control substance to another substrate, such as beads or a solid surface, and precipitating it in the purification or concentration step. Since the amount on the beads is known, it can then be used to control any loss in this step or variation in subsequent steps. Since the control is derived from, for example, an antibody that targets the analyte itself, there will be a direct relationship between the amount of control measured by the mass spectrometry spectrum and the amount of the target analyte measured by the same spectrum. Since the amount of control in the precipitation / concentration step is known, the value can be assigned to the level of the target analyte in the original sample.

[0007] For example, a capture antibody specific to the analyte, or actually another antibody, can be bound to the substrate. For instance, when the analyte bound to the capture antibody is eluted, for example, via acid elution, the binding occurs via the constant region of the heavy chain, releasing the light chain from the capture antibody or other antibody, which can then be detected by mass spectrometry.

[0008] The ability of these light chains, for example, immunoglobulins, to distinguish from the light chain of the analyte can be enhanced by selecting the light chain of a capture antibody or another antibody, resulting in them being heavier or lighter than the analyte's light chain. This can be achieved, for example, by selecting a monoclonal antibody and modifying the light chain of the monoclonal antibody by adding one or more amino acids, particularly 1 to 5 amino acids, to the light chain.

[0009] The applicant recognizes that other controls can be used, such as heteropolymers in which the detectable portion of the subunit is released by acid elution. For example, a non-immunoglobulin protein containing one or more subunits can be used, where the subunits are released by the elution step.

[0010] This has the advantage of controlling variations in each stage of elution and the subsequent detection stage.

[0011] The present invention provides a method for immunopurifying and characterizing analytes from a sample, and this method is (i) A step of providing a predetermined amount of a control substance bound to a substrate via a linkage cleavable by an acidic pH and / or a reducing agent, and an optional further analyte-specific antibody or fragment thereof bound to the substrate, wherein the control substance is analyte-specific or not analyte-specific; (ii) A step that, if present in the sample, enables the analyte to bind to a control substance or an optional further analyte-specific antibody or fragment, wherein the substrate-bound control substance can be provided after contacting the analyte with an optional further analyte-specific antibody; (iii) A step of washing away unbonded material from the substrate; (iv) Acid elution of an analyte bound to at least one substrate; (v) A step of performing mass spectrometry to identify two or more peaks, wherein at least one peak is associated with the presence of the analyte and at least a second peak is associated with at least a portion of the control substance; and (vi) A step that enables calibration of the first peak related to the analyte by comparing the size (such as the area under the curve) or intensity of the second peak with a predetermined calibration value.

[0012] The control substance and any analyte-specific antibody may be bound to the same or different substrates. For example, the control substance may be bound to a first group of beads, and additional analyte-specific antibodies may often be bound to a second set of beads. Alternatively, they may be bound to the same beads. The control substance may be an analyte-specific antibody or a fragment thereof. An alternative control may be, for example, an antibody or fragment thereof that is not specific to the analyte. In the latter example, any additional analyte-specific antibody or fragment is typically provided to bind the analyte to the substrate, usually before the washing step.

[0013] At least some, but typically virtually all, control substances are released from the substrate by the acid elution step.

[0014] One or more additional reducing agents, such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine hydrochloride (TCEP), may be optionally included (e.g., using disulfide bonds) to assist in reducing and cleaving the cleavable bonds.

[0015] The control material can potentially be any portion that can be detected by the mass spectrometry process when released. However, it is typically selected to have a similar mass and charge to the analyte being detected. Typically, the mass and charge are different so that the peak from the control material can be distinguished from the peak of the analyte at the baseline. The control material may also be selected to crystallize in a similar manner to the analyte placed (spotted) on the mass spectrometry target plate.

[0016] The analyte may be, for example, an analog of the analyte. This can be modified by isotopically altering its mass, or, for example, by increasing or decreasing its mass, or by adding or deleting one or more amino acids, for example, if the analyte is a protein or peptide.

[0017] If the analyte is an immunoglobulin light chain or heavy chain, the mass difference of the control substance, e.g., the control light chain or heavy chain, is at least 1000 Da to ensure that the MS peak of the control substance is separated from the analyte peak.

[0018] This linkage is cleaved by acid in an acid eluate. The linkage may also be cleavable by a reducing agent. Examples of linkages include disulfide bonds that are reduced by acid and / or a reducing agent to release a control substance.

[0019] The linkage may include, for example, a biotin molecule. One or the other of the substrate or control substance may comprise one portion of the linkage, while the other is present in the other portion of the substrate-control substance complex. Maleimide may also be used to bind proteins or peptides to substrates via reaction with sulfhydryl groups.

[0020] For example, other acid- or reducing agent-cleavable bonds that have been used in acid-cleavable drug delivery systems, which may still be useful in current systems, include, for example, orthoesters, acetals, hydrazones, imines, cis-aconityl, and trityl bonds (Binaud S. and Stenzel MH Chem Comm (2013), 49, 2082-2102).

[0021] The control substance is typically a macromolecule such as a peptide or protein, but may be any detectable compound containing nucleic acids such as DNA or RNA.

[0022] The control substance may be part of a heteropolymer. This may be any compound having two or more subunits, one of which is bound to the substrate, and the second subunit (acting as the control substance) is separable by acid elution and then detected as a second peak. Typically, the second subunit is selected to have substantially the same ionization properties as the analyte, and typically to have a mass similar to, but not identical to, the analyte to be detected. Accordingly, the second subunit is selected to generate one or more mass spectrometry peaks that do not substantially overlap with the mass spectrometry peak of the analyte, such that the first peak of the analyte and the second peak of at least a portion of the heteropolymer can be distinguished and measured.

[0023] The heteropolymer is typically a protein. The subunits may be, for example, subunits linked via one or more disulfide bonds, which can be cleaved by an acid elution step to release one or more subunits.

[0024] The heteropolymer can be an antibody or any fragment of an antibody. It may comprise at least one heavy chain or fragment thereof, and at least one light chain or fragment thereof, typically linked via one or more disulfide bonds.

[0025] The antibody or antibody fragment may itself be an analyte-specific antibody or fragment (e.g., F(ab')2) or a light chain, a portion of which is then detected as the second peak. Optional further analyte-specific antibodies are typically not used in that case.

[0026] When an antibody or fragment is used, the light chain can be modified by the addition or deletion of one or more (e.g., up to 5) amino acids to the C-terminus in order to increase the mass of the light chain.

[0027] An antibody or fragment can also be modified by adding a chemical modifying agent to increase the mass of the light chain. Examples include N-succinimidyl 3-(4-hydroxyphenyl)propionate, 6-biotinamidohexanoic acid N-succinimidyl ester, biotin-PEG36-pentafluorophenyl ester, and NHS-PEG4-biotin.

[0028] It has been found that kappa light chains are preferentially bound by several chemical modifying agents including biotin-PFP, for example on IgG kappa, and are also preferentially bound to lambda light chains. The level of binding to the kappa light chain can be controlled. Lambda light chain modification has also been observed by the present applicant.

[0029] The predetermined calibration value can be calculated by performing each of steps (i) to (v) in the absence of the analyte. That is, a predetermined amount of control substance bound to at least one substrate is not contacted with the antibody or fragment, but instead is contacted with a control substance solution in the absence of the analyte, and the washing step (iii) and the acid elution step (iv) are carried out in the same manner as described above. Next, the amount of the eluted control substance is quantified. This can be performed, for example, by liquid chromatography-mass spectrometry (LC-MS).

[0030] The amount of eluted antibody can be used to determine the calibration value for that sample.

[0031] The calibration step can be performed substantially simultaneously with performing the purification step and the characterization step using the analyte. Alternatively, it may be performed at the supplier's laboratory, for example, before supplying the analyte-specific antibody bound to the substrate. The calibration value can then be supplied with each batch of analyte-specific antibody bound to the substrate. This predetermined value provides a constant value that allows variations in the purification procedure due to the analyte to be calculated.

[0032] The analyte may be, for example, serum proteins such as immunoglobulins or their fragments, or other serum proteins such as albumin, coagulation factors, regulatory proteins, beta-2-microglobulin, C-reactive proteins, alpha-1 antitrypsin, or alpha-1 fetoprotein.

[0033] Examples of antibodies include polyclonal and monoclonal antibodies derived from sheep, cattle, horses, rats, mice, rabbits, camels, or recombinant and synthetic antibodies.

[0034] The antibody or its fragment may be an intact antibody or, for example, an F(ab')2 fragment.

[0035] The buffers used for washing, acid elution, and mass spectrometry are commonly known in the art, and the pH of the acid elution step is typically (but not exclusively) pH 7 or less, and typically less than 5 or greater than 1.5, such as 2-3. Such buffers are typically compatible with mass spectrometry methods such as MALDI-TOF, including:

[0036] Below are buffers suitable for use with MALDI-TOF, for example, peptides or proteins with typical maximum concentrations.

[0037] [Table 1]

[0038] Surfactant resistance to MALDI-TOF (especially in biological samples: peptides, proteins) Category A: Acceptable Category B: No or minimal negative impact. Reduction of Category C signals and their impact on quality Category D: Typically incompatible with MALDI TOF.

[0039] [Table 2]

[0040] Typically, MALDI-TOF mass spectrometry is used to generate at least one peak for the analyte (if any) and a second peak associated with at least one fragment of the control substance used in the purification process.

[0041] The second peak is typically the light chain or a fragment of the antibody. For example, if multiple charged antibody fragments or antibodies are produced by the mass spectrometry process, there may be two or more peaks. Acid elution typically dissociates the heavy chain from the light chain of the antibody.

[0042] When a separate control substance is used and the analyte is an immunoglobulin, the analyte-specific antibody can be crosslinked to prevent the release of light chains bound to the heavy chain, thereby preventing the sample from interfering with light chains released from, for example, the analyte-specific antibody or its fragments.

[0043] Therefore, analyte-specific antibodies or fragments thereof can be crosslinked, for example, by links that are stable under acid elution correlation.

[0044] WO2006 / 099481A describes the use of intrachain and interchain thioether crosslinking in a wide range of macromolecules, including polypeptides such as polyclonal antibodies, monoclonal antibodies, Fab, F(ab) and F(ab')2 fragments, single-chain antibodies, human antibodies, harmonic or chimeric antibodies, and epitope-binding fragments. The document states that the purpose of crosslinking is to enhance the stability and pharmacological and functional properties of the antibody or fragment. In particular, the purpose is to crosslink the heavy and light chains of different monoclonal antibodies, such as antiviral antigen antibodies, including anti-RSV antibodies. The stated purpose is to improve the pharmaceutical properties of the antibody.

[0045] WO00 / 44788 describes the use of thioethers to crosslink different antibody molecules with different specificities for the purpose of manufacturing improved therapeutic agents. Similarly, bispecific or trispecific F(ab)3 or F(ab)4 conjugates with various specificities are shown in WO91 / 03493.

[0046] Thioethers have been observed in therapeutic antibodies as their levels increase during storage (Zhang Q et al JBC manuscript (2013) M113.468367). Light-chain-heavy-chain disulfide (LC214-HC220) can be converted to thioether conjugates. One IgG1k therapeutic antibody was observed to be converted to a thioether at its site at a rate of 0.1% / day while circulating in the blood. Endogenous antibodies were also observed to form in healthy human subjects. Zhang et al. repeated thioether generation in vitro and used this to evaluate the safety impact of thioether conjugation on therapeutic monoclonal antibodies.

[0047] Crosslinking typically occurs within molecules, between chains of the same antibody.

[0048] Crosslinking typically involves thioether bonds.

[0049] Thioether crosslinks contain thioether bonds. This is a bond between antibody residues where the bond has a single sulfur bond rather than a disulfide bond. That is, a thioether crosslink does not contain a bond with two or more sulfur atoms, like a disulfide crosslink well known to those skilled in the art; instead, a thioether crosslink contains a single sulfur bond that crosslinks residues of a macromolecule. One or more additional non-sulfur atoms may further form the bond.

[0050] The residues linked by the thioether crosslink may be native or non-native residues. The formation of a thioether crosslink can result in the loss of atoms from the residues, as will be recognized by those skilled in the art. For example, the formation of a thioether crosslink between the side chains of two cysteine ​​residues can result in the loss of sulfur and hydrogen atoms from the residues, but the resulting thioether crosslink will be recognized by those skilled in the art as linking the cysteine ​​residues.

[0051] Thioether crosslinks can link any two residues of an antibody. One or more residues can be selected from, for example, cysteine, aspartic acid, glutamic acid, histidine, methionine, and tyrosine. Two residues can be selected from the group consisting of cysteine, aspartic acid, glutamic acid, histidine, methionine, and tyrosine. More typically, two residues are cysteine ​​residues. Typically, only one thioether crosslink exists between the heavy and light chains. Alternatively, two, three, or more thioether crosslinks may be used. The heavy chain pair of the antibody or a fragment thereof can also be linked by one or more non-disulfide crosslinks, such as thioether links.

[0052] Thioether crosslinking is described, for example, in WO2006 / 099481, Zhang et al (2013) J. Biol. Chem. vol 288(23), 16371-8 and Zhang & Flynn (2013) J. Biol. Chem, vol 288(43), 34325-35, which are incorporated herein by reference.

[0053] Phosphines and phosphites can also be used. Here, "phosphine" refers to any compound containing at least one functional group unit having the general formula R3P (where P = phosphorus and R = any other atom). In phosphites, the R position is specifically occupied by an oxygen atom. R3P-containing compounds act as potent nucleophiles that attack disulfide bonds. As a result, the disulfide may be reduced, but under certain conditions, a thioether bond may also be formed.

[0054] The compounds include the following: Tris(dimethylamino)phosphine (CAS number 1608-26-0) Tris(diethylamino)phosphine (CAS number 2283-11-6) Trimethyl phosphite (CAS number 121-45-9) Tributylphosphine (CAS number 998-40-3)

[0055] References: Bernardes et al. (2008) Angew. Chem. Int. Ed., vol 47, 2244-2247, incorporated herein by reference.

[0056] The crosslinking may also include a crosslinking agent, such as a maleimide crosslinking agent, which reacts with free thiols to crosslink the antibody molecule chain. This can be attached to one side of the thiol group and further to another side, such as a lysine carboxyl group, as described in WO00 / 44788.

[0057] A bifunctional crosslinking agent may include two reactive moieties linked to each other by a linker, particularly a flexible linker. The linker may include one or more carbon atoms covalently bonded in the chain, such as substituted or unsubstituted alkyl groups. Linkers, particularly C1-C10, most typically C2-C6 or C3-C6 linkers. The applicant has found that C2-C6 linkers containing crosslinking agents such as α,α'-dibromo-m-xylene, BMOE (bismaleimidoethane), or BMB (bismaleimidobutane) are particularly useful when the recovery level of crosslinked proteins is relatively high.

[0058] The size of an antibody or its fragment can be pre-selected to generate one or more peaks by mass spectrometry, which are separated by one or more peaks related to the analyte. This is possible, for example, by selecting the size of the antibody or fragment, or alternatively, by selecting the charge of the antibody or fragment.

[0059] When MALDI-TOF mass spectrometry is used, the peaks are typically m / z intensities.

[0060] The amount of control material, such as the initial antibody, added to the system is known. The expected amount of antibody without using a sample is also known for calibration. Therefore, the amount of antibody in the peak along with the analyte can be corrected by the calibration value to account for any material loss or, for example, pipetting errors. The m / z peak intensity ratio between the target and the antibody can be used to control, for example, sample loss, substrate loss, target capture, analyte elution, analyte spotting, and ionization.

[0061] The ability to precisely couple a known mass of antibody onto beads and subsequently quantify its elution and m / z peak intensity (by comparison with a target peak). This improves accuracy and precision, making it suitable for use in routine clinical procedures.

[0062] The substrate is typically a bead, such as a magnetic bead, latex bead, ceramic bead, polystyrene, or other substrate.

[0063] Magnetic and paramagnetic beads are generally known in the art.

[0064] The absorption of antibodies onto substrates such as beads is also commonly known in the art, and they may typically be covalently bonded via amino groups, carboxyl groups, epoxy groups, or thiol-activating groups. Passive adsorption can also be used.

[0065] When using magnetic beads, it is possible to mix the bound antibody in the sample containing the analyte with the beads and then precipitate the beads using a magnet. The antibody or fragment thereof may be a monoclonal antibody, a polyclonal antibody, or a mixture thereof.

[0066] Typically, antibodies or fragments are not enzymatically digested after the purification of the analyte.

[0067] The analyte can potentially be any suitable analyte. Typically, when applied to a mass spectrometry target, the analyte and immunoglobulin cocrystallize and ionize in substantially the same manner.

[0068] The sample may be from a mammal or, typically, a plant or animal such as a human. This could be a tissue or bodily fluid sample such as blood, serum, plasma, sweat, saliva, cerebrospinal fluid, or urine.

[0069] The analyte may be an immunoglobulin or a fragment thereof. For example, the immunoglobulin or fragment may be human IgG, IgA, IgM, lambda light chain, or kappa light chain.

[0070] The analyte may be a human analyte.

[0071] Typically, when the analyte is an immunoglobulin, the antibody or fragment thereof is a monoclonal antibody or fragment thereof. This is because the size of the monoclonal antibody can be adjusted, for example, by selecting the size of the monoclonal antibody, or alternatively, by adjusting the mass by deleting the antibody fragment or adding amino acids to the antibody, thereby ensuring that the antibody or fragment thereof does not overlap with, for example, the m / z peak observed for the analyte.

[0072] Immunoglobulins are inherently highly diverse. Therefore, a potential problem with some monoclonal antibodies is that they cannot detect all variants of an analyte, such as monoclonal antibodies themselves. This can be improved, for example, by coating a substrate with a predetermined amount of monoclonal antibody or a fragment thereof, and further coating it with multiple additional analyte-specific antibodies or fragments, such as polyclonal antibodies. This allows for broad specificity of analyte capture while simultaneously providing a predetermined antibody or fragment with, for example, a known peak or m / z size. For example, a ratio of 10-20% of a predetermined monoclonal antibody (or fragment) to 90-80% polyclonal antibody can be used.

[0073] The antibody or its fragment may be heavy chain class specific, light chain type specific, free light chain specific, or heavy chain class / light chain type specific.

[0074] This method can be used to detect or prognose diseases by detecting the presence of an analyte, or, for example, by detecting the concentration of an analyte in a sample. The disease may be any disease in which the analyte can be purified using an analyte-specific antibody. For example, B-cell related diseases or other immunoglobulin-related diseases. Other examples include acute-phase markers, liver diseases, and lung cancer biomarkers.

[0075] There are many proliferative disorders associated with antibody-producing cells.

[0076] In many such proliferative disorders, plasma cells proliferate to form monoclonal tumors of identical plasma cells. This results in the production of large amounts of identical immunoglobulins, a condition known as monoclonal immunoglobulinemia.

[0077] Diseases such as myeloma and primary systemic amyloidosis (AL amyloidosis) account for approximately 1.5% and 0.3% of cancer deaths in the UK, respectively. Multiple myeloma is the second most common hematological malignancy, after non-Hodgkin lymphoma. In the Caucasian population, the incidence is approximately 40 cases per million people per year. Traditionally, the diagnosis of multiple myeloma has been based on the presence of excess monoclonal plasma cells in the bone marrow, monoclonal immunoglobulins in the serum or urine, and associated organ or tissue damage such as hypercalcemia, renal failure, anemia, or bone lesions. While the normal plasma cell content in bone marrow is about 1%, in multiple myeloma, the content is typically over 10%, often over 30%, and can exceed 90%.

[0078] AL amyloidosis is a protein conformational disorder characterized by the accumulation of monoclonal free light chain fragments as amyloid deposits. Typically, these patients present with heart failure or renal failure, but peripheral nerves and other organs may also be affected.

[0079] Many other diseases can be identified by the presence of monoclonal immunoglobulins in a patient's bloodstream, or even in their urine. These include plasmacytoma and extramedullary plasmacytoma, plasma cell tumors that occur outside the bone marrow and can occur in any organ. If present, the monoclonal protein is typically IgA. Multiple solitary plasmacytomas can occur with or without evidence of multiple myeloma. Waldenström macroglobulinemia is a low-grade lymphoproliferative disorder associated with the production of monoclonal IgM. There are approximately 1,500 new cases per year in the United States and 300 in the United Kingdom. Quantification of serum IgM is important for both diagnosis and monitoring. B-cell non-Hodgkin lymphoma accounts for approximately 2.6% of all cancer deaths in the UK, and monoclonal immunoglobulins have been identified in the serum of approximately 10–15% of patients using standard electrophoresis. Initial reports have shown that monoclonal free light chains are detected in the urine of 60–70% of patients. In B-cell chronic lymphocytic leukemia, monoclonal proteins have been identified by free light chain immunoassay.

[0080] Furthermore, there is a condition known as MGUS, which is monoclonal immunoglobulinemia of unknown significance. This term refers to the unexpected presence of intact monoclonal immunoglobulins in patients without evidence of multiple myeloma, AL amyloidosis, or Waldenström macroglobulinemia. MGUS is found in 1% of the population aged 50 and over, 3% of the population aged 70 and over, and 10% of the population aged 80 and over. The majority of these are IgG or IgM-related, but IgA-related or diclonal are rare. Most MGUS patients die from unrelated diseases, but MGUS can transform into malignant monoclonal hypergammaglobulinemia.

[0081] In at least some cases of the diseases highlighted above, the disease presents with abnormal concentrations of monoclonal immunoglobulins or free light chains. When the disease causes abnormal replication of plasma cells, immunoglobulin production is often increased by these types of cells, so that their "monoclones" proliferate and appear in the blood.

[0082] A kit for use in the method of the present invention comprises at least one substrate, which includes a predetermined amount of several control substances and optionally an analyte-specific antibody or fragment thereof, further comprising a predetermined calibration value for calibrating the analyte to be calibrated. This is provided in combination with a reference value, which may be, for example, in the form of a numerical value programmed into a computer, or provided on an automated chip system for loading into a computer. Alternatively, it may be provided as a barcode or QR code for uploading to an assay device. The calibration value can be determined as described above. In such a kit, the control substance antibody or fragment thereof may be as defined above.

[0083] Further aspects of the present invention provide a mass spectrometer having means for performing steps (v) and (vi) of the present invention. A computer program is also provided which includes instructions for causing the mass spectrometer to perform steps (v) and (vi) of the present invention.

[0084] A computer-readable medium is also provided that, when executed on one or more processors, includes instructions for comparing the size or area of ​​a second peak obtained by the method of the present invention with a predetermined calibration value. This medium can also, for example, compare the peak size or m / z value of the second peak with the ratio of the analyte peak.

[0085] Substrates and methods for binding antibodies to substrates, as well as subsequent analytes and control materials, such as heteropolymers, and methods for eluting and detecting antibody peaks, are generally known in the art. Polyclonal anti-immunoglobulin antibodies, such as anti-light chain, anti-free light chain, anti-heavy chain, and anti-heavy chain class-light chain antibodies, are available, for example, from The Binding Site Group Limited, Birmingham, United Kingdom. Monoclonal anti-heavy chain and anti-light chain antibodies are also known in the art.

[0086] Herein, the present invention will be described by reference only to the following drawings. [Brief explanation of the drawing]

[0087] [Figure 1] Figure 1 illustrates the principle of using the antibody bound to the beads, following acid elution and MALDI-TOF. In this example, no antigen is present, and the concentration of the antibody eluted from the substrate is detected and quantified by chromatography-mass spectrometry. The MALDI-TOF mass spectrum of the antibody-derived light chain is also shown. [Figure 2] Figure 2 shows the use of a conjugated monoclonal antibody to detect proteins such as β2M on MALDI-TOF. [Figure 3]Figure 3 shows an example of using a light chain-specific monoclonal antibody to detect and quantify light chains from a sample. [Figure 4] Figure 4 shows the gradual biotinylation and mass shift of Daratumumab (IgGκCD38-specific monoclonal antibody) using PFP-Biotin. [Figure 5] Figure 5 shows the biotinylation and mass shift of polyclonal IgA (left panel) or polyclonal IgM (right panel) from healthy human serum. [Figure 6] Figure 6 shows the time course of biotinylation and mass shift of kappa light chains from polyclonal IgA and IgM using biotin-PEG36-PFP. [Figure 7a] Figure 7 shows the mass modification of the daratumumab kappa light chain using biotin-PEG36-PFP. Labeling was performed using a single dose of the reagent (Figure 7a) for 2 hours, or by adding a second dose of the reagent after 2 hours (Figure 7b) for 6 hours. [Figure 7b] Figure 7 shows the mass modification of the daratumumab kappa light chain using biotin-PEG36-PFP. Labeling was performed using a single dose of the reagent (Figure 7a) for 2 hours, or by adding a second dose of the reagent after 2 hours (Figure 7b) for 6 hours. [Figure 8a] Figure 8 shows the mass modifications and spectral shifts of monoclonal proteins. IgAK (A1K20, Figure 8a) and IgG2K (G2K14, Figure 8b) were labeled with biotin-PEG36-PFP. [Figure 8b] Figure 8 shows the mass modifications and spectral shifts of monoclonal proteins. IgAK (A1K20, Figure 8a) and IgG2K (G2K14, Figure 8b) were labeled with biotin-PEG36-PFP. [Figure 9] Figure 9 shows the effect of increased biotin-PEG24-TFP levels and reaction time (3 hours or overnight) on the mass modification of polyclonal immunoglobulin κ light chains in healthy serum. [Figure 10]Figure 10 shows the use of mass modification of the daratumumab kappa light chain using Biotin-PEG36-PFP as a mass standard. [Figure 11] Figure 11 shows dose-response data for MALDI-TOF signal intensity and daratumumab concentration (mass-modified (lower panel) and unmodified (upper panel) kappa light chains). [Figure 12] Figure 12 shows the QIP-MS (immunoprecipitation) reaction of monoclonal IgAL (A1L20) clinical specimens collected from multiple myeloma patients, with the addition of daratumumab. [Figure 13] Figure 13 shows the mass modification spike of daratumumab in the QIP-MS (immunoprecipitation) reaction of healthy IgA (UNP001) clinical specimens. [Modes for carrying out the invention]

[0088] Figure 1 is a schematic diagram illustrating, for illustrative purposes only, antibodies bound to a bead-like matrix. Antibodies can be attached using known techniques.

[0089] If no analyte is present, or if it is used to generate a control calibration value, the antibody may be eluted by acid elution, and the amount of antibody detected may be quantified by LC-MS. In this case, the mass spectrometer will produce two peaks based on the charge and mass of the light chain from the eluted antibody.

[0090] In Figure 2, the antibody is incubated with the patient sample to conjugate to an analyte such as β2M. After acid elution, the antibody and antigen are separated and detected using MALDI-TOF. In the example, the light chain is separated from the heavy chain, the light chain is detected and compared with the peak of the analyte sample. The sample volume can be corrected for a known amount of light chain generated by acid elution in the absence of antibody, and the antibody is used to generate a predetermined calibration value.

[0091] Figure 3 shows an example of a monoclonal antibody for detecting light chains from a patient sample. In this case, the size of the monoclonal antibody is heavier than the light chain of the sample being detected. This can be produced by selecting the size of the light chain in the monoclonal antibody, or by making the monoclonal antibody heavier, for example, by adding one or more additional amino acids to the N-terminus or C-terminus of the light chain residues of the monoclonal antibody. Techniques for producing such heavier monoclonal antibody light chains, or, if they are peaks detected by MALDI-TOF, actually monoclonal heavy chains, are generally known in the art.

[0092] Other control substances, such as those mentioned above, can be used in a similar manner. [Examples]

[0093] background Mass spectrometry (MS) allows for the separation of analytes by mass-to-charge ratio (m / z). Polyclonal immunoglobulin light chains, having various sets of masses, typically produce a normally distributed, bell-shaped curve in m / z with respect to signal intensity. Monoclonal light chains dissociate as sharp peaks extending out of the bell curve. We previously described a double-charged (light chain ion ([M+2H]) 2+ We observed that the best resolution is produced by MALDI-TOF in the m / z range of 10900–12300. The exemplified EXENT QIP-MS immunoassay preanalysis step comprises three main steps: (1) immunocapture of the analyte with magnetic beads, (2) co-elution and reduction of the analyte, and (3) spotting of the analyte onto a MALDI-TOF target plate. We have chosen to include, as an example, a mass-modified protein standard attached to magnetic beads that can be added during step 1, so that it can be amalgamated with the analyte before step 2 and spotted simultaneously. This is important because it can be used to control variability in step 1 and then to standardize or control the analyte spectral signal obtained from the MALDI-TOF mass spectrometer.

[0094] method Massive modification of immunoglobulins Immunoglobulins were modified with biotin or biotinylated PEG molecules via pentafluorophenyl (PFP) or tetrafluorophenyl (TFP) crosslinking agents. These target both primary and secondary amines in proteins and are more reactive and stable than the more commonly used N-hydroxysuccinimide (NHS) ester group of crosslinking agents. They have been shown to be suitable for biotin labeling of both proteins and amino acids and are commercially available (e.g., EZ-Link® PFP-Biotin, catalog no. 21218, Thermo Fisher Scientific, and Biotin-PEG36-PFP ester, catalog no. BP-24318, BroadPharm). Immunoglobulins at 5 mg / ml in PBS were incubated with varying amounts of PFP or TFP crosslinking agents dissolved in DMSO for varying periods (from several hours to several days) at room temperature on a shaker. The reaction was stopped by the addition of glycine (1:1 mol), and the unconjugated crosslinking agent was removed by dialyzing.

[0095] Preparation of mass spectral standard particles or beads To prepare mass spectrometry standard beads, 10 μl of modified immunoglobulin was diluted to 50 μl in PBS and incubated with anti-human IgG paramagnetic microparticles for 15 minutes. The beads were pelleted on a magnetic rack, the supernatant was removed, and the beads were washed three times with PBS-Tween and once with water before being stored until use.

[0096] EXENT QIP-MS Immunoassay Serum samples or pure proteins were diluted and captured in an EXENT QIP-MS immunoassay using paramagnetic microparticles containing antibodies specific to human immunoglobulin heavy and light chains (anti-IgG, IgA, IgM, total kappa, and total lambda). These microparticles were conjugated to either stabilized sheep polyclonal antibodies or recombinant camel VH domain antibodies (Thermo Fisher Scientific). The beads were pelletized and continuously washed with PBS-Tween. Mass spectrometry standard particle beads were added to the mixture, pelletized, and washed once with water. This was eluted with an acidic buffer containing both a reducing agent and an ionization control protein (see, e.g., WO2021 / 019211). Subsequently, the elutes were spotted onto a MALDI-TOF target plate in a sandwich containing a MALDI matrix (α-cyano-4-hydroxycinnamic acid) and dried. Mass spectra were obtained for dual charge ([M+2H]) 2+ The analytes (human kappa or lambda light chain) and mass spectra (std) were acquired in positive ion mode on a Bruker Microflex MALDI-TOF-MS covering the m / z range of 5000 to 30,000, including m / z 10900 to 12300.

[0097] result Massive modification of immunoglobulins To generate mass-shift molecules usable with the EXENT QIP-MS system, two parameters must be met: (1) modification of the immunoglobulin light chain that does not interfere with the immunoprecipitation or immunocapture of the molecule, and (2) addition (or actual removal) of a mass detectable as an m / z shift. Using biotin addition via PFP crosslinking to therapeutic monoclonal IgGK Daratumumab, we demonstrated that complete immunoglobulin modification with a corresponding m / z mass shift of the immunoglobulin light chain was observed by MALDI-TOF (Figure 4). Figure 5 shows that this mass shift due to biotination also works in polyclonal IgA and IgM samples. In all three cases, immunoprecipitation of these mass-modified proteins by camel antibody beads was unaffected. The mass increase observed with biotin alone is not sufficient to shift the mass beyond the expected biological range of +2 charged state ions; 10900–12300 Da; a larger molecule is required to achieve this. Biotin-PEG36-PFP was added to polyclonal IgA and IgM molecules, as well as molecules captured by sheep anti-kappa light chain beads. The time course showed that a 4-hour reaction time was sufficient to correct the mass of the kappa light chains to an average m / z of 11700–12650 (Figure 6). This study was expanded using biotin-PEG36-PFP modification of the daratumumab monoclonal protein (Figure 7). Two-step addition of the modifier over 6 hours shifted almost all of the mass of the kappa light chains compared to a single-step addition over 2 hours (Figure 7a) (Figure 7b). Biotin-PEG36-PFP can also be used to mass-shift light chains on myeloma-derived monoclonal immunoglobulins, IgAK (Figure 8a) and IgG2K (Figure 8b). Similar mass shifts of polyclonal immunoglobulin κ light chains in human serum could be obtained over 3 hours or overnight using the relevant crosslinking agent (biotin-PEG24-TFP) (Figure 9ab).

[0098] EXENT QIP-MS std To illustrate the use of mass-modified immunoglobulin as an in-situ MS control, daratumab was labeled with biotin-PEG36-PFP for 3 days. Analysis of the light chain m / z using an EXENT QIP-MS immunoassay with sheep anti-IgG beads suggested single-site modification (Figures 10 and 11). This single-site modification was sufficient to increase the +2 m / z from 11700 to 12600 without affecting the ability of IgGK immunoglobulin to be immunocaptured by anti-IgG beads. A dilution series of this molecule over a 10-fold range showed that this concentration was positively correlated with MS signal intensity (Figure 11).

[0099] Mass-modified daratumumab was conjugated to anti-IgG beads, and an EXENT QIP-MS immunoprecipitation IgA assay was performed, resulting in the simultaneous elution of the analyte and the mass-modified molecule. The latter mass-modified light chain +2 peaks were clearly distinguishable from those obtained from polyclonal IgA present in myeloma IgAL patients (Figure 12) or healthy samples (Figure 13).

Claims

1. A method for immunopurifying and characterizing analytes from a sample by mass spectrometry, wherein the method is (i) A step of providing a predetermined amount of a control substance bound to a first substrate via a linkage cleavable by an acidic pH and / or a reducing agent, and optionally a further analyte-specific antibody or fragment thereof bound to a first or second substrate, wherein the control substance is analyte-specific or not analyte-specific; (ii) A step that enables the analyte in the sample to bind to a control substance or any further analyte-specific antibody or fragment of the choice; (iii) A step of washing away unbonded material from the substrate or each substrate; (iv) A step of acid eluting the analyte bound to the substrate or each substrate to cleave at least a portion of the control substance, or, if the control substance is a heteropolymer of a protein comprising two or more separable protein subunits, a step of cleaving at least a portion of the protein subunits; (v) Perform mass spectrometry, At least one first peak associated with the presence of the analyte, and At least one second peak associated with at least a portion of the control substance A step of identifying two or more peaks including; and (vi) A step that enables the calibration of at least one first peak by comparing the size or intensity of at least one second peak with a predetermined calibration value. A method that includes this.

2. The method according to claim 1, wherein a control substance bound to a first substrate is provided after the analyte is contacted with a further analyte-specific antibody.

3. The method according to claim 1, wherein the control substance is a heteropolymer, which is a protein comprising two or more separable protein subunits, and the portion of the heteropolymer detected in at least one second peak is at least a part of one of the protein subunits.

4. The method according to claim 3, wherein the protein is an antibody or fragment thereof comprising at least one heavy chain or fragment thereof and at least one light chain or fragment thereof, and the subunit detected in at least one second peak is at least a portion of the light chain.

5. The method according to claim 4, wherein the antibody is specific to the analyte.

6. The method according to any one of claims 1 to 5, wherein the control substance is not specific to the analyte, and the substrate or each substrate comprises the further analyte-specific antibody.

7. A method for generating predetermined calibration values ​​for use in a method for immunopurifying and characterizing analytes from a sample by mass spectrometry, wherein the method is: (i) A step of providing a predetermined amount of a control substance bound to a first substrate via a linkage cleavable by an acidic pH and / or a reducing agent, and optionally a further analyte-specific antibody or fragment thereof bound to a first or second substrate; (ii) A step of adding a sample in which the analyte is not present in the sample; (iii) A step of washing away unbonded material from the substrate or each substrate; (iv) A step of acid eluting and cleaving at least a portion of the control substance, or, if the control substance is a heteropolymer of a protein comprising two or more separable protein subunits, a step of cleaving the substrate or at least a portion of the protein subunits from each substrate; (v) performing mass spectrometry to identify at least one second peak associated with at least a portion of the control substance; and (vi) A step of quantifying at least a portion of the control substance and generating a predetermined calibration value. A method that includes this.

8. The method according to claim 7, wherein at least a portion of the control substance is calibrated by liquid chromatography-mass spectrometry.

9. The method according to claim 7 or 8, wherein at least a portion of the control substance is an immunoglobulin light chain or a fragment thereof.

10. The method according to any one of claims 1 to 6, wherein, when the mass spectrometry step (v) is performed, the mass of at least a portion of the control substance is pre-selected to produce at least one second peak separated from at least one first peak.

11. The method according to any one of claims 1 to 10, wherein the mass spectrometry is MALDI-TOF mass spectrometry, and the peak or each peak is an m / z intensity.

12. The method according to any one of claims 1 to 6 or 7 to 11, wherein the control substance and / or further analyte-specific antibody or fragment thereof is a monoclonal antibody, a polyclonal antibody, or a mixture thereof.

13. The method according to any one of claims 1 to 6 or 11 to 12, wherein the immunopurified and characterized analyte is a serum protein.

14. The method according to claim 13, wherein the serum protein is an immunoglobulin or a fragment thereof.

15. The method according to claim 14, wherein the immunoglobulin or fragment thereof is human IgG, IgA, IgM, IgD, or IgE lambda light chain or kappa light chain.

16. The method according to claim 14 or 15, wherein the control substance and / or further analyte-specific antibody or fragment thereof is a monoclonal antibody or fragment thereof.

17. The method according to claim 16, wherein the monoclonal antibody or fragment thereof is selected to have different masses and / or charges when analyzed against an immunoglobulin analyte by mass spectrometry.

18. The method according to claim 17, wherein the monoclonal antibody or fragment thereof is modified in mass such that it has a different mass relative to the immunoglobulin analyte.

19. The method according to any one of claims 12 to 18, wherein the control substance and / or further analyte-specific antibody or fragment thereof is a heavy chain class-specific, light chain type-specific, free light chain type-specific, or heavy chain class / light chain type-specific antibody or fragment thereof.

20. The method according to any one of claims 1 to 19, wherein the substrate or each substrate comprises a predetermined amount of a control substance and a plurality of further analyte-specific antibodies or fragments thereof.

21. The method according to claim 20, wherein the control substance and / or analyte-specific antibody or fragment thereof is a polyclonal antibody or fragment thereof.

22. The method according to any one of claims 1 to 21, wherein the substrate or each substrate comprises a plurality of beads.

23. A kit for use in the method according to any one of claims 1 to 6, comprising at least one substrate comprising a predetermined amount of a control substance attached to a first substrate via an acid-cleavable linkage, wherein the control substance is specific to an analyte or not specific to an analyte, and if the control substance is not specific to an analyte, the kit further comprises a further analyte-specific antibody or fragment thereof conjugated to the first substrate or the second substrate.

24. The kit according to claim 23, wherein the multiple analyte-specific antibodies or fragments thereof are polyclonal, monoclonal, or mixtures thereof.

25. The kit according to claim 23, wherein the analyte is immunoglobulin or a fragment thereof.

26. The kit according to any one of claims 23 to 25, wherein the antibody or fragment thereof is heavy chain class specific, light chain type specific, free light chain type specific, or heavy chain class-light chain type specific.

27. A computer program comprising instructions for causing a mass spectrometer to perform steps (v) and (vi) as defined in any one of claims 1 to 22.

28. A computer-readable medium that, when executed on one or more processors, includes instructions for comparing the size or intensity of at least one second peak obtained by the method described in any one of claims 1 to 6 with a predetermined calibration value.

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