MASS SPECTROMETRY CONTROLS

Abstract

The Invention provides a method of immunopurifying and characterising an analyte from a sample comprising: (i) providing a predetermined amount of a control substance bound to a substrate via a linkage cleavable by acidic pH and/or reducing agents and optionally additional analyte specific antibodies or fragments thereof bound to a substrate, wherein the control substance is specific for the analyte or is not specific for the analyte; (ii) allowing analyte when present in the sample to bind to the control substance or said optional additional analyte-specific antibodies or fragments, wherein the control substance bound to the substrate (i) may be provided after contacting the analyte with the optional additional analyte-specific antibodies (ii); (iii) washing unbound material away from the substrate; (iv) acid eluting the analyte bound thereto, from at least one substrate; (v) performing mass spectrometry to identify two or more peaks, at least one peak of which is associated with the presence of the analyte and at least a second peak which is associated with at least a portion of the control substance; and (vi) comparing the size or intensity of the second peak to a predetermined calibration value to allow the first peak associated with the analyte to be calibrated.

Claims

1. A method of immunopurifying and characterising an analyte from a sample comprising: (i) providing a predetermined amount of a control substance bound to a substrate via a linkage cleavable by acidic pH and/or reducing agents and optionally additional analyte specific antibodies or fragments thereof bound to a substrate, wherein the control substance is specific for the analyte or is not specific for the analyte; (ii) allowing analyte when present in the sample to bind to the control substance or said optional additional analyte-specific antibodies or fragments, wherein the control substance bound to the substrate (i) may be provided after contacting the analyte with the optional additional analyte-specific antibodies (ii); (iii) washing unbound material away from the substrate; (iv) acid eluting the analyte bound thereto, from at least one substrate; (v) performing mass spectrometry to identify two or more peaks, at least one peak of which is associated with the presence of the analyte and at least a second peak which is associated with at least a portion of the control substance; and (vi) comparing the size or intensity of the second peak to a predetermined calibration value to allow the first peak associated with the analyte to be calibrated.

2. 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 the second peak is at least a portion of one of said protein subunits.

3. The method according to claim 2, wherein the protein is an antibody or fragment thereof comprising at least one heavy chain or fragments thereof and at least one light chain or a fragment thereof, and the subunit detected in the second peak is at least a portion of the light chain.

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

5. The method according to claim 1, wherein the control substance is not specific for the analyte and the substrate comprises said additional analyte specific antibodies.

6. The method according to claim 1, and further comprising performing the steps (i) to (v), without the presence of the analyte, and quantifying at least a portion of the control substance to produce the predetermined calibration value.

7. The method according to claim 1, wherein the at least a portion of the control substance is calibrated by liquid chromatography-mass spectrometry.

8. The method according to claim 1, wherein the portion of the control substance detected in the second peak are immunoglobulin light chains or fragments of light chains.

9. The method according to claim 1, wherein the size of portion of the control substance, such as the antibodies or fragments thereof are preselected to produce one or more peaks separated from one or more peaks associated with the analyte when the mass spectrometry step (vi) is performed.

10. The method according to claim 1, wherein the at least one peak and the at least second peak are determined by MALDI-TOF mass spectrometry and the peak is m/z intensity; or wherein the antibodies or fragments thereof are monoclonal antibodies or polyclonal antibodies; or wherein the analyte is a serum protein, for example, an immunoglobulin or fragment thereof, wherein the immunoglobulin or fragment thereof is optionally human IgG, IgA, IgM, IgD or IgE lambda light chains or kappa light chain.

11-13. (canceled)

14. The method according to claim 10, wherein the antibodies or fragments thereof are monoclonal antibodies or fragments thereof.

15. The method according to claim 14, wherein the monoclonal antibodies or fragments thereof are selected to have a different mass and/or charge when analysed by mass spectrometry to the immunoglobulin analyte.

16. The method according to claim 15, wherein the monoclonal antibodies or fragments thereof have had their mass modified to have a different mass to the immunoglobulin analyte.

17. The method according to claim 10, wherein the antibodies or fragments thereof are heavy chain class specific, light chain type specific, free light chain type specific, or heavy chain-class light chain type specific.

18. The method according to claim 1, wherein the substrate comprises a predetermined amount of the control substance and a plurality of additional analyte specific antibodies or fragments thereof which are preferably polyclonal antibodies or fragments thereof.

19. The method according to claim 1, wherein the substrate comprises a plurality of beads.

20. The method according to claim 1, and further comprising detecting, monitoring or prognosis of a disease by detecting the presence of an analyte according to claim 1, wherein the disease is optionally a B-cell related disease or other immune-related disease.

21. (canceled)

22. A kit comprising at least one substrate, comprising a predetermined amount of a control substance attached to the substrate via an acid cleavable linkage and optionally a plurality of analyte specific antibodies, or fragments thereof, for use in a method according to any preceding claim, additionally comprising a predetermined calibration value for calibrating the analyte to be calibrated; or comprising a plurality of polyclonal analyte-specific antibodies or fragments thereof, bound thereto and additionally a predetermined amount of a control substance; and optionally wherein the analyte is an immunoglobulin or fragment thereof; and optionally wherein the antibodies or fragments thereof are heavy chain class specific, light chain type specific, free light chain type specific or heavy chain class—light chain type specific.

23-25. (canceled)

26. A mass spectrometer having means to execute the steps (vi) and (vii) of claim 1.

27. A computer program comprising instructions to cause a mass spectrometer to perform steps (vi) and (vii) of claim 1; or comprising instructions which, when executed on one or more processors, compares the size or intensity of the second peak obtained by the method of claim 1 with a predetermined calibration value.

28. (canceled)

Description

[0094] The invention will now be described by way of example only with reference to the following figures.

[0095] FIG. 1 shows the principle of utilising an antibody attached to a bead, following by an acid elution and MALDI-TOF. In this example no antigen is present and the concentration of antibody eluted from the substrate is detected by chromatography-mass spectrometry and quantified. The mass spectra on MALDI-TOF of the light chain from the antibody is also shown.

[0096] FIG. 2 shows use of the bound monoclonal antibody to detect a protein, such as β2M, on MALDI-TOF.

[0097] FIG. 3 shows the example of using monoclonal antibody with light chain specificity to detect and quantify light chains from a sample.

[0098] FIG. 4 Progressive biotinylation and mass-shifting of Daratumumab (an IgG Kappa CD38 specific monoclonal antibody) using PFP-Biotin.

[0099] FIG. 5 Biotinylation and mass-shifting of polyclonal IgA (left panel) or polyclonal IgM (right panel) from healthy human serum.

[0100] FIG. 6 Time course of biotinylation and mass-shifting of kappa light chains from polyclonal IgA and IgM using Biotin-PEG36-PFP.

[0101] FIG. 7 Mass-modification of the Daratumumab kappa light chain using Biotin-PEG36-PFP. Labelling was performed for either 2 h using a single dose of the reagent (FIG. 7a) or 6 h with a second dose of the reagent added after 2 h (FIG. 7b).

[0102] FIG. 8 Mass-modification and spectral shifting of monoclonal proteins. An IgAK (A1K20, FIG. 8a) and IgG2K (G2K14, FIG. 8b) were labelled using Biotin-PEG36-PFP.

[0103] FIG. 9 Effect of increasing amounts of Biotin-PEG24-TFP and reaction time (3 h or overnight) on the mass-modification of polyclonal immunoglobulin kappa light chains in healthy serum.

[0104] FIG. 10 Mass-modification of the Daratumumab kappa light chain using Biotin-PEG36-PFP, to use as a Mass std.

[0105] FIG. 11 Dose response data for MALDI-TOF signal intensity and Daratumumab concentration for mass-modified (lower panel) and unmodified (upper panel) kappa light chains.

[0106] FIG. 12 Spiking of mass modified Daratumumab into the QIP-MS (immune-precipitation) reaction of a monoclonal IgAL (A1L20) clinical sample from a multiple myeloma patient.

[0107] FIG. 13 Spiking of mass modified Daratumumab into the QIP-MS (immuno-precipitation) reaction of a healthy IgA (UNP001) clinical sample.

[0108] FIG. 1 is a schematic diagram showing, by way of example only, antibodies attached to a matrix, such as a bead. The antibodies may be attached by known techniques.

[0109] Where no analyte is present, or alternatively where this is used to produce a control calibration value, the antibody may be eluted by acid elution and the amount of antibody detected is quantified by LC-MS. A mass spectrometer produces, in this case, two peaks based on the charge and mass of the light chain from the eluted antibody.

[0110] In FIG. 2, the antibody is incubated with a patient sample to bind to an analyte, such as β2M. After acid elution, the antibody and antigen are separated, and are detected using MALDI-TOF. In the example, the light chain is separated from the heavy chain and the light chain is detected and compared to the peak for the sample of the analyte. The amount of analyte may be corrected due to the known amount of light chain produced by acid elution in the absence of the antibody, which has been used to produce the predetermined calibration value.

[0111] FIG. 3 shows an example of a monoclonal antibody detecting a light chain from a patient sample. In this case the size of the monoclonal antibody is heavier that the light chain of the sample to be detected. This may be produced by either selecting the size of the light chain in the monoclonal antibody or alternatively making the monoclonal antibody heavier, for example by the addition of one or more additionally amino acids, for example, at the N or C terminus of the light chain residue of the monoclonal antibody. Techniques to produce such heavier monoclonal antibody light chains, or indeed monoclonal heavy chains should they be the peak being detected by MALDI-TOF, are generally known in the art.

[0112] Other control substances, such as those described above may also be used in a similar manner.

EXPERIMENTAL EXAMPLES

[0113] Background

[0114] Mass spectrometry (MS) allows the separation of analytes by mass-to-charge ratio (m/z). Polyclonal immunoglobulin light chains have a varied set of masses so typically produce a normally distributed bell-shaped curve of m/z against signal intensity. Monoclonal light chains resolve as a sharp peak extending out of the bell curve. We have previously observed that doubly charged (light chain ions ([M+2H].sup.2+) produce the best resolution by MALDI-TOF in the range m/z 10900 to 12300. The EXENT QIP-MS immunoassay pre-analytical phase exemplified has three main steps: (1) immunocapture of the analyte by magnetic beads, (2) simultaneous 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 a magnetic bead that can be added during step 1 so that it is amalgamated with the analyte prior to step 2 and can be spotted simultaneously with it. This is important since this can used to control for variability in step 1 and subsequently to standardise or control the resultant analyte spectral signal obtained from the MALDI-TOF mass spectrometer.

[0115] Methods

[0116] Mass Modification of Immunoglobulins

[0117] Immunoglobulins were modified using biotin or biotinylated-PEG molecules via pentafluorophenyl-ester (PFP) or tetrafluorophenyl-ester (TFP) crosslinkers. These target both primary and secondary amines in proteins, and are more reactive and more stable than the more commonly used N-hydroxysuccinimide (NHS) ester group of crosslinkers. They have been shown to be suitable for biotin labelling of both proteins and amino acids and are available commercially (e.g. EZ-Link™ PFP-Biotin, cat no. 21218, Thermo Fisher Scientific, and Biotin-PEG36-PFP ester, cat no. BP-24318, BroadPharm). Immunoglobulins at 5 mg/ml in PBS were incubated with different amounts of PFP or TFP crosslinkers dissolved in DMSO, at room temperature on a shaker for various durations (hours to days). Reactions were quenched by the addition of glycine (1:1 molar) and then dialysed to remove unconjugated cross-linker.

[0118] Preparation of Mass Spec Standard Particle or Bead

[0119] To prepare the Mass Spec std bead, 10 μl of the modified immunoglobulin was diluted to 50 μl with PBS-tween and incubated with an anti-human IgG paramagnetic microparticle for 15 min. The beads were pelleted on a magnetic rack, the supernatant was removed, and the bead washed thrice with PBS-tween and once with water and stored until use.

[0120] EXENT QIP-MS Immunoassay

[0121] Serum samples or pure proteins were diluted and captured during the EXENT QIP-MS immunoassay using a paramagnetic microparticle containing antibodies specific for human immunoglobulin heavy and light chains (anti-IgG, IgA, IgM, total kappa and total lambda). These microparticles were conjugated to either stabilised sheep polyclonal antibodies or recombinant Camelid VH domain antibodies (Thermo Fisher Scientific). The beads were pelleted and washed sequentially with PBS-tween. The Mass Spec std particle bead was added to the mixture, pelleted and washed once with water. This was eluted with an acidic buffer solution containing both reducing agent and an ionisation control protein (see for example WO2021/019211). The elution was subsequently spotted, in a sandwich with MALDI matrix (α-Cyano-4-hydroxycinnamic acid) onto a MALDI-TOF target plate and dried. Mass spectra were acquired in positive ion mode on a Bruker Microflex MALDI-TOF-MS covering the m/z range of 5000 to 30,000 which includes the doubly charged ([M+2H].sup.2+, m/z 10900-12300) ions of the analyte (human kappa or lambda light chains), and those of the Mass Spec std.

[0122] Results

[0123] Mass Modification of Immunoglobulins

[0124] To produce a mass shifted molecule that can be used in the EXENT QIP-MS system, two parameters are required to be met; (1) a modification of the immunoglobulin light chain that does not interfere with the immuno-precipitation or immunocapture of the molecule and (2) to add (or indeed alternatively remove) mass that can be detected as an m/z shift. The addition of biotin using PFP crosslinking to the therapeutic monoclonal IgGK Daratumumab was used to show that modification of intact immunoglobulins with a corresponding mass shift in the m/z of the immunoglobulin light chain could be observed by MALDI-TOF (FIG. 4). FIG. 5 shows that this mass-shift due to biotinylation also works with polyclonal IgA and IgM samples. In all 3 cases the immunoprecipitation of these mass-modified proteins by camelid-antibody beads is unaffected. The mass increases seen with biotin alone are not sufficient to shift the masses beyond the expected biological range for +2 charge state ion; 10900 to 12300 Da; to achieve this, larger molecules are required. Biotin-PEG36-PFP was added to polyclonal IgA and IgM and the molecules captured by sheep anti-kappa light chain beads. A time course shows that a 4-hour reaction time is sufficient to modify the masses of the kappa light chains from a mean m/z of 11700 to 12650 (FIG. 6). This work was expanded using Biotin-PEG36-PFP modification of Daratumumab monoclonal protein (FIG. 7). A 6-hour 2-step addition of the modifying substance shifted nearly all the mass of the kappa light chain (FIG. 7b) compared to one with a single step of 2-hour duration (FIG. 7a). Biotin-PEG36-PFP could also be used to mass-shift the light chains on myeloma-derived monoclonal immunoglobulin, IgAK (FIG. 8a) and IgG2K (FIG. 8b). A similar mass-shifting of polyclonal immunoglobulin kappa light chains in human serum could be obtained using a related cross-linker (Biotin-PEG24-TFP) over 3 h or overnight (FIG. 9ab).

[0125] EXENT QIP-MS Std

[0126] To illustrate the use of mass-modified immunoglobulins as in-situ MS controls, Daratumumab was labelled with Biotin-PEG36-PFP for 3 days. The resultant m/z of the light chain when analysed using the EXENT QIP-MS immunoassay using a sheep-anti-IgG bead suggested a single site had been modified (FIGS. 10 and 11). This single site modification was sufficient to increase the +2 m/z from 11700 to 12600 without effecting the ability of the IgGK immunoglobulin to be immuno-captured by the anti-IgG bead. A dilution series of this molecule over a 10-fold range showed that the concentration was positively associated with MS signal intensity (FIG. 11).

[0127] Spiking of the mass-modified Daratumumab bound to an anti-IgG bead into the EXENT QIP-MS immunoprecipitation IgA assay showed that simultaneous elution of the analyte and the mass modified molecule could be obtained. The mass-modified light chain +2 peak of the latter is clearly distinguishable from that obtained from a myeloma IgAL patient (FIG. 12) or the polyclonal IgA present in a healthy sample (FIG. 13).