US-20260126453-A1 - Mass Spectrometry Kit Including a Cross-linked Antibody or Fragment Thereof

Abstract

An anti-immunoglobulin specific antibody (or fragment thereof), characterised that the antibody or fragment thereof comprises one or more non-disulphide cross-links between at least one heavy chain or fragment thereof and at least one light chain or fragment thereof of the antibody or fragment thereof. A method purifying an anti-immunoglobulin specific antibody (or fragment thereof), characterised that the antibody or fragment thereof comprises one or more non-disulphide cross-links between at least one heavy chain or fragment thereof and at least one light chain or fragment thereof of the antibody or fragment thereof. A method of quantifying an amount of a subject analyte, or a fragment of an analyte in a sample from the subject comprising: (i) adding to the sample a predetermined amount of one or more control analytes or fragments thereof, which are distinguishable from the equivalent subject analyte or fragment; (ii) measuring the relative amount of the subject analyte or fragment and the amount of the control analyte or fragment in the sample; and (iii) comparing the relative amount of subject analyte or fragment to the relative amount of control analyte or fragment, to quantify the amount of analyte or fragment in the original subject sample.

Inventors

• David L. Murray
• Stephen Harding
• David R. Barnidge
• Gregg WALLIS
• John Mills
• Jamie ASHBY

Assignees

• THE BINDING SITE GROUP LIMITED
• MAYO FOUNDATION FOR MEDICAL EDUCATION AND RESEARCH

Dates

Publication Date

20260507

Application Date

20250916

Priority Date

20160225

Claims (19)

1. 1 . A method of purifying or characterizing an immunoglobulin, comprising: contacting a sample containing the immunoglobulin with a composition comprising a population of anti-immunoglobulin specific antibodies or anti-immunoglobulin specific fragments thereof, wherein at least 70% of the antibodies or fragments thereof of the population comprise one or more non-reducible thioether containing cross-links between at least one heavy chain or fragment thereof and at least one light chain or fragment thereof of the antibodies or fragments thereof, wherein said one or more non-reducible thioether containing cross-links replace one or more naturally occurring disulphide bonds between said at least one heavy chain or fragment thereof, and said at least one light chain or fragment thereof, and further wherein said non-reducible thioether containing cross-links are introduced by a reagent comprising a bis-maleimide cross-linker; allowing immunoglobulin to bind to the antibodies or fragments thereof; washing unbound material away from the immunoglobulin bound to the antibodies or fragments thereof; and removing the bound immunoglobulin from the antibodies or fragments thereof to produce purified immunoglobulin or immunoglobulin fragment.
2. 2 . The method according to claim 1 , further comprising characterizing the purified immunoglobulin by mass-spectroscopy.
3. 3 . A method of preparing a sample for analyzing by mass spectrometry comprising: (i) providing a sample from a subject, the sample comprising immunoglobulins or fragments thereof; (ii) adding to the sample a predetermined amount of a control immunoglobulin or fragment thereof having a predetermined molecular weight; (iii) copurifying the control immunoglobulin or fragment thereof with the immunoglobulin or fragment thereof by immunopurification using the composition of claim 9 .
4. 4 . The method according to claim 3 , wherein the control immunoglobulin or fragment thereof has a higher or lower molecular weight than an immunoglobulin or fragment thereof of interest to be measured by the mass spectrometry.
5. 5 . The method according to claim 3 , additionally comprising the step of subjecting a target comprising the copurified sample to mass spectrometry to analyze the sample.
6. 6 . A composition comprising a population of anti-immunoglobulin specific antibodies or anti-immunoglobulin specific fragments thereof, wherein at least 70% of the antibodies or fragments thereof of the population comprise one or more non-reducible thioether containing cross-links between at least one heavy chain or fragment thereof and at least one light chain or fragment thereof of the antibodies or fragments thereof; wherein said one or more non-reducible thioether containing cross-links replace one or more naturally occurring disulphide bonds between said at least one heavy chain or fragment thereof, and said at least one light chain or fragment thereof; and further wherein said non-reducible thioether containing cross-links are introduced by a reagent comprising a bis-maleimide cross-linker.
7. 7 . The composition according to claim 6 , wherein the anti-immunoglobulin antibodies or anti-immunoglobulin specific fragments thereof are anti-free light chain specific, anti-heavy chain class specific, anti-heavy chain subclass specific, anti-heavy chain class-light chain type specific, or anti-light chain type specific.
8. 8 . The composition according to claim 6 , attached to a support.
9. 9 . The composition according to claim 6 , comprising anti-immunoglobulin specific F(ab′) 2 fragments.
10. 10 . The composition of claim 9 , wherein the anti-immunoglobulin specific F(ab′)2 fragments are selected from the group consisting of: anti-free kappa F(ab′)2 fragments, anti-free lambda F(ab′)2 fragments, and anti-total lambda F(ab′)2 fragments.
11. 11 . The composition of claim 6 , wherein the bismaleimide cross-linker is bismaleimidoethane (BMOE).
12. 12 . An immunoglobulin purification and assay kit for use in mass spectrometry, comprising the composition of claim 6 and one or more mass spectrometry standards.
13. 13 . An assay kit, comprising: the composition of claim 6 and an immunoglobulin or fragment thereof mass spectrometry standard.
14. 14 . The assay kit according to claim 12 comprising a mass spectrometry target.
15. 15 . A reagent for detecting a target immunoglobulin analyte in a biological sample by mass spectrometry, comprising: a population of anti-immunoglobulin specific monomeric antibodies or anti-immunoglobulin specific fragments thereof wherein at least 70% of the antibodies or fragments thereof of the population comprise one or more non-reducible thioether containing cross-links between at least one heavy chain or fragment thereof and at least one light chain or fragment thereof of the anti-immunoglobulin specific monomeric antibodies or anti-immunoglobulin specific fragments thereof; wherein said one or more non-reducible thioether containing cross-links replace one or more naturally occurring disulphide bonds between said at least one heavy chain or fragment thereof, and said at least one light chain or fragment thereof; and further wherein the non-reducible thioether containing cross-links are introduced by a reagent comprising a bis-maleimide cross-linker.
16. 16 . The reagent of claim 15 , wherein the population of antibodies consists of anti-immunoglobulin G (IgG) antibodies.
17. 17 . The reagent of claim 16 , wherein the population of antibodies consists of anti-IgG3 antibodies.
18. 18 . The reagent of claim 15 , wherein the anti-immunoglobulin specific monomeric antibodies or anti-immunoglobulin specific fragments thereof are selected from the group consisting of: anti-free kappa light chain antibodies or F(ab′)2 fragments thereof, anti-free lambda chain antibodies or F(ab′)2 fragments thereof, and anti-lambda total antibodies or F(ab′)2 fragments thereof.
19. 19 . The reagent of claim 15 , wherein the bismaleimide cross-linker is bismaleimidoethane (BMOE).

Description

This application claims priority from U.S. Provisional Application Ser. No. 62/368,606 filed Jul. 29, 2016, and is a continuation U.S. patent application Ser. No. 16/078,986, which is a national stage entry of International Patent Application No. PCT/GB2017/050489 filed Feb. 24, 2017. The invention relates to anti-immunoglobulin specific antibodies (or fragments thereof) which comprise one or more non-disulphide cross-links between at least one heavy chain and at least one light chain of the antibodies. These are particularly useful in the isolation or purification of immunoglobulins from samples. Control immunoglobulins are also provided for incorporation into samples to be used as standards in mass spectrometry. Antibody molecules (also known as immunoglobulins) have a twofold symmetry and typically are composed of two identical heavy chains and two identical light chains, each containing variable and constant domains. The variable domains of the heavy and light chains combine to form an antigen-binding site, so that both chains contribute to the antigen-binding specificity of the antibody molecule. The basic tetrameric structure of antibodies comprises two heavy chains covalently linked by a disulphide bond. Each heavy chain is in turn attached to a light chain, again via a disulphide bond. This produces a substantially “Y”-shaped molecule. Heavy chains are the larger of the two types of chain found in antibodies, with typical molecular mass of 50,000-77,000 Da, compared with the smaller light chain (25,000 Da). There are five main classes or class or classes of heavy chain which are γ, α, μ, δ and ε which are the constituents heavy chains for: IgG, IgA, IgM, IgD and IgE respectively. IgG is the major immunoglobulin of normal human serum, accounting for 70-75% of the total immunoglobulin pool. This is the major antibody of secondary immune responses. It forms a single tetramer of two heavy chains plus two light chains. IgM accounts for approximately 10% of the immunoglobulin pool. The molecules, together with J-chains, form a pentamer of five of the basic 4-chain structures. The individual heavy chains have a molecular weight of approximately 65,000 Da and the whole molecule has a molecular weight of about 970,000 Da. IgM is largely confined to the intravascular pool and is the predominant early antibody. IgA represents 15-20% of human serum immunoglobulin pool. More than 80% of IgA occurs as a monomer. However, some of the IgA (secretory IgA) exists as a dimeric form. IgD accounts for less than 1% of the total plasma immunoglobulin. IgD is found on the surface membrane of maturing B-cells. IgE, although scarce in normal serum, is found on the surface membrane of basophils and mast-cells. It is associated with allergic diseases such as asthma and hay-fever. In addition to the five main class or classes, there are four subclasses for IgG (IgG1, IgG2, IgG3 and IgG4). Additionally there are two subclasses for IgA (IgAQ1 and IgA2). There are two types of light chain: lambda (λ) and kappa (κ). There are approximately twice as many κ as λ molecules produced in humans, but this is quite different in some mammals. Each chain contains approximately 220 amino acids in a single polypeptide chain that is folded into one constant and one variable domain. Plasma cells produce one of the five heavy chain types together with either κ or λ molecules. There is normally approximately 40% excess free light chain production over heavy chain synthesis. Where the light chain molecules are not bound to heavy chain molecules, they are known as “free light chain molecules”. The K light chains are usually found as monomers. The A light chains tend to form dimers. There are a number of proliferative diseases associated with antibody producing cells. In many such proliferative diseases a plasma cell proliferates to form a monoclonal tumour of identical plasma cells. This results in production of large amounts of identical immunoglobulins and is known as a monoclonal gammopathy. Diseases such as myeloma and primary systemic amyloidosis (AL amyloidosis) account for approximately 1.5% and 0.3% respectively of cancer deaths in the United Kingdom. Multiple myeloma is the second-most common form of haematological malignancy after non-Hodgkin lymphoma. In Caucasian populations the incidence is approximately 40 per million per year. Conventionally, the diagnosis of multiple myeloma is based on the presence of excess monoclonal plasma cells in the bone marrow, monoclonal immunoglobulins in the serum or urine and related organ or tissue impairment such as hypercalcaemia, renal insufficiency, anaemia or bone lesions. Normal plasma cell content of the bone marrow is about 1%, while in multiple myeloma the content is typically greater than 10%, frequently greater than 30%, but may be over 90%. AL amyloidosis is a protein conformation disorder characterised by the accumulation of monoclonal free light chain fragments as amyloid deposits. Typicall