WO2019209730A1 - Removal of interfering factors from serum protein electrophoresis profiles - Google Patents

Abstract

A method for precluding an interfering factor from creating a false result in a serum protein electrophoresis profile by mixing a serum protein sample with a binding partner that forms a complex with at least one predetermined interfering factor present in the sample. The binding partner includes a binding segment having specificity for the interfering factor and a mobility shifting segment comprising a polyanionic polymer. The sample mixture is separated by electrophoresis. In the resulting protein separation profile, the complex formed by the binding partner and the interfering factor immunodisperses the interfering factor.

Description

REMOVAL OF INTERFERING FACTORS FROM

SERUM PROTEIN ELECTROPHORESIS PROFILES

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Serial No. 62/664,816 filed on April 24, 2018, the contents of which are hereby incorporated.

BACKGROUND OF THE INVENTION

Field of the Invention:

[001] The present invention relates to methods and systems for improving diagnostic protein electrophoresis processes. More particularly, the invention relates to methods and systems for mitigating the interference of potentially interfering substances from qualitative and/or quantitative diagnostic electrophoretic testing methods by dispersing the potentially interfering substances.

Description of the Related Art:

[002] Electrophoresis is one of the most common methods of separating proteins, nucleotides and other biological macromolecules. Methods vary depending in the medium being used (e.g. agarose, acrylamide, cellulose, capillary) and chemical interactions used during the process (e.g. capillary zone, immunofixation, immunosubtraction, etc.). While there are many different types of electrophoresis, all separate a sample into its individual constituents by applying electric voltage across a medium into which a sample is placed. The electrophoretic mobility of a molecule depends on the type of medium used, pH, and the characteristics of each molecule such as its size, shape, charge and interactions with other molecules in medium selected. Different types of electrophoresis are generally named based on the method that is used to separate and differentiate the various serum components. In Capillary Zone Electrophoresis (CZE), for example, different proteins are separated into concentrated zones within a capillary by sandwiching the sample between different types of electrolytes.

[003] In the simplest form of electrophoresis, the macromolecules of a sample are separated across a gel or other medium, a stain is applied, revealing a sample separation profile of bands of each distinct constituent of the sample. The densities and width of these bands are measured and used as quantitative and/or qualitative measurements of the various macromolecules in a sample. In some instances, a single band includes signals from several similar but distinct macromolecules. Further identification and characterization of macromolecules generating a signal within each band can be achieved by removing selected bands and performing further diagnostic tests.

[004] Serum Protein Electrophoresis (SPE or SPEP), developed in the l930s is one of the oldest clinical diagnostic tools in wide use today. Generally, SPE may be performed on a sample of blood from a patient with little or no preparation of the sample itself. A sample may be placed directly into a well in an agarose gel, which may be run an appropriate amount of time, stained with Coomassie blue or other common protein stain, and protein separation profile, comprising characteristic bands of serum proteins are readily observed. In some cases, the sample may be first diluted or treated to avoid clotting, but otherwise no preparation is required. The separation pattern resulting from SPEP depends on the fractions of two major types of proteins: albumin and globulins that are present in the sample. Both the albumin and globulin bands contain signals from many similar but distinct proteins.

[005] Albumin, a major protein component of serum, is produced by the liver under normal physiologic conditions, and makes up the largest and most anodal peak in an SPE test. The albumin level is decreased under circumstances in which there is less production of the protein by the liver or in which there is increased loss or degradation of this protein. Malnutrition, significant liver disease, renal loss (e.g., in nephrotic syndrome), hormone therapy, and pregnancy may account for a low albumin level. Burns also may result in a low albumin level. Increased levels of albumin are observed for example in patients with a relative reduction in serum water (e.g., abrupt dehydration).

[006] Globulins form five bands cathodal to the albumin band. These five globulin peaks, labeled alpha- 1, alpha-2, beta-l, beta-2, and gamma, are usually the primary focus when analyzing SPEP. The alpha 1 -protein fraction is comprised of alpha 1 antitrypsin, thyroid-binding globulin, and transcortin. Malignancy and acute inflammation (resulting from acute-phase reactants) can increase the alpha- 1 protein abundance. A decreased alpha- 1 protein band may occur because of alpha- 1 -antitrypsin deficiency or decreased production of the globulin as a result of liver disease. Ceruloplasmin, alpha-2 -macroglobulin, and haptoglobin contribute to the alpha-2 band. The alpha- 2 component is increased as part of an acute phase response. The beta fraction has two peaks labeled beta-l and beta-2. Beta-l is composed mostly of transferrin and beta-lipoprotein (LDL).

[007] The gamma region of an SPE is primarily composed of immunoglobulins (Ig's), a/k/a antibodies (Ab's). Plasma cells produce immunoglobulins (also called gamma-globulins), which consist of a heavy chain (IgG, IgA, IgM, IgD or IgE) and a light chain (kappa or lambda) linked together. One plasma cell produces one type of immunoglobulin (for instance, IgA kappa or IgG kappa). Normally the body contains a variety of different plasma cells ("polyclonal"), thus the immunoglobulins in the serum also represent a broad spectrum of different formats and specificities (polyclonal). In the case of multiple myeloma, the malignant cells are copies of only one or only a few distinct plasma cell(s) and the Immunoglobulin secreted by this or these cell(s) is considered as monoclonal. It should be noted that low levels of immunoglobulins often can be found throughout the electrophoretic pattern. For example, C-reactive protein (CRP) is located in the area between the beta and gamma components. In addition, some antigen-specific antibodies, due to their unique epitope binding regions, may migrate anywhere within the globulin region upon protein electrophoresis. However, the majority of antibodies migrate in the gamma region.

[008] A normal gamma band is commonly referred to as the "polyclonal background" and indicates a normal amount of several different antibodies present in the blood. An increase in the polyclonal background indicates a secondary disease state due to clinical disorders such as chronic liver diseases, collagen disorders, rheumatoid arthritis and chronic infections.

[009] In some instances, a significant spike is observed the gamma region of a SPE protein separation profile. This is known as a "monoclonal gammopathy" or simply a "gammopathy" and is caused by the overexpression of a single monoclonal antibody (mAh), designated as the M- protein or paraprotein of the gammopathy. The M-protein may consist of a heavy chain (most often IgG or IgA but also IgM, IgD or IgE) and a light chain (kappa or lambda) or truncated forms of these immunoglobulins.

[0010] When a gammopathy is observed, additional testing, is required, because a simple SPE separation profile provides no information regarding the identity of the actual proteins comprising the various bands. A more complex diagnostic tool, Immunofixation Electrophoresis (IFE) is often used to identify a specific mAb responsible for a gammopathy. IFE is a two-stage procedure combining SPE with an immunoprecipitation procedure. First, SPE is performed over an agarose gel. Next, an antisera comprising antibodies to specific immunoglobulin classes (e.g. IgG, IgA, IgM, kappa and lambda) and/or to specific monoclonal antibodies applied to the gel. The gel and antisera are incubated, during which time immune complexes form between the specific immunoglobulins and the antibodies. The location of such immune complexes is visualized by staining, revealing the I_g type of the M protein. IFE is performed in the following four steps:

[0011] 1) Separation: During electrophoresis on agarose, for example, the sample is simultaneously electrophoresed in several tracks in parallel. After the electrophoresis, one track serves as a reference (containing the total protein fraction) providing a complete electrophoretic pattern of the proteins in the sample. In capillary electrophoresis, the sample is electrophoresed through a narrow bore capillary and protein absorbance observed for each fraction.

[0012] 2) Antigen-Antibody reaction: Immunofixation (immunoprecipitation) of the electrophoresed proteins occurs by incubating the appropriate electrophoretic migration tracks with individual antisera. The antisera diffuse into the gel and precipitate the corresponding antigens when present. The proteins in the reference track are fixed with a fixative agent. Immunodisplacement on capillary electrophoresis is performed by pre-incubation of the protein sample with specific antisera before Step 1. The antisera-target protein is displaced from the capillary electrophoretic spectrophotometric absorbance pattern.

[0013] 3) Visualization: For immunofixation, the precipitated proteins are visualized by a staining methodology (e.g., acid violet stain). For capillary immunodisplacement, the proteins remaining after antibody treatment are "visualized" by spectrophotometric absorbance.

[0014] 4) Interpretation: On agarose, for example, the immunofixed bands are then compared with the bands in the reference pattern - the identified M-proteins should have the same migration position if present in sufficient quantity in the reference pattern. In capillary electrophoresis, the resulting patterns are compared to the reference pattern as well. However, the M-proteins identified are based on removal of proteins during capillary electrophoresis and lack of detection in subsequent protein absorbance measurements.

[0015] Alternatively, Immunosubtraction Electrophoresis (ISE) may be used to identify immunoglobulins or other proteins. In (ISE), a sample is incubated with a binding partner specific to a target protein (e.g. an M-protein responsible for a gammopathy) prior to SPE. The binding partner and target protein form an insoluble complex. When SPE is performed, the signal generated by the target protein will be absent. Those skilled in the art will appreciate that there are many other types of electrophoresis, and other tests generally that may be used to identify a specific target protein such as an M-protein responsible for a gammopathy. By way of example, various types of electrophoresis may be performed on a capillary tube rather than a gel and spectroscopic analysis of a sample as it passes through capillary tube may be used to observe signals from the various proteins or other macromolecules.

[0016] The most common cause of monoclonal gammopathy is multiple myeloma (MM). Multiple myeloma is a hematological cancer that involves the clonal expansion of malignant plasma cells. MM is the most common malignant plasma cell tumor and the second most common hematologic malignancy in the ETnited States. The U.S. age-adjusted incidence rate is 5.5 cases per 100,000 and the annual incidence reaches approximately 6 to 7 per 100,000 in the United Kingdom.

[0017] Multiple staging systems are currently used for the diagnosis and monitoring of responses in multiple myeloma: a) the Durie and Salmon Staging System, b) the International Staging System (ISS), and the International Myeloma Working Group (IMWG). The Durie and Salmon staging system involves features that assess tumor cells mass, elevated serum immunoglobulin IgG levels, end-organ damage, and osteolytic bone lesions. The ISS places more emphasis on the disease burden based on 2-microglobulin levels and serum albumin levels. The IMWG takes into account both molecular and cytogenetic abnormalities, specifically, M-protein reduction over time is one of the most important factors and is used to assess the progress of disease and treatment success.

[0018] Protein manifestations characteristic of multiple myeloma include increases of M-protein concentrations (IgG, IgA, IgA, IgD), light chain concentrations (including kappa [K] and lambda[A]), abnormal 2-microglobulin, serum albumin, creatinine, and hemoglobin levels, and findings of bone marrow plasma cells (of greater than or equal to 5%). Measurement of the protein manifestations (such as M-protein) produced by patients can be achieved by numerous methods. Tests that measure M-proteins are the 24-hour urine collection test, urine protein electrophoresis (UPEP), SPEP, IFE, and serum free light chain (sFLC) assay.

[0019] As is known in the art, an increasingly common method of treating a variety of diseases is to use a therapeutic mAh. For example, daratumumab is a commercially available mAh for treating multiple myeloma. However, treatment with a therapeutic mAh such as daratumumab causes problems in monitoring the efficacy of that treatment when it is used against a disease that causes overexpression of monoclonal antibodies. Because daratumumab is itself a mAh in the blood serum of an MM patient, it interferes with diagnostic tests by migrating within the gamma region and contributes to the observed M-protein spike. As a result, the gammopathy shown in that patient's SPE and other tests are artificially elevated by the therapeutic mAh, masking the true amount of M-protein present in the patient's blood serum and obfuscating diagnostic tests used to monitor the current state of a patient's disease.

[0020] One approach to resolving this problem is to treat a sample of the patient's blood serum with a binding partner specific to daratumumab which forms a complex having a substantially different electrophoretic mobility than the therapeutic mAh alone. This interaction shifts the daratumumab out of the IgG region to a more anodal position preferably in or near the albumin region. This approach, known as Immunodisplacement Electrophoresis (IDE), is described in WO publication WO 2017/149122, which is International Patent Application PCT/EP2017/055011 (Morphosys AG), the entirety of which is hereby incorporated by reference, has a priority date of March 4, 2016. Sebia, in collaboration with Janssen, has recently developed an anti-daratumumab assay using a mouse anti-daratumumab antibody labeled with biotin or Alexa-fluor tags to shift the complex on IFE. This approach also shifts the daratumumab signal from the IgG region to a more anodal position.

[0021] However, such IDE methods are not without problems. Any degradation of the binding partner results in a free immunoglobulin that can increase the signal of the M-protein. If insufficient binding partner is used during incubation, excess target protein can also increase the signal of the M-protein. Furthermore, shifting the target proteins signal often results in the shifted signal interfering with other bands found during SPE. This prevents accurate analysis of these additional bands.

[0022] ISE may be used to remove a target protein, such as an interfering therapeutic mAb, from a sample prior to performing SPE or other electrophoresis methods. IFE also may be utilized to separate an M-protein from an interfering therapeutic mAb. However, both of these procedures require adding several steps to the diagnostic testing process, increasing the time required and complexity. For diagnostic testing, simplicity, speed and overall efficiency as well as accuracy are of paramount importance. Thus, both ISE and IFE are undesirable for high-volume, high throughput diagnostic testing facilities.

[0023] The above-described deficiencies of today's systems are merely intended to provide an overview of some of the problems of conventional systems, and are not intended to be exhaustive. Other problems with the state of the art and corresponding benefits of some of the various non limiting embodiments may become further apparent upon review of the following detailed description.

[0024] In view of the foregoing, it is desirable to provide a method for eliminating a signal from an interfering substance such as a therapeutic mAb from an electrophoresis diagnostic test.

BRIEF SUMMARY OF THE INVENTION

[0025] Disclosed is a method for electrophoretic analysis of a sample comprising one or more proteins, wherein the sample is selected from the group consisting of blood serum and urine. First a mixture is prepared by combining the sample with a binding partner. The binding partner is a macromolecule comprising a binding segment, usually an antibody having antigenic specificity for a predetermined interfering factor, covalently linked to a substances such as a polyanionic poly(amino acid). When the binding partner and the interfering factor are mixed, they form a complex having an electrophoretic mobility such that it is dispersed such that the complex is below the detection level. The sample mixture is then deposited on an electrophoretic agarose gel. The sample is electrophoresed and stained to obtain a protein separation profile of the sample. The complexed binding partner and interfering factor are immune-dispersed and/or may be displaced in the separation profile, outside the gamma zone of the protein separation. Optionally, the sample and the binding partner may be mixed within a well in the agarose gel immediately prior to running the gel. Other electrophoretic systems are disclosed.

[0026] It is therefore an object of the present invention to provide methods for improving serum protein electrophoresis by eliminating the result of interfering factors from the protein separation profiles.

[0027] These and other objects and advantages of the present invention will become apparent from a reading of the attached specification and appended claims. There has thus been outlined, rather broadly, the more important features of the invention in order that the detailed description thereof that follows may be better understood, and in order that the present contribution to the art may be better appreciated. There are features of the invention that will be described hereinafter and which will form the subject matter of the claims appended hereto.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] A more complete understanding of the present invention, and the attendant advantages and features thereof, will be more readily understood by reference to the following detailed description when considered in conjunction with the accompanying drawings wherein:

[0029] Figure 1 illustrates the result of electrophoresis blood samples of three patients as will be described in greater detail below; and

[0030] Figure 2 illustrates the result of electrophoresis of blood samples of a patient with myeloma with and without monoclonal antibody (mAB) treatment as will be described in greater detail below.

DETAILED DESCRIPTION

[0031] The invention is not limited in its application to the details of construction and to the arrangements of the components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced and carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting. The disclosed subject matter is described with reference to the drawings, wherein like reference numerals are used to refer to like elements throughout. In the following description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the various embodiments of the subject disclosure. It may be evident, however, that the disclosed subject matter may be practiced without these specific details.

[0032] Unless otherwise indicated, all numbers expressing quantities of ingredients, dimensions reaction conditions and so forth used in the specification and claims are to be understood as being modified in all instances by the term“about”. The term“a” or“an” as used herein means“at least one” unless specified otherwise. In this specification and the claims, the use of the singular includes the plural unless specifically stated otherwise. In addition, use of“or” means“and/or” unless stated otherwise. Moreover, the use of the term“including”, as well as other forms, such as “includes” and“included”, is not limiting. Also, terms such as“element” or“component” encompass both elements and components comprising one unit and elements and components that comprise more than one unit unless specifically stated otherwise. The term "antibody" includes antibody fragments, and particularly antibody fragments which include the antigen binding region. "M protein" and "paraprotein" are used interchangeably to refer to a globulin responsible for a gammopathy.

[0033] Various embodiments of the disclosure could also include permutations of the various elements recited in the claims as if each dependent claim was a multiple dependent claim incorporating the limitations of each of the preceding dependent claims as well as the independent claims. Such permutations are expressly within the scope of this disclosure.

[0034] Disclosed are methods and systems for removing the effect of one or more interfering factors from a serum protein sample analyzed by gel electrophoresis, usually agarose gel electrophoresis (AGE). The interfering factor is typically a compound that co-migrates with the M-protein of a gammopathy. A binding partner specific to the interfering factor is mixed with a biological sample, usually blood serum or urine, prior to electrophoresis. The binding partner forms a complex with the interfering factor, dispersing it within the electrophoretic system thus reducing its interference with co-migrating compounds being analyzed by AGE.

[0035] The interfering factor may be for example a therapeutic mAb present in the blood of a patient undergoing mAb therapy to treat a gammopathy. The therapeutic mAb co-migrates with the M protein of the gammopathy, thus disturbing quantitative analysis of the M protein. A serum protein sample from a patient undergoing mAb therapy is mixed with a binding partner specific to the therapeutic mAb and which forms a complex with the interfering therapeutic mAb. Formation of the complex with the binding partner disperses the interfering mAB such that it does not appear in the gamma region, either by shifting out of that region and/or by dispersion such that its presence is below the detection level and thus does not interfere. Thus, when SPE is subsequently performed on the serum sample mixed with the binding partner, the resulting protein separation profile includes a band for the gammopathy's M protein which is substantially free of the interfering factor.

[0036] The binding partner in accordance with the principles of the invention is a synthetic macromolecule that includes a binding segment having a high degree of specificity for the interfering factor and a mobility shifting segment covalently linked to the binding segment. Typically, the binding segment is an antibody or antigen binding fragment of an antibody. Where the interfering factor is a therapeutic mAb, the binding segment is an antibody specific to the therapeutic mAb, or an antigen binding fragment thereof. A high degree of specificity for the interfering factor is typically desirable to avoid interactions between the binding segment and other serum proteins such as the M protein being quantified. A mAb specific to an interfering therapeutic mAb, or an antigen binding segment thereof, is typically a suitable binding segment for use in a binding partner used to remove any interfering therapeutic mAb.

[0037] In one embodiment, the interfering factor is daratumumab. Serum protein electrophoresis is typically used to monitor the progress of multiple myeloma and the efficacy of a patient's treatment. Daratumumab is a therapeutic mAb used to treat multiple myeloma. However, daratumumab co-migrates with the M-protein indicative of multiple myeloma, interfering with quantitative analysis of the M-protein and obfuscating test results from SPE monitoring of a patient's multiple myeloma treatment. A suitable binding partner for daratumumab includes a binding segment comprising an anti-daratumumab mAb, or the antigen binding portion thereof. The same method of the invention may be employed to remove any interfering factor, such as for example a different therapeutic mAb, by utilizing an antibody specific to that therapeutic mAb, or an antigen binding derivative thereof, as the binding segment of the binding partner in accordance with the principles of the invention.

[0038] The mobility shifting segment can be any macromolecule that sufficiently shifts the electrophoretic mobility of the complex between daratumumab and the binding partner. Suitable mobility shifting segments include polyanionic polymers having a pKa around or below the pH at which the binding partner is used, preferably at least two pH units below the optimal pH of the serum protein electrophoresis procedure. “Shifting” in this context means that the interfering substance is dispersed such that it does not appear in the result of the electrophoretic test. Therefore,“shifting” may refer to dilution as well as movement of a sufficient quantity of the interfering substance such that the interfering substance does not impact the result of the electrophoresis process.

[0039] In one embodiment, the mobility shifting segment comprises a polyanionic polymer comprising a plurality of acid groups having a pKa of 7 or less. A portion of the monomeric units need not comprise an ionizable group, however it is preferred that 50-100%, in particular 75-100%, more in particular 90-100% of the monomeric groups comprise one or more groups that are ionizable to form an anionic group at alkaline pH.

[0040] Examples of ionized groups of a polyanionic polymer in particular include, but are not limited to, carboxylate groups, sulphate groups, sulphonate groups, phosphate groups and phosphonate groups.

[0041] In one embodiment, the mobility shifting segment is a polyanionic polymer selected from the group of polyanionic poly(amino acids), poly(carboxylic acids), poly(sulphonic acids), polynucleotides, carboxylated polysaccharides, sulphated polysaccharides and phosphorylated polysaccharides, including copolymers thereof.

[0042] In one embodiment, the mobility shifting segment is a poly(amino acid). The poly(amino acid) usually has a plurality of acid side-groups. These side-groups may be a side-group of a natural amino acid having a carboxylic acid side-group, such as glutamic acid or aspartic acid, or another acidic group, such as a hydroxyl group (as in tyrosine, having a pKa of about 10). In one embodiment of the invention, the mobility shifting segment is a poly(amino acid) of which a plurality of amine side-groups (e.g. a plurality of lysine amino acid residues) have been derivatized to form an acid group. To achieve this, such side-groups are reacted with a polyacid or anhydride thereof, e.g. dicarboxylic acid, a tricarboxylic acid or a carboxylic acid having more than three carboxylic acid functional groups. In particular, the amine side group may have been derivatized with succinic acid, mellitic acid, benzene tricarboxylic acid, a sulphonic acid a phosphoric acid, or an anhydride of any of these. Such poly(amino acids) may be purchased commercially or derivatized in a manner known in the art.

[0043] In one embodiment, the mobility shifting segment is a poly(amino acid) segment comprising a plurality of lysine residues of which at least the majority of the amine-side-groups have been transformed into anionic side groups, preferably by carboxylation, e.g. by succinylation, in particular polylysine wherein 90-100%, or preferentially 98-100% of the amine side groups have been transformed into anionic side groups. A binding partner comprising such a segment has been found particularly advantageous for use in a method wherein a sample is analyzed for the presence of a serum protein.

[0044] In one embodiment a mobility shifting segment comprising polyaspartic acid or polyglutamic acid is provided. In another embodiment the mobility shifting segment comprises a polyarginine, polyasparagine, polyglutamine or polyhistidine segment of which at least the majority of the amine-side-groups (preferably 90-100%) have been transformed into anionic side groups, preferably by reaction with succinic acid or another polycarboxylic acid or anhydride thereof.

[0045] A mobility shifting segment comprising an anionic polysaccharide segment may be selected from the group of carboxyalkyl celluloses, such as carboxymethyl celluloses; heparins; sulphated dextrans; hyaluronic acids and the like.

[0046] A suitable polysulphonic acid is poly(4-styrenesulphonic acid). Suitable polycarboxylic acids may be selected from the group consisting of polymaleic acids, polyacrylic acids, polymethacrylic acids and polyfumaric acids, including copolymers thereof. A suitable copolymer is, for instance, a poly(4-styrenesulphonic acid-co-maleic acid) copolymer. [0047] The average size (molecular weight) of the mobility shifting segment may be chosen within wide limits, in particular depending on its intended use. It has been found that the effective mobility of a complex of a interfering factor and the binding partner may increase (i.e. becomes more negative) with increasing polyanionic polymer segment molecular mass (at a similar mass over charge ratio of the segment). Thus, it is contemplated that by choosing the average molecular weight of the polyanionic polymer segment the desired mobility of the interfering factor-binding partner complex may be fine-tuned.

[0048] As used herein the (average) molecular weight is the (average) molecular weight based on matrix assisted light scattering spectrometry (MALLS) or on viscosity (as specified by the supplier if a commercially obtained polymer is used to prepare the binding partner), or the (average) molecular weight as determined by analytical ultracentrifugation (AUC). The number average molecular weight (which is determinable by AUC) of the polyanionic polymer segment may be at least 1 kg/mol, at least 10 kg/mol or at least 20 kg/mol. In certain embodiments, the number average molecular weight is at least 40 kg/mol, more preferably at least 50 kg/mol or at least 75 kg/mol.

[0049] The upper-limit is primarily defined by solubility/dispersibility of the binding partner. Further, the larger the polymeric segment, the higher the viscosity tends to be. Thus, usually the polymeric segment chosen has an average molecular weight such that the viscosity of the sample comprising the binding partner is still easy to handle. The skilled person will know how to determine a suitable upper viscosity and molecular weight. In general, the number average molecular weight will by 10000 kg/mol or less, preferably 5000 kg/mol or less, in particular 1000 kg/mol or less, more in particular 750 kg/mol or less.

[0050] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.

[0051] Example 1, Immunodisplacement CE Using Binding Partner Having an Anti- Daratumumab Antibody Binding Segment and a Poly(Amino Acid) Mobility Shifting Segment. To form the binding partner, the following procedure was used:

[0052] Solution A preparation: A solution containing 8 mg/ml of an anti-daratumumab monoclonal antibody was dialysed overnight into 100 mM Sodium Phosphate, 900 mM NaCl pH 7.4. 500 pl of this antibody solution were used per reaction.

[0053] Solution B preparation: 10 mg poly(amino acid) was dissolved in 250 ml 100 mM Sodium Phosphate, 900 mM NaCl, pH 7.4.

[0054] Solution C preparation: 10 mg NHS(N-hydroxysulphosuccinimide sodium salt) was dissolved in 40 mΐ 100 mM Sodium Phosphate, 100 mM NaCl, pH 7.4.

[0055] Solution D preparation: 10 mg EDC(N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) dissolved in 40 mΐ 100 mM Sodium Phosphate, 100 mM NaCl, pH 7.4.

[0056] Solutions A, B, C and D were combined and mixed to form a reaction mixture by vortexing. The reagents in the mixture were allowed to react overnight at room temperature (about 20° C.) whilst being mixed end-over-end on a rotator, thereby forming an antibody to which poly(amino acid) is covalently linked, i.e. the modified antibody or binding partner.

[0057] Thereafter the reaction mixture was dialysed against a 50 mM Sodium Phosphate solution, 200 mMNaCl, pH 8.1, using 100 kg/mol molecular weight cut-off dialysis concentrated by placing the dialysis tubing containing the modified antibody in Spectra/Gel absorbent (Spectrum Labs, Rancho Dominguez, Calif., USA.)

[0058] After the concentration step, a solution of 50 mM Sodium Phosphate, 200 mM NaCl, pH 8.1 was added to the reaction mixture, up to a volume of approximately 5004 ml.

[0059] Sample Preparation for Immunodisplacement CE:

[0060] 2 mΐ of human blood serum was added to 98 mΐ of CE buffer solution. The buffer was 200 mM CAPS, 66 mM TAPS in water, pH 9.7, and mixed. Thereafter 20 mΐ modified antibody solution was added. After mixing, the sample was ready for separation by CE.

[0061] Immunodisplacement Electrophoresis:

[0062] The CE system configuration was as follows:

[0063] Instrument: PrinCE CEC 760, DAx 3D 8.1 software; [0064] Capillary: fused silica (no internal coating);

[0065] Capillary length to detector: 23 cm.;

[0066] Total capillary length: 30 cm.;

[0067] Capillary internal diameter: 50 pm.

[0068] A sample was injected using pressure injection (25 mbar, 6 sec) and for the separation 13 kV was applied. The temperature was 25° C.

[0069] Further, a serum sample was prepared and separated in the same way, with the exception of adding the modified antibody.

[0070] Example 1A: Immunodisplacement CE ETsing Binding Partner Having an Anti- Daratumumab Binding Segment and a Polylysine succinylate Mobility Shifting Segment.

[0071] An anti-daratumumab mAh was modified with poly-L-Lysine succinate (Sigma-Aldrich, catalogue nr. P3513, Mw>50 000 g/mol, Mw based on poly-L-lysine viscosity, also assessed by MALLS) as described above.

[0072] The binding partner was effective in binding to immunoglobulins in the serum and neither the binding partner itself nor the complex of binding partner with human immunoglobulins migrated within or near the serum protein bands. The separation was stopped after 4 min (about 45 sec after the last band (albumin) had fully migrated past the detector), at which time neither the complex nor uncomplexed binding partner had yet migrated past the detector. This illustrates that a binding partner according to the present invention is particularly suitable for use in the analysis of serum proteins.

[0073] Example 1B: Immunodisplacement CE ETsing Binding Partner having an Anti- Daratumumab Binding Segment and a Poly-L-glutamic Acid Mobility Shifting Segment:

[0074] An anti-daratumumab mAh was modified with poly-L-glutamic acid (Sigma-Aldrich catalogue number P4886, molecular weight 50000-100000 g/mol, 64000 g/mol average, determination based on viscosity and by MALLS) as described above. Thereafter a human blood serum sample with an abnormal gamma-band level (a so-called abnormal control sample) to which poly-L-glutamic acid modified binding partner had been added (as above) and a sample to which no modified antibody had been added where separated with CE (as above). It was found that the modified antibody was effective in almost completely removing the abnormal human immunoglobulin in the gamma band. The last serum protein band (albumin) fully migrated within 3 min 15 sec. After 4 minutes the separation was stopped, at which time no modified antibody or complex thereof with human immunoglobulin had been detected.

[0075] Example 1C: Immunodisplacement CE ETsing Binding Partner having an Anti- Daratumumab Binding Segment and a Poly-gamma Gutamic Acid Mobility Shifting Segment.

[0076] An anti-daratumumab mAh was modified with poly -gamma-glutamic acid (PGGA) (Natto Biosciences, Montreal, Canada), with molecular weights of 390 kg/mol or 2250 kg/mol as described above using 5 mg poly(amino acid) per modification reaction.

[0077] In each of the examples, the modified antibody is effective in removing human daratumumab from the gamma band and that the immunodisplaced daratumumab as well as the binding partner migrate after the last serum protein band (albumin).

[0078] Example 2, Binding Partner having an Anti-Daratumumab Binding Segment and a Carboxylated Polysaccharide Mobility Shifting Segment:

[0079] The same protocol was used as described in Example 1, with the proviso that the amounts of polyanionic polymer, in this case two different carboxymethyl celluloses (CMC), were varied as follows:

[0080] A CMC sodium salt, average Mw 90 000 g/mol (Aldrich product number 419273) 2, 5 or 10 mg were used.

[0081] A CMC sodium salt average Mw 250 000 g/mol (Aldrich product number 419303) 2, 4 or 6 mg were used.

[0082] Sample Preparation for Immunodisplacement CE:

[0083] Serum samples were prepared as described above, with and without binding partner. [0084] Immunodisplacement Electrophoresis:

[0085] CE separations were carried out as described above. It was found that binding partners modified with either type of CMC were effective in binding to immunoglobulins in the serum samples and causing the immunoglobulins bound to the binding partners to migrate after the last serum protein band (albumin). It was found that the binding partner comprising CMC having an average molecular weight of 90,000 g/mol (complexed with human immunoglobulin) migrated closer to the last serum protein band than the binding partner comprising CMC having an average molecular weight of 250,000 g/mol (complexed with human immunoglobulin), although for both types of binding partners a baseline separation between the binding partner (complex) and albumin was feasible.

[0086] It was further found that the resolution between binding partner (complex) and albumin was higher when for the binding partner obtained by adding 4, 5, 6 or 10 mg CMC than for the binding partner obtained by adding only 2 mg CMC.

[0087] Example 3, Immunodisplacement CE ETsing an Binding Partner Having an Anti- Daratumumab Binding Segment and a Sulphonic Acid Carboxylic Acid Copolymer Mobility Shifting Segment

[0088] Two poly(4-styrenesulphonic acid-co-maleic acid) polymers (PSSA-MA) were used to modify an anti-daratumumab murine/human antibody. The PSSA-MAs, obtained from Sigma- Aldrich, both had an average molecular weight of about 20,000 g/mol. One PSSA-MA had a styrene sulphonic acid to maleic acid molar ratio of 1 : 1 (Aldrich catalogue number 434558), the other had a styrene sulphonic acid to maleic acid molar ratio of 3 : 1 (Aldrich catalogue number 434566)

[0089] The same protocol was used as described in Example 1, with the proviso that the amounts were 15 mg or 20 mg, instead of 10 mg.

[0090] Sample Preparation for Immunodisplacement CE:

[0091] Serum samples were prepared as described above, with and without binding partner. [0092] Immunodisplacement Electrophoresis:

[0093] CE separations were carried out as described above.

[0094] It was found that the binding partners modified with either type of PSSA-MA were effective in binding to immunoglobulins in the serum samples and causing the immunoglobulins bound to the binding partners to migrate after the last serum protein band (albumin).

[0095] Alternatively, CZE (Capillary Zone Electrophoresis) may be employed utilizing, for example, the V8 System manufactured by Helena Laboratories Corporation of Beaumont, Texas (ET.S.) and/or the V8 Nexus Clinical Chemistry Analyser from Helena Biosciences Europe (EG.K.).

[0096] Example 4: Serum Protein Agarose Gel Electrophoresis ETsing a Binding Partner Having an Anti-Daratumumab Antibody Binding Segment and a Polylysine succinylate Mobility Shifting Segment:

[0097] Figure 1 shows an agarose gel immunodisplacement assay of samples from three patients. Patient 1 was not diagnosed with myeloma. Patients 2 and 3 were diagnosed with myeloma. All three patient samples were treated with daratumumab. Blood samples were taken from each of the three patients and two serum protein electrophoresis samples were prepared for each patient. For each patient, one of the two samples (the left-side scan for each patient) is indicated as not including the binding partner (-) and the second of the two samples (the right-side scan for each patient) is indicated as being mixed with the binding partner (+) as described in Example 1 A above. Two samples from patient 1 were presented in lanes 2 and 3 (one free of binding partner (-) and the other mixed with binding partner (+)); two samples from patient 2 were presented in lanes 6 and 7 (one free of binding partner (-) and the other mixed with binding partner (+)); and two samples from patient 3 were presented in lanes 10 and 11 (one free of binding partner (-) and the other mixed with binding partner (+)). Thus, the samples in lanes 2, 6 and 10 were free of binding partner and the samples in lanes 3, 7 and 11 were mixed with a binding partner.

[0098] Approximately 9 pL of binding partner solution was mixed with approximately 42 pL of serum sample from the patient to form a + sample mixture. This mixture was incubated at room temperature for approximately 10 minutes and then applied to lanes 3, 7 and 11. An electric potential was applied to the gel according to a standard serum protein electrophoresis protocol. The gel was then stained with Coomassie blue and the image shown in Figure 1 was captured. A distinct daratumumab band 18 is clearly visible in the gamma regions of lanes 2, 6 and 10. In contrast, no daratumumab band is visible in lanes 3, 7, and 11, which were loaded with the sample mixtures containing both a serum protein sample and a binding partner in accordance with the principles of the invention. Thus, even for a patient that does not have myeloma (Patient 1), treatment with daratumumab results in a positive daratumumab band 18 (lane 2) unless the sample is mixed with a binding partner (lane 3).

[0099] Figure 2 shows an agarose gel immunodisplacement and immunofixation assay from a patient undergoing monoclonal antibody therapy in which the patient is receiving regular doses of daratumumab. A blood serum sample from the patient was divided into two separate samples. One of the samples was mixed with a binding partner prepared as described in Example 1A above. Approximately one microliter of a binding partner solution was added for every 5 pL of blood serum sample to one of the samples to form a sample mixture. The sample mixture was allowed to stand for about 10 minutes. The two separate samples were divided into G and K lanes. Daratumumab is known to appear in the G and K lanes, so for purposes of this experiment the A, M and L lanes were omitted. A standard immunofixation assay was then performed on the gel 20. The characteristic daratumumab band 22 is clearly visible in the G and K lanes of the untreated sample. No daratumumab bands are visible in the G and K lanes of the sample mixture that includes the binding partner in accordance with principles of the invention.

[00100] In an alternative embodiment, the binding partner is utilized to remove the interfering factor from the sample such as by precipitation prior to the electrophoresis step. Prior to the present approach, the targeting and removal of an interfering drug from a sample was not known, rather, only the concept of modification of the sample such that the interfering factor will bind to a different component in the sample as described in the Morphosys AG International Patent Application referred to above.

[00101] In the broadest sense, an interfering therapeutic mAh (or other interfering factor) may appear at any location during any type of electrophoresis. For example, daratumumab may appear in the IgG and IgK regions of a separated sample. The present approach is not limited to reducing interference within those specific regions nor is the present approach limited to any specific interfering factor or medical conditions or form of electrophoresis.

[00102] Terms such as mitigation, migration and dispersion are sufficiently broad so as to encompass a process with the result that if mAB or other interfering factor is present (e.g., within the regions under consideration such as Gamma and Kappa) the presence is sufficiently minimal so as not to adversely impact the result of electrophoresis. Similarly, the term“mobility shifting” is sufficiently broad so as to encompass a process with the result that mAB or other interfering factor is present (e.g., within the regions under consideration such as Gamma and Kappa) the presence is sufficiently minimal so as not to adversely impact the result of electrophoresis.

[00103] One non-limiting aspect is a method of evaluating a blood sample obtained from a patient undergoing treatment for a gammopathy comprising the steps of:

[00104] obtaining a blood sample from said patient;

[00105] incubating the blood sample with a binding partner, such that the binding partner binds to the interfering factor in the patient sample, and precipitates the interfering factor in the sample;

[00106] evaluating the sample such as by performing immunofixation electrophoresis (IFE), Immunodisplacement, Serum Protein Electrophoresis, Capillary Electrophoresis (CE), Capillary Zone Electrophoresis (CZE) and/or Chromatography; and

[00107] reporting the results of the evaluation.

[00108] "Evaluating a sample" means (for example in the context of a blood sample) evaluating the blood or portion of the blood sample most relevant for the method. Currently immunofixation electrophoresis is done on the serum component of blood. If, however, in the future a different blood component is evaluated, the invention is directed to a method evaluating that blood component. Blood components include, for example, plasma, serum, cells, e.g. red and white cells, and platelets. Plasma includes proteins, such as globulins, and clotting factors, and salts, sugars, fat, hormones and vitamins. The sample need not be blood but may be any appropriate bodily fluid.

[00109] It is beneficial to increase the net charge of the binding partner such as by derivatization to facilitate target protein precipitation so that the target will not appear in the resulting electrophoretic separation. However, it should be appreciated that the binding partner may be manufactured with a sufficient net charge such that a separate derivatization step may not be necessary. Another benefit of derivatization is that if excess binding partner is present, it will not be observed in (or will not interfere with) the electrophoretic result.

[00110] As described above, the antisera may be derivatized by the addition of poly-L-lysine succinylate onto the antisera via chemical linkage using EDC and NHS which is commonly used to link to antisera.

[00111] The interfering factor and the binding partner are combined to cause the interfering factor to disperse, partially precipitate out of the specimen or fully precipitate out of the specimen. Typically, when the specimen is human blood, the specimen is placed in a vial which may include a preservative to prevent clotting. If the particular interfering factor is known in advance, e.g., if it is known that the patient is being treated with daratumumab, the binding partner may also be placed in the vial before the blood is withdrawn. The present approach is not limited, therefore, to the sequence of placing the components in the specimen container.

[00112] The binding partner may be derivatized to increase the net charge although, as noted above, the binding partner may be manufactured with a sufficient net charge so that a separate derivatization step may not be required. What is important is that the binding partner have a will bind to substantially all of the interfering factor in the sample, and the resulting bound complex will precipitate out of the specimen. If derivatization is appropriate, a suitable technique may be employed and is not limited to the addition of poly-L-lysine succinylate onto the antisera via chemical linkage.

[00113] In the broadest sense, the interfering substance may be a humanized monoclonal antibody or proteinaceous molecule that is used in the treatment of a diagnosed medical condition. This includes, but is not limited to, daratumumab and elotuzumab.

[00114] In another alternative embodiment, the binding partner comprises a heterogenous mixture of binding partners constructed by covalently attaching one or more mobility shifting segments to a binding segment specific to one or more interfering factors such as a therapeutic mAh. Typically, the binding segment is either an antibody or a segment of an antibody specific to the interfering factor. In either case, the binding segment binds to and forms a complex with the interfering factor when mixed with a sample containing the interfering factor.

[00115] The one or more mobility shifting segments covalently attached to the binding segment are typically polymers constructed from one or more anionic monomers polymerized to form either single chains and/or dendrimers of varying lengths. The polymerization process for generating the heterogenous mixture of binding partners is regulated to generate binding partners having a wide range of electrophoretic mobilities. Polymerization reactions are usually controlled in order to minimize polydispersity so that the vast majority of reaction products fall within a narrow range of molecular weight. Polymerization processes in accordance with the principles of the invention however, referred to herein as heterogenous polymerization reactions, are designed in an opposite manner, providing a heterogenous mixture of binding partners having a wide range of molecular weights and/or charges without a high concentration of reaction products within any single range of molecular weights. As used herein, the term "heterogenous mixture of binding partners" refers to a mixture of binding partners, all specific to an interfering therapeutic mAh, but having different electrophoretic mobilities. The heterogenous mixture of binding partners may be subdivided into groups of heterogenous binding partner types, where each type has an electrophoretic mobility in a range that is a subset of the total range of electrophoretic mobilities of the heterogenous mixture of binding partners. Preferably, the binding partner types are evenly distributed across a range of electrophoretic mobilities. Electrophoretic mobility is determined by size and charge of a molecule. Therefore, a polymerization reaction that produces binding partner types in substantially equal amounts across a wide range of molecular weight and/or charge results in a mixture of binding partners having electrophoretic mobilities substantially equally dispersed across a range of electrophoretic mobilities.

[00116] In one alternative embodiment, the electrophoretic mobility of the binding partners extends across a range equal to or greater than the electrophoretic mobility of albumin or of beta- 2 globulins. Prior to SPE, a protein sample is incubated with the heterogenous mixture of binding partners. To ensure that all of the interfering antibody reacts with the heterogenous mixture, an excess of the heterogenous mixture of binding partners is used for the incubation. This ensures that substantially all the interfering antibody is complexed with the binding partners. All of the complexes formed will migrate to positions anodal to the gamma region. Any unbound binding partners will also migrate anodal to the gamma region.

[00117] When the interfering therapeutic mAh forms complexes with the heterogenous mixture of binding partners, it does not result in the typical immunodisplacement where the entire signal from the target protein is shifted to a single region where it forms its own peak. Instead, the signal is substantially equally dispersed across a wide range of electrophoretic mobilities. As a result, the signal from the target protein, in this case the interfering therapeutic mAh, essentially disappears into the background noise. To distinguish the process of the present invention from immunodisplacement and immunosubtraction, the inventors describe the process of the present invention as immunodispersion, because it disperses the interfering substance throughout a lane in an agarose gel.

[00118] A heterogenous polymerization reaction may be based on covalently attaching one or more polyanionic poly(amino acid) to the binding segment as described above. The poly(amino acid) usually has a plurality of acid side-groups. These side-groups may be a side-group of a natural amino acid having a carboxylic acid side-group, such as glutamic acid or aspartic acid, or another acidic group, such as a hydroxyl function (as in tyrosine, having a pKa of about 10). In one embodiment of the invention, the polyanionic polymer segment is a poly(amino acid) segment, of which a plurality of amine side-groups (e.g. a plurality of lysine amino acid residues) have been derivatized to form an acid group. To achieve this, such acid groups may be reacted with a polyacid or anhydride thereof, e.g. dicarboxylic acid, a tricarboxylic acid or a carboxylic acid having more than three carboxylic acid groups. In particular, the amine side group may have been derivatised with succinic acid, mellitic acid, benzene tricarboxylic acid, a sulphonic acid a phosphoric acid, or an anhydride of any of these. Such poly(amino acids) may be purchased commercially or derivatized in a manner known described above.

[00119] The method of this embodiment has been described above in relation to an interfering therapeutic mAh. This is only one nonlimiting example of how immunodispersion can be used to remove an unwanted, interfering substance during electrophoresis. The immunodispersion methods in accordance with the present invention can also be used to target other interfering substances when diagnosing or monitoring gammopathy. The methods of the invention can also be used to remove interfering substances from other electrophoretic processes. [00120] The present invention has been described in relation to the drawings attached hereto, it should be understood that other and further modifications, apart from those shown or suggested herein, may be made within the spirit and scope of this invention. Descriptions of the embodiments shown in the drawings should not be construed as limiting or defining the ordinary and plain meanings of the terms of the claims unless such is explicitly indicated.

[00121] As such, those skilled in the art will appreciate that the conception, upon which this disclosure is based, may readily be utilized as a basis for the designing of other structures, methods and systems for carrying out the several purposes of the present invention. It is important, therefore, that the claims be regarded as including such equivalent constructions insofar as they do not depart from the spirit and scope of the present invention.

Claims

1. A method for removing an interfering factor from an electrophoresis profile of a sample comprising one or more proteins, wherein the sample is selected from the group consisting of blood serum and urine, the method comprising the steps of:

a. mixing the sample with a binding partner, to form a sample mixture,

where the binding partner is a macromolecule comprising:

(i) a binding segment comprising an antibody having antigenic specificity for a predetermined interfering factor that may be present in the sample; and

(ii) a mobility shifting segment comprising a polyanionic poly(amino acid);

wherein the binding segment and the mobility shifting segment are covalently linked; wherein the mixing of the binding partner and the sample produces a complex of the binding partner with at least one of the predetermined interfering factors that may be present in the sample;

b. electrophoresing the sample mixture to obtain a protein separation profile of the biological sample, the binding partner precluding detection of the at least one predetermined interfering factor that may be present in the biological sample within the gamma zone of the protein profile during the electrophoretic migration; and,

d. evaluating the protein profile of the electrophoresed sample mixture.

2. The method according to claim 1 wherein the presence of the interfering factor in the gamma zone is below the detection level.

3. The method according to claim 1 where there interfering factor is absent from the gamma zone.

4. The method according to claim 1 wherein the detection of more than one interfering factor is precluded.

5. The method for removing an interfering factor from an electrophoresis profile of a sample comprising one or more proteins of claim 1 wherein the binding segment of the binding partner comprises a humanized anti-daratumumab antibody.

6. The method for removing an interfering factor from an electrophoresis profile of a sample comprising one or more proteins of claim 1 wherein the mobility shifting segment is a poly(amino acid).

7. The method for removing an interfering factor from an electrophoresis profile of a sample comprising one or more proteins of claim 6 wherein the poly(amino acid) is selected from the group consisting of poly-L-lysine, poly-L-glutamic acid, and Poly-gamma-glutamic acid.

8. The method for removing an interfering factor from an electrophoresis profile of a sample comprising one or more proteins of claim 1 wherein the mobility shifting segment is a carboxylated polysaccharide, and/or a sulphonic acid carboxylic acid copolymer.

9. The method for removing an interfering factor from an electrophoresis profile of a sample comprising one or more proteins of claim 1 wherein the predetermined interfering factor is a humanized monoclonal antibody or proteinaceous molecule used in the treatment of a diagnosed condition.

10. The method for removing an interfering factor from an electrophoresis profile of a sample comprising one or more proteins of claim 1 wherein the electrophoresing the sample mixture is performed by a method selected from the group consisting of capillary electrophoresis, capillary zone electrophoresis, and immunofixation electrophoresis (IFE), Immunodisplacement, Serum Protein Electrophoresis, agarose gel electrophoresis.

11. A method for treating at least one interfering substance in a specimen in a container prior to the analysis of the specimen comprising the steps of:

a. selecting an antisera to the interfering substance;

b. placing the antisera in a container such that the antisera binds to the interfering substance in the container to disperse the interfering substance within the specimen; and

c. evaluating the sample substantially free of the interfering substance.

12. The method of claim 11 wherein the specimen is placed in the container prior to the step of placing the antisera in the container.

13. The method of claim 11 wherein the specimen is placed in the container after the step of placing the antisera in the container.

14. The method of any of the preceding claims where the antisera are derivatized prior to binding to the interfering substance.

15. The method of any of the preceding claims where the interfering substance is a humanized monoclonal antibody or proteinaceous molecule.

16. The method of any of the preceding claims where the interfering substance is

daratumumab.

17 The method of any of the preceding claims where the interfering substance is used during patient diagnosis, therapy, or maintenance, and can potentially interfere with the assessment of patient disease/condition status.

18. The method of any of the preceding claims where the evaluation step is one or more of chromatography, gel plate electrophoresis, capillary electrophoresis, capillary zone

electrophoresis and immunofixation electrophoresis.