WO2010088547A1 - Assays for detecting pegylated blood coagulation factors - Google Patents
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- WO2010088547A1 WO2010088547A1 PCT/US2010/022630 US2010022630W WO2010088547A1 WO 2010088547 A1 WO2010088547 A1 WO 2010088547A1 US 2010022630 W US2010022630 W US 2010022630W WO 2010088547 A1 WO2010088547 A1 WO 2010088547A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
Definitions
- This invention relates generally to methods for measuring PEGylated blood coagulation factor concentration and activity in a sample using an anti-polyethylene glycol (PEG) capture agent.
- PEG polyethylene glycol
- Blood coagulation is a complex chemical and physical reaction that occurs when blood comes into contact with an activating agent.
- the blood coagulation process can be generally viewed as three activities: platelet adhesion, platelet aggregation, and formation of a fibrin clot.
- platelets flow through the blood vessels in an inactivated state because the blood vessel lining, the endothelium, prevents activation of platelets.
- the endothelium loses its integrity and platelets are activated by contact with tissue underlying the damaged site. Activation of the platelets causes them to become “sticky” and adhere together. Additional platelets then adhere to the activated platelets and also become activated. This process continues until a platelet "plug" is formed. This platelet plug then serves as a matrix upon which blood clotting or coagulation proceeds.
- thrombin is then produced that causes fibrinogen to convert to fibrin, which forms the major portion of the clot mass.
- additional platelets are activated and trapped in the forming clot, further contributing to clot formation.
- polymerization and cross-linking of fibrin results in the permanent clot.
- Clotting may be initiated by either the intrinsic pathway, which involves the activation of Factor XII, or by the extrinsic pathway, which involves the release of tissue factor.
- Hemophilia A is a congenital bleeding disorder resulting from an X-chromosome-linked deficiency of Factor VIII (FVIII), occuring with a frequency of 1 in 5000 males. It is caused by either a quantitative or a qualitative deficiency in FVIII, a critical component of the intrinsic pathway of blood coagulation.
- FVIII Factor VIII
- Human FVIII is synthesized as a single-chain precursor of approximately 300 kD. It consists of the structural domains A1-A2-B-A3-C1-C2 (Thompson, Semin Thromb Hemost 29:11-22, 2003).
- FVIII can be further processed into two polypeptide chains consisting of a 200 kD heavy chain (A1-A2-B) and an 80 kD light chain (A3-C1-C2). Processing of FVIII occurs in the Golgi apparatus, and the resulting heavy and light chains are held together by metal ions.
- A1-A2-B 200 kD heavy chain
- A3-C1-C2 80 kD light chain
- Processing of FVIII occurs in the Golgi apparatus, and the resulting heavy and light chains are held together by metal ions.
- FVIII Upon secretion, FVIII circulates in the blood as a heterodimer of heavy and light chains, and binds tightly and non-covalently to von Willebrand factor (vWF).
- vWF binding sites on FVIII include FVIII residues 1649-1689 in the A3 domain (Foster, et al., J. Biol. Chem. 264:5230-5234, 1988), and parts of the Cl (Jacquemin, et al., Blood 96:958-965, 2000) and C2 domains (Spiegel, et al., 279:53691-53698, 2004).
- a FVIII binding defect on vWF or severe deficiency of vWF causes a hemophilia- like form of von Willebrand disease (type 2N or type 3 vWD) (Gu, et al., Blood 89:3263-3269, 1997; Sadler, et al., J Thromb Hemost 4:2103-2114, 2006).
- FVIII is activated by thrombin, which cleaves peptide bonds after residues 372, 740, and 1689 to generate a heterotrimer of Al, A2, and A3-C1-C2 domains (Pittman, et al., Proc. Natl. Acad. Sci. USA 85:2429-2433, 1988).
- FVIII dissociates from vWF and is concentrated at the cell surface of platelets by binding to phospholipid. Phospholipid binding involves FVIII residues 2199, 2200, 2251, and 2252 (Gilbert, et al., J. Biol. Chem. 277:6374-6381, 2002).
- FVIII binds to Factor IX (FIX) through interactions with FVIII residues 558-565 (Fay, et al., J. Biol. Chem. 266:8957-8962, 1991; Regan, et al., J. Biol. Chem. 269:9445- 9452, 1984) and 1811-1818 (Lenting, et al., J. Biol. Chem. 271 :1935-1940, 1996) and to Factor X (FX) through interactions with FVIII residues 349-372 (Nogami, et al., J. Biol. Chem. 279:15763- 15771, 2004).
- FVIII also acts as a cofactor for FIX activation of FX, an essential component of the intrinsic coagulation pathway.
- Activated FVIII (FVIIIa) is partially inactivated by the protease activated protein C (APC) through cleavage after FVIII residues 336 and 562 (Regan, et al., J. Biol. Chem. 271 :3982-3987, 1996).
- APC protease activated protein C
- the predominant determinant of inactivation is the dissociation of the A2 domain from Al and A3-C1-C2 (Fay, et al., J. Biol. Chem. 266:8957-8962, 1991).
- PEG polyethylene glycol
- blood coagulation factors such as FVIII
- PEGylation is the covalent attachment of one or more long-chained polyethylene glycol (PEG) molecules to a protein.
- PEGylation has been demonstrated to reduce immunogenicity, prevent protease digestion, and block entry of the protein into the kidney filtrate (Harris, et al., Clin Pharmacokinet 40:539-551, 2001). PEGylation may also increase the overall stability and solubility of the protein.
- Randomly PEGylated proteins such as interferon-alpha (Kozlowski, et al., BioDrugs 15:419-429, 2001) have been approved as therapeutics.
- Various site-directed protein PEGylation strategies have been summarized in a recent review (Kochendoerfer, et al., Opin Chem Biol 9:555-560, 2005).
- FVIII muteins that are covalently bound at a predefined site to one or more biocompatible polymers such as polyethylene glycol (PEG) are described in U.S. Patent Application No. 20060115876.
- the mutein conjugates retain FVIII procoagulant activity and have improved pharmacokinetic properties.
- a FVIII molecule having at least a portion of the B domain intact, which is conjugated to a water-soluble polymer such as polyethylene glycol (PEG) having a molecular weight of greater than 10 kD is described in U.S. Patent Application No. 20070244301.
- the construct has a biological activity of at least 80% of the biological activity of native FVIII, and the in vivo half- life of the construct is increased by at least 1.5 fold as compared to the in vivo half-life of native FVIII.
- AGP3 and El l which are able to detect PEG-modified proteins by ELISA (Cheng, et al., Bioconj Chem 16:1225-1231, 2005; Tsai, et al, BioTechniques 30:396-402, 2001).
- AGP3 and El 1 have also been used to detect B 16/DNS mammalian cells incubated with DNS-PEG conjugates and for in vivo clearance of a PEGylated protein in mice (Cheng, et al., 2005).
- FXa hydrolyses the chromogenic substrate S-2765 (Z-D- Arg-Gly-Arg-pNA (para-nitroaniline)) thus liberating the chromophoric group, pNA.
- the intensity of color generated is proportional to the concentration of FVIII in the test sample.
- This assay is further described, for example, in U.S. Patent No. 6,100,050.
- Assays for other blood coagulation factors are also available, such as the Coaset® assay for analysis of FVII activity (Chromogenix, Milan, Italy). PEGylation of a protein can often reduce the sensitivity of these activity assays. Therefore, there is a need for increasing the sensitivity of these assays for the analysis of PEGylated blood coagulation factors.
- a method for detecting PEGylated blood coagulation factors has been developed.
- the present invention provides a method for detecting a PEGylated blood coagulation factor or a PEGylated fragment thereof in a sample, comprising: simultaneously or in any sequence contacting a sample comprising PEGylated blood coagulation factor or a PEGylated fragment thereof with a capture agent comprising at least one first reporter function observable at at least one first emission wavelength and at least one probe agent comprising at least one second reporter function observable at at least one second emission wavelength; illuminating at least a portion of said capture agent and at least one probe agent to excite the at least one first reporter function and the at least one second reporter function; and detecting coincidence of emission signals from the capture agent by at least one first emission wavelength detector and the at least one probe agent by at least one second emission wavelength detector, thereby detecting the PEGylated blood coagulation factor or a PEGylated fragment thereof, wherein the capture agent is capable of binding PEG and the capture
- the invention also provides a method for detecting a PEGylated blood coagulation factor or a PEGylated fragment thereof in a sample, the method comprising: contacting the sample with a capture antibody that is capable of binding PEG, said capture antibody being attached to a bead having at least one detectable characteristic, wherein the PEGylated blood coagulation factor or PEGylated fragment thereof binds to the capture antibody and forms a complex therewith; contacting the complex formed in the first step with a probe antibody that is capable of binding a non-PEGylated blood coagulation factor, said probe antibody being labeled with a detectable marker, to form a complex which includes the capture antibody of the first step, the PEGylated blood coagulation factor or PEGylated fragment, and the probe antibody; and detecting the probe antibody and the bead attached to the capture antibody by flow cytometry, thereby detecting the presence of PEGylated blood coagulation factor or a PEGylated fragment thereof in
- the present invention provides a method for determining blood coagulation factor activity of a sample using a capture agent that is capable of binding PEG, the method comprising: contacting a sample comprising PEGylated blood coagulation factor or a PEGylated fragment thereof with the capture agent under conditions such that the capture agent binds to the PEGylated blood coagulation factor or a PEGylated fragment thereof; removal of the sample components that are not bound by the capture agent; and measuring the blood coagulation factor activity of the sample components bound by the capture agent.
- the capture agent is capable of binding with high affinity.
- the blood coagulation factor may be Factor I, Factor II, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII, as well as variants, muteins, and biologically active fragments.
- the probe agent may comprise two probe agents, for example, directed to different epitopes of the blood coagulation factor.
- the sample may be a biological tissue or fluid, for example, such sample may include, but are not limited to, blood, serum, plasma, tissue or biopsy samples, sputum, urine, peritoneal fluid, pleural fluid as well as cell lysates or products from tissue culture.
- Figure 1 is a model of a dual antibody immunoassay for detection of PEGylated FVIII.
- Figure 2 is a model of a Coatest® assay for measuring activity of PEGylated FVIII.
- FIG. 3 shows the quantification of PEGylated FVIII in plasma from different species.
- a monoclonal anti-PEG antibody (10F05) was used for capture and biotinylated polyclonal anti- FVIII antibody was used for detection.
- Mean fluorescence intensity (MFI) was quantified by flow cytometry.
- Figure 4 shows a comparison of the anti-FVIII mAb capture antibody and anti-PEG mAb capture antibody in several plasma samples.
- Figure 5 illustrates the anti-PEG monoclonal antibody in the Coatest® assay.
- Figure 6 demonstrates the effect of substrate concentration in the Coatest® assay.
- Figure 7 shows quantification of PEGylated FVIII in an ELISA assay.
- Figure 8 shows quantification of PEGylated FIX in an ELISA assay.
- Blood coagulation factors include proteins involved in blood coagulation and clotting and biologically active fragments thereof.
- the invention can be used to detect PEGylated blood coagulation factors of the intrinsic and extrinsic pathways, including but not limited to Factors I (fibrinogen), II (prothrombin), V, VII, VIII, IX, X, XI, and XII.
- FIX means Coagulation Factor IX, which is also known as Human Clotting Factor IX, or Plasma Thromboplastin Component
- FX means Coagulation Factor X, which is also known by the names Human Clotting Factor X and by the eponym Stuart-Prower factor.
- Blood clotting FVIII is a glycoprotein synthesized and released into the bloodstream by the liver. In the circulating blood, it is bound to von Willebrand factor (vWF, also known as FVIII -related antigen) to form a stable complex. Upon activation by thrombin, it dissociates from the complex to interact with other clotting factors in the coagulation cascade, which eventually leads to the formation of a thrombus.
- Factor VIII may include, for example, the full-length FVIII as well as allelic variants and muteins.
- blood coagulation factor includes Factor I, Factor II, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII, as well as variants, muteins, and biologically active fragments.
- B domain deleted FVIII is a Factor VIII molecule in which all or part of the B domain has been deleted.
- the B domain of FVIII is dispensable since BDD has also been shown to be effective as a replacement therapy for hemophilia A.
- PEG and "polyethylene glycol” as used herein are interchangeable and include any water-soluble poly(ethylene oxide).
- PEGs may comprise the following structure "-(OCH 2 CH 2 ) n ⁇ " where (n) is 2 to 4000.
- PEG also includes "-CH 2 CH 2 - 0(CH 2 CH 2 O) n -CH 2 CH 2 -” and "-(OCH 2 CH 2 ) n O-,” depending upon whether or not the terminal oxygens have been displaced.
- PEG includes structures having various terminal or "end capping" groups, such as without limitation a hydroxyl or a Ci -2 O alkoxy group.
- PEG also means a polymer that contains a majority, that is to say, greater than 50%, of -OCH 2 CH 2 — repeating subunits.
- the PEG can take any number of a variety of molecular weights, as well as structures or geometries such as branched, linear, forked, and multifunctional.
- a PEG-conjugated protein is a protein which is covalently attached to one or more polyethylene glycol (PEG) molecules.
- PEG polyethylene glycol
- PEG-conjugated and “PEGylated” are used herein interchangeably.
- This invention relates to PEG-conjugated blood coagulation factors. Methods for PEG conjugation of blood coagulation factors are described, for example, in US Patent No. 5,766,897; US Patent No. 6,753,165; WO 90/12874; and US Application No. 2006/0115876.
- Proteins can be PEG-conjugated in several different ways, for example, by random modification of primary amines (N-terminus and lysines) or by site-directed strategies such as incorporation of unnatural amino acids followed by the addition of a PEG derivative that will react specifically with the unnatural amino acid. Another approach to site-specific PEG- conjugation of proteins is by targeting N-terminal backbone amine with PEG-aldehydes. Proteins can also be PEG-conjugated by inserting or substituting a cysteine for another amino acid, then adding a PEG moiety that has a sulfhydryl reactive group.
- a mutein is a genetically engineered protein arising as a result of a mutation to a protein or polypeptide.
- a variant refers to a polypeptide having an amino acid sequence in which one or more amino acid residues is altered.
- the variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties (e.g. , replacement of: leucine with isoleucine).
- a variant may also have "nonconservative” changes (e.g., replacement of glycine with tryptophan).
- Analogous minor variations may include amino acid deletions or insertions, or both.
- a biologically active fragment denotes a fragment of a coagulation factor which possesses functional activity.
- the sample which is analyzed is a biological fluid or tissue.
- suitable biological fluids may include, but are not limited to, blood, serum, plasma, tissue or biopsy samples, sputum, urine, peritoneal fluid, pleural fluid as well as cell lysates or products from tissue culture.
- the biological fluid comprises serum or plasma.
- the term plasma generally refers to a solution comprising proteins having procoagulant activity. Proteins in the plasma may include blood clotting factors, thrombin, and fibrinogen. The plasma may also contain other plasma proteins, sugars, and/or salts.
- the plasma can be whole plasma that is obtained from humans or other animals.
- the plasma may also be deficient in one or more blood clotting factors, for example, FVIII- deficient plasma from a hemophilia A patient.
- the plasma can also be a plasma derivative that has procoagulant activity and is derived from one or more whole plasmas.
- the plasma derivative can be, for example, a plasma fraction or a plasma that has been purified or otherwise treated to remove some protein, sugar, salt, or other components thereof.
- the plasma can alternatively be a plasma substitute formed from components obtained from separate sources, including natural or man-made components.
- man-made components include plasma proteins that are substantially isolated and/or purified from natural sources and plasma proteins that are prepared using recombinant technology.
- blood generally refers to whole blood, citrated blood, platelet concentrate, or control mixtures of plasma and blood cells.
- the samples analyzed by the methods of the present invention may be obtained from a vertebrate species.
- the vertebrate species may be a warm-blooded vertebrate species.
- Such warm-blooded vertebrate species include, but are not limited to, human, canine, porcine, bovine, guinea pig, horse, cat, monkey, sheep, rat, mouse, goat, rabbit, and chicken.
- the subject may be a human having or at risk of developing a bleeding disorder.
- Anti-PEG and anti-blood coagulation factor antibodies may be polyclonal or monoclonal.
- the interaction of the capture and probe agents with the PEGylated blood coagulation factors can be characterized in terms of a binding affinity.
- high affinity binding results from greater intermolecular force between the PEGylated protein and the capture and probe agents, while low affinity ligand binding involves less intermolecular force.
- high affinity binding also involves a longer residence time for the PEGylated blood coagulation factor at the binding site of the capture or probe agent than is the case for low affinity binding.
- high affinity binding results from specific interactions between the PEGylated blood coagulation factor and the capture and probe agents at specific sites on each molecule.
- any art-recognized method can be used to generate an anti-blood coagulation factor antibody.
- a blood coagulation factor protein, or fragment thereof can be used to immunize a suitable subject, (e.g., rabbit, goat, mouse or other mammal or vertebrate).
- a suitable subject e.g., rabbit, goat, mouse or other mammal or vertebrate.
- the immunogenic preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent.
- Immunization of a suitable subject with a blood coagulation factor protein or fragment thereof induces a polyclonal anti-blood coagulation factor antibody response.
- the anti-blood coagulation factor antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized blood coagulation factor protein or peptide.
- ELISA enzyme linked immunosorbent assay
- the antibody molecules directed against a blood coagulation factor antigen can be isolated from the immunized mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction.
- antibody-producing cells can be obtained from the subject and used to prepare, for example, monoclonal antibodies by standard techniques, such as the hybridoma technique described by Brown, et al. (J. Biol. Chem. 255:4980-83, 1980) and Yeh, et al. (Proc. Natl. Acad. Sci. USA 76:2927-2933, 1976).
- standard techniques such as the hybridoma technique described by Brown, et al. (J. Biol. Chem. 255:4980-83, 1980) and Yeh, et al. (Proc. Natl. Acad. Sci. USA 76:2927-2933, 1976).
- the technology for producing monoclonal antibody hybridomas is well known.
- an immortal cell line typically a myeloma
- lymphocytes typically splenocytes
- the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds a blood coagulation factor antigen.
- Anti-PEG mAbs can be generated as described, for example, in US Patent No. 7,320,791; Cheng, et al., (Bioconj. Chem. 10:520-528, 1999), and Wunderlich, et al, 2007.
- mice are immunized with keyhole limpet hemocyanin (KLH) derivitized with 2 kD mPEG prepared by nonspecifically cross-linking succinimidyl propionate-derivitized PEG to the amino groups of KLH.
- KLH keyhole limpet hemocyanin
- Hybridomas are generated by fusion of immune spleen cells with F/O mouse myeloma cells. Hybridomas that survive selection are tested by enzyme immunoassay for the presence of antibodies reactive with PEG and non-reactive with KLH-PEG.
- Hybridomas secreting anti-PEG IgG are expanded and subjected to single cell cloning by limiting dilution.
- an anti-PEG mAb (e.g., 10F05).
- This antibody was shown to be capable of binding a PEGylated protein with high affmitiy.
- this antibody is able to recognize a PEGylated peptide in plasma at concentrations as low as 3 pg/mL.
- Anti-PEG mAbs are also commercially available from, for example, Epitomics (Burlingame, CA) and Academia Sinica (Taipei, Taiwan).
- the capture agent comprises an anti-PEG antibody for binding of PEGylated blood coagulation factor.
- the antibody is directed to a PEG molecule covalently attached to the blood coagulation factor protein or fragment thereof such that upon contact, the blood coagulation factor protein or fragment specifically binds to the antibody and forms a complex with it. In this manner the antibody "captures" the blood coagulation factor protein or fragment, removing it from the sample.
- the capture antibody may be a monoclonal antibody or a polyclonal antibody.
- Capture agents may also comprise other proteins that are capable of binding PEG, including but not limited to engineered antibody constructs, such as single-chain Fv fragments, chimeric antibodies, diabodies, triabodies, tetravalent antibodies, peptabodies and hexabodies. Engineered antibody constructs are described, for example, in US Patent No. 6,830,752 and US Patent Application No. 2003/0226155.
- a capture agent comprises a protein capable of binding PEG which is attached to a particle, such as a bead, as described in Example 1.
- a capture agent that is capable of binding PEG for detecting PEGylated blood coagulation factors provides several advantages.
- One advantage is that the same capture agent can be used for different PEGylated blood coagulation factors, eliminating the need to develop separate capture agents for each coagulation factor.
- Capture of PEGylated blood coagulation factors also allows for distinction between an endogenous coagulation factor and a PEGylated coagulation factor administered to a subject. This distinction would be especially useful in preclinical and clinical trials for measuring concentration and activity of an administered coagulation factor in plasma.
- Use of a capture agent before detection also allows for concentration and purification of the PEGylated blood coagulation factor from the sample, increasing the sensitivity of the assay and reducing the potential negative effects of other sample components.
- the capture agent also comprises a fluorescent marker for detection.
- a fluorescent marker for detection.
- the capture agent is labeled by direct conjugation of a fluorophore to a protein capable of binding PEG. Kits for fluorescent labeling of proteins are commercially available (e.g., Thermo Scientific, Rockford, IL).
- the protein capable of binding PEG is labeled by conjugation to a bead containing one or more fluorophores.
- the protein capable of binding PEG may be labeled by conjugation to a molecule that binds a luminescent marker, for example, N-hydroxysuccinimidobiotin (biotin). Biotinylated proteins can then be incubated with a fluorescently labeled molecule that binds biotin, for example, avidin or streptavidin, for detection.
- the capture agent comprises a biotinylated protein capable of binding PEG and a fluorescently labeled molecule capable of binding biotin.
- FITC fluorescein isothiocyanate
- PE phycoerythrin
- the methods of the present invention further include the use of a probe agent also directed to a blood coagulation factor protein.
- the probe agent can comprise any protein that is capable of binding a non-PEGylated blood coagulation factor, including but not limited to monoclonal antibodies, polyclonal antibodies, and the engineered antibody constructs described above.
- the probe agent recognizes an epitope of the non-PEGylated blood coagulation factor protein and is therefore different from the capture agent, which recognizes PEG.
- the probe agent upon binding to its recognition site on the captured blood coagulation factor protein, contributes to a complex that includes the capture agent, the probe agent, and the PEGylated blood coagulation factor protein.
- the capture agent and probe agent are labeled with different fluorescent markers that emit light of different wavelengths.
- the probe agent can be labeled by several different methods including but not limited to direct conjugation of a fluorophore or by biotinylation as described above. Lasers can be used to excite the fluorescent dyes of the capture and probe agents, and different wavelengths of light emitted from the dyes can be detected. In this way, the presence of both the capture agent and the probe agent in a complex can be detected, thereby increasing the specificity of the assay. Fluorescent light detection systems are described in more detail below.
- the capture agent comprises a protein capable of binding PEG attached to a particle.
- the particle may be a bead or "microsphere" having physical characteristics and fluorescent properties suitable for use in applications such as flow cytometry.
- Beads suitable for the invention are generally known in the art and may be obtained from manufacturers such as Spherotech (Libertyville, IL), Molecular Probes (Eugene, OR), and Luminex (Austin, TX).
- the bead may comprise at least one appropriate fluorescing compound.
- Flow cytometry analysis of the beads operates in a conventional manner. That is, the beads are processed by illuminating them, essentially one at a time, with a laser beam. Measurements of the scattered laser light are obtained for each illuminated bead by a plurality of optical detectors. If a bead contains at least one appropriate fluorescing compound, it will fluoresce when illuminated. A plurality of optical detectors within the flow analyzer measure fluorescence at a plurality of wavelengths. Typical measured bead characteristics include, but are not limited to, forward light scatter, side light scatter, red fluorescence, green fluorescence, and orange fluorescence.
- Luminex Austin, TX
- U.S. Patent No. 5,981,180 An exemplary flow cytometric system for simultaneous assay of multiple analytes in a sample, including antigens bound to antibodies conjugated to fluorescent beads is marketed by Luminex (Austin, TX) and is described, for example, in U.S. Patent No. 5,981,180.
- One method for detecting a PEGylated blood coagulation factor or fragment comprises contacting a sample with an anti-PEG capture antibody attached to a bead having at least one detectable characteristic, such as a first identifiable spectral property.
- anti-PEG capture antibody complexes with PEGylated blood coagulation factor in the sample.
- the complexes formed on the beads comprising capture anti-PEG antibodies are detected by contacting the respective beads with a second (probe) antibody directed to a blood coagulation factor and labeled with a detectable marker.
- This second probe antibody recognizes an epitope of the blood coagulation factor protein and is therefore capable of binding to a non-PEGylated form of the coagulation factor.
- Complexes which include the bead bound to the capture antibody, the PEGylated blood coagulation factor, and the probe antibody are then detected, and the amount of PEGylated blood coagulation factor in the sample is determined.
- a bead-based fluorescent immunoassay embodiment is useful, due to its very high level of sensitivity, for detection of blood coagulation factors in human plasma from normal subjects and those with blood clotting and autoimmune disorders.
- the immunoassays of the invention are also suitable for analysis of the concentration of blood coagulation factors in commercial products.
- Other methods of the invention can be used to measure the activity of PEGylated blood coagulation factors in a sample. In these methods, a capture agent that is capable of binding PEG is used to separate the PEGylated blood coagulation factor from the sample. The activity of the blood coagulation factor is then determined.
- the PEGylated blood coagulation factor is PEGylated FVIII.
- FVIII activity is determined by the Coatest® assay (Rosen, et al., Thromb Hemost 54:818-823, 1985). In this assay, plasma from individuals is mixed with FIXa/FX complex. In the presence of calcium and phospholipids, FX is activated to FXa by Factor IXa. This activation is greatly stimulated by FVIII. The rate of activation of FX is solely dependent on the amount of FVIII. In the Coatest® assay, FXa hydro lyses a chromogenic substrate, thus liberating a chromophoric group. The intensity of color generated is proportional to the concentration of FVIII in the test sample. This assay is further described, for example, in U.S. Patent No. 6,100,050.
- phospholipids may be such representative compounds as phosphotidyl choline, phosphotidyl serine, or cholesterol and mixtures thereof in various proportions. Other lipid and phospholipid compositions may be substituted as well.
- Any chemical source of calcium cation may be used to effect the conversion of FX. Sufficient calcium ion may be added to the original incubation mixture to drive the reaction converting FX to FXa, or a second amount of calcium ion may be added at the time FX is to be converted.
- Sources of calcium cation (Ca ++ ) may be CaCl 2 , Ca(NO 2 ) 2 , CaSO/i, or other inorganic calcium cation containing compounds.
- An indicator agent capable of reacting with blood coagulation FXa can be used for detection of blood coagulation factor activity. In such reaction, by-products of chemical reaction must be generated which produce a measurable signal moiety.
- US Patent Nos. 4,480,030 and 4,666,831 describe a class of chromogenic compounds capable of reacting with FXa.
- a suitable chromogenic compound is the indicator CH OCO-D-CHG-GIy- Arg-pNA- AcOH (Pentapharm, Basel, Switzerland).
- An example of a chromogenic compound is S-2765 (Chromogenix, Milan, Italy). Upon reaction of S-2765 with FXa, a signal molecule P-nitroaniline (pNA) is released, which may be conveniently measured by spectrophotometric determination at 405 nm.
- a kit for performing a Coatest® assay on a sample comprises: a sufficient amount of FIXa to saturate all FVIIIa in a sample, a sufficient amount of a thrombin inhibitor to inhibit thrombin activity without affecting FXa activity, a sufficient amount of phospholipid and calcium ion to facilitate the conversion of FX to FXa, a sufficient amount of FX to saturate the complex of FIXa and FVIII and phospholipids, and a sufficient quantity of an indicator agent capable of reacting with FXa.
- FVII blood coagulation factor
- the activity of FVII may be determined by the Coaset® assay (Chromogenix, Milan, Italy). This assay is based on a two-step process. In the first step, FX is activated to FXa via the extrinsic pathway, that is, through the action of FVII and thromboplastin. In the second step the generated FXa hydrolyzes S-2675, thus liberating pNA which can be measured by spectrophotometric determination at 405 nM.
- Factor IX coagulation activity may be determined using an activated partial thromboplastin time assay (aPTT) (described by, e.g., Proctor, et al., Am. J. Clin. Pathol. 36:212, 1961). Activity assays for other blood coagulation factor are well known in the art.
- aPTT activated partial thromboplastin time assay
- polypeptides, materials, compositions, and methods described herein are intended to be representative examples of the invention, and it will be understood that the scope of the invention is not limited by the scope of the examples. Those skilled in the art will recognize that the invention may be practiced with variations on the disclosed polypeptides, materials, compositions and methods, and such variations are regarded as within the ambit of the invention.
- a series of buffers and solutions were prepared for use in a dual antibody immunoassay for quantification of FVIII.
- the following buffers were prepared and stored at 4°C for up to one week, except for the standard dilution buffer which was prepared on the day the assay was run: plate pre-wet buffer: 0.1% BSA/IX PBS/0.02% NaN 3 ; binding buffer: IX PBS/0.5% BSA/0.02% NaN 3 ; washing buffer: IX PBS/0.02% NaN 3 /0.05% TWEEN®-20 (polyoxyethylenesorbitan monolaurate); and standard dilution buffer: 1% plasma/lX PBS/0.5% BSA/0.02% NaN 3 .
- Plasma standard solutions were prepared from the stock solutions for FVIII, BDD, and PEG-FVIII.
- FVIII and BDD a 1000 ng/mL plasma standard solution was prepared by adding 10 ⁇ L each of 25 ⁇ g/mL stock solution to 240 ⁇ L of 100% plasma. Following addition, the sample was mixed well. This 1000 ng/mL plasma standard was diluted 10-fold with binding buffer to obtain 100 ng/mL in 10% plasma, then further diluted 10-fold with binding buffer to obtain 10 ng/mL at 1% plasma.
- a 750 ng/mL plasma standard solution was prepared by adding 10 ⁇ L each of 17 ⁇ g/mL stock solution to 216.7 ⁇ L of 100% plasma. Following addition, the sample was mixed well. This 750 ng/mL plasma standard was diluted 10- fold with binding buffer to obtain 75 ng/mL in 10% plasma, then further diluted 10-fold with binding buffer to obtain 7.5 ng/mL in 1% plasma.
- Plasma QC standards at 40, 200, and 500 ng/mL were prepared by parallel dilution of the same stock solution in rabbit, rat, and dog plasma. Aliquots of 50 ⁇ L/vial were stored at -80 0 C. Two-step 10-fold dilution of all controls with binding buffer was performed on the same day of the assay to obtain a 1/100 dilution.
- Blood samples were collected as one part anticoagulant to 9 parts blood (v/v) with 3.8% sodium citrate or 5% sodium citrate (for rat) as anticoagulant. After centrifugation, plasma samples were diluted 1/100 in binding buffer using a two-step dilution.
- Normal New Zealand White (NZW) female rabbit plasma samples were pooled from 20 animals.
- Rat blood samples were collected from 10-week-old normal Sprague-Dawley (SD) rats (Charles River). Rat blood was drawn under 2.5% isoflurane and then centrifuged for 10 minutes at 3,000 rpm. Normal beagle female dog plasma was pooled from 8 animals.
- Anti-PEG mAb (10F05) and biotinylated anti-FVIII pAb (R8B12) were used.
- Microspheres Luminex, Austin, TX
- Microspheres were coated with antibody according to manufacturer's instructions (PolyLink Protein Coupling Kit, Bangs Laboratories, Inc., Fishers, IN). Briefly, the microspheres were resuspended by votex and sonication for 1 minute, and transferred (5 x 10 6 ) to a microcentrifuge tube. The microspheres were pelleted by centrifuge for 3 minutes at 12,000 rpm and resuspended in 400 ⁇ L PolyLink coupling buffer.
- microspheres were pelleted again via centrifugation for 3 minutes at 12,000 rpm and resuspended in 170 ⁇ L PolyLink coupling buffer.
- EDAC 20 ⁇ L, 200 mg/mL carbodiimide
- antibody 200 ⁇ L, 0.97 ⁇ g/ ⁇ L
- the microspheres were incubated for 5 hours at ambient temperature with rotation in the dark.
- the microspheres were then centrifuged at 12,000 rpm for 2 minutes and resuspended with 400 ⁇ L PolyLink washing buffer.
- the microspheres were again centrifuged at 12,000 rpm for 2 minutes, resuspended with 500 ⁇ L PolyLink washing buffer, and stored at 4°C.
- Assays were performed in 96-well Multiscreen-BV plates with a pore size of 1.2 ⁇ m (Millipore, Billerica, MA). The plates were pre-wet with IX PBS/0.1%BSA buffer for at least 10 minutes, and the buffer was aspirated by a vacuum manifold before addition of the microspheres. The microspheres (50 ⁇ L; 100 beads/ ⁇ L dilution) were added to each well, and then 50 ⁇ L FVIII- containing standards, controls, or plasma samples diluted in binding buffer (plasma final concentration: 1%) were also added to each well. The plates were covered and placed on a shaker for 2 hours at 37°C with constant shaking.
- Biotinylated anti-FVIII pAb was diluted in binding buffer to a concentration of 10 ⁇ g/mL, and 100 ⁇ L of the dilution was added to each well.
- the plates were covered and incubated for 1 hour at room temperature with constant shaking.
- Plasma samples from several animal species were analyzed using anti-PEG mAb (10F05) as capture antibody.
- Two-fold serial dilutions of PEGylated FVIII were made in plasma from Hem A mouse (FVIII- deficient plasma), New Zealand white female rabbit, Sprague-Dawley male rat, and beagle female dog. Sensitivity of the assay was relatively high for the plasmas tested, indicating that this assay could be used for samples containing plasma. Results are shown in Figure 3. Each point is a mean of three samples and error bars represent the standard deviation of the mean.
- each assay plate was first prepared with capture antibody.
- Each well of the microtiter plate was coated with 100 ⁇ L anti-PEG mAb (10F05) diluted in coating buffer.
- the antibody was prepared at concentrations of 5.0 or 2.5 ⁇ g/mL.
- the plate was sealed and incubated on a platform shaker at 200 rpm for about 1 hour at 37°C.
- the well contents were removed by aspiration.
- the wells were washed by filling each well completely with wash buffer, letting it stand for 10 seconds and aspirating again. The wash procedure was repeated 3 additional times for a total of 4 washes.
- an automated plate washer such as the Skanwasher® 300 (Skatron Instruments AS, Lier, Norway) or equivalent can be used. After washing, 300 ⁇ L blocking buffer was added to each well and the plates were incubated on a platform shaker at 200 rpm for about 1 hour at 37°C. [079] To prepare the assay standards, one vial of calibrator (standard) was placed in a 37°C water bath until just thawed and then transferred to ice. The calibrator was used within 1 hour of thawing. A 10-fold dilution of monkey or other appropriate plasma was prepared in binding buffer. This solution was used as the diluent for the standards.
- Skanwasher® 300 Skatron Instruments AS, Lier, Norway
- a 2-fold serial dilution of the calibrator was prepared with a starting concentration of 20 mIU/mL for monkey plasma and 10 mlU/mL for HemA mouse plasma.
- the calibrator was mixed prior to dilution by gently pipetting up and down.
- Concentration of the standards ranged from 0.313 mIU/mL to 20 mIU/mL for monkey plasma or 0.156 mIU/mL to 10 mIU/mL for Hem A mouse plasma. Volume of the standard at each concentration was 400 ⁇ L.
- a zero mIU/mL aliquot (without FVIII) was also included in the standard curve.
- the Coatest® reagents were prepared as per package instructions.
- One part assay buffer (supplied with the Coatest® kit) was diluted with 9 parts water to make IX Coatest® assay buffer.
- the FIXa/FX (supplied with the Coatest® kit) was rehydrated with 10 mL water.
- One mL phospholipid solution (supplied with the Coatest® kit) was added per 5 mL FIXa/FX and mixed well by pipetting. The phospholipids solution was kept on ice and used within 30 minutes of preparation. The plate was washed as described above and 50 ⁇ L IX Coatest® assay buffer was added to each well.
- the FIXa/FX/phospho lipid solution was mixed again just before use and 50 ⁇ L was added to each well. Mixing of the FIXa/FX/phospho lipid mixture just prior to use and during the incubation minimizes sample variability.
- the plates were incubated at 37°C for exactly 10 minutes on a platform shaker at a setting of 2. Twenty- five ⁇ L CaCl 2 solution (supplied with the Coatest® kit) was added to each well and the plates were incubated at 37°C for exactly 10 minutes on a heater block on a platform shaker set to 2. A 25 mg vial of S-2765 was rehydrated per manufacturer's instruction, and then 50 ⁇ L was added to each well.
- the plate was incubated at 37°C with continued shaking for approximately 20 minutes. The plate was then transferred to a plate reader at ambient room temperature, and the entire plate was scanned for OD 40S - OD 490 .
- the specificity of a anti-PEG mAb in the Coatest® assay is shown in Figure 5. Each point is a mean of three samples and error bars represent the standard deviation of the mean.
- the Factor Xa substrate S-2765 was added to the reaction mixture at either a IX (as per kit instructions) or 2X concentration.
- a 25 mg vial of S-2765 was rehydrated with 19.5 mL Milli-Q® water (Millipore, Billerica, MA), and then 12 mL of this S-2765 rehydrate was added to the Coatest® substrate which contains S-2765 and 1-2581.
- This addition of S-2765 doubled the concentration of S-2765 in the Coatest® substrate stock solution from 2.7 to 5.4 mM.
- Coatest® substrate 50 ⁇ L was added to each well to create a total reaction volume of 175 ⁇ L and a final S-2765 concentration of 0.77 mM (IX) or 1.54 mM (2X).
- the plate was incubated at 37°C with continued shaking for approximately 20 minutes.
- the plate was transferred to a plate reader at ambient room temperature, and the entire plate was scanned for OD 405 - OD 490 . Further plate development may be stopped at this point by addition of acetic acid for later plate scans.
- SOFTmax® Pro (Molecular Devices, Sunnyvale, CA) was used for data analysis (e.g., 4- Parameter from the standard curve fit). The "zero FVIII" was used as one of the standards and was not subtracted from the well readings. The CV of triplicate wells for standards, controls, and unknowns was verified to be less than 25%. Precision and accuracy (% recovery, and absolute % residual error) were calculated as described in Example 1. Calibration standards and quality control samples were excluded from further evaluation if their experimentally determined concentrations varied more than ⁇ 25 % from the nominal values.
- Anti-PEG mAb, anti-FVIII mAb, and anti-FVIII pAb were coated in 96-well plate at 5 ⁇ g/mL. PEGylated-FVIII was serial titrated in 5% rat plasma starting at 10 ng/mL, and incubated for 2 hours. The plate was washed for 3 times, and incubated with biotin-anti-FVIII pAb at 1 ug/ml for 1 hour. After washing, Streptavidin-HRP (1/200) was added to each well and incubated for 30 minutes at room temperature.
- the plate was developed for 20 minutes after adding TMB (3,3',5,5'-tetramethylbenzidine) substrate and read at 450 nm immediately after adding 3 M H 2 SO 4 .
- the results are shown in Figure 7. Each point is a mean of three samples and error bars represent the standard deviation of the mean.
- Anti-PEG mAb and anti-FIX mAb were coated in a 96-well plate at 5 ⁇ g/mL. PEGylated- FIX was serial titrated in 10% rat plasma starting at 6.25 ng/mL, and incubated for 2 hours. The plate was washed 4 times, and incubated with horseradish peroxidase-conjugated anti-FIX pAb at 1 ⁇ g/mL for 1.5 hours. After washing, the plate was developed for 20 minutes after adding OPD (o-phenylenediamine dihydrochloride) substrate and read at 490 nm after adding 3 M H 2 SO 4 . The results are shown in Figure 8. Each point is a mean of three samples and error bars represent the standard deviation of the mean.
- OPD o-phenylenediamine dihydrochloride
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Abstract
The invention relates generally to methods for detecting PEGylated blood coagulation factors in a sample. These methods utilize anti-polyethylene glycol (PEG) capture agents and anti-blood coagulation factor probe agents for detection. Methods for determining the activity of PEGylated blood coagulation factors in a sample are also provided.
Description
ASSAYS FOR DETECTING PEGYLATED BLOOD COAGULATION FACTORS
[001] This application claims benefit of U.S. Provisional Application Serial No. 61/148,058; filed on January 29, 2009, the contents of which are incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[002] This invention relates generally to methods for measuring PEGylated blood coagulation factor concentration and activity in a sample using an anti-polyethylene glycol (PEG) capture agent.
BACKGROUND OF THE INVENTION
[003] Blood coagulation is a complex chemical and physical reaction that occurs when blood comes into contact with an activating agent. The blood coagulation process can be generally viewed as three activities: platelet adhesion, platelet aggregation, and formation of a fibrin clot. In vivo, platelets flow through the blood vessels in an inactivated state because the blood vessel lining, the endothelium, prevents activation of platelets. When a blood vessel is damaged, however, the endothelium loses its integrity and platelets are activated by contact with tissue underlying the damaged site. Activation of the platelets causes them to become "sticky" and adhere together. Additional platelets then adhere to the activated platelets and also become activated. This process continues until a platelet "plug" is formed. This platelet plug then serves as a matrix upon which blood clotting or coagulation proceeds.
[004] If the chemical balance of the blood is suitable, thrombin is then produced that causes fibrinogen to convert to fibrin, which forms the major portion of the clot mass. During clotting, additional platelets are activated and trapped in the forming clot, further contributing to clot formation. As clotting proceeds, polymerization and cross-linking of fibrin results in the permanent clot. Clotting may be initiated by either the intrinsic pathway, which involves the activation of Factor XII, or by the extrinsic pathway, which involves the release of tissue factor.
[005] Several different blood coagulation factors are involved in the formation of a blood clot. These factors include Factors I (fibrinogen), II (prothrombin), V, VII, VIII, IX, X, XI, and XII. In the intrinsic pathway, coagulation factors circulate in the form of inactive precursors which are converted into an active form, which in turn activates the next clotting factor in sequence. For example, proenzyme Factor XII is converted to its enzyme XIIa which in turn converts the zymogen Factor XI to the enzyme Factor XIa, which then activates Factor IX in the presence of calcium. The enzyme Factor IXa in the presence of Factor VIII and phospholipids activates Factor
X. This reaction is greatly increased by the prior exposure of Factor VIII to thrombin or Factor Xa.
[006] Deficiencies in blood coagulation factors can result in severe hematological disorders. Hemophilia A is a congenital bleeding disorder resulting from an X-chromosome-linked deficiency of Factor VIII (FVIII), occuring with a frequency of 1 in 5000 males. It is caused by either a quantitative or a qualitative deficiency in FVIII, a critical component of the intrinsic pathway of blood coagulation. Human FVIII is synthesized as a single-chain precursor of approximately 300 kD. It consists of the structural domains A1-A2-B-A3-C1-C2 (Thompson, Semin Thromb Hemost 29:11-22, 2003). FVIII can be further processed into two polypeptide chains consisting of a 200 kD heavy chain (A1-A2-B) and an 80 kD light chain (A3-C1-C2). Processing of FVIII occurs in the Golgi apparatus, and the resulting heavy and light chains are held together by metal ions. (Anderson, et al., Proc. Natl. Acad. Sci. USA 83:2979-2984, 1986; Kaufman, et al., J. Biol. Chem. 263:6352-6362, 1988).
[007] Upon secretion, FVIII circulates in the blood as a heterodimer of heavy and light chains, and binds tightly and non-covalently to von Willebrand factor (vWF). vWF binding sites on FVIII include FVIII residues 1649-1689 in the A3 domain (Foster, et al., J. Biol. Chem. 264:5230-5234, 1988), and parts of the Cl (Jacquemin, et al., Blood 96:958-965, 2000) and C2 domains (Spiegel, et al., 279:53691-53698, 2004). A FVIII binding defect on vWF or severe deficiency of vWF causes a hemophilia- like form of von Willebrand disease (type 2N or type 3 vWD) (Gu, et al., Blood 89:3263-3269, 1997; Sadler, et al., J Thromb Hemost 4:2103-2114, 2006).
[008] FVIII is activated by thrombin, which cleaves peptide bonds after residues 372, 740, and 1689 to generate a heterotrimer of Al, A2, and A3-C1-C2 domains (Pittman, et al., Proc. Natl. Acad. Sci. USA 85:2429-2433, 1988). Upon activation, FVIII dissociates from vWF and is concentrated at the cell surface of platelets by binding to phospholipid. Phospholipid binding involves FVIII residues 2199, 2200, 2251, and 2252 (Gilbert, et al., J. Biol. Chem. 277:6374-6381, 2002). At the cell surface, FVIII binds to Factor IX (FIX) through interactions with FVIII residues 558-565 (Fay, et al., J. Biol. Chem. 266:8957-8962, 1991; Regan, et al., J. Biol. Chem. 269:9445- 9452, 1984) and 1811-1818 (Lenting, et al., J. Biol. Chem. 271 :1935-1940, 1996) and to Factor X (FX) through interactions with FVIII residues 349-372 (Nogami, et al., J. Biol. Chem. 279:15763- 15771, 2004). FVIII also acts as a cofactor for FIX activation of FX, an essential component of the intrinsic coagulation pathway. Activated FVIII (FVIIIa) is partially inactivated by the protease activated protein C (APC) through cleavage after FVIII residues 336 and 562 (Regan, et al., J. Biol. Chem. 271 :3982-3987, 1996). The predominant determinant of inactivation, however, is the dissociation of the A2 domain from Al and A3-C1-C2 (Fay, et al., J. Biol. Chem. 266:8957-8962, 1991).
[009] Several different forms of recombinant FVIII have been developed for the treatment of hemophilia. Current effective treatment for severe hemophilia A requires FVIII infused at a dose of , for example, 20-40 IU/kg three times per week. Frequent dosaging such as this presents an inconvenience for patients and may interfere with patient complaince with dosage recommendations. An additional disadvantage to the current therapy is that approximately 25- 30% of patients develop antibodies that inhibit FVIII activity (Saenko, et al., Hemophilia 8:1-11, 2002). Antibody development prevents the use of FVIII as a replacement therapy for these patients, forcing them to seek an even more expensive treatment with high-dose recombinant Factor Vila and immune tolerance therapy.
[010] For the reasons stated above, recent research has focused on developing an improved FVIII variant that possesses greater duration of action in vivo and reduced immunogenicity, while retaining functional activity (Lusher, et al., Therapy 3:699-708, 2006; Saenko, et al., Hemophilia 12:42-51, 2006). Furthermore, it is desirable that such a protein can be produced as a homogeneous product in a consistent manner.
[011] Like many proteins, conjugation of polyethylene glycol (PEG) to blood coagulation factors, such as FVIII, extends half-life in animals, potentially increasing their efficacy as therapeutic agents. PEGylation is the covalent attachment of one or more long-chained polyethylene glycol (PEG) molecules to a protein. In addition to increasing half-life, PEGylation has been demonstrated to reduce immunogenicity, prevent protease digestion, and block entry of the protein into the kidney filtrate (Harris, et al., Clin Pharmacokinet 40:539-551, 2001). PEGylation may also increase the overall stability and solubility of the protein. Randomly PEGylated proteins, such as interferon-alpha (Kozlowski, et al., BioDrugs 15:419-429, 2001) have been approved as therapeutics. Various site-directed protein PEGylation strategies have been summarized in a recent review (Kochendoerfer, et al., Opin Chem Biol 9:555-560, 2005). FVIII muteins that are covalently bound at a predefined site to one or more biocompatible polymers such as polyethylene glycol (PEG) are described in U.S. Patent Application No. 20060115876. The mutein conjugates retain FVIII procoagulant activity and have improved pharmacokinetic properties. A FVIII molecule having at least a portion of the B domain intact, which is conjugated to a water-soluble polymer such as polyethylene glycol (PEG) having a molecular weight of greater than 10 kD is described in U.S. Patent Application No. 20070244301. The construct has a biological activity of at least 80% of the biological activity of native FVIII, and the in vivo half- life of the construct is increased by at least 1.5 fold as compared to the in vivo half-life of native FVIII.
[012] Antibodies that recognize PEG have been developed. Cheng, et al. disclose the anti-PEG mouse monoclonal antibodies AGP3 and El l which are able to detect PEG-modified proteins by
ELISA (Cheng, et al., Bioconj Chem 16:1225-1231, 2005; Tsai, et al, BioTechniques 30:396-402, 2001). AGP3 and El 1 have also been used to detect B 16/DNS mammalian cells incubated with DNS-PEG conjugates and for in vivo clearance of a PEGylated protein in mice (Cheng, et al., 2005). Since PEGylation often reduces the immunogenicity of an antigen, developing an antibody that binds PEG with high affinity can be extremely challenging. Wunderlich, et al. recently developed the anti-PEG IgG mouse monoclonal antibody 10F05 (Hybridoma 26:168-172, 2007). This antibody reacts with PEG regardless of the linker used for PEG attachment, and is able to recognize a PEGylated peptide in plasma.
[013] Precise measurement of blood coagulation factors is critical for both preclinical and clinical applications. Quantification of blood coagulation factor plasma levels is crucial for accurate measurement of pharmacokinetics in preclinical models and disease severity in clinical applications. For example, accurate determination of the level of procoagulant FVIII is necessary in order to assess severity in hemophilia A patients and to correlate clinical phenotype with circulating FVIII levels. Several different methods have been developed to quantify FVIII concentration in plasma, including clotting methods, chromogenic assays, thrombin generation assays, and activated partial thromboplastin time clot waveform analysis. Since the current assays involve bioassays, they can exhibit problems of poor reproducibility due to complex reaction kinetics. These techniques also possesses limitations that reduce sensitivity and precision at FVIII levels of <0.01 IU/ml. Since PEGylation can also reduce assay sensitivity, standard assays are often not effective in measuring PEGylated blood coagulation factors. Therefore, there is a need to develop a simple and accurate method that can detect and quantify very low levels of PEGylated coagulation factors in plasma.
[014] Measurement of blood coagulation factor activity is also critical in treating hematological disorders and the development of new therapeutics. The most common technique for measuring FVIII activity in plasma is the Coatest® assay (Rosen, et al., Thromb Hemost 54:818-823, 1985). In this assay, plasma from individuals is mixed with FIXa/FX complex. In the presence of calcium and phospholipids, FX is activated to FXa by Factor IXa. As mentioned above, this activation is greatly stimulated by FVIII. The rate of activation of FX is solely dependent on the amount of FVIII. In the Coatest® assay, FXa hydrolyses the chromogenic substrate S-2765 (Z-D- Arg-Gly-Arg-pNA (para-nitroaniline)) thus liberating the chromophoric group, pNA. The intensity of color generated is proportional to the concentration of FVIII in the test sample. This assay is further described, for example, in U.S. Patent No. 6,100,050. Assays for other blood coagulation factors are also available, such as the Coaset® assay for analysis of FVII activity (Chromogenix, Milan, Italy). PEGylation of a protein can often reduce the sensitivity of these
activity assays. Therefore, there is a need for increasing the sensitivity of these assays for the analysis of PEGylated blood coagulation factors.
[015] Numerous assay kits have been designed around the particle-based flow cytometry assay platform technology (e.g., xMAP® Technology, Luminex Corporation, Austin, TX). This technology uses fluorescently labeled microspheres as a solid surface to attach specific reagents for bioassays. The attached reagents then serve to bind and capture analytes from heterogeneous samples. These bioassays are performed in 96-well filter bottom microplates, which serve as fluid reservoirs for the test reagents. This approach has been used to measure numerous types of soluble analytes, including antibodies specific to various antigens, and to quantify the cell surface expression of various receptors (Vignali, et al., J. Immunol. Methods 243:243-255, 2000). Parhami-Seren and colleagues developed a particle-based flow cytometric assay to simultaneously measure human tissue factor and FVIII in human plasma (WO 2007/011746; Parhami-Seren, et al., J. Thromb. Hemost. 4:1747-1755, 2006). Both of these studies utilized simple capture-type assays where the desired analytes are directly detected in a sample. WO 2007/011746 discloses antibodies that selectively bind to FVIII and highly sensitive immunological assays comprising these antibodies. The assays involve contacting a sample comprising FVIII protein with a reducing agent that releases FVIII from a FVIII-binding molecule. The assays have a wide array of applications including accurate monitoring of FVIII concentration in pharmaceutical products for treatment of blood coagulation disorders, and determination of FVIII levels in plasma of human patients, including those with blood coagulation disorders such as hemophilia.
[016] Although immunoassays for the detection of non-PEGylated FVIII have been developed, detection of PEGylated proteins presents distinct challenges. Conjugation of PEG molecules to a protein can often result in reduced antibody binding, due to interference of the PEG molecules with protein: antibody interactions. Therefore, many antibodies that bind blood coagulation factors will not bind the PEGylated form. Considering the importance of accurately measuring blood coagulation factors, and the potential advantages of PEGylation, a need exists for a method of detecting PEGylated blood coagulation factors with high sensitivity.
SUMMARY OF THE INVENTION
[017] A method for detecting PEGylated blood coagulation factors has been developed. The present invention provides a method for detecting a PEGylated blood coagulation factor or a PEGylated fragment thereof in a sample, comprising: simultaneously or in any sequence contacting a sample comprising PEGylated blood coagulation factor or a PEGylated fragment thereof with a capture agent comprising at least one first reporter function observable at at least
one first emission wavelength and at least one probe agent comprising at least one second reporter function observable at at least one second emission wavelength; illuminating at least a portion of said capture agent and at least one probe agent to excite the at least one first reporter function and the at least one second reporter function; and detecting coincidence of emission signals from the capture agent by at least one first emission wavelength detector and the at least one probe agent by at least one second emission wavelength detector, thereby detecting the PEGylated blood coagulation factor or a PEGylated fragment thereof, wherein the capture agent is capable of binding PEG and the at least one probe agent is capable of binding non-PEGylated blood coagulation factor or a fragment thereof. In a further embodiment, the capture agent and/or probe agent are capable of binding with high affinity.
[018] The invention also provides a method for detecting a PEGylated blood coagulation factor or a PEGylated fragment thereof in a sample, the method comprising: contacting the sample with a capture antibody that is capable of binding PEG, said capture antibody being attached to a bead having at least one detectable characteristic, wherein the PEGylated blood coagulation factor or PEGylated fragment thereof binds to the capture antibody and forms a complex therewith; contacting the complex formed in the first step with a probe antibody that is capable of binding a non-PEGylated blood coagulation factor, said probe antibody being labeled with a detectable marker, to form a complex which includes the capture antibody of the first step, the PEGylated blood coagulation factor or PEGylated fragment, and the probe antibody; and detecting the probe antibody and the bead attached to the capture antibody by flow cytometry, thereby detecting the presence of PEGylated blood coagulation factor or a PEGylated fragment thereof in the sample, wherein the capture antibody is capable of binding PEG. In a further embodiment, the capture agent is capable of binding with high affinity.
[019] An assay for measuring the activity of PEGylated blood coagulation factor has also been developed. The present invention provides a method for determining blood coagulation factor activity of a sample using a capture agent that is capable of binding PEG, the method comprising: contacting a sample comprising PEGylated blood coagulation factor or a PEGylated fragment thereof with the capture agent under conditions such that the capture agent binds to the PEGylated blood coagulation factor or a PEGylated fragment thereof; removal of the sample components that are not bound by the capture agent; and measuring the blood coagulation factor activity of the sample components bound by the capture agent. In a further embodiment, the capture agent is capable of binding with high affinity.
[020] In other embodiments of the invention, the blood coagulation factor may be Factor I, Factor II, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII, as well as variants, muteins, and biologically active fragments. The probe agent may comprise two probe
agents, for example, directed to different epitopes of the blood coagulation factor. The sample may be a biological tissue or fluid, for example, such sample may include, but are not limited to, blood, serum, plasma, tissue or biopsy samples, sputum, urine, peritoneal fluid, pleural fluid as well as cell lysates or products from tissue culture.
DESCRIPTION OF THE FIGURES
[021] Figure 1 is a model of a dual antibody immunoassay for detection of PEGylated FVIII. [022] Figure 2 is a model of a Coatest® assay for measuring activity of PEGylated FVIII.
[023] Figure 3 shows the quantification of PEGylated FVIII in plasma from different species. A monoclonal anti-PEG antibody (10F05) was used for capture and biotinylated polyclonal anti- FVIII antibody was used for detection. Mean fluorescence intensity (MFI) was quantified by flow cytometry.
[024] Figure 4 shows a comparison of the anti-FVIII mAb capture antibody and anti-PEG mAb capture antibody in several plasma samples.
[025] Figure 5 illustrates the anti-PEG monoclonal antibody in the Coatest® assay. [026] Figure 6 demonstrates the effect of substrate concentration in the Coatest® assay.
[027] Figure 7 shows quantification of PEGylated FVIII in an ELISA assay. [028] Figure 8 shows quantification of PEGylated FIX in an ELISA assay.
DESCRIPTION OF THE INVENTION
[029] Blood coagulation factors include proteins involved in blood coagulation and clotting and biologically active fragments thereof. The invention can be used to detect PEGylated blood coagulation factors of the intrinsic and extrinsic pathways, including but not limited to Factors I (fibrinogen), II (prothrombin), V, VII, VIII, IX, X, XI, and XII. As used herein, FIX means Coagulation Factor IX, which is also known as Human Clotting Factor IX, or Plasma Thromboplastin Component and FX means Coagulation Factor X, which is also known by the names Human Clotting Factor X and by the eponym Stuart-Prower factor.
[030] Blood clotting FVIII is a glycoprotein synthesized and released into the bloodstream by the liver. In the circulating blood, it is bound to von Willebrand factor (vWF, also known as FVIII -related antigen) to form a stable complex. Upon activation by thrombin, it dissociates from the complex to interact with other clotting factors in the coagulation cascade, which eventually
leads to the formation of a thrombus. Factor VIII may include, for example, the full-length FVIII as well as allelic variants and muteins.
[031] As used herein, blood coagulation factor includes Factor I, Factor II, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII, as well as variants, muteins, and biologically active fragments.
[032] As used herein B domain deleted FVIII (BDD) is a Factor VIII molecule in which all or part of the B domain has been deleted. The B domain of FVIII is dispensable since BDD has also been shown to be effective as a replacement therapy for hemophilia A.
[033] "PEG" and "polyethylene glycol" as used herein are interchangeable and include any water-soluble poly(ethylene oxide). As an example, PEGs may comprise the following structure "-(OCH2CH 2)n~" where (n) is 2 to 4000. As used herein, PEG also includes "-CH2CH2- 0(CH2CH2O)n-CH2CH2-" and "-(OCH2CH2)nO-," depending upon whether or not the terminal oxygens have been displaced. Throughout the specification and claims, it should be remembered that the term "PEG" includes structures having various terminal or "end capping" groups, such as without limitation a hydroxyl or a Ci-2O alkoxy group. The term "PEG" also means a polymer that contains a majority, that is to say, greater than 50%, of -OCH 2CH2— repeating subunits. With respect to specific forms, the PEG can take any number of a variety of molecular weights, as well as structures or geometries such as branched, linear, forked, and multifunctional.
[034] A PEG-conjugated protein is a protein which is covalently attached to one or more polyethylene glycol (PEG) molecules. The terms "PEG-conjugated" and "PEGylated" are used herein interchangeably. This invention relates to PEG-conjugated blood coagulation factors. Methods for PEG conjugation of blood coagulation factors are described, for example, in US Patent No. 5,766,897; US Patent No. 6,753,165; WO 90/12874; and US Application No. 2006/0115876. Proteins can be PEG-conjugated in several different ways, for example, by random modification of primary amines (N-terminus and lysines) or by site-directed strategies such as incorporation of unnatural amino acids followed by the addition of a PEG derivative that will react specifically with the unnatural amino acid. Another approach to site-specific PEG- conjugation of proteins is by targeting N-terminal backbone amine with PEG-aldehydes. Proteins can also be PEG-conjugated by inserting or substituting a cysteine for another amino acid, then adding a PEG moiety that has a sulfhydryl reactive group.
[035] A mutein is a genetically engineered protein arising as a result of a mutation to a protein or polypeptide.
[036] A variant refers to a polypeptide having an amino acid sequence in which one or more amino acid residues is altered. For example, the variant may have "conservative" changes,
wherein a substituted amino acid has similar structural or chemical properties (e.g. , replacement of: leucine with isoleucine). A variant may also have "nonconservative" changes (e.g., replacement of glycine with tryptophan). Analogous minor variations may include amino acid deletions or insertions, or both.
[037] A biologically active fragment denotes a fragment of a coagulation factor which possesses functional activity.
[038] In certain embodiments of the invention, the sample which is analyzed is a biological fluid or tissue. Suitable biological fluids, for example, may include, but are not limited to, blood, serum, plasma, tissue or biopsy samples, sputum, urine, peritoneal fluid, pleural fluid as well as cell lysates or products from tissue culture. In one embodiments of the invention, the biological fluid comprises serum or plasma.
[039] As used herein, the term plasma generally refers to a solution comprising proteins having procoagulant activity. Proteins in the plasma may include blood clotting factors, thrombin, and fibrinogen. The plasma may also contain other plasma proteins, sugars, and/or salts. The plasma can be whole plasma that is obtained from humans or other animals. The plasma may also be deficient in one or more blood clotting factors, for example, FVIII- deficient plasma from a hemophilia A patient. The plasma can also be a plasma derivative that has procoagulant activity and is derived from one or more whole plasmas. The plasma derivative can be, for example, a plasma fraction or a plasma that has been purified or otherwise treated to remove some protein, sugar, salt, or other components thereof. The plasma can alternatively be a plasma substitute formed from components obtained from separate sources, including natural or man-made components. Exemplary man-made components include plasma proteins that are substantially isolated and/or purified from natural sources and plasma proteins that are prepared using recombinant technology. As used herein "blood" generally refers to whole blood, citrated blood, platelet concentrate, or control mixtures of plasma and blood cells.
[040] The samples analyzed by the methods of the present invention may be obtained from a vertebrate species. The vertebrate species may be a warm-blooded vertebrate species. Such warm-blooded vertebrate species include, but are not limited to, human, canine, porcine, bovine, guinea pig, horse, cat, monkey, sheep, rat, mouse, goat, rabbit, and chicken. As an example, the subject may be a human having or at risk of developing a bleeding disorder.
[041] Anti-PEG and anti-blood coagulation factor antibodies may be polyclonal or monoclonal. The term "monoclonal antibody" as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of an antigen, whereas the term "polyclonal antibody" refers to a population of antibody
molecules that contain multiple species of antigen binding sites capable of interacting with a particular antigen.
[042] The interaction of the capture and probe agents with the PEGylated blood coagulation factors can be characterized in terms of a binding affinity. In general, high affinity binding results from greater intermolecular force between the PEGylated protein and the capture and probe agents, while low affinity ligand binding involves less intermolecular force. In general, high affinity binding also involves a longer residence time for the PEGylated blood coagulation factor at the binding site of the capture or probe agent than is the case for low affinity binding. Furthermore, high affinity binding results from specific interactions between the PEGylated blood coagulation factor and the capture and probe agents at specific sites on each molecule.
[043] Any art-recognized method can be used to generate an anti-blood coagulation factor antibody. For example, a blood coagulation factor protein, or fragment thereof (alone, or linked to a hapten or protein carrier) can be used to immunize a suitable subject, (e.g., rabbit, goat, mouse or other mammal or vertebrate). For example, the methods described in U.S. Patent Nos. 5,422,110; 5,837,268; 5,708,155; 5,723,129; and 5,849,531 can be used. The immunogenic preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with a blood coagulation factor protein or fragment thereof induces a polyclonal anti-blood coagulation factor antibody response. The anti-blood coagulation factor antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized blood coagulation factor protein or peptide. The antibody molecules directed against a blood coagulation factor antigen can be isolated from the immunized mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction.
[044] At an appropriate time after immunization, for example, when the anti-blood coagulation factor antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare, for example, monoclonal antibodies by standard techniques, such as the hybridoma technique described by Brown, et al. (J. Biol. Chem. 255:4980-83, 1980) and Yeh, et al. (Proc. Natl. Acad. Sci. USA 76:2927-2933, 1976). The technology for producing monoclonal antibody hybridomas is well known. Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with a blood coagulation factor immunogen as described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds a blood coagulation factor antigen.
[045] Anti-PEG mAbs can be generated as described, for example, in US Patent No. 7,320,791; Cheng, et al., (Bioconj. Chem. 10:520-528, 1999), and Wunderlich, et al, 2007. In the methods of Wunderlich, et al., mice are immunized with keyhole limpet hemocyanin (KLH) derivitized with 2 kD mPEG prepared by nonspecifically cross-linking succinimidyl propionate-derivitized PEG to the amino groups of KLH. Hybridomas are generated by fusion of immune spleen cells with F/O mouse myeloma cells. Hybridomas that survive selection are tested by enzyme immunoassay for the presence of antibodies reactive with PEG and non-reactive with KLH-PEG. Hybridomas secreting anti-PEG IgG are expanded and subjected to single cell cloning by limiting dilution. The methods described in Wunderlich, et al., were used to generate an anti-PEG mAb (e.g., 10F05). This antibody was shown to be capable of binding a PEGylated protein with high affmitiy. For example, this antibody is able to recognize a PEGylated peptide in plasma at concentrations as low as 3 pg/mL. Anti-PEG mAbs are also commercially available from, for example, Epitomics (Burlingame, CA) and Academia Sinica (Taipei, Taiwan).
[046] In certain embodiments of the present invention, the capture agent comprises an anti-PEG antibody for binding of PEGylated blood coagulation factor. The antibody is directed to a PEG molecule covalently attached to the blood coagulation factor protein or fragment thereof such that upon contact, the blood coagulation factor protein or fragment specifically binds to the antibody and forms a complex with it. In this manner the antibody "captures" the blood coagulation factor protein or fragment, removing it from the sample. The capture antibody may be a monoclonal antibody or a polyclonal antibody. Capture agents may also comprise other proteins that are capable of binding PEG, including but not limited to engineered antibody constructs, such as single-chain Fv fragments, chimeric antibodies, diabodies, triabodies, tetravalent antibodies, peptabodies and hexabodies. Engineered antibody constructs are described, for example, in US Patent No. 6,830,752 and US Patent Application No. 2003/0226155. In one embodiment, a capture agent comprises a protein capable of binding PEG which is attached to a particle, such as a bead, as described in Example 1.
[047] Use of a capture agent that is capable of binding PEG for detecting PEGylated blood coagulation factors provides several advantages. One advantage is that the same capture agent can be used for different PEGylated blood coagulation factors, eliminating the need to develop separate capture agents for each coagulation factor. Capture of PEGylated blood coagulation factors also allows for distinction between an endogenous coagulation factor and a PEGylated coagulation factor administered to a subject. This distinction would be especially useful in preclinical and clinical trials for measuring concentration and activity of an administered coagulation factor in plasma. Use of a capture agent before detection also allows for concentration
and purification of the PEGylated blood coagulation factor from the sample, increasing the sensitivity of the assay and reducing the potential negative effects of other sample components.
[048] In addition to the protein capable of binding PEG, the capture agent also comprises a fluorescent marker for detection. Several different methods can be used for labeling of the capture agent with the fluorescent marker. In one embodiment of the invention, the capture agent is labeled by direct conjugation of a fluorophore to a protein capable of binding PEG. Kits for fluorescent labeling of proteins are commercially available (e.g., Thermo Scientific, Rockford, IL). In another embodiment, the protein capable of binding PEG is labeled by conjugation to a bead containing one or more fluorophores. In an additional embodiment, the protein capable of binding PEG may be labeled by conjugation to a molecule that binds a luminescent marker, for example, N-hydroxysuccinimidobiotin (biotin). Biotinylated proteins can then be incubated with a fluorescently labeled molecule that binds biotin, for example, avidin or streptavidin, for detection. In this embodiment, the capture agent comprises a biotinylated protein capable of binding PEG and a fluorescently labeled molecule capable of binding biotin.
[049] The most common fluorophores are FITC (fluorescein isothiocyanate) and PE (phycoerythrin), but there are many others available. FITC is a small, charged molecule that can be easily conjugated to protein through an isothiocyanate group. It has an excitation maximum at 495 nm and emits green light (520 nm). It can be excited by a 488 nm argon-ion laser. R- phycoerythrin is a phycobiloprotein extracted from red algae. Its excitation maxima are 564 and 495 nm, and emission maximum is 576 nm. This is also excited by a 488 nm argon-ion laser.
[050] The methods of the present invention further include the use of a probe agent also directed to a blood coagulation factor protein. The probe agent can comprise any protein that is capable of binding a non-PEGylated blood coagulation factor, including but not limited to monoclonal antibodies, polyclonal antibodies, and the engineered antibody constructs described above. The probe agent recognizes an epitope of the non-PEGylated blood coagulation factor protein and is therefore different from the capture agent, which recognizes PEG. The probe agent, upon binding to its recognition site on the captured blood coagulation factor protein, contributes to a complex that includes the capture agent, the probe agent, and the PEGylated blood coagulation factor protein.
[051] The capture agent and probe agent are labeled with different fluorescent markers that emit light of different wavelengths. The probe agent can be labeled by several different methods including but not limited to direct conjugation of a fluorophore or by biotinylation as described above. Lasers can be used to excite the fluorescent dyes of the capture and probe agents, and different wavelengths of light emitted from the dyes can be detected. In this way, the presence of
both the capture agent and the probe agent in a complex can be detected, thereby increasing the specificity of the assay. Fluorescent light detection systems are described in more detail below.
[052] In one embodiment, the capture agent comprises a protein capable of binding PEG attached to a particle. As an example, the particle may be a bead or "microsphere" having physical characteristics and fluorescent properties suitable for use in applications such as flow cytometry. Beads suitable for the invention are generally known in the art and may be obtained from manufacturers such as Spherotech (Libertyville, IL), Molecular Probes (Eugene, OR), and Luminex (Austin, TX). The bead may comprise at least one appropriate fluorescing compound.
[053] Flow cytometry analysis of the beads operates in a conventional manner. That is, the beads are processed by illuminating them, essentially one at a time, with a laser beam. Measurements of the scattered laser light are obtained for each illuminated bead by a plurality of optical detectors. If a bead contains at least one appropriate fluorescing compound, it will fluoresce when illuminated. A plurality of optical detectors within the flow analyzer measure fluorescence at a plurality of wavelengths. Typical measured bead characteristics include, but are not limited to, forward light scatter, side light scatter, red fluorescence, green fluorescence, and orange fluorescence. An exemplary flow cytometric system for simultaneous assay of multiple analytes in a sample, including antigens bound to antibodies conjugated to fluorescent beads is marketed by Luminex (Austin, TX) and is described, for example, in U.S. Patent No. 5,981,180.
[054] One method for detecting a PEGylated blood coagulation factor or fragment comprises contacting a sample with an anti-PEG capture antibody attached to a bead having at least one detectable characteristic, such as a first identifiable spectral property. Upon contact with the sample, anti-PEG capture antibody complexes with PEGylated blood coagulation factor in the sample. The complexes formed on the beads comprising capture anti-PEG antibodies are detected by contacting the respective beads with a second (probe) antibody directed to a blood coagulation factor and labeled with a detectable marker. This second probe antibody recognizes an epitope of the blood coagulation factor protein and is therefore capable of binding to a non-PEGylated form of the coagulation factor. Complexes which include the bead bound to the capture antibody, the PEGylated blood coagulation factor, and the probe antibody are then detected, and the amount of PEGylated blood coagulation factor in the sample is determined.
[055] A bead-based fluorescent immunoassay embodiment is useful, due to its very high level of sensitivity, for detection of blood coagulation factors in human plasma from normal subjects and those with blood clotting and autoimmune disorders. In addition to their uses for detection of blood coagulation factors in biological samples, the immunoassays of the invention are also suitable for analysis of the concentration of blood coagulation factors in commercial products.
[056] Other methods of the invention can be used to measure the activity of PEGylated blood coagulation factors in a sample. In these methods, a capture agent that is capable of binding PEG is used to separate the PEGylated blood coagulation factor from the sample. The activity of the blood coagulation factor is then determined. In one embodiment, the PEGylated blood coagulation factor is PEGylated FVIII. In another embodiment, FVIII activity is determined by the Coatest® assay (Rosen, et al., Thromb Hemost 54:818-823, 1985). In this assay, plasma from individuals is mixed with FIXa/FX complex. In the presence of calcium and phospholipids, FX is activated to FXa by Factor IXa. This activation is greatly stimulated by FVIII. The rate of activation of FX is solely dependent on the amount of FVIII. In the Coatest® assay, FXa hydro lyses a chromogenic substrate, thus liberating a chromophoric group. The intensity of color generated is proportional to the concentration of FVIII in the test sample. This assay is further described, for example, in U.S. Patent No. 6,100,050.
[057] As mentioned above, the conversion of Factor X to Xa in the Coatest® assay proceeds most efficiently in the presence of phospholipids. These phospholipids may be such representative compounds as phosphotidyl choline, phosphotidyl serine, or cholesterol and mixtures thereof in various proportions. Other lipid and phospholipid compositions may be substituted as well.
[058] Any chemical source of calcium cation may be used to effect the conversion of FX. Sufficient calcium ion may be added to the original incubation mixture to drive the reaction converting FX to FXa, or a second amount of calcium ion may be added at the time FX is to be converted. Sources of calcium cation (Ca++) may be CaCl2, Ca(NO2)2, CaSO/i, or other inorganic calcium cation containing compounds.
[059] An indicator agent capable of reacting with blood coagulation FXa can be used for detection of blood coagulation factor activity. In such reaction, by-products of chemical reaction must be generated which produce a measurable signal moiety. US Patent Nos. 4,480,030 and 4,666,831 describe a class of chromogenic compounds capable of reacting with FXa. One example of a suitable chromogenic compound is the indicator CH OCO-D-CHG-GIy- Arg-pNA- AcOH (Pentapharm, Basel, Switzerland). An example of a chromogenic compound is S-2765 (Chromogenix, Milan, Italy). Upon reaction of S-2765 with FXa, a signal molecule P-nitroaniline (pNA) is released, which may be conveniently measured by spectrophotometric determination at 405 nm.
[060] A kit for performing a Coatest® assay on a sample comprises: a sufficient amount of FIXa to saturate all FVIIIa in a sample, a sufficient amount of a thrombin inhibitor to inhibit thrombin activity without affecting FXa activity, a sufficient amount of phospholipid and calcium ion to facilitate the conversion of FX to FXa, a sufficient amount of FX to saturate the complex of FIXa
and FVIII and phospholipids, and a sufficient quantity of an indicator agent capable of reacting with FXa.
[061] Other assays may be used to determine the activity of other blood coagulation factors. For example, the activity of FVII may be determined by the Coaset® assay (Chromogenix, Milan, Italy). This assay is based on a two-step process. In the first step, FX is activated to FXa via the extrinsic pathway, that is, through the action of FVII and thromboplastin. In the second step the generated FXa hydrolyzes S-2675, thus liberating pNA which can be measured by spectrophotometric determination at 405 nM. Factor IX coagulation activity may be determined using an activated partial thromboplastin time assay (aPTT) (described by, e.g., Proctor, et al., Am. J. Clin. Pathol. 36:212, 1961). Activity assays for other blood coagulation factor are well known in the art.
[062] The polypeptides, materials, compositions, and methods described herein are intended to be representative examples of the invention, and it will be understood that the scope of the invention is not limited by the scope of the examples. Those skilled in the art will recognize that the invention may be practiced with variations on the disclosed polypeptides, materials, compositions and methods, and such variations are regarded as within the ambit of the invention.
[063] The following examples are presented to illustrate the invention described herein, but should not be construed as limiting the scope of the invention in any way.
EXAMPLES
[064] In order that this invention may be better understood, the following examples are set forth. These examples are for the purpose of illustration only, and are not to be construed as limiting the scope of the invention in any manner. All publications mentioned herein are incorporated by reference in their entirety.
Example 1. Dual Antibody Immunoassay
[065] A series of buffers and solutions were prepared for use in a dual antibody immunoassay for quantification of FVIII. The following buffers were prepared and stored at 4°C for up to one week, except for the standard dilution buffer which was prepared on the day the assay was run: plate pre-wet buffer: 0.1% BSA/IX PBS/0.02% NaN3; binding buffer: IX PBS/0.5% BSA/0.02% NaN3; washing buffer: IX PBS/0.02% NaN3/0.05% TWEEN®-20 (polyoxyethylenesorbitan monolaurate); and standard dilution buffer: 1% plasma/lX PBS/0.5% BSA/0.02% NaN3.
[066] Stock solutions were prepared for FVIII, B domain deleted FVIII (BDD), and PEGylated FVIII (PEG-FVIII). Stock solutions were prepared in formulation buffer (20 mM MOPS, 150 mM NaCl, 1% sucrose, 100 ppm TWEEN®-80 (polyoxyethylenesorbitan monooleate, pH 7.0), stored at -800C, and thawed at 37°C prior to use. Control solutions were also prepared by adding known amounts of FVIII to animal plasma.
[067] Plasma standard solutions were prepared from the stock solutions for FVIII, BDD, and PEG-FVIII. For FVIII and BDD, a 1000 ng/mL plasma standard solution was prepared by adding 10 μL each of 25 μg/mL stock solution to 240 μL of 100% plasma. Following addition, the sample was mixed well. This 1000 ng/mL plasma standard was diluted 10-fold with binding buffer to obtain 100 ng/mL in 10% plasma, then further diluted 10-fold with binding buffer to obtain 10 ng/mL at 1% plasma. For PEG-FVIII, a 750 ng/mL plasma standard solution was prepared by adding 10 μL each of 17 μg/mL stock solution to 216.7 μL of 100% plasma. Following addition, the sample was mixed well. This 750 ng/mL plasma standard was diluted 10- fold with binding buffer to obtain 75 ng/mL in 10% plasma, then further diluted 10-fold with binding buffer to obtain 7.5 ng/mL in 1% plasma.
[068] Plasma QC standards at 40, 200, and 500 ng/mL were prepared by parallel dilution of the same stock solution in rabbit, rat, and dog plasma. Aliquots of 50 μL/vial were stored at -800C. Two-step 10-fold dilution of all controls with binding buffer was performed on the same day of the assay to obtain a 1/100 dilution.
[069] Blood samples were collected as one part anticoagulant to 9 parts blood (v/v) with 3.8% sodium citrate or 5% sodium citrate (for rat) as anticoagulant. After centrifugation, plasma
samples were diluted 1/100 in binding buffer using a two-step dilution. Normal New Zealand White (NZW) female rabbit plasma samples were pooled from 20 animals. Rat blood samples were collected from 10-week-old normal Sprague-Dawley (SD) rats (Charles River). Rat blood was drawn under 2.5% isoflurane and then centrifuged for 10 minutes at 3,000 rpm. Normal beagle female dog plasma was pooled from 8 animals.
[070] Anti-PEG mAb (10F05) and biotinylated anti-FVIII pAb (R8B12) were used. Microspheres (Luminex, Austin, TX) were coated with antibody according to manufacturer's instructions (PolyLink Protein Coupling Kit, Bangs Laboratories, Inc., Fishers, IN). Briefly, the microspheres were resuspended by votex and sonication for 1 minute, and transferred (5 x 106) to a microcentrifuge tube. The microspheres were pelleted by centrifuge for 3 minutes at 12,000 rpm and resuspended in 400 μL PolyLink coupling buffer. The microspheres were pelleted again via centrifugation for 3 minutes at 12,000 rpm and resuspended in 170 μL PolyLink coupling buffer. EDAC (20 μL, 200 mg/mL carbodiimide) was added to the microspheres and mixed gently. Then, antibody (200 μL, 0.97 μg/μL) was added to the microspheres and mixed gently. The microspheres were incubated for 5 hours at ambient temperature with rotation in the dark. The microspheres were then centrifuged at 12,000 rpm for 2 minutes and resuspended with 400 μL PolyLink washing buffer. The microspheres were again centrifuged at 12,000 rpm for 2 minutes, resuspended with 500 μL PolyLink washing buffer, and stored at 4°C.
[071] Assays were performed in 96-well Multiscreen-BV plates with a pore size of 1.2 μm (Millipore, Billerica, MA). The plates were pre-wet with IX PBS/0.1%BSA buffer for at least 10 minutes, and the buffer was aspirated by a vacuum manifold before addition of the microspheres. The microspheres (50 μL; 100 beads/μL dilution) were added to each well, and then 50 μL FVIII- containing standards, controls, or plasma samples diluted in binding buffer (plasma final concentration: 1%) were also added to each well. The plates were covered and placed on a shaker for 2 hours at 37°C with constant shaking. The plates were then washed 3 times with washing buffer using a vacuum manifold. Biotinylated anti-FVIII pAb was diluted in binding buffer to a concentration of 10 μg/mL, and 100 μL of the dilution was added to each well. The plates were covered and incubated for 1 hour at room temperature with constant shaking.
[072] The plates were washed 2 times with washing buffer using a vacuum manifold. For quantification of the FVIII antigen, R-phycoerythrin-streptavidin fluorophore (Invitrogen, Carlsbad, CA) was diluted in binding buffer to a concentration of 5 μg/mL, and 100 μL of the dilution was added to each well. The plates were incubated for 30 minutes at ambient temperature with constant shaking. The liquid was aspirated by vacuum manifold and the plates were washed one time with washing buffer. Buffer (100 μL, IX PBS/0.02% NaN3) was added to each well and the plates were shaken for 2 minutes at ambient temperature.
[073] The samples were then analyzed on the Luminex® 200™ instrument. "Calibration 1" microspheres of known fluorescent light intensities were used to calibrate the settings for both classification channels and the doublet discrimination channel. "Calibration 2" microspheres of known fluorescent light intensities were used to calibrate the settings for the reporter channel. "Control 1" microspheres were used to verify the calibration and optical integrity for both the classification channels and doublet discrimination channel. "Control 2" microspheres were used to verify the calibration and optical integrity for the reporter channel. The reading time was set at 10-60 sec per analytical run. The gate was set at 6,000 to 15,000.
[074] For data evaluation, all calculations were performed by MasterPlex® QT 2.5 software (MiraiBio, Hitachi) and additional data calculations and summaries were performed by Microsoft Excel. The standard curve was generated by plotting the known concentration of standards versus the median fluorescence intensity (MFI) using a 5-parameter logistic curve fit model:
F(X) = ((A-D) / (1+(X/C)ΛB)ΛE) + D
A = Lower Asymptote
B = Slope at inflection point
C = X value corresponding to the Y value that is halfway between A and D
D = Upper Asymptote
E = Parameter used for asymmetry correction of the 5-parameter fit generated from software
X = Values of concentration.
[075] Plasma concentrations were calculated using the standard curve. For calculation of precision (CV percentage) and accuracy (absolute residual error percentage and recovery percentage), equations (1), (2), (3) were used, respectively:
Standard deviation
(1) CV [%] = x 100 mean
(2) T R? pecsiirdiiuiQail F Errrrmor- π (K? mb) r [°//<> 1 J - - Mean - Nominal content x 100
Nominal content
mean
(3) Recovery (accuracy) [%] = x 100.
Nominal content
[076] Plasma samples from several animal species were analyzed using anti-PEG mAb (10F05) as capture antibody. Two-fold serial dilutions of PEGylated FVIII were made in plasma from Hem A mouse (FVIII- deficient plasma), New Zealand white female rabbit, Sprague-Dawley male rat, and beagle female dog. Sensitivity of the assay was relatively high for the plasmas tested, indicating that this assay could be used for samples containing plasma. Results are shown in Figure 3. Each point is a mean of three samples and error bars represent the standard deviation of the mean.
[077] To compare anti-FVIII mAb capture antibody and anti-PEG mAb capture antibody, PEGylated FVIII was added to plasma samples: rabbit, rat, dog, human SHP (severe hemophilic), and human vWD (von Willebrand disease). Results are shown in Figure 4. Each bar is a mean of three samples and error bars represent the standard deviation of the mean. The anti-PEG mAb capture assay showed an increased sensitivity as compared to anti-FVIII mAb capture assay.
Example 2. Modified Coatest® Assay for Quantifying Activity of FVIII
[078] To perform the modified Coatest® assay, each assay plate was first prepared with capture antibody. Each well of the microtiter plate was coated with 100 μL anti-PEG mAb (10F05) diluted in coating buffer. The antibody was prepared at concentrations of 5.0 or 2.5 μg/mL. The plate was sealed and incubated on a platform shaker at 200 rpm for about 1 hour at 37°C. The well contents were removed by aspiration. The wells were washed by filling each well completely with wash buffer, letting it stand for 10 seconds and aspirating again. The wash procedure was repeated 3 additional times for a total of 4 washes. Alternatively, an automated plate washer such as the Skanwasher® 300 (Skatron Instruments AS, Lier, Norway) or equivalent can be used. After washing, 300 μL blocking buffer was added to each well and the plates were incubated on a platform shaker at 200 rpm for about 1 hour at 37°C.
[079] To prepare the assay standards, one vial of calibrator (standard) was placed in a 37°C water bath until just thawed and then transferred to ice. The calibrator was used within 1 hour of thawing. A 10-fold dilution of monkey or other appropriate plasma was prepared in binding buffer. This solution was used as the diluent for the standards. A 2-fold serial dilution of the calibrator was prepared with a starting concentration of 20 mIU/mL for monkey plasma and 10 mlU/mL for HemA mouse plasma. The calibrator was mixed prior to dilution by gently pipetting up and down. Concentration of the standards ranged from 0.313 mIU/mL to 20 mIU/mL for monkey plasma or 0.156 mIU/mL to 10 mIU/mL for Hem A mouse plasma. Volume of the standard at each concentration was 400 μL. A zero mIU/mL aliquot (without FVIII) was also included in the standard curve.
[080] To prepare the assay controls, one vial of High Control (80 mIU/mL prepared in approximately 100% monkey plasma) and Low Control (8 and 4 mIU/mL prepared in approximately 100% plasma) were placed in a 37°C water bath until just thawed, then stored on ice. The controls were used within 1 hour of thawing. After thawing, the controls were diluted 10-fold in binding buffer.
[081] To prepare the samples, the samples were placed in a 37°C water bath until just thawed and transferred to ice. Each sample was diluted 10-fold by adding 40 μL sample to 360 μL of binding buffer in a cluster tube and mixing gently by pipetting up and down at least 6 times. The sample was further diluted in 10% plasma diluent to appropriate dilution scheme as determined by the pilot plate.
[082] To perform the capture step, the samples were washed as described above and 100 μL of each standard, diluted control, or diluted sample was added to three replicate wells. The plates were incubated on a shaker platform for about 2 hours at 37°C.
[083] The Coatest® reagents were prepared as per package instructions. One part assay buffer (supplied with the Coatest® kit) was diluted with 9 parts water to make IX Coatest® assay buffer. The FIXa/FX (supplied with the Coatest® kit) was rehydrated with 10 mL water. One mL phospholipid solution (supplied with the Coatest® kit) was added per 5 mL FIXa/FX and mixed well by pipetting. The phospholipids solution was kept on ice and used within 30 minutes of preparation. The plate was washed as described above and 50 μL IX Coatest® assay buffer was added to each well. The FIXa/FX/phospho lipid solution was mixed again just before use and 50 μL was added to each well. Mixing of the FIXa/FX/phospho lipid mixture just prior to use and during the incubation minimizes sample variability. The plates were incubated at 37°C for exactly 10 minutes on a platform shaker at a setting of 2. Twenty- five μL CaCl2 solution (supplied with the Coatest® kit) was added to each well and the plates were incubated at 37°C for exactly 10 minutes on a heater block on a platform shaker set to 2. A 25 mg vial of S-2765 was rehydrated
per manufacturer's instruction, and then 50 μL was added to each well. The plate was incubated at 37°C with continued shaking for approximately 20 minutes. The plate was then transferred to a plate reader at ambient room temperature, and the entire plate was scanned for OD40S - OD490. The specificity of a anti-PEG mAb in the Coatest® assay is shown in Figure 5. Each point is a mean of three samples and error bars represent the standard deviation of the mean.
[084] To test the effect of different Factor Xa substrate concentrations, the Factor Xa substrate S-2765 was added to the reaction mixture at either a IX (as per kit instructions) or 2X concentration. A 25 mg vial of S-2765 was rehydrated with 19.5 mL Milli-Q® water (Millipore, Billerica, MA), and then 12 mL of this S-2765 rehydrate was added to the Coatest® substrate which contains S-2765 and 1-2581. This addition of S-2765 doubled the concentration of S-2765 in the Coatest® substrate stock solution from 2.7 to 5.4 mM. Coatest® substrate (50 μL) was added to each well to create a total reaction volume of 175 μL and a final S-2765 concentration of 0.77 mM (IX) or 1.54 mM (2X). The plate was incubated at 37°C with continued shaking for approximately 20 minutes. The plate was transferred to a plate reader at ambient room temperature, and the entire plate was scanned for OD405 - OD490. Further plate development may be stopped at this point by addition of acetic acid for later plate scans.
[085] The effect of FXa substrate concentration is shown in Figure 6. Anti-PEG mAb (5 μg/mL) was coated in 96-well plates, and PEGylated FVIII was titrated in 10% monkey plasma starting at 20 mlU/mL. Each point is a mean of three samples and error bars represent the standard deviation of the mean.
[086] SOFTmax® Pro (Molecular Devices, Sunnyvale, CA) was used for data analysis (e.g., 4- Parameter from the standard curve fit). The "zero FVIII" was used as one of the standards and was not subtracted from the well readings. The CV of triplicate wells for standards, controls, and unknowns was verified to be less than 25%. Precision and accuracy (% recovery, and absolute % residual error) were calculated as described in Example 1. Calibration standards and quality control samples were excluded from further evaluation if their experimentally determined concentrations varied more than ± 25 % from the nominal values.
Example 3. ELISA assay for Factor VIII
[087] Anti-PEG mAb, anti-FVIII mAb, and anti-FVIII pAb were coated in 96-well plate at 5 μg/mL. PEGylated-FVIII was serial titrated in 5% rat plasma starting at 10 ng/mL, and incubated for 2 hours. The plate was washed for 3 times, and incubated with biotin-anti-FVIII pAb at 1 ug/ml for 1 hour. After washing, Streptavidin-HRP (1/200) was added to each well and incubated for 30 minutes at room temperature. The plate was developed for 20 minutes after adding TMB (3,3',5,5'-tetramethylbenzidine) substrate and read at 450 nm immediately after adding 3 M H2SO4. The results are shown in Figure 7. Each point is a mean of three samples and error bars represent the standard deviation of the mean.
Example 4. ELISA assay for Factor XI
[088] Anti-PEG mAb and anti-FIX mAb were coated in a 96-well plate at 5 μg/mL. PEGylated- FIX was serial titrated in 10% rat plasma starting at 6.25 ng/mL, and incubated for 2 hours. The plate was washed 4 times, and incubated with horseradish peroxidase-conjugated anti-FIX pAb at 1 μg/mL for 1.5 hours. After washing, the plate was developed for 20 minutes after adding OPD (o-phenylenediamine dihydrochloride) substrate and read at 490 nm after adding 3 M H2SO4. The results are shown in Figure 8. Each point is a mean of three samples and error bars represent the standard deviation of the mean.
[089] All publications and patents mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the described compositions and methods of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments.
Claims
1. A method for detecting a PEGylated blood coagulation factor or a PEGylated fragment thereof in a sample, comprising: a) contacting a sample comprising PEGylated blood coagulation factor or a PEGylated fragment thereof with a capture agent comprising at least one first reporter function and at least one probe agent comprising at least one second reporter function, and b) illuminating at least a portion of said capture agent and at least one probe agent to excite the at least one first reporter function and the at least one second reporter function, and c) detecting coincidence of emission signals from the capture agent by at least one first emission wavelength detector and the at least one probe agent by at least one second emission wavelength detector, thereby detecting the PEGylated blood coagulation factor or a PEGylated fragment thereof, wherein the capture agent is capable of binding PEG and the at least one probe agent is capable of binding non-PEGylated blood coagulation factor or a fragment thereof .
2. The method of claim 1 , wherein the blood coagulation factor is Factor I, Factor II, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII, or variants, muteins, and biologically active fragments thereof.
3. The method of claim 1, wherein the capture agent comprises a particle.
4. The method of claim 1, wherein the capture agent comprises an antibody attached to a bead.
5. The method of claim 1, wherein the capture agent and a probe agent are detected by flow cytometry.
6. The method of claim 1, wherein a probe agent comprises an antibody.
7. The method of claim 1, wherein a probe agent comprises two probe agents directed to different epitopes of the PEGylated blood coagulation factor.
8. The method of claim 1, wherein the sample is a biological fluid.
9. The method of claim 8, wherein the biological fluid is blood, serum, plasma, sputum, urine, peritoneal fluid, or pleural fluid.
10. The method of claim 9, wherein the sample is plasma.
11. A method for detecting a PEGylated blood coagulation factor or a PEGylated fragment thereof in a sample, the method comprising: a) contacting the sample with a capture antibody that is capable of binding PEG, said capture antibody being attached to a bead having at least one detectable characteristic, wherein the PEGylated blood coagulation factor or PEGylated fragment thereof binds to the capture antibody and forms a complex therewith, and b) contacting said complex with a probe antibody that is capabe of binding a non-PEGylated blood coagulation factor, said probe antibody being labeled with a detectable marker, to form a complex which includes the capture antibody, the PEGylated blood coagulation factor or PEGylated fragment, and the probe antibody, and c) detecting the probe antibody and the bead attached to the capture antibody by flow cytometry, thereby detecting the presence of PEGylated blood coagulation factor or a PEGylated fragment thereof in the sample, wherein the capture antibody is capable of binding PEG.
12. A method for determining blood coagulation factor activity of a sample using a capture agent that is capable of binding PEG, the method comprising: a) contacting a sample comprising PEGylated blood coagulation factor or a PEGylated fragment thereof with the capture agent under conditions such that the capture agent binds to the PEGylated blood coagulation factor or a PEGylated fragment thereof, and b) removal of the sample components that are not bound by the capture agent, and c) measuring the blood coagulation factor activity of the sample components bound by the capture agent.
13. The method of claim 12, wherein the blood coagulation factor is Factor I, Factor II, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII, or variants, muteins, and biologically active fragments thereof.
14. The method of claim 13, wherein Factor VIII activity of the sample components bound by the capture agent is measured by a kit comprising Factor XIa, Factor X, calcium, phospholipids, and a chromogenic substrate of FXa.
15. The method of claim 12, wherein the sample is a biological fluid.
16. The method of claim 15, wherein the biological fluid is blood, serum, plasma, sputum, urine, peritoneal fluid, or pleural fluid.
7. The method of claim 16, wherein the sample is plasma.
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2015154090A1 (en) * | 2014-04-04 | 2015-10-08 | Bloodworks | Routine laboratory and point-of-care (poc) testing for hemostasis |
US9804170B2 (en) | 2015-02-09 | 2017-10-31 | Bristol-Myers Squibb Company | Antibodies to polyethylene glycol |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6100050A (en) * | 1989-08-17 | 2000-08-08 | Dade Behring Ag | Factor VIII:Ca chromogenic assay |
US20050239143A1 (en) * | 2002-08-30 | 2005-10-27 | Capaldi Roderick A | Immunocapture-based measurements of mammalian pyruvate dehydrogenase complex |
WO2007011746A2 (en) * | 2005-07-15 | 2007-01-25 | The University Of Vermont And State Agriculture College | Highly sensitive immunoassays and antibodies for detection of blood factor viii |
US20070259381A1 (en) * | 2006-02-21 | 2007-11-08 | The Trustees Of Tufts College | Methods and arrays for target analyte detection and determination of reaction components that affect a reaction |
US20090005297A1 (en) * | 2004-07-13 | 2009-01-01 | Kevin Lumb | Aprotinin Variants |
-
2010
- 2010-01-29 WO PCT/US2010/022630 patent/WO2010088547A1/en active Application Filing
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6100050A (en) * | 1989-08-17 | 2000-08-08 | Dade Behring Ag | Factor VIII:Ca chromogenic assay |
US20050239143A1 (en) * | 2002-08-30 | 2005-10-27 | Capaldi Roderick A | Immunocapture-based measurements of mammalian pyruvate dehydrogenase complex |
US20090005297A1 (en) * | 2004-07-13 | 2009-01-01 | Kevin Lumb | Aprotinin Variants |
WO2007011746A2 (en) * | 2005-07-15 | 2007-01-25 | The University Of Vermont And State Agriculture College | Highly sensitive immunoassays and antibodies for detection of blood factor viii |
US20070259381A1 (en) * | 2006-02-21 | 2007-11-08 | The Trustees Of Tufts College | Methods and arrays for target analyte detection and determination of reaction components that affect a reaction |
Non-Patent Citations (1)
Title |
---|
STARLING ET AL.: "Development of a Dual Label Fluorescence Technique That Can Be Utilized to Elucidate the Mechanism of Action of Monoclonal Antibody-Drug Conjugates.", CANCER RESEARCH., vol. 48, no. 21, November 1988 (1988-11-01), pages 6211 - 6216 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2015154090A1 (en) * | 2014-04-04 | 2015-10-08 | Bloodworks | Routine laboratory and point-of-care (poc) testing for hemostasis |
US20170184617A1 (en) * | 2014-04-04 | 2017-06-29 | Bloodworks | Routine Laboratory and Point-of-Care (POC) Testing for Hemostasis |
US11181534B2 (en) | 2014-04-04 | 2021-11-23 | Bloodworks | Routine laboratory and point-of-care (POC) testing for hemostasis |
US9804170B2 (en) | 2015-02-09 | 2017-10-31 | Bristol-Myers Squibb Company | Antibodies to polyethylene glycol |
US10451634B2 (en) | 2015-02-09 | 2019-10-22 | Bristol-Myers Squibb Company | Method of using human FC-bearing IGG antibodies to polyethylene glycol |
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