Modification-dependent activity assays

Abstract

To provide a method for measuring the existence and / or the activity of recombinant polypeptide including modification, a system and a kit.SOLUTION: A method for detecting the existence of recombinant polypeptide including modification includes: a process for performing incubation under a condition that a scavenger for selectively coupling a specimen including the recombinant polypeptide including the modification to the modification, and a scavenger with respect to the modification can be selectively coupled to each other, and forming a polypeptide-agent complex by the incubation; a process for refining the polypeptide-agent complex from the specimen; and a process for assaying the existence of the recombinant polypeptide and / or polypeptide activity. The modification is related to recombinant protein by at least one of HES, sylation or polysialylation, the scavenger is a modification recognition antibody, and the detection of the recombinant polypeptide and / or the polypeptide activity indicates the existence of the recombinant polypeptide including the modification.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] Claiming priority This patent application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 61 / 487,612, filed May 18, 2011, the entirety of which is incorporated herein by reference.

[0002] Field of the Disclosure Disclosed herein are methods, systems and kits that enable modification-dependent activity assays. [Background technology]

[0003] Many diseases or disorders are caused by insufficient levels of a particular polypeptide in the body or by the production of a defective form of this polypeptide. With the advent of genetic engineering and molecular biology, it is now possible to treat such diseases and disorders by replacement polypeptide therapy. For example, administration of a polypeptide produced by recombinant technology can treat a disease or disorder by supplementing low levels of endogenous polypeptide or by replacing a defective polypeptide produced by the body.

[0004] One element essential to the design of an effective recombinant polypeptide therapy is to increase the circulating half-life of the polypeptide once administered to the body. The length of time that a polypeptide remains active in the body can be extended by, for example, modifying the polypeptide using a variety of functional groups that increase the half-life of the polypeptide. Such modifications can protect the polypeptide against proteolysis, increase its stability, enhance or promote its interaction with another molecule, reduce its antigenicity, and / or reduce its rate of removal from the body. Exemplary modifications useful for extending the circulating half-life of administered recombinant polypeptides include, but are not limited to, PEGylation, polysialylation, HESylation, sylation, and citrullination. Summary of the Invention [Problem to be solved by the invention]

[0005] An important aspect of developing a recombinant polypeptide therapy for a disease or disorder is the ability to measure the activity of the polypeptide after modification and / or administration into an individual. However, this ability is often hindered by the presence of endogenous polypeptides that interfere with the specificity and accuracy of the assay used to detect the presence or activity of the recombinant polypeptide. Thus, there is a need to develop methods to assess the presence and / or activity of a recombinant polypeptide. [Means for solving the problem]

[0006] Described herein are methods, systems and kits, referred to as Modification Dependent Activity Assays (MDAA). MDAA utilizes a modification-recognizing capture agent that selectively associates with a polypeptide that contains a modification, even in the presence of an endogenous polypeptide, an unmodified version of the same or a similar polypeptide, or a polypeptide that contains a different pattern or degree of modification. The presence or activity of the captured polypeptide can then be measured as a method of detecting the presence of a polypeptide that contains a modification.

[0007] Aspects of the present specification disclose a method for detecting the presence of a recombinant polypeptide comprising a modification. The method may include incubating a sample comprising the recombinant polypeptide comprising the modification with a capture agent that selectively binds to the modification under conditions that allow selective binding of the capture agent to the modification, thereby forming a polypeptide-agent complex, purifying the polypeptide-agent complex from the sample, and assaying for the presence of the recombinant polypeptide, where detection of the recombinant polypeptide indicates the presence of the recombinant polypeptide comprising the modification. Alternatively or simultaneously, the method may assay for polypeptide activity, where detection of the polypeptide activity indicates the presence of the recombinant polypeptide comprising the modification. The recombinant polypeptide comprising the modification may be a PEGylated, polysialylated, HESylated, or sylated recombinant polypeptide. The capture agent may be an antibody, an aptamer, a synthetic peptide, a binding molecule, and a nucleic acid.

[0008] Another aspect of the present disclosure discloses a method for detecting the presence of a recombinant coagulation factor comprising a modification. The method may include incubating a sample comprising a recombinant coagulation factor comprising a modification with a capture agent that selectively binds to the modification under conditions that allow selective binding of the capture agent to the modification, thereby forming a factor-agent complex, purifying the factor-agent complex from the sample, and assaying for the presence of the recombinant coagulation factor, where detection of the recombinant coagulation factor indicates the presence of the recombinant coagulation factor comprising the modification. Alternatively or simultaneously, the method may assay for coagulation factor activity, where detection of the coagulation factor activity indicates the presence of the recombinant coagulation factor comprising the modification. The coagulation factor comprising a modification may be PEGylated recombinant factor VII, polysialylated recombinant factor VII, HESylated factor VII, silylated recombinant factor VII, PEGylated recombinant factor VIII, polysialylated recombinant factor VIII, HESylated factor VIII, silylated recombinant factor VIII, PEGylated recombinant factor IX, polysialylated recombinant factor IX, HESylated factor IX, and / or silylated recombinant factor IX.

[0009] Another aspect of the present specification discloses a kit that includes one or more components useful for carrying out the methods disclosed herein and instructions for carrying out the methods. The kit may include one or more capture agents disclosed herein, one or more solid supports, and / or one or more reagents necessary to detect the presence and / or activity of a recombinant polypeptide that includes a modification disclosed herein. [Brief description of the drawings]

[0010] [Figure 1] 1 is a graph of the concentration response curve of MDAA for PEGylated recombinant FVIII obtained using a PEGylated FVIII preparation and a human reference plasma preparation. [Diagram 2] FIG. 1 is a graph of MDAA dose-response curves for PEGylated recombinant FVIII in sample matrices with different complexities (buffer vs. plasma). [Diagram 3] Graph of mean calibration curve and percent agreement of back-fitted assay standards demonstrating the precision and accuracy of MDAA for PEGylated recombinant FVIII. Error bars indicate standard deviation of the single mean. [Figure 4] 1 is a graph demonstrating the specificity of MDAA for PEGylated recombinant FVIII using competition with PEG5000. [Diagram 5] 1 is a graph demonstrating the specificity of MDAA for PEGylated recombinant FVIII using competition with an anti-PEG antibody. [Figure 6] 1 is a dilution linearity graph demonstrating the accuracy and precision of MDAA for PEGylated recombinant FVIII. [Figure 7] 1 is a graph of MDAA concentration response curves for polysialylated recombinant FVIII using polysialylated FVIII preparations at FVIII activities ranging from 78 to 2.4 mIU / mL and the deficiency response of human plasma containing unmodified FVIII. [Figure 8]1 is a graph of MDAA concentration response curves for polysialylated recombinant FVIII using polysialylated recombinant FVIII spiked into plasma from different animal species compared to that measured in buffer. [Figure 9] 1 is a graph of concentration-response curves demonstrating the performance and sensitivity of MDAA for polysialylated recombinant FVIII. [Figure 10] 1 is a graph of the average calibration curve demonstrating the accuracy and precision of MDAA for polysialylated recombinant FVIII. [Figure 11] 1 is a graph of the percent agreement between nominal and backfit concentrations for five calibrators for each curve, illustrating that the backfit concentrations were within ±10% of the nominal concentrations over the entire range. [Figure 12] FIG. 1 is a graph of the pharmacokinetic profile obtained with MDAA for polysialylated recombinant FVIII administered to rats containing normal levels of endogenous FVIII. [Figure 13] 1 is a graph of the average calibration curve demonstrating the accuracy and precision of MDAA for polysialylated recombinant FVIII. Error bars indicate the standard deviation of the single mean value. [Figure 14] 1 is a graph demonstrating the specificity of MDAA for polysialylated recombinant FVIII using competition with polysialic acid. [Figure 15] 1 is a graph demonstrating the precision of MDAA for polysialylated recombinant FVIII. The highlighted areas indicate the 2-SD range of the mean obtained. [Figure 16] 1 is a graph of dose-response curves in animal plasma samples demonstrating the accuracy and precision of MDAA for polysialylated recombinant FVIII. [Figure 17] 1 is a graph of MDAA concentration response curves for PEGylated recombinant FIX obtained using PEGylated FIX preparations and human reference plasma preparations. [Figure 18] FIG. 13 is a graph of MDAA for PEGylated recombinant FVIII using a clotting assay format. [Figure 19]FIG. 13 is a graph of MDAA for polysialylated recombinant FVIII using a clotting assay format. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] An important aspect of developing therapeutic compounds for hemophilia or other disorders is the ability to measure the activity of the therapeutic compound after modification and / or administration in its natural therapeutic environment. However, this ability is often hindered by the presence of similar compounds in the therapeutic environment that interfere with the specificity and accuracy of the activity assay test results.

[0012] For example, systems and methods are described herein, referred to as Modification-Dependent Activity Assays (MDAA), that allow for the separation and detection of recombinant polypeptides in the presence of unmodified versions of the same or similar polypeptides, including naturally occurring or endogenous polypeptides produced from the genome of the individual being treated. As a non-limiting example, in the development of hemophilia treatments, it may be necessary to measure the activity of a PEGylated recombinant Factor VIII compound after administration. Before the compound is administered to a human with Factor VIII deficiency, it is often administered to an experimental animal that may or may not be Factor VIII deficient. Without the use of the MDAA of the present disclosure, it is not possible to determine whether the presence or activity measured after administration is due to the administered PEGylated recombinant Factor VIII or due to naturally occurring Factor VIII. The MDAA disclosed herein allows for such discrimination.

[0013] In certain embodiments, the MDAA of the present disclosure comprises a capture agent bound to a solid support.A test sample can be incubated with the solid support, in which the modified compound is selectively bound by the immobilized capture agent.In certain embodiments, all other compounds, including endogenous unmodified compounds, can be removed by washing.An activity assay can be carried out on the captured modified compound.

[0014] The steps of the MDAA disclosed herein may include one or more of the following: binding an antibody to a solid support, incubating a sample on the surface of the solid support, washing the solid support, and performing a chromogenic assay on the solid support. In certain embodiments, the method includes only the steps of incubating, washing, and / or performing a chromogenic assay. The binding step may include binding the antibody to the plate at a neutral to slightly alkaline pH.

[0015] Aspects of the present specification disclose recombinant polypeptides. Recombinant polypeptides are those synthesized using molecular biology techniques. Any recombinantly expressed polypeptide containing modifications can be detected in the methods disclosed herein. The terms "polypeptide", "peptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers, those containing modified residues, and non-naturally occurring amino acid polymers.

[0016] Typically, recombinant polypeptides are expressed from recombinant polynucleotides that are introduced into cells suitable for culturing. In general, recombinant polynucleotides include expression vectors that contain an open reading frame that encodes the polypeptide to be expressed and specialized regulatory coding sequences involved in DNA replication, polypeptide expression, antibiotic resistance, genome integration, as well as other features. For example, prokaryotic expression vectors typically include an origin of replication, appropriate promoter and / or enhancer elements, and also sites required for ribosome binding, polyadenylation, transcription termination, as well as 5' flanking nontranscribed sequences and other nontranscribed genetic elements. Exemplary prokaryotic vectors include pET and pRSET, which use promoters such as the bacteriophage T7 promoter.

[0017] Eukaryotic expression vectors typically include an origin of replication, suitable promoter and / or enhancer elements, and also sites required for ribosome binding, polyadenylation, splicing, transcription termination, as well as 5' flanking nontranscribed sequences and other nontranscribed genetic elements. Exemplary yeast vectors include pAO, pMET, pPIC, pPICZ, and pYES, using promoters such as AOX1, AUG1, GAP, and GAL1. Exemplary insect vectors include pAc5, pBAC, pIB, pMIB, pMT, using promoters such as PH, p10, MT, Ac5, OpIE2, gp64, and poIh. Exemplary mammalian vectors include pBPV, pCMV, pCMVTNT, pDNA, pDisplay, pMSG, pOG44, pQBI25, pRc / RSV, pSECTag, pSECTag2, pSG, pSV2cat, pSVK3, pSVL, pUCIG-MET, pVAX1, pWLneo, and pXT1, using promoters such as beta-casein, beta-lactoglobulin, whey acid promoter, HSV thymidine kinase, early and late simian virus 40 (SV40), LTRs from retrovirus, and mouse metallothionein-1. Selectable markers include ampicillin, chloramphenicol transferase, kanamycin, neomycin, and tetracycline. Suitable expression vectors are known in the art and commercially available.

[0018] Insect cells and cell lines derived from insects include, for example, cells derived from Spodoptera frugiperda, Trichoplusia ni, Drosophila melanogaster, and Manduca sexta. Non-limiting examples of insect cell lines include High-Five, Kc, Schneider's Drosophila strain 2 (S2), SF9, and SF21 cell lines. Mammalian cells and cell lines derived from mammalian cells include, for example, cells derived from mouse, rat, hamster, pig, cow, horse, primate, and human. Non-limiting examples of mammalian cell lines include 1A3, 3T3, 6E6, 10T1 / 2, APRT, BALB / 3T3, BE(2)-C, BHK, BT, C6, C127, CHO, CHP3, COS-1, COS-7, CPAE, ESK-4, FB2, GH1, GH3, HeLa, HEK-293, HepG2, HL-60, IMR-32, L2, LLC-PK1, LM, MCF-7, NB4, NBL-6, NCTC, Neuro2A, NIE-115, NG108-15, NIH3T3, PC12, PK15, SBAC, SH-SY5Y, SK-Hep, SK-N-DZ, SK-N-F1, SK-N-SH, ST, SW-13, and VV-1 cell lines. Cell lines may be obtained from the American Type Culture Collection, the European Collection of Cell Cultures and / or the German Collection of Microorganisms and Cell Cultures.

[0019] A variety of prokaryotic and / or eukaryotic expression systems can be utilized to recombinantly express the proteins disclosed herein. Expression systems can include any of a variety of characteristics, including, but not limited to, inducible expression, non-inducible expression, constitutive expression, tissue-specific expression, cell-specific expression, viral-mediated expression, stably integrated expression, and transient expression. Methods for making and using such expression systems are known in the art.

[0020] The recombinant polypeptides disclosed herein are typically therapeutic polypeptides. Non-limiting examples of therapeutic polypeptides include factor IX (FIX), factor VIII (FVIII), factor VIIa (FVIIa), von Willebrand factor (VWF), factor V (FV), factor X (FX), factor XI (FXI), factor XII (FXII), thrombin (FII), protein C, protein S, tPA, PAI-1, tissue factor (TF), ADAMTS13 protease, IL-1 alpha, IL-1 beta, IL-3, IL-4, IL-5, IL-6, IL-11, colony-stimulating factor-1 (CSF- 1), M-CSF, SCF, GM-CSF, granulocyte colony stimulating factor (G-CSF), EPO, interferon-α (IFN-α), consensus interferon, IFN-β, IFN-γ, IFN-ω, IL-7, IL-8, IL-9, IL-10, IL-12 , IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-31, IL-32 alpha, IL-33, thrombopoietin (TPO), Ang-1, Ang-2 , Ang-4, Ang-Y, angiopoietin-like polypeptide 1 (ANGPTL1), angiopoietin-like polypeptide 2 (ANGPTL2), angiopoietin-like polypeptide 3 (ANGPTL3), angiopoietin-like polypeptide 4 (ANGPTL4), angiopoietin-like polypeptide 5 (ANGPTL5), angiopoietin-like polypeptide 6 (ANGPTL6), angiopoietin-like polypeptide 7 (ANGPTL7), vitronectin, vascular endothelial growth factor (VEGF), angiogenin, activin A, activin B, Activin C, bone morphogenetic protein-1, bone morphogenetic protein-2, bone morphogenetic protein-3, bone morphogenetic protein-4, bone morphogenetic protein-5, bone morphogenetic protein-6, bone morphogenetic protein-7, bone morphogenetic protein-8, bone morphogenetic protein-9, bone morphogenetic protein-10, bone morphogenetic protein-11, bone morphogenetic protein-12, bone morphogenetic protein-13, bone morphogenetic protein-14, bone morphogenetic protein-15, bone morphogenetic protein receptor IA, bone morphogenetic protein receptor IB,Bone morphogenetic protein receptor II, brain-derived neurotrophic factor, cardiotrophin-1, ciliary neurotrophic factor, ciliary neurotrophic factor receptor, crypto, cryptic, cytokine-induced neutrophil chemotactic factor 1, cytokine-induced neutrophil chemotactic factor 2α, cytokine-induced neutrophil chemotactic factor 2β, β-endothelial growth factor, endothelin 1, epidermal growth factor, epigen, epiregulin, epithelial-derived neutrophil inducer, fibroblast growth factor 4, fibroblast growth factor 5, fibroblast growth factor 6, fibroblast growth factor 7 , fibroblast growth factor 8, fibroblast growth factor 8b, fibroblast growth factor 8c, fibroblast growth factor 9, fibroblast growth factor 10, fibroblast growth factor 11, fibroblast growth factor 12, fibroblast growth factor 13, fibroblast growth factor 16, fibroblast growth factor 17, fibroblast growth factor 19, fibroblast growth factor 20, fibroblast growth factor 21, acidic fibroblast growth factor, basic fibroblast growth factor, glial cell line-derived neurotrophic factor receptor alpha 1, glial cell line-derived neurotrophic factor receptor alpha 2, growth-associated protein, growth-associated protein alpha, growth-associated protein β, growth-related protein γ, heparin-binding epidermal growth factor, hepatocyte growth factor, hepatocyte growth factor receptor, hepatoma-derived growth factor, insulin-like growth factor I, insulin-like growth factor receptor, insulin-like growth factor II, insulin-like growth factor binding protein, keratinocyte growth factor, leukemia inhibitory factor, leukemia inhibitory factor receptor α, nerve growth factor, nerve growth factor receptor, neuropoietin, neurotrophin-3, neurotrophin-4, oncostatin M (OSM), placental growth factor, placental growth factor 2, platelet-derived endothelial cell growth factor, platelet-derived growth factor, platelet derived growth factor A chain, platelet-derived growth factor AA, platelet-derived growth factor AB, platelet-derived growth factor B chain, platelet-derived growth factor BB, platelet-derived growth factor receptor alpha, platelet-derived growth factor receptor beta, pre-B cell growth-stimulating factor, stem cell factor (SCF), stem cell factor receptor, TNF, TNF0, TNF1, TNF2, transforming growth factor alpha, transforming growth factor beta, transforming growth factor beta 1, transforming growth factor beta 1.2, transforming growth factor beta 2, transforming growth factor beta 3, transforming growth factor beta 5, latent transforming growth factor beta 1, transforming growth factor beta binding protein I,Transforming growth factor beta binding protein II, transforming growth factor beta binding protein III, thymic stromal lymphopoietin (TSLP), tumor necrosis factor receptor type I, tumor necrosis factor receptor type II, urokinase-type plasminogen activator receptor, phospholipase activating protein (PUP), insulin, lectin, ricin, prolactin, chorionic gonadotropin, follicle stimulating hormone, thyroid stimulating hormone, tissue plasminogen activator, IgG, IgE, IgM, IgA, and IgD, alpha-galactosidase, beta-galactosidase, DNAse, fetuin, luteinizing hormone, estrogen, insulin, albumin, lipoprotein, fetoprotein, transferrin, thrombopoietin, urokinase, integrin, thrombin, leptin, Humira (adalimumab), Prolia (denosumab), Enbrel (etanercept), or a biologically active fragment, derivative, or variant thereof. Other therapeutic polypeptides are described in Table 1 of Siekmann et al., Nucleophilic Catalysts for Oxime Linkage, U.S. Patent Application Publication No. 2012 / 0035344, which is incorporated by reference in its entirety.

[0021] Recombinant polypeptides disclosed herein include, but are not limited to, growth factors, cytokines, immunomodulators, hormones, antibodies, enzymes, enzyme inhibitors, proteases, protease inhibitors, esterases, transferases, oxidoreductases, hydrolases, asparaginases, adenosine deaminases, neurotoxins, liver proteins, pancreatic proteins, muscle proteins, brain proteins, lung proteins, and blood proteins.

[0022] In aspects of this embodiment, the esterase may include, but is not limited to, butyrylcholinesterase or acetylcholinesterase.

[0023] In aspects of this embodiment, cytokines may include, but are not limited to, chemokines, lymphokines, tumor necrosis factors, hematopoietic factor-like granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor.

[0024] In aspects of this embodiment, immunomodulators may include, but are not limited to, interleukins and interferons.

[0025] In aspects of this embodiment, the blood protein may be an erythropoiesis stimulating agent, including but not limited to erythropoietin, erythropoietin, erthropoietin, and darbepoietin.

[0026] In aspects of this embodiment, blood proteins include, but are not limited to, ADAMTS-13, alpha 1-antiplasmin, alpha 2-antiplasmin, antithrombin, antithrombin III, cancer procoagulant, erythropoietin, factor II, factor IIa, factor V, factor Va, factor VI, factor VIa, factor VII, factor VIIa, factor VIII, factor VIIIa, factor IX, factor IXa, factor X, factor Xa, factor XI, factor XIa, factor XII, factor XIIa, factor XIII, factor XIIIa, fibronectin, fibrinogen (factor I), heparin coagulant, factor I protein, factor II protein, factor II protein, factor V ... These may include cytochrome P450, cytochrome P450, cytochrome P450+ ...

[0027] In an aspect of this embodiment, the blood protein may be a blood clotting protein, including both its inactivated and active forms. Blood clotting factors refer to factors of the blood clotting pathway, including components in the intrinsic, extrinsic, and common clotting pathways. The term encompasses such factors, whether they are present in the sample as intrinsic components (i.e., inherent in the blood sample) or whether they are added as exogenous factors. Phospholipid(s) may also be included as clotting factors when added to a method that utilizes either the intrinsic, extrinsic, or common pathway to activate clotting. In an aspect of this embodiment, the blood protein may be a blood clotting factor, including, but not limited to, Factor II, Factor VII, Factor VIII, Factor IX, and Factor X.

[0028] In aspects of this embodiment, the blood protein may be a protease inhibitor, including, but not limited to, alpha 1-antitrypsin, alpha 1-antichymotrypsin, C1-inhibitor, and alpha 2-antiplasmin, antithrombin.

[0029] In an embodiment of the invention, the blood protein may be a protease, including, but not limited to, trypsin, chymotrypsin, elastase, pepsin, and ADAMTS13.

[0030] Aspects of the present specification disclose modifications. The modifications disclosed herein relate to the recombinant polypeptides disclosed herein. Any modification having a binding site or moiety to which a capture agent can selectively bind can be used in the methods disclosed herein. Thus, any modification for which a naturally occurring capture agent exists or for which a capture agent can be prepared is useful in the methods disclosed herein. Modifications disclosed herein include those that occur during or after expression of the recombinant polypeptides disclosed herein.

[0031] In one embodiment, the modification may be a post-translational modification. A post-translational modification is a chemical modification of a polypeptide, typically by attaching a biochemical functional group to an amino acid of the polypeptide. The recombinant polypeptides disclosed herein may be modified by linking the polypeptide to any of these biochemical functional groups, depending on the particular modification of the polypeptide to be captured, as will be understood by those skilled in the art.

[0032] Examples of modifications include, but are not limited to, acetate, phosphate, lipid, or carbohydrate groups, myristate, palmitate, isoprenoid groups such as farnesol and geranylgeraniol, glycosylphosphatidylinositol (GPI) groups, lipoate, flavin, heme C, 4'-phosphopantetheinyl, retinylidene, diphthamide, ethanolamine phosphoglycerol, hypusine, acetyl, formyl, alkyl, methyl, amide, amino acid, butyl, carboxyl, glycosyl, polysialic acid (PSA) groups, hydroxyl, malonyl, iodine, phosphate, adenylyl, succinyl, sulfate, selenium, carbohydrate groups, starch groups, hydroxyl-ethyl starch (HES) groups, polysaccharide groups, sugar groups, polyethylene Examples of the polyoxyethylene (PEG) group, ubiquitin group, pullulane group, chitosan group, hyaluronic acid group, chondroitin sulfate group, dermatan sulfate group, dextran group, carboxymethyl-dextran group, polyalkylene oxide (PAO) group, polyalkylene glycol (PAG) group, polypropylene glycol (PPG) group, polyoxazoline group, polyacryloylmorpholine group, polyvinyl alcohol (PVA) group, polycarboxylate group, polyvinylpyrrolidone (PVP) group, polyphosphazene group, polyoxazoline group, polyethylene-co-maleic anhydride group, polystyrene-co-maleic anhydride group, poly(1-hydroxymethylethylene hydroxymethylformal) (PHF) group, and 2-methacryloyloxy-2'-ethyltrimethylammonium-phosphate (MPC) group.

[0033] Known processes for attaching biochemical functional groups to amino acids of a polypeptide include, but are not limited to, myristoylation, palmitoylation, isoprenylation (prenylation), glypiation, lipoylation, flavinylation, phosphopantetheinylation, retinylidenylation, diphthamidylation, ethanolamine phosphoglycerylation, and the like. phosphoglycerylation, hypusinylation, acylation, acetylation, formylation, alkylation, amidation, arginylation, polyglutamylation, polyglycylation, butyrylation, gamma-carboxylation, glycosylation, polysialylation, malonylation, hydroxylation, iodination, nucleosylation, oxidation, phosphoroesterification, phosphoramidation, phosphorylation, adenylylation, propionylation , pyroglutamylation, S-glutathionylation, S-nitrosylation, succinylation, sulfation, selenoylation, glycosylation, biotinylation, acylation, PEGylation, HESylation, silylation (starchylation), citrullination, deamidation, eliminylation, carbamylation, deimination, pupylation, NEDDylation, ubiquitination, SUMOylation, and ISGylation.

[0034] Aspects of the present disclosure include, in part, a sample comprising a recombinant polypeptide disclosed herein. A sample may be any material to be tested for the presence or activity of a recombinant polypeptide disclosed herein. A variety of samples may be assayed according to the methods disclosed herein, including, but not limited to, purified, partially purified, or unpurified recombinant polypeptides disclosed herein, formulated recombinant polypeptide products, such as crude, fractionated or partially purified, or purified cell lysates from bacterial, yeast, insect, or mammalian sources, and cell, tissue, or organ samples. A sample may be from any subject, including, but not limited to, insects or mammals, such as humans, birds, pigs, horses, cows, mice, cats, rats, dogs, or sheep.

[0035] In one aspect of this embodiment, the sample may be a biological sample that contains or may contain a recombinant polypeptide disclosed herein. Biological samples may include any cell, tissue, organ sample obtained directly from an individual. Biological samples may also be any sample of bodily fluid obtained directly from an individual, including but not limited to blood, urine, sputum, semen, feces, saliva, bile, cerebrospinal fluid, nasal swab, urogenital swab, nasal aspirate, spinal fluid, etc. Biological samples may also include any preparation derived from a sample obtained directly from an individual, including but not limited to the plasma fraction of a blood sample, the serum fraction of a blood sample, or an eluate from a purification process. A blood sample refers to any sample obtained or derived from blood, such as a whole blood sample, a plasma sample, or a serum sample.

[0036] The sample may be treated in a manner that improves the detectability of the recombinant polypeptide disclosed herein or its activity in the sample. Such treatment may, for example, reduce the viscosity of the sample or purify the component fractions of the sample. Treatment methods may include dissolution, dilution, purification, extraction, filtration, distillation, separation, concentration, inactivation of interfering components, and addition of reagents. In addition, solid materials suspected of containing the recombinant polypeptide disclosed herein can be used as test samples once modified to form a liquid medium or release the recombinant polypeptide. Selection and pretreatment of biological samples prior to testing are well known in the art and need not be described further.

[0037] In processes involving extraction, the extraction buffer may, in certain embodiments, comprise about 0.75 to about 1.125M salt in the buffer, although this is a non-limiting range and other molar concentrations, both less than 0.75M and / or greater than 1.25M, may also be used. In one embodiment, the salt in the buffer is about 0.75M, 1M, 1.1M or 1.125M. In further embodiments, a zwitterionic agent (e.g., Zwittergent 3 / 12) is provided to enhance extraction of one or more modified components. For example, a zwitterionic agent is provided in the extraction buffer at about 0.1% to about 1.5%. In still further embodiments, the zwittergent is at a concentration of about 0.1%, 0.15%, 0.175%, 0.2%, 0.225%, 0.25%, 0.275%, 0.3%, 0.325%, 0.350%, 0.375%, 0.4%, 0.425%, 0.450%, 0.475%, 0.5%, 0.525%, 0.550%, 0.575%, 0.6%, 0.7%, 0.75%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2.0%. Examples of zwitterionic agents include Zwittergent 3 / 12; many amino acids at physiological pH used as buffers in Good's buffers: amino-sulfonic acid based MES, MOPS, HEPES, PIPES or CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate); amino-carboxylic acid (amino acid) based glycine, its derivatives bicine and tricine, and alanine; CHAPSO (3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate); CAPSO (3-cyclohexylamino)-2-hydroxy-1-propanesulfonic acid); natural products such as the alkaloids psilocybin and lysergic acid; betaine; quinonoid zwitterions; drugs such as fexofenadine (Allegra) and cephaloridine;2-(N-morpholino)ethanesulfonic acid, (3-[N-morpholino])propanesulfonic acid, 2-[(2-amino-2-oxoethyl)amino]-ethanesulfonic acid, piperazine-N,N'-bis(2-ethanesulfonic acid), 3-(N-morpholino)-2-hydroxypropanesulfonic acid, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 3-(N-morpholino)propanesulfonic acid, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid), N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid, 3-[N-tris(hydroxymethyl)methylamino)-2-hydro 2-(N-cyclohexylamino)ethanesulfonic acid, 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid, 2-amino-2-methyl-1-propanol, 3-(cyclohexylamino)-1-propanesulfonic acid, or mixtures thereof. The zwitterionic agent and / or detergent selected should not include the specific entities (i.e., PEG, HES, etc.) used to modify the compound being measured in a particular assay;

[0038] Aspects of the present specification disclose capture agents. A capture agent or modification recognition capture agent refers to any molecule that can selectively or substantially selectively (i.e., have limited cross-reactivity) bind to a moiety present in a modification disclosed herein or otherwise associate with a modification disclosed herein. As used herein, the term "selective" refers to having a unique effect or affecting or reacting in only one way or with only one thing. As used herein, the term "selectively binds" when referring to a capture agent refers to differential binding of the capture agent to a designated target epitope such that the antibody does not substantially cross-react with non-target epitopes. Any capture agent that can selectively bind to a modification present in a recombinant polypeptide disclosed herein may be used in the methods disclosed herein. Capture agents generally have a single specificity, although capture agents with multiple specificities for two or more recombinant polypeptides disclosed herein may be used. Non-limiting examples of capture agents include antibodies, aptamers, synthetic peptides, binding molecules, and nucleic acids.

[0039] The selective binding of a capture agent includes binding characteristics such as binding affinity, binding specificity, and binding avidity. Binding affinity refers to the length of time that the capture agent is present at its binding site or moiety and can be considered as the strength with which the capture agent binds to its binding site or moiety. Binding affinity can be expressed as the equilibrium dissociation constant (KD) of the capture agent, which is defined as the ratio Kd / Ka at equilibrium. Here, Ka is the association rate constant of the capture agent and kd is the dissociation rate constant of the capture agent. Binding affinity is determined by both association and dissociation, and high affinity cannot be ensured by either high association or low dissociation alone. The association rate constant (Ka), or on-rate constant (Kon), measures the number of binding events per unit time, or the tendency of the capture agent and its binding site or moiety to reversibly associate into its agent-moiety complex. The association rate constant is expressed as M -1 s -1It is expressed symbolically as: [CA] x [BS] x Kon. The larger the association rate constant, the more rapidly the capture agent binds to its binding site or moiety, or the higher the binding affinity between the capture agent and its binding site or moiety. The dissociation rate constant (Kd), or off-rate constant (Koff), measures the number of dissociation events per unit time, the tendency of an agent-moiety complex to reversibly separate (dissociate) into its component molecules, i.e., the capture agent and its binding site or moiety. The dissociation rate constant is s -1 The equilibrium dissociation constant is expressed as M and is symbolized as follows: [CA+BS]×Koff. The smaller the dissociation rate constant, the stronger the capture agent binds to its binding site or moiety, or the higher the binding affinity between the capture agent and its binding site or moiety. The equilibrium dissociation constant (KD) measures the rate at which the rate at which new agent-moiety complexes are formed equals the rate at which agent-moiety complexes dissociate at equilibrium. The equilibrium dissociation constant is expressed as M and is defined as Koff / Kon=[CA]×[BS] / [CA+BS], where [CA] is the molar concentration of the capture agent, [BS] is the molar concentration of the binding site or moiety, and [CA+BS] is the molar concentration of the agent-moiety complex, all concentrations being those of such components when the system is in equilibrium. The smaller the equilibrium dissociation constant, the stronger the capture agent binds to its binding site or moiety, or the higher the binding affinity between the capture agent and its binding site or moiety.

[0040] In one embodiment, the binding affinity of a capture agent disclosed herein is, for example, 1×10 5 M -1 s -1 Less than 1×10 6 M -1 s -1 Less than 1×10 7 M -1 s -1 Less than or 1×10 8 M -1 s -1 In another embodiment, the binding affinity of the capture agents disclosed herein may be, for example, 1×10 5 M -1s -1 Super, 1×10 6 M -1 s -1 Super, 1×10 7 M -1 s -1 Over or 1×10 8 M -1 s -1 In other embodiments, the binding affinity of the capture agents disclosed herein may be greater than 1×10 5 M -1 s -1 ~1×10 8 M -1 s -1、 1×10 6 M -1 s -1 ~1×10 8 M -1 s -1 , 1×10 5 M -1 s -1 ~1×10 7 M -1 s -1 , or 1 × 10 6 M -1 s -1 ~1×10 7 M -1 s -1 may have an association measure constant of

[0041] In another embodiment, the binding affinity of the capture agents disclosed herein is greater than or equal to 1×10 -3 s -1 Less than 1×10 -4 s -1 Less than or 1×10 -5 s -1 In another aspect of this embodiment, the binding affinity of the capture agents disclosed herein may be, for example, 1.0×10 -4 s -1 Less than 2.0×10 -4 s -1 Less than 3.0×10 -4 s -1 Less than 4.0×10 -4 s -1 Less than 5.0×10 -4 s -1 Less than 6.0×10-4 s -1 Less than 7.0×10 -4 s -1 Less than 8.0×10 -4 s -1 Less than or equal to 9.0 x 10 -4 s -1 In another embodiment, the binding affinity of the capture agents disclosed herein may be, for example, 1×10 -3 s -1 Super, 1×10 -4 s -1 Over or 1×10 -5 s -1 In another aspect of this embodiment, the binding affinity of the capture agents disclosed herein may be greater than, for example, 1.0×10 -4 s -1 Super, 2.0×10 -4 s -1 Super, 3.0×10 -4 s -1 Super, 4.0×10 -4 s -1 Super, 5.0×10 -4 s -1 Super, 6.0×10 -4 s -1 Super, 7.0×10 -4 s -1 Super, 8.0×10 -4 s -1 Over 9.0 x 10 -4 s -1 The compound may have a dissociation rate constant greater than 0.05.

[0042] In another embodiment, the binding affinity of the capture agents disclosed herein may have an equilibrium dissociation constant of less than 0.500 nM. In aspects of this embodiment, the binding affinity of the capture agents disclosed herein may have an equilibrium dissociation constant of, for example, less than 0.500 nM, less than 0.450 nM, less than 0.400 nM, less than 0.350 nM, less than 0.300 nM, less than 0.250 nM, less than 0.200 nM, less than 0.150 nM, less than 0.100 nM, or less than 0.050 nM. In another embodiment, the binding affinity of the capture agents disclosed herein may have an equilibrium dissociation constant of more than 0.500 nM. In aspects of this embodiment, the binding affinity of a capture agent disclosed herein may have an equilibrium dissociation constant of, for example, greater than 0.500 nM, greater than 0.450 nM, greater than 0.400 nM, greater than 0.350 nM, greater than 0.300 nM, greater than 0.250 nM, greater than 0.200 nM, greater than 0.150 nM, greater than 0.100 nM, or greater than 0.050 nM.

[0043] In yet another embodiment, the binding affinity of the capture agents disclosed herein is, for example, 1×10 0 M -1 s -1 Less than 1×10 1 M -1 s -1 Less than 1×10 2 M -1 s -1 Less than 1×10 3 M -1 s -1 Less than or 1×10 4 M -1 s -1 In another embodiment, the binding affinity of the capture agents disclosed herein may be, for example, less than 1×10 0 M -1 s -1 Below, 1×10 1 M -1 s -1 Below, 1×10 2 M -1 s -1 Below, 1×10 3 M -1 s-1 or less, or 1×10 4 M -1 s -1 The following association rate constants for polypeptides without the modifications or with different patterns or degrees of modifications may be present:

[0044] Binding specificity is the ability of a capture agent to distinguish between molecules that contain the binding site or moiety and those that do not. One way to measure binding specificity is to compare the K on association rate of the capture agent for molecules that contain the binding site or moiety to the K on association rate of the capture agent for molecules that do not contain the binding site or moiety. For example, the association rate constant (Ka) of a capture agent for a recombinant polypeptide that contains a modification is compared to a recombinant polypeptide that does not have such a modification. In aspects of this embodiment, a capture agent that selectively binds to a recombinant polypeptide that contains a modification will selectively bind to a recombinant polypeptide that does not have such a modification, for example, at least 1×10 0 M -1 s -1 Less than 1×10 1 M -1 s -1 Less than 1×10 2 M -1 s -1 Less than 1×10 3 M -1 s -1 Less than or 1 x 10 4 M -1 s -1 In another aspect of this embodiment, a capture agent that selectively binds a recombinant polypeptide that includes a modification has an association rate constant (Ka) of, for example, 1×10 0 M -1 s -1 Below, 1×10 1 M -1 s -1 Below, 1×10 2 M -1 s -1 Below, 1×10 3 M-1 s -1 or less, or 1×10 4 M -1 s -1 In yet another aspect of this embodiment, a capture agent that selectively binds to a recombinant polypeptide that includes a modification has an association rate constant (Ka) for a recombinant polypeptide that includes a modification that is less than or equal to 1×10 compared to the association rate constant (Ka) of the capture agent for a recombinant polypeptide that does not have such a modification and / or the association rate constant (Ka) of the capture agent for a recombinant polypeptide that has a different pattern or degree of modification. 0 M -1 s -1 Large, 1×10 1 M -1 s -1 Large, 1×10 2 M -1 s -1 Large, 1×10 3 M -1 s -1 Large or 1×10 4 M -1 s -1 big.

[0045] In further aspects of this embodiment, a capture agent that selectively binds a recombinant polypeptide comprising a modification has an association rate constant (Ka) that is, for example, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, or at least 9-fold greater than a recombinant polypeptide that does not have such a modification. In further aspects of this embodiment, a capture agent that selectively binds a recombinant polypeptide comprising a modification has an association rate constant (Ka) that is, for example, at least 10-fold greater, at least 100-fold greater, at least 1,000-fold greater, or at least 10,000-fold greater than a recombinant polypeptide that does not have such a modification. In yet other aspects of this embodiment, a capture agent that selectively binds a recombinant polypeptide comprising a modification has an association rate constant (Ka) that is, for example, up to 1-fold greater, up to 2-fold greater, up to 3-fold greater, up to 4-fold greater, up to 5-fold greater, up to 6-fold greater, up to 7-fold greater, up to 8-fold greater, or up to 9-fold greater than a recombinant polypeptide that does not have such a modification. In yet other aspects of this embodiment, a capture agent that selectively binds to a recombinant polypeptide that contains a modification has an association rate constant (Ka) that is, for example, up to 10-fold greater, up to 100-fold greater, up to 1,000-fold greater, or up to 10,000-fold greater, compared to a recombinant polypeptide that does not have such a modification.

[0046] The binding specificity of a capture agent may also be characterized as the ratio of binding specificity of a recombinant polypeptide that includes a modification to a recombinant polypeptide that does not have such a modification. In aspects of this embodiment, the binding specificity ratio of a capture agent that includes a recombinant polypeptide that does not have such a modification is, for example, at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 64:1, at least 7:1, at least 8:1, at least 9:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 35:1, or at least 40:1. In yet other aspects of this embodiment, the binding specificity ratio of the capture agent for a recombinant polypeptide that includes a modification to a recombinant polypeptide that does not have such a modification is, for example, at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, at least 8:1, at least 9:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 35:1, or at least 40:1. In yet other aspects of this embodiment, the binding specificity ratio of the capture agent for a recombinant polypeptide that includes a modification to a recombinant polypeptide that does not have such a modification is, for example, at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 64:1, at least 7:1, at least 8:1, at least 9:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 35:1, or at least 40:1.

[0047] Binding affinity, also known as functional group affinity, refers to the sum of functional group binding strengths between a multivalent capture agent and its binding site or moiety. A capture agent may have more than one binding site, and many modifications contain more than one binding site or moiety. Although the binding affinity of a capture agent depends on the binding affinity of the individual capture agent binding sites, binding affinity is greater than binding affinity because all agent-moiety interactions must be broken simultaneously for the capture agent to completely dissociate. It is envisioned that a capture agent can selectively bind to any and all binding sites or moieties for that capture agent.

[0048] Typically, the capture agent can distinguish between a recombinant polypeptide containing the modifications disclosed herein and the same polypeptide but without the modifications or with a different pattern or degree of the same modifications. Unmodified polypeptide and polypeptide but without the modifications refer to a polypeptide that does not contain the modifications present in the recombinant peptide disclosed herein. Unmodified polypeptides may be present in a sample, but will not selectively bind to the capture agents disclosed herein, since the polypeptide lacks the modifications required for capture. One non-limiting example of an unmodified polypeptide is a naturally occurring or endogenous polypeptide expressed from the genome of an individual from whom the sample was obtained directly or from whom the sample was derived. Another non-limiting example of an unmodified polypeptide is a recombinant polypeptide expressed from a prokaryotic expression system.

[0049] A polypeptide comprising a different pattern or degree of modification refers to a polypeptide having a modification present in a recombinant polypeptide disclosed herein, but present in a pattern or degree that allows the methods disclosed herein to distinguish between the two polypeptides. For example, a polypeptide having a different pattern or degree of modification may be present in a sample, but will not selectively bind to a capture agent disclosed herein, because the polypeptide lacks the pattern or degree of modification required for capture. One non-limiting example of a polypeptide having a different pattern or degree of modification is a naturally occurring or endogenous polypeptide expressed from the genome of an individual from whom the sample was obtained directly or from whom the sample was derived. Another non-limiting example of a polypeptide having a different pattern or degree of modification is a recombinant polypeptide expressed from a prokaryotic expression system. Yet another non-limiting example of a polypeptide having a different pattern or degree of modification is a recombinant polypeptide expressed from cells of a cell culture system different from the cells of the cell culture system used to express the recombinant polypeptide disclosed herein. Yet another non-limiting example of a polypeptide having a different pattern or degree of modification is a recombinant polypeptide expressed using a production process different from the production process used to express the recombinant polypeptides disclosed herein.

[0050] In one embodiment, the capture agent is an antibody. An antibody refers to a molecule generated by the immune system in response to a particular antigen and specifically binds to that antigen, including both naturally occurring and non-naturally occurring antibodies. The antibody may be a polyclonal antibody, a monoclonal antibody, a dimer, a multimer, a monospecific antibody, a bispecific antibody, such as a disulfide-stabilized Fv fragment, a scFv tandem [(scFv)2 fragment], a multispecific antibody, a multivalent antibody, a humanized antibody, a camelized antibody, a chimeric antibody, a bifunctional antibody, a cell-bound antibody-like Ig receptor, a linear antibody, a diabody, a tribody, a tetrabody, a minibody, or a derivative or analog thereof, and a single chain derivative thereof, so long as the fragment exhibits the desired biological activity. An antibody may be a V H and V L domain, as well as the light chain constant domain (C L ) and a heavy chain constant domain, C H1 , C H2 and C H3 or an immunologically active fragment of a full-length immunoglobulin molecule, such as a Fab fragment, F(ab') 2 The antibody may be an Fc fragment, an Fd fragment, an Fv fragment, a single chain Fv (scFv), etc. The antibody may be from any vertebrate species (e.g., human, goat, horse, donkey, mouse, rat, rabbit, or chicken) and may be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgA, IgD, IgE, IgG, and IgM) or subclass (IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0051] Antibodies useful for practicing the methods disclosed herein are commercially available or may be produced according to methods well known in the art. For example, monoclonal antibodies may be produced by hybridoma technology. Antibody fragments may be produced by proteolysis of intact antibodies or may be produced directly by recombinant host cells. For example, Fab' fragments may be directly recovered from E. coli or chemically coupled to produce F(ab')2 fragments. In another embodiment, F(ab')2 may be produced using the leucine zipper GCN4 to promote assembly of F(ab')2 molecules. According to another approach, Fv, Fab or F(ab')2 fragments may be directly isolated from recombinant host cell culture. Other techniques for producing antibody fragments will be apparent to those skilled in the art.

[0052] Examples of antibodies suitable for the methods disclosed herein include, but are not limited to, anti-acetate antibodies, anti-phosphate antibodies, anti-lipid or anti-carbohydrate antibodies, anti-myristate antibodies, anti-palmitate antibodies, anti-farnesol antibodies, and anti-geranylgeraniol antibodies, anti-glycosylphosphatidylinositol (GPI) antibodies, anti-lipoate antibodies, anti-flavin antibodies, anti-heme C antibodies, anti-4'-phosphopantetheinyl antibodies, anti-retinylidene antibodies, anti-diphthamide antibodies, anti-ene antibodies, and the like. ethanolamine phosphoglycerol antibody, antihypusine antibody, antiacetyl antibody, antiformyl antibody, antialkyl antibody, antimethyl antibody, antiamide antibody, antiamino acid antibody, antibutyl antibody, anticarboxyl antibody, antiglycosyl antibody, antipolysialic acid antibody, antihydroxyl antibody, antimalonyl antibody, antiiodine antibody, antiphosphate antibody, antiadenylyl antibody, antisuccinyl antibody, antisulfate antibody, antiselenium antibody, anticarbohydrate antibody, antipolysaccharide antibody, antistarch (anti-S) antibody, antihydroxyl-ethyl decasaccharide antibody, anti-glucose ... starch (HES) antibody, anti-saccharide antibody, anti-polyethylene glycol (PEG) antibody, anti-ubiquitin antibody, anti-pullulan antibody, anti-chitosan antibody, anti-hyaluronic acid antibody, anti-chondroitin sulfate antibody, anti-dermatan sulfate antibody, anti-dextran antibody, anti-carboxymethyl-dextran antibody, anti-polyalkylene oxide (PAO) antibody, anti-polyalkylene glycol (PAG) antibody, anti-polypropylene glycol (PPG) antibody, anti-polyoxazoline antibody, anti-polyacryloyl mol These include holin antibodies, anti-polyvinyl alcohol (PVA) antibodies, anti-polycarboxylate antibodies, anti-polyvinylpyrrolidone (PVP) antibodies, anti-polyphosphazene antibodies, anti-polyoxazoline antibodies, anti-polyethylene-co-maleic anhydride antibodies, anti-polystyrene-co-maleic anhydride antibodies, anti-poly(1-hydroxymethylethylene hydroxymethylformal) (PHF) antibodies, and anti-2-methacryloyloxy-2'-ethyltrimethylammonium-phosphate (MPC) antibodies.

[0053] The capture agents disclosed herein may be bound to a solid phase as a support for the capture agents. As used herein, the term "solid phase support" is synonymous with "solid phase" and refers to any matrix that can be used to immobilize the capture agents disclosed herein. The solid phase may be constructed using any suitable material that has sufficient surface affinity to bind the capture agent. The solid phase support selected may have physical properties that allow it to be easily separated from soluble or unbound materials, and typically allow unbound materials, such as excess reagents, reaction by-products, or solvents, to be separated or otherwise removed (e.g., by washing, filtration, centrifugation, etc.) from the assay components to which the solid phase support is bound. Non-limiting examples of methods for making and using solid phase supports are described, for example, in Molecular Cloning, A Laboratory Manual, supra, (2001), and Current Protocols in Molecular Biology, supra, (2004), each of which is incorporated herein by reference in its entirety.

[0054] Useful solid supports include natural polymeric carbohydrates and their synthetically modified cross-linked or substituted derivatives, such as agar, agarose, cross-linked alginic acid, substituted and cross-linked guar gum, dextran, diazocellulose, carbohydrates, starches, cellulose esters, especially cellulose esters mixed with nucleic acids and carboxylic acids, and cellulose ethers; nitrogen-containing natural polymers, such as cross-linked or modified gelatin, proteins and derivatives; natural hydrocarbon polymers, such as latex and rubber; synthetic polymers, such as vinyl polymers, polyacrylamides, polymethacrylates, copolymers of the above polycondensates, and terpene, including polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyvinyl acetate and partially hydrolyzed derivatives thereof. Polymers include polymers such as polyesters, polyamides, and other polymers such as polyurethanes or polyepoxides; inorganic materials such as barium sulfate, calcium sulfate, calcium carbonate, alkali and alkaline earth metal silicates, alkaline earth metal and magnesium sulfates or carbonates, including aluminum and magnesium; and aluminum or silicon oxides or aluminum or silicon hydrates, such as clay, alumina, talc, kaolin, zeolite, silica gel, or glass (these materials can be used as filters with the above polymeric materials); and mixtures or copolymers of the above classes, such as graft copolymers obtained by initiating the polymerization of synthetic polymers on existing natural polymers. Nitrocellulose and nylon may also be used. All of these materials may be used in suitable shapes, such as films, sheets, tubes, columns; pins or "dipsticks"; magnetic particles, particles, microparticles, beads, or plates, or they may be coated, bonded, or laminated onto suitable inert carriers, such as paper, glass, plastic films, fibers, etc.

[0055] Alternatively, the solid phase may be composed of microparticles. Suitable microparticles include those made of polystyrene, polymethacrylate, polypropylene, latex, polytetrafluoroethylene, polyacrylonitrile, polycarbonate, or similar materials. Furthermore, the microparticles may be magnetic or paramagnetic to facilitate manipulation of the microparticles in a magnetic field. The microparticles may be suspended in the mixture of soluble reagents and biological sample, or may be retained and immobilized by a support material. In the latter case, the microparticles on or in the support material cannot substantially move to a position elsewhere in the support material. Alternatively, the microparticles may be separated from suspension in the mixture of soluble reagents and biological sample by sedimentation or centrifugation. If the microparticles are magnetic or paramagnetic, they may be separated from suspension in the mixture of soluble reagents and biological sample by a magnetic field.

[0056] If the capture agent is held by hydrophobic forces, the capture agent may be bound to the solid phase by adsorption. Alternatively, the surface of the solid phase may be activated by a chemical process that causes the covalent attachment of the capture agent to the support. If the capture agent is held by a charged polymer, the capture agent may be bound to the solid phase by ion capture.

[0057] After the incubation step disclosed herein, a purification step is performed to concentrate the capture agent complex, such as, for example, recombinant polypeptide-agent complex, coagulation factor-agent complex, factor VII-antibody complex, factor VIII-antibody complex, and factor IX-antibody complex. Typically, complex purification may involve capture of the complex to a more concentrated form, intermediate purification steps to remove impurities, and / or polishing to remove further impurities and protein variants. See, for example, Current Protocols in Protein Science, "Conventional chromatographic Seperations", Ch. 8-9, (John Wiley & Sons Inc., Hoboken, NJ, 1995). Common purification methods include, but are not limited to, affinity chromatography, gel filtration, precipitation, and / or size exclusion chromatography. Processes useful as intermediate or polishing steps include cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, and ceramic hydroxyapatite chromatography, reversed-phase HPLC, gel filtration, precipitation, diafiltration, affinity chromatography, or chromatofocusing.

[0058] Detection of the presence or activity of a recombinant polypeptide disclosed herein can be accomplished by any assay capable of qualitatively or quantitatively measuring a characteristic indicative of the presence or activity associated with the monitored polypeptide, including, but not limited to, in vitro assays, cell-based assays, or in vivo assays. In addition, the assay may be a non-specific polypeptide assay, such as, for example, a UV absorption assay or a chemical-based assay such as a Bradford assay or a Biuret assay, or a specific polypeptide assay, such as, for example, a chromogenic assay, a colorimetirc assay, a chronometric assay, a chemiluminescent assay, an electrochemiluminescent assay, a bioluminescent assay, a fluorescent assay, a resonance energy transfer assay, a plane polarization assay, a flow cytometric assay, an immuno-based assay, or an activity assay such as an enzymatic activity, an inhibitory activity, a coagulation activity, or a polymerization activity. The actual assay used to detect a characteristic of a polypeptide disclosed herein can be determined by one of skill in the art by considering factors including, but not limited to, the polypeptide being assayed, the amount of polypeptide present, the characteristic being assayed, and the preference of the artisan. Detection of the presence or activity of a recombinant polypeptide disclosed herein may be performed in a singleplex or multiplex format.

[0059] Detecting the presence or activity of the polypeptide indicates the presence of the recombinant polypeptide containing the modification.

[0060] Non-limiting examples of immune-based assays include immunoblot analysis such as Western blot and dot blot, immunoprecipitation analysis, enzyme-linked immunosorbent assay (ELISA), and sandwich ELISA. Signal detection can be achieved using autoradiography (AU) with imaging or phosphorimaging, chemiluminescence (CL), electrochemiluminescence (ECL), bioluminescence (BL), fluorescence, resonance energy transfer, plane polarization, colorimetry, or flow cytometry (FC).Details of immune-based detection systems are found, for example, in Michael M. Rauhut, Chemiluminescence, In Kirk-Othmer Concise Encyclopedia of Chemical Technology (Ed. Grayson, 3rd ed., John Wiley and Sons, 1985); A. W. Knight, A Review of Recent Trends in Analytical Applications of Electrogenerated Chemiluminescence, Trends Anal. Chem. 18(1):47-62(1999); K. A. Fahnrich et al., Recent Applications of Electrogenerated Chemiluminescence in Chemical Analysis, Talanta 54(4):531-559(2001); Commonly Used Techniques in Molecular Cloning, pages A8.1-A8-55 (Sambrook & Russell, eds., Molecular Cloning A Laboratory Manual, Vol. 3, 3rd ed., 2001); Detection Systems, pages A9.1-A9-49 (Sambrook & Russell, eds., Molecular Cloning A Laboratory Manual, Vol. 3, 3rd Edition, 2001), Electrogenerated Chemiluminescence, (Ed. Allen J. Bard, Marcel Dekker, Inc., 2004), each of which is incorporated herein by reference in its entirety.

[0061] Chromogenic assays use peptide substrates consisting of specific oligopeptide or polypeptide moieties and chromophores (dye carriers) and are routinely used to determine factors possessing protease activity, for example, for determining coagulation factors in blood and plasma samples. The chromogenic peptide substrate, which is initially colorless, is cleaved depending on the amount and / or activity of the recombinant polypeptide disclosed herein present in the sample, thereby releasing the chromophore. Cleavage changes the optical properties of the product, which differ from those of the uncleaved substrate, and can be measured spectrophotometrically. Non-limiting examples of chromogenic groups that can be attached to the peptide substrate are para-nitroaniline (pNA), 5-amino-2-nitrobenzoic acid (ANBA), 7-amino-4-methoxycoumarin (ANC), quinonylamide (QUA), dimethyl 5-aminoisophthalate (DPA) and their derivatives. Fluorescent substrates include, but are not limited to, Z-Gly-Pro-Arg-AMC [Z=benzyloxycarbonyl, AMC=7-amino-4-methylcoumarin], homovanillic acid, 4-hydroxy-3-methoxyphenylacetic acid, reduced phenoxazine, reduced benzothiazine, Amplex®, resorufin β-D-galactopyranoside, fluorescein diglucuronide (FDG), fluorescein diglucuronide, and structural modifications thereof. variants (U.S. Pat. Nos. 5,208,148, 5,242,805, 5,362,628, 5,576,424, and 5,773,236, which are incorporated by reference), 4-methylumbelliferyl β-D-galactopyranoside, carboxyumbelliferyl β-D-galactopyranoside, and fluorinated coumarin β-D-galactopyranoside (U.S. Pat. No. 5,830,912, which is incorporated by reference).

[0062] A non-limiting activity assay is a chromogenic assay based on the blood coagulation cascade that can be used to detect FVIII activity. In this assay, thrombin-activated factor VIII forms a complex with factor IXa, which subsequently activates factor X. Activated factor X activity can be assessed by hydrolysis of a chromogenic substrate, such as p-nitro-aniline (pNA), which releases a chromogenic group. The initial rate of pNA release, determined by the change in absorbance / min measured at 405 nm with dOD, is proportional to the factor Xa activity, and subsequently the FVIII activity, in the sample. By using excess factors IXa and X, the rate of activation of factor X is proportional only to the amount of factor VIII cleaved by thrombin present in the sample. Alternatively, factor IXa activity may be determined by changing the conditions such that factors VIII and X are in excess, and thus factor IXa is rate limiting. Similarly, factor X activity may be determined by changing the conditions so that factors VIII and IXa are in excess and thus factor X is rate limiting. Thus, factor VIII activity, as well as factors IXa and X, can be detected using chromogenic assays based on the blood coagulation cascade.

[0063] Another non-limiting activity assay is a chromogenic assay based on the blood coagulation cascade that can be used to detect thrombin activity. Such an assay can use the pNA-linked peptide substrate Ala-Gly-Arg-pNA (PEFACHROM® TG, Pentapharm Ltd., Basel, Switzerland) or the AMC-linked peptide substrate Gly-Cly-Arg-AMC (Bachem). Other suitable peptide substrates that are cleaved by thrombin have the general formula Msc-Val-Xaa-R 1 where Msc is methylsulfonyl-ethyloxycarbonyl, Val is the amino acid valine, and Xaa is an amino acid residue which contains a terminal guanidino or ureido group separated from the peptide backbone by at least two carbon atoms, and where R 1 is the chromophore, and the peptide is Msc-Val-Arg-R 1or Msc-Val-Arg-pNA. Other examples of chromogenic peptide substrates with specificity for different proteases can be found, for example, in U.S. Patent No. 4,508,644, which is incorporated by reference herein in its entirety.

[0064] A non-limiting activity assay is a one-stage clotting assay that applies the activated partial thromboplastin time (APTT) that can be used to detect FVIII activity. In this time-measurement assay, a sample containing factor VIII is diluted with CaCl 2 is added to factor VIII-deficient plasma to promote clotting, and the effect of this sample on the APTT clotting time of the plasma can be determined on an MDA-II instrument (BioMerieux, Marcy-I'Etoile), which is a measure of factor VIII activity. The activity of an unknown sample is calculated by comparing the observed factor VIII activity with a standard curve generated from samples of known factor VIII activity. The clotting activity can be measured by a timed assay on a robotic platform, and the timed activity of the modified compound can be compared to the activity of wild-type FVIII used as an internal standard. This blood clotting assay may also be used for any other protein involved in the blood clotting cascade by using plasma defects in the protein to be assayed.

[0065] The methods disclosed herein may be evaluated by several parameters, including, for example, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ), range, specificity, selectivity, linearity, ruggedness, and system suitability. Method precision is a measure of the accuracy of an analytical method, or the closeness of agreement between measured values ​​and values ​​traditionally accepted as true or accepted reference values. Method precision is the degree of agreement between individual test results when a procedure is applied repeatedly to multiple samplings of a homogenous sample. Thus, precision evaluates 1) within assay variability, 2) within day variability (repeatability), and 3) between day variability (intermediate precision), and 4) between experiment variability (reproducibility). Coefficient of variation (CV%) is a quantitative measure of precision expressed relative to the observed or theoretical mean value.

[0066] The methods disclosed herein must be capable of detecting the presence or activity of the recombinant polypeptide disclosed herein over background. The limit of detection (LOD) of the method refers to the concentration of the recombinant polypeptide disclosed herein that produces a signal that is significantly different from a negative control or blank and represents the minimum concentration of the recombinant polypeptide disclosed herein that can be distinguished from the background.

[0067] Thus, in one embodiment, the methods disclosed herein can detect an LOD of a recombinant polypeptide disclosed herein at an amount significantly different from a negative control or blank. In aspects of this embodiment, the methods disclosed herein have an LOD of, for example, 10 ng or less, 9 ng or less, 8 ng or less, 7 ng or less, 6 ng or less, 5 ng or less, 4 ng or less, 3 ng or less, 2 ng or less, or 1 ng or less of a recombinant polypeptide disclosed herein. In yet other aspects of this embodiment, the methods disclosed herein have an LOD of, for example, 900 pg or less, 800 pg or less, 700 pg or less, 600 pg or less, 500 pg or less, 400 pg or less, 300 pg or less, 200 pg or less, or 100 pg or less of a recombinant polypeptide disclosed herein. In further aspects of this embodiment, the methods disclosed herein have an LOD of, for example, 90 pg or less, 80 pg or less, 70 pg or less, 60 pg or less, 50 pg or less, 40 pg or less, 30 pg or less, 20 pg or less, or 10 pg or less of a recombinant polypeptide disclosed herein. In other aspects of this embodiment, the methods disclosed herein have an LOD of, for example, 9 pg or less, 8 pg or less, 7 pg or less, 6 pg or less, 5 pg or less, 4 pg or less, 3 pg or less, 2 pg or less, or 1 pg or less of a recombinant polypeptide disclosed herein. In yet other aspects of this embodiment, the methods disclosed herein have an LOD of, for example, 0.9 pg or less, 0.8 pg or less, 0.7 pg or less, 0.6 pg or less, 0.5 pg or less, 0.4 pg or less, 0.3 pg or less, 0.2 pg or less, or 0.1 pg or less of a recombinant polypeptide disclosed herein.

[0068] In another aspect of this embodiment, the methods disclosed herein have an LOD of, for example, 10 nM or less or less, 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, or 1 nM or less of the recombinant polypeptides disclosed herein. In another aspect of this embodiment, the methods disclosed herein have an LOD of, for example, 900 pM or less, 800 pM or less, 700 pM or less, 600 pM or less, 500 pM or less, 400 pM or less, 300 pM or less, 200 pM or less, or 100 pM or less of the recombinant polypeptides disclosed herein. In other aspects of this embodiment, the methods disclosed herein have, for example, an LOD of 100 pM or less, 90 pM or less, 80 pM or less, 70 pM or less, 60 pM or less, 50 pM or less, 40 pM or less, 30 pM or less, 20 pM or less, or 10 pM or less for the recombinant polypeptides disclosed herein. In yet other aspects of this embodiment, the methods disclosed herein have, for example, an LOD of 10 pM or less, 9 pM or less, 8 pM or less, 7 pM or less, 6 pM or less, 5 pM or less, 4 pM or less, 3 pM or less, 2 pM or less, or 1 pM or less for the BoNT / A for the recombinant polypeptides disclosed herein. In yet other aspects of this embodiment, the methods disclosed herein have an LOD of, for example, 1000 fM or less, 900 fM or less, 800 fM or less, 700 fM or less, 600 fM or less, 500 fM or less, 400 fM or less, 300 fM or less, 200 fM or less, or 100 fM or less for the recombinant polypeptides disclosed herein. In yet other aspects of this embodiment, the methods disclosed herein have an LOD of, for example, 100 fM or less, 90 fM or less, 80 fM or less, 70 fM or less, 60 fM or less, 50 fM or less, 40 fM or less, 30 fM or less, 20 fM or less, or 10 fM or less for the recombinant polypeptides disclosed herein. In yet other aspects of this embodiment, the methods disclosed herein provide, for example, a recombinant polypeptide disclosed herein having an LOD of 10 fM or less, 9 fM or less, 8 fM or less, 7 fM or less, 6 fM or less, 5 fM or less, 4 fM or less, 3 fM or less, 2 fM or less, or 1 fM or less.

[0069] Limit of quantification (LOQ) is the minimum and maximum concentration of a recombinant polypeptide disclosed herein in a sample or specimen that can be measured with an acceptable level of accuracy and precision. Lower limit of quantification refers to the lowest dose that a detection method can consistently measure from background. Upper limit of quantification is the highest dose that a detection method can consistently measure before signal saturation occurs. The linear range of the method is the area between the lower limit of quantification and the upper limit of quantification. The linear range is calculated by subtracting the lower limit of quantification from the upper limit of quantification. As used herein, the term "signal to noise ratio for the lower asymptote" refers to the signal detected by a method at the lower limit of detection divided by the background signal. As used herein, the term "signal to noise ratio for the upper asymptote" refers to the signal detected by a method at the upper limit of detection divided by the background signal.

[0070] Thus, in one embodiment, the method disclosed herein can detect an LOQ of a recombinant polypeptide disclosed herein at an amount significantly different from a negative control or blank. In aspects of this embodiment, the method disclosed herein has an LOQ of, for example, 10 ng or less, 9 ng or less, 8 ng or less, 7 ng or less, 6 ng or less, 5 ng or less, 4 ng or less, 3 ng or less, 2 ng or less, or 1 ng or less of a recombinant polypeptide disclosed herein. In yet other aspects of this embodiment, the method disclosed herein has an LOQ of, for example, 900 pg or less, 800 pg or less, 700 pg or less, 600 pg or less, 500 pg or less, 400 pg or less, 300 pg or less, 200 pg or less, or 100 pg or less of a recombinant polypeptide disclosed herein. In further aspects of this embodiment, the methods disclosed herein have an LOQ of, for example, 90 pg or less, 80 pg or less, 70 pg or less, 60 pg or less, 50 pg or less, 40 pg or less, 30 pg or less, 20 pg or less, or 10 pg or less of a recombinant polypeptide disclosed herein. In other aspects of this embodiment, the methods disclosed herein have an LOQ of, for example, 9 pg or less, 8 pg or less, 7 pg or less, 6 pg or less, 5 pg or less, 4 pg or less, 3 pg or less, 2 pg or less, or 1 pg or less of a recombinant polypeptide disclosed herein. In yet other aspects of this embodiment, the methods disclosed herein have an LOQ of, for example, 0.9 pg or less, 0.8 pg or less, 0.7 pg or less, 0.6 pg or less, 0.5 pg or less, 0.4 pg or less, 0.3 pg or less, 0.2 pg or less, or 0.1 pg or less of a recombinant polypeptide disclosed herein.

[0071] In another aspect of this embodiment, the methods disclosed herein have an LOQ of, for example, 10 nM or less, 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, or 1 nM or less for a recombinant polypeptide disclosed herein. In another aspect of this embodiment, the methods disclosed herein have an LOQ of, for example, 900 pM or less, 800 pM or less, 700 pM or less, 600 pM or less, 500 pM or less, 400 pM or less, 300 pM or less, 200 pM or less, or 100 pM or less for a recombinant polypeptide disclosed herein. In other aspects of this embodiment, the methods disclosed herein have an LOQ of, for example, 100 pM or less, 90 pM or less, 80 pM or less, 70 pM or less, 60 pM or less, 50 pM or less, 40 pM or less, 30 pM or less, 20 pM or less, or 10 pM or less for a recombinant polypeptide disclosed herein. In yet other aspects of this embodiment, the methods disclosed herein have an LOQ of, for example, 10 pM or less for a recombinant polypeptide disclosed herein, 9 pM or less, 8 pM or less, 7 pM or less, 6 pM or less, 5 pM or less, 4 pM or less, 3 pM or less, 2 pM or less, or 1 pM or less for a recombinant polypeptide disclosed herein. In yet other aspects of this embodiment, the methods disclosed herein have an LOQ of, for example, 1000 fM or less, 900 fM or less, 800 fM or less, 700 fM or less, 600 fM or less, 500 fM or less, 400 fM or less, 300 fM or less, 200 fM or less, or 100 fM or less for a recombinant polypeptide disclosed herein. In yet other aspects of this embodiment, the methods disclosed herein have an LOQ of, for example, 100 fM or less, 90 fM or less, 80 fM or less, 70 fM or less, 60 fM or less, 50 fM or less, 40 fM or less, 30 fM or less, 20 fM or less, or 10 fM or less for a recombinant polypeptide disclosed herein. In yet other aspects of this embodiment, the methods disclosed herein have an LOQ of, for example, 10 fM or less, 9 fM or less, 8 fM or less, 7 fM or less, 6 fM or less, 5 fM or less, 4 fM or less, 3 fM or less, 2 fM or less, or 1 fM or less for the recombinant polypeptides disclosed herein.

[0072] The methods disclosed herein may have an accuracy of 50% or less. In aspects of this embodiment, the methods disclosed herein may have an accuracy of 50% or less, 40% or less, 30% or less, or 20% or less. In other aspects of this embodiment, the methods disclosed herein may have an accuracy of 15% or less, 10% or less, or 5% or less. In other aspects of this embodiment, the methods disclosed herein may have an accuracy of 4% or less, 3% or less, 2% or less, or 1% or less.

[0073] The methods disclosed herein may have an accuracy of at least 50%. In aspects of this embodiment, the methods disclosed herein may have an accuracy of at least 50%, at least 60%, at least 70%, or at least 80%. In other aspects of this embodiment, the methods disclosed herein may have an accuracy of at least 85%, at least 90%, or at least 95%. In other aspects of this embodiment, the methods disclosed herein may have an accuracy of at least 96%, at least 97%, at least 98%, or at least 99%.

[0074] The methods disclosed herein may have a signal to noise ratio for the lower asymptote that is statistically significant and a signal to noise ratio for the upper asymptote that is statistically significant. In aspects of this embodiment, the methods disclosed herein may have a signal to noise ratio for the lower asymptote of, for example, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, at least 8:1, at least 9:1, at least 10:1, at least 15:1, or at least 20:1. In other aspects of this embodiment, the methods disclosed herein may have a signal to noise ratio for the upper asymptote of, for example, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 35:1, at least 40:1, at least 45:1, at least 50:1, at least 60:1, at least 70:1, at least 80:1, at least 90:1, or at least 100:1, at least 150:1, at least 200:1, at least 250:1, at least 300:1, at least 350:1, at least 400:1, at least 450:1, at least 500:1, at least 550:1, or at least 600:1.

[0075] The specificity of the methods disclosed herein defines the ability of the method to measure a recombinant polypeptide disclosed herein to the exclusion of other related components, such as, for example, partially active or inactive recombinant polypeptides disclosed herein. The selectivity of the methods disclosed herein indicates the ability of the method to distinguish between different substances in a sample. The linearity of the methods disclosed herein indicates the ability of the method to derive a result that is directly or by a well-defined mathematical transformation that is proportional to the concentration of the recombinant polypeptide disclosed herein in the sample. Thus, in one embodiment, the methods disclosed herein can distinguish a recombinant polypeptide disclosed herein from a partially active recombinant polypeptide disclosed herein, for example, having 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less of the activity of a fully active recombinant polypeptide.

[0076] Durability of the methods disclosed herein indicates the reproducibility of results obtained for the same sample under normal (but variable) conditions of the method. Robustness of the methods disclosed herein indicates the ability of the measurement method to remain unaffected by small but intended changes in method parameters, giving an indication of its reliability in normal use. Thus, durability evaluates unavoidable changes, whereas robustness evaluates intended changes. Typical parameters evaluated by durability and robustness include the effects of freeze / thaw, incubation time, incubation temperature, reagent life, sample preparation, sample storage, cell passage number, many toxins, variability between purifications, and variability between cleavage reactions. Robustness parameters for the methods disclosed herein include cell bank (start, middle, and end of freezing), cell passage level, cell seeding density, cell stock density (days of culture), cell age in flask (time to wait before seeding), incubation time, different plates, excess amount of serum, and reagent source. The system suitability of the methods disclosed herein refers to the ability of the method to determine the method performance, including the performance of the reagents and the equipment over time by the analysis of the reference standard. System suitability refers to the fact that the equipment, electronics, assay performance, and the sample being analyzed constitute an integrated system. System suitability may be evaluated by testing parallelism, where serial dilutions of the reference and serial dilutions of the sample should produce parallel curves when plotting log dose vs. response.

[0077] Aspects of the present specification disclose kits that include one or more components useful for carrying out the methods disclosed herein. One or more components of the kit may include one or more capture agents disclosed herein, one or more solid supports, and / or one or more reagents necessary to detect the presence and / or activity of a recombinant polypeptide comprising a modification disclosed herein. The kits disclosed herein may include a solid phase and a capture agent bound to the solid phase. The kits disclosed herein may also include a capture agent and / or a recombinant polypeptide comprising a modification useful as a positive control for the assay step. If desired, the components may be included in the test kit at multiple concentrations to facilitate the creation of a standard curve against which signals detected in test samples can be compared.

[0078] A kit generally includes a package having one or more containers holding the components as one or more separate compositions, or optionally as a mixture if compatibility of the reagents permits. The kit may also include other material(s) that may be desirable from a user's perspective, such as buffer(s), diluent(s), standard(s), and / or any other material useful for performing sample processing, washing, or any other step of the assay. The kits disclosed herein may also include instructions for performing one or more of the methods disclosed herein. The instructions included in the kits disclosed herein may be affixed to the packaging material or may be included as a package insert. The instructions are typically written or printed materials, but are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by the embodiments disclosed herein. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term "instructions" may include an internet address providing the instructions.

[0079] The embodiments herein can also be described as follows: 1. A method for detecting the presence of a recombinant polypeptide comprising a modification, comprising the steps of incubating a sample comprising a recombinant polypeptide comprising said modification with a capture agent that selectively binds to said modification under conditions that allow for selective binding of the capture agent to said modification, thereby forming a polypeptide-agent complex, purifying said polypeptide-agent complex from said sample, and assaying for the presence of said recombinant polypeptide and / or polypeptide activity, wherein detection of said recombinant polypeptide and / or said polypeptide activity indicates the presence of a recombinant polypeptide comprising said modification.

[0080] 2. The method of embodiment 1, wherein said sample comprises polypeptides that do not have said modification and / or polypeptides that have different patterns of degree of modification.

[0081] 3. The method of embodiment 2, wherein the recombinant polypeptide is a growth factor, a cytokine, an immunomodulator, a hormone, an antibody, an enzyme, an enzyme inhibitor, a protease, a protease inhibitor, an esterase, a transferase, an oxidoreductase, a hydrolase, an asparaginase, an adenosine deaminase, a neurotoxin, a liver protein, a pancreatic protein, a muscle protein, a brain protein, a lung protein, or a blood protein.

[0082] 4. The method of embodiment 3, wherein the esterase is butyrylcholinesterase or acetylcholinesterase.

[0083] 5. The method of embodiment 3, wherein the cytokine is a chemokine, lymphokine, tumor necrosis factor, or hematopoietic factor.

[0084] 6. The method of embodiment 3, wherein the immunomodulatory agent is an interleukin or an interferon.

[0085] 7. The method of embodiment 3, wherein the blood protein is an erythropoiesis stimulator, a protease, a protease inhibitor, or a clotting factor.

[0086] 8. The method of embodiment 7, wherein said erythropoiesis-stimulating agent is erythropoietin, erythropoietin, erythropoietin, or darbepoietin.

[0087] 9. The method of embodiment 7, wherein the protease is trypsin, chymotrypsin, elastase, pepsin, or ADAMTS13.

[0088] 10. The method of embodiment 7, wherein the protease inhibitor is alpha1-antitrypsin, alpha1-antichymotrypsin, C1-inhibitor, or alpha2-antiplasmin, antithrombin.

[0089] 11. The method of embodiment 7, wherein the coagulation factor is Factor II, Factor IIa, Factor VII, Factor VIIa, Factor VIII, Factor VIIIa, Factor IX, Factor IXa, Factor X, or Factor Xa.

[0090] 12. The blood protein is selected from the group consisting of ADAMTS-13, α1-antiplasmin, α2-antiplasmin, antithrombin, antithrombin III, cancer procoagulant, erythropoietin, factor II, factor IIa, factor V, factor Va, factor VI, factor VIa, factor VII, factor VIIa, factor VIII, factor VIIIa, factor IX, factor IXa, factor X, factor Xa, factor XI, factor XIa, factor XII, factor XIIa, factor XIII, factor XIIIa, fibronectin, fibrinogen (factor I), heparin cofactor II, high molecular weight kininogen, and the like. 4. The method of embodiment 3, wherein the immunoglobulin A is selected from the group consisting of heparin, heparin, cysteine, heparin, cysteine, heparin activator inhibitor ...

[0091] 13. A method for detecting the presence of a recombinant coagulation factor comprising the steps of: incubating a sample containing the recombinant coagulation factor comprising the modification with a capture agent that selectively binds to the modification under conditions that allow selective binding of the capture agent to the modification, thereby forming a factor-agent complex; purifying the factor-agent complex from the sample; assaying for the presence and / or activity of said recombinant coagulation factor; wherein detection of said recombinant coagulation factor and / or said coagulation factor activity indicates the presence of a recombinant coagulation factor comprising said modification.

[0092] 14. The method according to embodiment 13, wherein the sample further comprises coagulation factors without said modification and / or coagulation factors with different patterns of degree of modification.

[0093] 15. The method according to any one of embodiments 13-14, wherein the coagulation factor is Factor II, Factor IIa, Factor VII, Factor VIIa, Factor VIII, Factor VIIIa, Factor IX, Factor IXa, Factor X, or Factor Xa.

[0094] 16. The modification is an acetate group, a phosphate group, a lipid group, or a carbohydrate group, a myristate group, a palmitate group, an isoprenoid group such as a farnesol group and a geranylgeraniol group, a glycosylphosphatidylinositol (GPI) group, a lipoate group, a flavin group, a heme C group, a 4'-phosphopantetheinyl group, a retinylidene group, a diphthamide group, an ethanolamine phosphoglycerol group, a hypusine group, an acetyl group, a formyl group, an alkyl group, a methyl group, an amide group, an amino acid, a butyl group, a carboxyl group, a glycosyl group, a polysialic acid (PSA) group, a hydroxyl group, a malonyl group, an iodine group, a phosphate group, an adenylyl group, a succinyl group, a sulfate group, a selenium group, a carbohydrate group, a starch group, a hydroxyl-ethyl starch (HES) group, a polysaccharide group, a saccharide group, a polyethylene glycol (PEG) group, an ubiquitin group, 16. The method according to any one of the preceding embodiments, wherein the carboxyl group is a carboxymethyl-2-methyl-1,2-dimethyl-2,3-dimethyl-1,3-dimethyl-2,4 ...

[0095] 17. The modification is myristoylation, palmitoylation, isoprenylation (prenylation), glycolipidation, lipoylation, flavinylation, phosphopantetheinylation, retinylidenation, diphthamidylation, ethanolamine phosphoglycerylation, hypusination, acylation, acetylation, formylation, alkylation, amidation, arginylation, polyglutamylation, polyglycylation, butyrylation, gamma-carboxylation, glycosylation, polysialylation, malonylation, hydroxylation, iodination, nucleosylation, 17. The method of any one of embodiments 1 to 16, wherein the recombinant polypeptide is associated with the polypeptide by oxidation, phosphoroesterification, phosphoramidation, phosphorylation, adenylylation, propionylation, pyroglutamylation, S-glutathionylation, S-nitrosylation, succinylation, sulfation, selenoylation, glycosylation, biotinylation, acylation, PEGylation, HESylation, silylation, citrullination, deamidation, eliminylation, carbamylation, deimination, pupylation, NEDDylation, ubiquitination, sumoylation, or ISGylation.

[0096] 18. The method of any one of embodiments 1-17, wherein the sample comprises a purified preparation of the recombinant polypeptide, a partially purified preparation of the recombinant polypeptide, an unpurified preparation of the recombinant polypeptide, a formulated preparation of the recombinant polypeptide, a crude extract of the recombinant polypeptide, a fractionated extract of the recombinant polypeptide, a cell lysate comprising the recombinant polypeptide, or a biological sample.

[0097] 19. The method of embodiment 18, wherein said biological sample comprises cells, tissue samples, blood samples, body fluid samples, or organ samples obtained directly from an individual.

[0098] 20. The method of embodiment 19, wherein said bodily fluid is urine, sputum, semen, feces, saliva, bile, cerebrospinal fluid, nasal swab, urogenital swab, nasal aspirate, or spinal fluid.

[0099] 21. The method of embodiment 18, wherein the biological sample is a preparation derived from a sample obtained directly from an individual.

[0100] 22. The method of embodiment 21, wherein the preparation derived from a sample obtained directly from the individual is the plasma fraction of a blood sample, the serum fraction of a blood sample, or an eluate from a purification process.

[0101] 23. The method according to any one of the preceding embodiments, wherein the sample is treated to increase the detectability of the recombinant polypeptide or to increase the activity of the recombinant polypeptide.

[0102] 24. The method of embodiment 23, wherein the processing comprises dissolving, diluting, purifying, extracting, filtering, distilling, separating, concentrating, inactivating interfering components, adding reagents, or any combination thereof.

[0103] 25. The capture agent is 1×10 5 M -1 s -1 Super, 1×10 6 M -1 s -1 Super, 1×10 7 M -1 s -1 Over or 1×10 8 M -1 s -1 25. The method of any one of embodiments 1 to 24, wherein the polypeptide has an association rate constant for the polypeptide comprising the modification of greater than or equal to 1.

[0104] 26. The capture agent is 1×10 -3 S -1 Less than 1×10 -4 S -1 Less than or 1×10 -5 S -1 26. The method of any one of the preceding claims, wherein the polypeptide has a dissociation rate constant for the polypeptide comprising said modification of less than 1.

[0105] 27. The method of any one of embodiments 1 to 26, wherein the capture agent has an equilibrium dissociation constant for a polypeptide comprising the modification of less than 0.500 nM, less than 0.450 nM, less than 0.400 nM, less than 0.350 nM, less than 0.300 nM, less than 0.250 nM, less than 0.200 nM, less than 0.150 nM, less than 0.100 nM, or less than 0.050 nM.

[0106] 28. The capture agent is 1×10 0 M -1 s -1 Less than 1×10 1 M -1 s -1 Less than 1×10 2 M -1 s -1 Less than 1×10 3 M -1 s -1 Less than or 1×10 4 M -1 s -1 28. The method according to any one of the preceding embodiments, wherein the polypeptide has an association rate constant for a polypeptide without the modification or a polypeptide having a different pattern or degree of modification of less than 1.

[0107] 29. The association rate constant (Ka) of the capture agent for a recombinant polypeptide containing the modification is greater than or equal to 1×10 compared to the association rate constant (Ka) of the capture agent for a recombinant polypeptide not having the modification and / or the association rate constant (Ka) of the capture agent for a recombinant polypeptide having a different pattern or degree of modification. 0 M -1 s -1 Large, 1×10 1 M -1 s -1 Large, 1×10 2 M -1 s -1 Large, 1×10 3 M -1 s -1 Large or 1×10 4 M -1 s -1 The method according to any one of embodiments 1 to 28.

[0108] 30. The method of any one of embodiments 1-28, wherein the association rate constant (Ka) of the capture agent for a recombinant polypeptide comprising the modification is at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 100-fold greater, at least 1,000-fold greater, or at least 10,000-fold greater than the association rate constant (Ka) of the capture agent for a recombinant polypeptide without the modification and / or compared to the association rate constant (Ka) of the capture agent for a recombinant polypeptide having a different pattern or degree of modification.

[0109] 31. The method of any one of embodiments 1-28, wherein the ratio of the binding specificity of the capture agent to a recombinant polypeptide comprising the modification to the binding specificity of the capture agent to a recombinant polypeptide not comprising the modification and / or having a different pattern or degree of modification is at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 64:1, at least 7:1, at least 8:1, at least 9:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 35:1, or at least 40:1.

[0110] 32. The method of any one of the preceding embodiments, wherein the capture agent is a multivalent capture agent.

[0111] 33. The method according to any one of the preceding embodiments, wherein the capture agent distinguishes between the recombinant polypeptide comprising a modification and the same polypeptide but without the modification.

[0112] 34. The method of any one of the preceding embodiments, wherein the capture agent distinguishes between a recombinant polypeptide containing a modification and the same polypeptide but having a different pattern or degree of the same modification.

[0113] 35. The method of any one of embodiments 1 to 34, wherein the capture agent is an antibody.

[0114] 36. The antibody is an anti-acetate antibody, an anti-phosphate antibody, an anti-lipid antibody, or an anti-carbohydrate antibody, an anti-myristate antibody, an anti-palmitate antibody, an anti-farnesol antibody, and an anti-geranylgeraniol antibody, an anti-glycosylphosphatidylinositol (GPI) antibody, an anti-lipoate antibody, an anti-flavin antibody, an anti-heme C antibody, an anti-4'-phosphopantetheinyl antibody, an anti-retinylidene antibody, an anti-diphthamide antibody, an anti-ethanolamine phosphoglycerol antibody, Antihypusine, antiacetyl, antiformyl, antialkyl, antimethyl, antiamide, antiamino acid, antibutyl, anticarboxyl, antiglycosyl, antipolysialic acid, antihydroxyl, antimalonyl, antiiodine, antiphosphate, antiadenylyl, antisuccinyl, antisulfate, antiselenium, anticarbohydrate, antipolysaccharide, antistarch, antihydroxyl-ethyl starch (HES), antisaccharide, antipoly ... 36. The method of embodiment 35, wherein the antibody is an anti-polyethylene glycol (PEG) antibody, an anti-ubiquitin antibody, an anti-pullulan antibody, an anti-chitosan antibody, an anti-hyaluronic acid antibody, an anti-chondroitin sulfate antibody, an anti-dermatan sulfate antibody, an anti-dextran antibody, an anti-carboxymethyl-dextran antibody, an anti-polyalkylene oxide (PAO) antibody, an anti-polyalkylene glycol (PAG) antibody, an anti-polypropylene glycol (PPG) antibody, an anti-polyoxazoline antibody, an anti-polyacryloylmorpholine antibody, an anti-polyvinyl alcohol (PVA) antibody, an anti-polycarboxylate antibody, an anti-polyvinylpyrrolidone (PVP) antibody, an anti-polyphosphazene antibody, an anti-polyoxazoline antibody, an anti-polyethylene-co-maleic anhydride antibody, an anti-polystyrene-co-maleic anhydride antibody, an anti-poly(1-hydroxymethylethylene hydroxymethyl formal) (PHF) antibody, or an anti-2-methacryloyloxy-2'-ethyltrimethylammonium-phosphate (MPC) antibody.

[0115] 37. The method according to any one of embodiments 1 to 36, wherein the recombinant polypeptide comprising the modification or the recombinant coagulation factor comprising the modification is PEGylated Factor II, PEGylated Factor IIa, polysialylated Factor II, polysialylated Factor IIa, HES-linked Factor II, HES-linked Factor IIa, silylated Factor II, or silylated Factor IIa.

[0116] 38. The method according to any one of embodiments 1 to 36, wherein the recombinant polypeptide comprising the modification or the recombinant coagulation factor comprising the modification is PEGylated Factor VII, PEGylated Factor VIIa, polysialylated Factor VII, polysialylated Factor VIIa, HES-linked Factor VII, HES-linked Factor VIIa, silylated Factor VII, or silylated Factor VIIa.

[0117] 39. The method according to any one of embodiments 1 to 36, wherein the recombinant polypeptide comprising the modification or the recombinant coagulation factor comprising the modification is PEGylated Factor VIII, PEGylated Factor VIIIa, polysialylated Factor VIII, polysialylated Factor VIIIa, HES-linked Factor VIII, HES-linked Factor VIIIa, silylated Factor VIII, or silylated Factor VIIIa.

[0118] 40. The method according to any one of embodiments 1 to 36, wherein the recombinant polypeptide comprising the modification or the recombinant coagulation factor comprising the modification is PEGylated Factor IX, PEGylated Factor IXa, polysialylated Factor IX, polysialylated Factor IXa, HESylated Factor IX, HESylated Factor IXa, silylated Factor IX, or silylated Factor IXa.

[0119] 41. The method of any one of the preceding embodiments, wherein the capture agent is bound to a solid support.

[0120] 42. The method of embodiment 41, wherein the solid support is a multiwell plate, a film, a tube, a sheet, a column, or a microparticle.

[0121] 43. A method for detecting the presence of PEGylated recombinant Factor VII, comprising the steps of incubating a sample containing said PEGylated recombinant Factor VII with an anti-PEG antibody under conditions allowing selective binding of the anti-PEG antibody to said PEGylated recombinant Factor VII, thereby forming a Factor VII-antibody complex, purifying said Factor VII-antibody complex from said sample, and assaying for the presence of said recombinant Factor VII and / or VII activity, wherein detection of said Factor VII and / or said Factor VII activity indicates the presence of said PEGylated recombinant Factor VII, and said PEGylated recombinant Factor VII is Factor VII and / or Factor VIIa.

[0122] 44. A method for detecting the presence of polysialylated recombinant Factor VII, comprising the steps of incubating a sample containing said polysialylated recombinant Factor VII with an anti-PSA antibody under conditions allowing selective binding of the anti-PSA antibody to said polysialylated recombinant Factor VII, thereby forming a Factor VII-antibody complex, purifying said Factor VII-antibody complex from said sample, and assaying for the presence of said recombinant Factor VII and / or Factor VII activity, wherein detection of said Factor VII and / or said Factor VII activity indicates the presence of said polysialylated recombinant Factor VII, and said polysialylated recombinant Factor VII is Factor VII and / or Factor VIIa.

[0123] 45. A method for detecting the presence of HES-modified recombinant factor VII, comprising the steps of incubating a sample containing the HES-modified recombinant factor VII with an anti-S antibody under conditions allowing selective binding of the anti-S antibody to the HES-modified recombinant factor VII, thereby forming a factor VII-antibody complex, purifying the factor VII-antibody complex from the sample, and assaying for the presence of the recombinant factor VII and / or factor VII activity, wherein detection of the factor VII and / or the factor VII activity indicates the presence of the HES-modified recombinant factor VII, and the HES-modified recombinant factor VII is factor VII and / or factor VIIa.

[0124] 46. ​​A method for detecting the presence of silylated recombinant Factor VII, comprising the steps of incubating a sample containing the silylated recombinant Factor VII with an anti-S antibody under conditions allowing selective binding of the anti-S antibody to the silylated recombinant Factor VII, thereby forming a Factor VII-antibody complex, purifying the Factor VII-antibody complex from the sample, and assaying for the presence of the recombinant Factor VII and / or Factor VII activity, wherein detection of the Factor VII and / or the Factor VII activity indicates the presence of the silylated recombinant Factor VII, and the silylated recombinant Factor VII is Factor VII and / or Factor VIIa.

[0125] 47. A method for detecting the presence of PEGylated rFVIII, comprising the steps of incubating a sample containing the PEGylated rFVIII with an anti-PEG antibody under conditions allowing selective binding of the anti-PEG antibody to the PEGylated rFVIII, thereby forming a factor VIII-antibody complex, purifying the factor VIII-antibody complex from the sample, and assaying for the presence of the rFVIII and / or factor VIII activity, wherein detection of the factor VIII and / or the factor VIII activity indicates the presence of the PEGylated rFVIII, and the PEGylated rFVIII is factor VII and / or factor VIIIa.

[0126] 48. A method for detecting the presence of polysialylated recombinant factor VIII, comprising the steps of incubating a sample containing said polysialylated recombinant factor VIII with an anti-PSA antibody under conditions that allow selective binding of the anti-PSA antibody to said polysialylated recombinant factor VIII, thereby forming a factor VIII-antibody complex, purifying said factor VIII-antibody complex from said sample, and assaying for the presence of said recombinant factor VIII and / or factor VIII activity, wherein detection of said factor VIII and / or said factor VIII activity indicates the presence of said polysialylated recombinant factor VIII, and said polysialylated recombinant factor VIII is factor VII and / or factor VIIIa.

[0127] 49. A method for detecting the presence of HES-modified recombinant factor VIII, comprising the steps of incubating a sample containing the HES-modified recombinant factor VIII with an anti-S antibody under conditions allowing selective binding of the anti-S antibody to the HES-modified recombinant factor VIII, thereby forming a factor VIII-antibody complex, purifying the factor VIII-antibody complex from the sample, and assaying for the presence of the recombinant factor VIII and / or factor VIII activity, wherein detection of the factor VIII and / or the factor VIII activity indicates the presence of the HES-modified recombinant factor VIII, and the HES-modified recombinant factor VIII is factor VII and / or factor VIIIa.

[0128] 50. A method for detecting the presence of silylated recombinant factor VIII, comprising the steps of incubating a sample containing the silylated recombinant factor VIII with an anti-S antibody under conditions allowing selective binding of the anti-S antibody to the silylated recombinant factor VIII, thereby forming a factor VIII-antibody complex, purifying the factor VIII-antibody complex from the sample, and assaying for the presence of the recombinant factor VIII and / or factor VIII activity, wherein detection of the factor VIII and / or the factor VIII activity indicates the presence of the silylated recombinant factor VIII, and the silylated recombinant factor VIII is factor VII and / or factor VIIIa.

[0129] 51. A method for detecting the presence of PEGylated recombinant factor IX, comprising the steps of incubating a sample containing said PEGylated recombinant factor IX with an anti-PEG antibody under conditions allowing selective binding of the anti-PEG antibody to said PEGylated recombinant factor IX, thereby forming a factor IX-antibody complex, purifying said factor IX-antibody complex from said sample, and assaying for the presence of said recombinant factor IX and / or factor IX activity, wherein detection of said factor IX and / or said factor IX activity indicates the presence of said PEGylated recombinant factor IX, and wherein the PEGylated recombinant factor IX is factor IX and / or factor IXa.

[0130] 52. A method for detecting the presence of polysialylated recombinant factor IX, comprising the steps of incubating a sample containing said polysialylated recombinant factor IX with an anti-PSA antibody under conditions allowing selective binding of the anti-PSA antibody to said polysialylated recombinant factor IX, thereby forming a factor IX-antibody complex, purifying said factor IX-antibody complex from said sample, and assaying for the presence of said recombinant factor IX and / or factor IX activity, wherein detection of said factor IX and / or said factor IX activity indicates the presence of said polysialylated recombinant factor IX, and wherein said polysialylated recombinant factor IX is factor IX and / or factor IXa.

[0131] 53. A method for detecting the presence of a HES-modified recombinant factor IX, comprising the steps of incubating a sample containing the HES-modified recombinant factor IX with an anti-S antibody under conditions allowing selective binding of the anti-S antibody to the HES-modified recombinant factor IX, thereby forming a factor IX-antibody complex, purifying the factor IX-antibody complex from the sample, and assaying for the presence of the recombinant factor IX and / or factor IX activity, wherein detection of the factor IX and / or the factor IX activity indicates the presence of the HES-modified recombinant factor IX, and the HES-modified recombinant factor IX is factor IX and / or factor IXa.

[0132] 54. A method for detecting the presence of silylated recombinant factor IX, comprising the steps of incubating a sample containing said silylated recombinant factor IX with an anti-S antibody under conditions allowing selective binding of the anti-S antibody to said silylated recombinant factor IX, thereby forming a factor IX-antibody complex, purifying said factor IX-antibody complex from said sample, and assaying for the presence of said recombinant factor IX and / or factor IX activity, wherein detection of said factor IX and / or said factor IX activity indicates the presence of said silylated recombinant factor IX, and wherein said silylated recombinant factor IX is factor IX and / or factor IXa.

[0133] 55. The method of any one of the preceding embodiments, wherein the assaying step is carried out using a qualitative or quantitative assay.

[0134] 56. The method of any one of the preceding embodiments, wherein the assaying step is carried out using an in vitro assay, a cell-based assay, or an in vivo assay.

[0135] 57. The method according to any one of the preceding embodiments, wherein the assaying step is carried out using a non-specific polypeptide assay or a specific polypeptide assay.

[0136] 58. The method of embodiment 57, wherein the non-specific polypeptide assay is a UV absorption assay, a Biuret assay, or a Bradford assay.

[0137] 59. The method of embodiment 57, wherein the specific polypeptide assay is a chromogenic assay, a colorimetric assay, a time-measured assay, a chemiluminescent assay, an electrochemiluminescent assay, a bioluminescent assay, a fluorescent assay, a resonance energy transfer assay, a plane polarization assay, a flow cytometric assay, an immune-based assay or an activity assay.

[0138] 60. The method of embodiment 59, wherein the activity assay is an enzymatic activity assay, an inhibitory activity assay, a coagulation activity assay, or a polymerization activity assay.

[0139] 61. The method of any one of the preceding embodiments, wherein selective binding of the capture agent occurs at neutral to alkaline pH.

[0140] 62. The method of any one of the preceding embodiments, wherein the recombinant polypeptide is a therapeutic polypeptide.

[0141] 63. The therapeutic polypeptide is selected from the group consisting of factor IX (FIX), factor VIII (FVIII), factor VIIa (FVIIa), von Willebrand factor (VWF), factor V (FV), factor X (FX), factor XI (FXI), factor XII (FXII), thrombin (FII), protein C, protein S, tPA, PAI-1, tissue factor (TF), ADAMTS13 protease, IL-1 alpha, IL-1 beta, IL-3, IL-4, IL-5, IL-6, IL-11, colony-stimulating factor-1 (CSF-1), M-CSF, SCF, G M-CSF, granulocyte colony-stimulating factor (G-CSF), EPO, interferon-α (IFN-α), consensus interferon, IFN-β, IFN-γ, IFN-ω, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-31, IL-32 alpha, IL-33, thrombopoietin (TPO), Ang-1, Ang-2, Ang-4, Ang-Y, angiopoietin-like Angiopoietin-like polypeptide 1 (ANGPTL1), Angiopoietin-like polypeptide 2 (ANGPTL2), Angiopoietin-like polypeptide 3 (ANGPTL3), Angiopoietin-like polypeptide 4 (ANGPTL4), Angiopoietin-like polypeptide 5 (ANGPTL5), Angiopoietin-like polypeptide 6 (ANGPTL6), Angiopoietin-like polypeptide 7 (ANGPTL7), Vitronectin, Vascular endothelial growth factor (VEGF), Angiogenin, Activin A, Activin B, Activin C, Bone morphogenetic protein-1, Bone morphogenetic protein -2, bone morphogenetic protein-3, bone morphogenetic protein-4, bone morphogenetic protein-5, bone morphogenetic protein-6, bone morphogenetic protein-7, bone morphogenetic protein-8, bone morphogenetic protein-9, bone morphogenetic protein-10, bone morphogenetic protein-11, bone morphogenetic protein-12, bone morphogenetic protein-13, bone morphogenetic protein-14, bone morphogenetic protein-15, bone morphogenetic protein receptor IA, bone morphogenetic protein receptor IB, bone morphogenetic protein receptor II, brain-derived neurotrophic factor, cardiotrophin-1,Ciliary neurotrophic factor, Ciliary neurotrophic factor receptor, Crypt, Cryptic, Cytokine-induced neutrophil chemotactic factor 1, Cytokine-induced neutrophil chemotactic factor 2α, Cytokine-induced neutrophil chemotactic factor 2β, β-endothelial growth factor, Endothelin 1, Epidermal growth factor, Epigen, Epiregulin, Epithelial-derived neutrophil inducer, Fibroblast growth factor 4, Fibroblast growth factor 5, Fibroblast growth factor 6, Fibroblast growth factor 7, Fibroblast growth factor 8, Fibroblast growth factor 8b, Fibroblast growth factor 8c, Fibroblast growth factor 9, Fibroblast growth factor 10, Fibroblast growth factor 10, Fibroblast growth factor 11, Fibroblast growth factor 12, Fibroblast growth factor 13, Fibroblast growth factor 14, Fibroblast growth factor 15, Fibroblast growth factor 16, Fibroblast growth factor 17, Fibroblast growth factor 18, Fibroblast growth factor 19, Fibroblast growth factor 20, Fibroblast growth factor 21, Fibroblast growth factor 22, Fibroblast growth factor 23, Fibroblast growth factor 24, Fibroblast growth factor 25, Fibroblast growth factor 26, Fibroblast growth factor 27, Fibroblast growth factor 28, Fibroblast growth factor 29, Fibroblast growth factor 30, Fibroblast growth factor 31, Fibroblast growth factor 32, Fibroblast growth factor 33, Fibroblast growth factor 34, Fibroblast growth factor 35, Fibroblast growth factor 36, Fibroblast growth factor 37, Fibroblast growth factor 38, Fibroblast growth Fibroblast growth factor 11, fibroblast growth factor 12, fibroblast growth factor 13, fibroblast growth factor 16, fibroblast growth factor 17, fibroblast growth factor 19, fibroblast growth factor 20, fibroblast growth factor 21, acidic fibroblast growth factor, basic fibroblast growth factor, glial cell line-derived neurotrophic factor receptor alpha 1, glial cell line-derived neurotrophic factor receptor alpha 2, growth-associated protein, growth-associated protein alpha, growth-associated protein beta, growth-associated protein gamma, heparin-binding epidermal growth factor, hepatocyte growth factor, hepatocyte growth factor receptor, hepatoma-derived growth factor, insulin -like growth factor I, insulin-like growth factor receptor, insulin-like growth factor II, insulin-like growth factor binding protein, keratinocyte growth factor, leukemia inhibitory factor, leukemia inhibitory factor receptor alpha, nerve growth factor, nerve growth factor receptor, neuropoietin, neurotrophin-3, neurotrophin-4, oncostatin M (OSM), placental growth factor, placental growth factor 2, platelet-derived endothelial cell growth factor, platelet-derived growth factor, platelet-derived growth factor A chain, platelet-derived growth factor AA, platelet-derived growth factor AB, platelet-derived growth factor B chain, platelet-derived growth factor BB, Platelet-derived growth factor receptor α, Platelet-derived growth factor receptor β, Pre-B cell growth-stimulating factor, Stem cell factor (SCF), Stem cell factor receptor, TNF, TNF0, TNF1, TNF2, Transforming growth factor α, Transforming growth factor β, Transforming growth factor β1, Transforming growth factor β1.2, Transforming growth factor β2, Transforming growth factor β3, Transforming growth factor β5, Latent transforming growth factor β1, Transforming growth factor β binding protein I, Transforming growth factor β binding protein II, Transforming growth factor β binding protein III, Thymic stromal lymphopoietin (TSLP),64. The method of embodiment 63, wherein the antibody is selected from the group consisting of tumor necrosis factor receptor type I, tumor necrosis factor receptor type II, urokinase-type plasminogen activator receptor, phospholipase-activating protein (PUP), insulin, lectin, ricin, prolactin, chorionic gonadotropin, follicle-stimulating hormone, thyroid-stimulating hormone, tissue plasminogen activator, IgG, IgE, IgM, IgA, and IgD, alpha-galactosidase, beta-galactosidase, DNAse, fetuin, luteinizing hormone, estrogen, insulin, albumin, lipoprotein, fetoprotein, transferrin, thrombopoietin, urokinase, integrin, thrombin, leptin, Humira (adalimumab), Prolia (denosumab), Enbrel (etanercept), or a biologically active fragment, derivative, or variant thereof.

[0142] 64. A kit comprising one or more components useful for carrying out the method according to any one of embodiments 1 to 64.

[0143] 65. The kit of embodiment 64, wherein the one or more components comprise one or more capture agents, one or more solid supports, and / or one or more reagents necessary for detecting the presence and / or activity of the recombinant polypeptide.

[0144] 66. The kit of embodiment 65, wherein the capture agent is bound to the solid phase.

[0145] 67. The kit according to any one of embodiments 64 to 66, wherein the kit further comprises a capture agent and / or a recombinant polypeptide comprising a modification useful as a positive control for the assaying step. EXAMPLES

[0146] The following non-limiting examples are provided for illustrative purposes only to facilitate a more complete understanding of the representative embodiments contemplated herein, and should not be construed as limiting any of the embodiments described herein, including those related to the modification-dependent activity assay methods disclosed herein and the products processed using those methods.

[0147] [Example 1] MDAA for PEGylated FVIII in PBS This example illustrates that MDAA for PEGylated FVIII can be performed in a buffer.

[0148] To attach the modified recognition antibody to the solid support, 0.43 mg / mL of rabbit anti-PEG antibody (B47-2061-1, Epitomics, Inc., Burlingame, CA) was dissolved in 0.1 M NaHCO 3 -Na 2 CO 3 The antibodies were diluted 1 / 50 in PBS, pH 9.5, and incubated in wells of an F96 Maxisorp plate (100 μL / well) overnight at 0±10° C. The plates were then washed with phosphate buffered saline (PBS, 8 g / L NaCl, 0.2 g / L KCl, 0.2 g / L KH2PO4, 1.26 g / L Na 2 The wells were then washed with wash buffer containing 4×2HPO (natural pH) and 0.05% Tween 20. The wells of the plate were then blocked by incubation of 200 μL / well of dilution buffer containing PBS and 10 mg / mL human serum albumin for 60±10 minutes at 37±5° C. The blocked wells were then washed with wash buffer.

[0149] To selectively bind the samples to the solid support, 100 μL of the following samples were added to the wells: 1) a dilution series of PEGylated recombinant FVIII standard prepared using PBS containing 10 mg / mL human serum albumin (HSA) or 2) a dilution series of human reference plasma prepared using PBS containing 10 mg / mL HSA. A PEGylated recombinant FVIII with a degree of PEGylation of about 2 was used, which had a FVIII:C activity of 2333 IU FVIII / mL as measured using a chromogenic method and contained 56.2 μg / mL bound PEG / mL. The samples were loaded into the plate and incubated at about 18° C. to about 26° C. for 60±10 minutes. Under these conditions, the PEGylated recombinant FVIII was selectively bound to the solid support by its PEG moiety using an anti-PEG antibody. The plates were then washed six times with washing buffer and then incubated with 200 μL / well of FVIII dilution buffer containing 3.4 g / L imidazole, 5.85 g / L NaCl, 10 mg / mL HSA, pH 7.4. This equilibration was carried out for 3 min at room temperature. After a washing step with washing buffer, the wells were emptied and the FVIII activity was measured using a chromogenic assay after a standard color development procedure using the Immunochrom FVIII kit (Technoclone, GmbH, Vienna, Austria). The wells were filled with 20 μL of FVIII dilution buffer, after which 20 μL of reagent A and reagent B were added successively. The plates were then incubated for 5 min at 37±5° C. A pre-warmed substrate solution was then added (100 μL / well) and incubated for 5 min at 37±5° C. Finally, the reaction was stopped by adding 20% ​​acetic acid (40 μL / well). The plates were then read at 405 nm (reference wavelength 620 nm) using an ELISA reader.

[0150] FIG. 1 shows the concentration-response curves obtained for the PEGylated FVIII preparation and the human reference plasma preparation. The dose-response curve obtained for the PEGylated FVIII preparation was deemed suitable for estimating the samples, since it met the FVIII activity range of 7.3-233 mIU / mL and the acceptable requirements for accuracy, precision and linearity. In particular, the correlation coefficient of the log-log regression curve was 0.9993, with a mean accuracy of 100.1%, calculated as the average relative agreement of the backfit concentrations with the nominal ones, and a precision of 4.9%, expressed as the standard deviation of this mean. Alternatively, a calibration curve based on lin-lin regression analysis is equally feasible. In this case, the four-point calibration curve ranged only from 29.2 to 233 mIU / mL, with a mean accuracy of 104.4% and a precision of 6.2%. These data support the log-log approach for constructing the calibration curve, since this approach offers a higher sensitivity along with a higher accuracy and precision. Furthermore, the data demonstrated the absolute specificity of the approach to measure only modified FVIII. Human reference plasma containing unmodified native human FVIII at a concentration of 1 IU / mL did not elicit any significant signal. These data demonstrated that the FVIII activity of PEGylated FVIII can be measured sensitively and specifically.

[0151] [Example 2] MDAA for PEGylated FVIII in human plasma This example illustrates that MDAA for PEGylated FVIII can be performed in the presence of plasma.

[0152] The modified recognition antibody was prepared by mixing 4 μg / mL of rabbit anti-PEG antibody (B47-2061-1, Epitomics, Inc., Burlingame, CA), 0.1 M NaHCO 3 -Na 2 CO 3 The antibodies were attached to the solid support as described in Example 1, except that 100 μL / well of coating antibody solution containing 10 μL of 0.1% EDTA, pH 9.5 was used.

[0153] The samples were selectively bound to a solid support and FVIII activity was measured using the chromogenic assay described in Example 1 except that: 1) a first dilution series of recombinant PEGylated FVIII was prepared by diluting in PBS containing 30 mg / mL skim milk to obtain a dilution series of 1 / 10,000 to 1 / 320,000; 2) a second dilution series of recombinant PEGylated FVIII was prepared by diluting in human plasma; and 3) FVIII activity was measured in a chromogenic assay using a 15 minute incubation time for the substrate reaction at 37±5° C.

[0154] Figure 2 shows the dose-response curves for the two preparations with different purities. The two concentration-response curves were very similar as shown by their slopes differing by less than 1%. These data demonstrated that the modification-dependent activity assay for PEGylated FVIII performed well in human plasma when diluted 1 / 200. Non-PEGylated FVIII contained in plasma did not contribute to the signal as shown by overlaying the concentration-response curves.

[0155] [Example 3] Accuracy and precision of MDAA for PEGylated FVIII This example illustrates the accuracy and precision of MDAA for PEGylated FVIII.

[0156] To bind the modified recognition antibody to the solid support, 4 μg / mL of rabbit anti-PEG antibody (B47-2061-1, Epitomics, Inc., Burlingame, CA), 0.1 M NaHCO 3 -Na 2 CO 3100 μL / well of coating antibody solution containing PBS, 3% skim milk, and 50 mM benzamidine was incubated in the wells of an F96 Maxisorp plate overnight at 0±10° C. The plate was then washed with a wash buffer containing PBS and 0.05% Tween 20. The wells of the plate were then blocked by incubation of 200 μL / well of blocking buffer containing PBS, 3% skim milk, and 50 mM benzamidine for 60±10 minutes at about 18° C. to about 26° C. The blocked wells were then washed with wash buffer.

[0157] To selectively bind samples to the solid support, 100 μL of the following samples were added to the wells: 1) a dilution series of PEGylated recombinant FVIII prepared using 1 / 10 human plasma solution diluted in PBS, 3% skim milk, and 50 mM benzamide, spanning FVIII concentrations ranging from 2.2 mU / mL to 69 mU / mL, 2) a dilution series of human plasma prepared using 1 / 10 human plasma solution diluted in PBS, 3% skim milk, and 50 mM benzamide, or 3) a dilution series of test subject prepared using 1 / 10 human plasma solution diluted in PBS, 3% skim milk, and 50 mM benzamidine. A PEGylated recombinant FVIII with a degree of PEGylation of approximately 2 was used, with a FVIII:C activity of 2333 IU FVIII / mL as measured using a chromogenic method, and contained 56.2 μg / mL of bound PEG / mL. The samples were loaded onto the plate and incubated for 120±10 minutes at about 18° C. to about 26° C. Under these conditions, the PEGylated recombinant FVIII was selectively bound to the solid support by its PEG moiety using an anti-PEG antibody. The plate was then washed six times with wash buffer, followed by incubation with 200 μL / well of FVIII dilution buffer containing 3.4 g / L imidazole, 5.85 g / L NaCl, 10 mg / mL HSA, pH 7.4. The equilibration was carried out for 5-10 minutes at about 18° C. to about 26° C.

[0158] After a washing step with washing buffer, the wells were emptied and FVIII activity was measured using a chromogenic assay after a standard color development procedure using the Immunochrome FVIII Kit (Technoclone, GmbH, Vienna, Austria). The wells were filled with 20 μL of FVIII dilution buffer, after which 20 μL of reagent A and reagent B were added successively. The plate was placed on a mixer set at 500 rpm and incubated for 15 min at about 18° C. to about 26° C. Then 100 μL / well of substrate solution was added and incubated for 45 min at about 18° C. to about 26° C. Finally, the reaction was stopped by adding 40 μL / well of 20% acetic acid. The optical density (OD) was measured in an ELISA reader at 405 nm (reference wavelength 620 nm). The quantitative evaluation is based on a double logarithmic calibration curve. The blank-corrected mean OD of the individual calibration curve standards is related to their FVIII concentration. If the OD of the sample is within the OD range covered by the calibration curve, the resulting calibration curve is used to calculate the FVIII concentration of the sample. Three of these different analyses were included in this study to investigate the operator-dependent effects.

[0159] Table 1 shows the original data averages (n=24) of the calibration curves and their characteristics. In particular, it shows the average of the blank-corrected ODs measured for the six calibration curve standards D1-D6, with FVIII concentrations ranging from 2.2 mU / mL to 69 mU / mL, and for the corresponding blanks. Apart from these direct assay readouts, the calibration curve characteristic slope, y-intercept and correlation coefficient of the obtained calibration curves are shown. Finally, the relative total error (RTE), a combined measure of the accuracy and precision of the curve fit, is shown. The RTE was calculated by backfitting the ODs measured for each calibration curve standard. The concentrations thus obtained were multiplied by the dilution factor and finally averaged to obtain the average backfit concentration. The RTE is then the sum of the absolute differences between the nominal concentration and the average backfit concentration of the assay standards, and is calculated by multiplying the double standard deviation of the backfit average concentration by the nominal concentration.

number

number

number

number

[0160] The individual direct readouts, i.e. blank-corrected ODs, showed a certain acceptable variation between the different assays with RSDs of 18.3% to 27.8% for the blank-corrected ODs measured for the individual assay calibration standards D1 to D6, whereas the blanks showed a low RSD of 6.6%. To compensate for these differences in the direct readouts, calibration curves are constructed on each single plate. The low RSDs determined for the mean slopes and mean y-intercepts indicate that, despite the differences in ODs measured for the assay standards, the calibration curves obtained have a very similar shape, verifying that this procedure is effective in compensating for the differences between the absolute readouts. Thus, we found RSDs of 3.7% and 8.3% for the mean slopes and mean y-intercepts of the calibration curves. Their linearity was adequate as shown by the correlation coefficient r=0.9994 with all individual values ​​above 0.9978. Since the correlation coefficient can be a spurious measure of linearity and precision, we also calculated the RTE of the curves. The average RTE of 8.3% confirmed a high correlation coefficient as an indicator for the precision and linearity of the calibration curve. The individual values ​​for RTE ranged from 2.0% to 17.6%. The fact that only one of the 24 RTEs was higher than 15% and 16 of the 24 RTEs were lower than 10% further illustrated the suitability of the log-log calibration model selected to represent the relationship between the signal and FVIII activity. In addition, it should be noted that the calibration curve consists of six individual points, although only the pseudo-linear part of the dose-response relationship was combined. With this design, the calibration curve complies with the EMA guidelines for bioanalytical method validation, which require a minimum number of six calibration points for LBAs, regardless of the calibration model selected. Although we used a linear evaluation model with a narrow range, our calibration curve nevertheless complies with this requirement. Figure 3 shows the average calibration curve obtained over 24 trials. The insert shows the agreement of the backfit assay calibration standards as a percentage of their respective nominal concentrations. Current guidelines for bioanalytical method validation require that 75% of the backfit concentrations should be within ±20%.

[0161] The mean backfit concentrations differed by less than 5% for all six calibration curve standards. There was no tendency for the relative error to depend on the FVIII concentration, but rather a stationary distribution over the entire calibration curve range. It is noteworthy that the mean RE determined for standard D6, with the lowest FVIII concentration of 2.2 mU / mL, did not differ from those determined for the other assay standards. This clearly supports that the measurement of a sample that was only diluted once and had a PEGylated recombinant FVIII concentration close to the lower limit of quantification, yields a signal within the range covered by the calibration curve. The individual REs were within ±12%, thus meeting the acceptability criteria of the validation protocol.

[0162] Overall, all the data confirmed the accuracy, precision and robustness of constructing a calibration curve for MDAA to measure PEGylated recombinant FVIII.

[0163] [Example 4] Specificity of MDAA for PEGylated FVIII This example illustrates how to confirm the specificity of MDAA for PEGylated FVIII using a competition assay.

[0164] The modified recognition antibody was bound to a solid support as described in Example 3.

[0165] Samples were selectively bound to a solid support and FVIII activity was measured using the chromogenic assay described in Example 3, except that the recombinant PEGylated FVIII samples were diluted to a concentration of 69 mU / mL and then mixed 1+1 with PEG5000 to achieve final PEG5000 concentrations ranging from 100 μg / mL to 0.012 μg / mL (14 dilutions).

[0166] Each competing sample was measured four times in one run. The average signal obtained was then related back to that obtained from the sample containing no competitor for the calculated relative signal. Figure 4 shows the competition curve obtained by this approach and further shows the IC 50, i.e., a PEG5000 concentration that provides a semi-competitive, and this IC was calculated using GraphPad Prism version 5.00 for Windows, GraphPad Software. 50 The corresponding confidence interval CI calculated for 95% The data clearly confirmed the specificity of the capture step, since we clearly observed a dose-dependent decrease in the signal measured in the presence of PEG5000. Half-competition was reached under the given assay conditions with a PEG concentration of 2.4 μg / mL.

[0167] [Example 5] Specificity of MDAA for PEGylated FVIII This example illustrates the specificity of MDAA for PEGylated FVIII using a competition assay.

[0168] The modified recognition antibody was bound to a solid support as described in Example 3.

[0169] Samples were selectively bound to a solid support and FVIII activity was measured using the chromogenic assay described in Example 3, with the exception that 1) recombinant PEGylated FVIII samples were diluted to a concentration of 40 mU / mL and then mixed 1+1 with a rabbit anti-PEG antibody (PEG-B-47, Epitomics, Inc., Burlingame, CA) to achieve final concentrations ranging from 605 μg / mL to 0.00007 μg / mL.

[0170] Each competing sample was measured twice in one run. The average signal obtained was then related back to that obtained from samples containing no competitor, and the relative signal was calculated. Figure 5 shows the competition curves obtained by this approach, and furthermore, the IC 50 , i.e., the antibody concentration that provides the semi-competitive IC, and this IC was calculated using GraphPad Prism version 5.00 for Windows, GraphPad Software. 50 The corresponding confidence interval CI calculated for 95% We show that the coefficient of determination R 2A common competition curve was found that was characterized by a ΔP < 0.095 μg / mL anti-PEG concentration, which caused a half-maximal decrease in signal under the given assay conditions, confirming the selectivity of the capture step.

[0171] [Example 6] Accuracy and precision of MDAA for PEGylated FVIII in plasma This example illustrates the accuracy and precision of the MDAA using a spike recovery assay for PEGylated FVIII in plasma.

[0172] The modified recognition antibody was bound to a solid support as described in Example 3.

[0173] Samples were selectively bound to a solid support and FVIII activity was measured using the chromogenic assay described in Example 3, except that 1) samples consisted of normal human plasma (NHP) spiked with five concentrations of PEGylated recombinant FVIII ranging from 0.07 to 2 U / mL, or 2) samples consisted of eight commercially available FVIII-deficient plasma samples, measured before and after spiking with PEGylated recombinant FVIII at 0.07 U / mL and 0.5 U / mL. Eight FVIII-deficient plasmas were obtained from George King Bio-Medical Inc. (Chelsea, MA): GK897-1966 (#1), GK892-2056 (#2), GK896-2031 (#3), Pool-1928 (#4), Pool-1930 (#5), GK893-1964 (#6), GK895-1959 (#7), and GK894-1965 (#8).

[0174] The intra-run precision (n=6) and inter-run precision (n=6) were determined for spiked plasma and buffer samples, while the precision of the assay was determined by calculating the recovery of the spiked amount. Finally, the total error was calculated as the sum of the inter-run precision and precision. Current EMA guidelines propose that this total error should not exceed 30% or 40% at the lower limit of quantification (LLOQ) of the assay. Table 2 summarizes the above data. [Table 2]

[0175] High quality data were obtained by determining the precision and accuracy of the assay in normal human plasma and in buffer matrices, and the total error of MDAA for PEGylated recombinant FVIII was clearly less than 20%, even at the assay's LLOQ of 0.07 U / mL. These data validate MDAA for PEGylated recombinant FVIII as a precise and accurate method suitable for use as a bioanalytical method.

[0176] Table 3 shows the results of the spike recovery study carried out in eight FVIII-deficient plasma samples. The recovery of spiked PEGylated recombinant FVIII was also good in the FVIII-deficient plasma samples. Mean recoveries of 91.3% and 98.5% were detected when 0.07 U / mL and 0.5 U / mL of PEGylated recombinant FVIII were spiked, respectively. Figure 6 shows representative curves for four of the eight FVIII-deficient plasma samples spiked at 0.5 U / mL. The high parallelism of the spiked sample curves to the assay standards is evident: the slopes obtained for the spiked sample curves differed by less than ±10% from the assay calibration curves indicating the excellent linearity of the dose-response curves obtained for the spiked FVIII-deficient plasma samples. [Table 3]

[0177] [Example 7] MDAA for polysialylated FVIII in PBS or human plasma This example illustrates that MDAA for polysialylated FVIII can be performed in a buffer or in the presence of plasma.

[0178] To bind the modified recognition antibody to the solid support, a 1 / 400 dilution of mouse anti-polysialic acid NCAM antibody (MAB5324-clone 2-2B, Millipore, Inc.,) in 0.1 M NaHCO3 -Na 2 CO 3 100 μL / well of coating antibody solution containing 100 μL / well of PBS, pH 9.5 was incubated in the wells of an F96 Maxisorp plate overnight at 0±10° C. The plate was then washed with wash buffer containing PBS and 0.05% Tween 20. The wells of the plate were then blocked by incubation of 200 μL / well of dilution buffer containing PBS and 10 mg / mL human serum albumin for 60±10 minutes at 37±5° C. The blocked wells were then washed with wash buffer.

[0179] To selectively bind samples to the solid support, 100 μL of the following samples were added to the wells: 1) a 1 / 40,000 to 1 / 1,280,000 dilution series of polysialylated recombinant FVIII standard prepared using PBS containing 10 mg / mL HSA or 2) a 1 / 20 to 1 / 320 dilution series of human reference plasma prepared using PBS containing 10 mg / mL HSA. Polysialylated recombinant FVIII was prepared by conjugating a 20 kDa polysialic acid reagent containing active aminooxy groups to the oxidized N-glycans of FVIII (see U.S. Patent Application Publication Nos. 2011 / 0027350 and 2011 / 0028693, each of which is incorporated herein by reference in its entirety). The preparation had a FVIII:C activity of 3110 IU FVIII / mL, as measured by chromogenic assay, and contained 483 μg / mL N-acetylneuraminic acid resulting in a degree of polysialylation of about 7-8. Samples were loaded onto the plate and incubated at about 18° C. to about 26° C. for 60±10 minutes. Under these conditions, polysialylated recombinant FVIII was selectively bound to the solid support by its polysialic acid (PSA) moiety using anti-PSA antibody. The plate was then washed six times with wash buffer, followed by incubation with 200 μL / well of FVIII dilution buffer containing 3.4 g / L imidazole, 5.85 g / L NaCl, 10 mg / mL HSA, pH 7.4. The equilibration was carried out for 3 minutes at room temperature. After a washing step with washing buffer, the wells were emptied and the FVIII activity was measured by chromogenic assay after a standard color development procedure using the Immunochrome FVIII Kit (Technoclone, GmbH, Vienna, Austria). The wells were filled with 20 μL of FVIII dilution buffer, after which 20 μL of Reagent A and Reagent B were added successively. The plate was then incubated for 5 min at 37±5° C. Then pre-warmed substrate solution was added (100 μL / well) and incubated for 15 min at 37±5° C. Finally, the reaction was stopped by adding 20% ​​acetic acid (40 μL / well). The plate was then measured at 405 nm (reference wavelength 620 nm) using an ELISA reader.

[0180] FIG. 7 shows the concentration-response curves obtained for polysialylated FVIII preparations in the FVIII activity range of 2.4-78 mIU / mL. The concentration-response curves for polysialylated FVIII showed good linearity over the entire FVIII concentration range after logarithmic transformation. This was shown by a correlation coefficient r=0.9989 and supported by the backfit concentrations calculated for the individual points of the calibration curve, which differed from the nominal by less than 12% over the entire range (range: 89.8%-108.5%, mean 100.2%). The data further confirmed the specificity of MDAA, since human plasma containing unmodified FVIII showed no response when measured at FVIII concentrations similar to those performed for polysialylated FVIII.

[0181] The effect of human plasma on MDAA was further investigated by measuring the response for polysialylated FVIII diluted in human plasma prediluted 1 / 200. Table 4 directly compares the average blank-corrected optical density (OD) measured for polysialylated FVIII diluted in buffer and human plasma. It also gives the backfit FVIII concentrations for the two dilution series in buffer and human plasma, respectively, as well as the percent agreement with the nominal FVIII concentrations, given as a % of the nominal concentration. [Table 4]

[0182] The concentration-response curves obtained for the polysialylated FVIII preparations were very similar in buffer and human plasma as shown by their slopes differing by less than 3%. The recovery of polysialylated FVIII activity in human plasma was as good as in buffer. The average recovery was 104.2% with individual values ​​between 96.6 and 111.4% for the polysialylated preparations diluted in human plasma and 100.2% for the same preparations diluted in buffer. These data demonstrated that the modification-dependent activity assay for polysialylated FVIII performed well in human plasma when diluted 1 / 200. The non-polysialylated FVIII contained in plasma did not contribute to the signal, as expected, demonstrating the absolute specificity of MDAA for polysialylated FVIII.

[0183] [Example 8] MDAA for polysialylated FVIII in plasma from different animal species This example illustrates that MDAA for polysialylated FVIII can be performed in the presence of plasma.

[0184] The modified recognition antibody was bound to a solid support as described in Example 7.

[0185] Samples were selectively bound to a solid support and FVIII activity was measured using a chromogenic assay as described in Example 7, except that 1) recombinant polysialylated FVIII was diluted 1 / 20,000 in PBS containing 10 mg / mL HSA and 50 mM benzamidine, then further diluted 1+1 with 1 / 5 pre-diluted plasma samples of the different species and finally diluted 1+1 directly on the plate (final concentration in plasma corresponded to a 1 / 20 dilution), and 2) the preparation was diluted with plasma from the following species: rat, mouse, FVIII-deficient mouse, cynomolgus monkey and human reference plasma.

[0186] FIG. 8 shows the concentration-response curves obtained for polysialylated FVIII in different plasma matrices and gives their slopes relative to those determined in buffer. Linear concentration-response curves were obtained for the investigated plasma samples. In addition, these curves were very parallel to those obtained in buffer and their slopes differed only slightly, i.e., less than 4%, from those determined in buffer. Thus, these data demonstrate that the plasma from the different species investigated did not interfere with MDAA for polysialylated FVIII when diluted 1 / 20. Furthermore, these data support the preparation of an MDAA calibration curve in dilution buffer and the use of this dilution series to measure plasma samples.

[0187] [Example 9] Performance and sensitivity of MDAA for polysialylated FVIII This example illustrates the performance and sensitivity of MDAA for polysialylated FVIII using a spike recovery assay.

[0188] The modified recognition antibody was bound to a solid support as described in Example 7.

[0189] The samples were selectively bound to a solid support, and the lyophilized polysialylated recombinant FVIII was dissolved to obtain a solution with 250 IU / mL, diluted to nominal FVIII concentrations of 10, 5, 2.5, 1 and 0.5 IU / mL with dilution buffer containing PBS, 0.05% Tween 20, 10 mg / mL HSA, and 50 mM benzamidine, and then further diluted 1 / 10 with rat plasma, followed by 1 / 10 dilution with buffer, to measure FVIII activity using a chromogenic assay as described in Example 7. These samples were then further diluted 1+1 directly on the plate to obtain a final rat plasma concentration corresponding to a 1 / 20 dilution. In addition, FVIII activity was measured in imuno using a 15 minute incubation time for the substrate reagent and a 45 minute incubation time for the substrate solution.

[0190] FIG. 9 shows the concentration-response curves for these samples containing polysialylated FVIII concentrations ranging from 1 to 0.05 IU / mL. The concentration-response curves for spiked rat plasma samples were linear and parallel to those obtained for assay standards diluted in buffer when they contained more than three data points. In such cases, the slopes differed by less than 7% from those of the dilution series measured for samples containing buffer only. Even samples spiked with 0.05 IU / mL polysialylated FVIII showed linear concentration-response curves. The slopes differed by only 13.5% from those of the buffer samples, demonstrating that MDAA also performed adequately in rat plasma at very low concentrations of polysialylated FVIII, close to the estimated limit of quantitation. The recoveries of spiked polysialylated FVIII were within 100±20% of the nominal concentration. These data demonstrated that MDAA for polysialylated FVIII performed acceptably in rat plasma samples at a minimum dilution of 1 / 20.

[0191] [Example 10] Accuracy and precision of MDAA for polysialylated FVIII. This example illustrates the accuracy and precision of MDAA for polysialylated FVIII.

[0192] The modified recognition antibody was bound to a solid support as described in Example 7.

[0193] The samples were selectively bound to a solid support and FVIII activity was measured using the chromogenic assay described in Example 9.

[0194] Table 5 gives the original data and calibration curve characteristics obtained for 16 calibration curves constructed on different days. Blank-corrected optical densities (OD) of calibrators D1-D5 cover the polysialylated FVIII activity range of 1.6-25 mIU / mL, blank, slope, y-intercept, correlation coefficient r and relative total error (RTE).

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[0195] The mean OD of the five calibrators was inversely proportional to their concentrations, but still showed an acceptable RSD of less than 20% even for the lowest FVIII concentration. The calibration curves had mean RSDs of 6.8% and 5.6% for the slope and y-intercept, respectively. In addition, their linearity was acceptably good, with a correlation coefficient higher than 0.9983 and an RTE lower than 13.0%.

[0196] Figure 10 shows the average calibration curve. The data demonstrates the robustness of the assay and shows that calibration curves can be constructed with acceptable reproducibility. Furthermore, the accuracy of the curves was checked by backfitting the measured ODs and calculating the percent agreement with the nominal values. Figure 11 shows these data for the five calibrators of the individual curves and illustrates that the backfit concentrations are within ±10% of the nominal concentrations over the entire range.

[0197] [Example 11] Detection of polysialylated FVIII from blood samples using MDAA This example illustrates the in vivo detection of blood proteins using MDAA.

[0198] Polysialylated recombinant FVIII was prepared as described in Example 7. The preparation contained 483 μg / mL N-acetylneuraminic acid, yielding a degree of polysialylation of approximately 7 to 8, with a FVIII:C activity of 3110 IU FVIII / mL, as measured by chromogenic assay. Polysialylated recombinant FVIII was administered to CD rats, and FVIII activity was monitored using MDAA for polysialylated FVIII in rat citrated plasma samples obtained pre-dose and 0.08, 0.5, 2, 6, 12, 24, 36, and 48 hours post-dose.

[0199] The modified recognition antibody was bound to a solid support as described in Example 7.

[0200] The samples were selectively bound to the solid support and FVIII activity was measured using the chromogenic assay described in Example 7.

[0201] Figure 12 shows the pharmacokinetic profile obtained. The data demonstrated that the MDAA for polysialylated FVIII was suitable for determining the pharmacokinetic profile of polysialylated FVIII in animal models containing endogenous FVIII. There was no interference from endogenous unmodified rat FVIII, and polysialylated FVIII could be measured with very high sensitivity.

[0202] [Example 12] Accuracy and precision of MDAA for polysialylated FVIII. This example illustrates the accuracy and precision of MDAA for polysialylated FVIII.

[0203] To bind the modified recognition antibody to the solid support, a 1 / 1,000 dilution of mouse anti-polysialic acid NCAM antibody (MAB5324-clone 2-2B, Millipore, Inc.,) in 0.1 M NaHCO 3 -Na 2 CO3 100 μL / well of coating antibody solution containing 100 μL / well of PBS, pH 9.5 was incubated in the wells of an F96 Maxisorp plate overnight at 0±10° C. The plate was then washed with wash buffer containing PBS and 0.05% Tween 20. The wells of the plate were then blocked by incubation of 200 μL / well of dilution buffer containing PBS and 10 mg / mL human serum albumin for 60±10 minutes at 37±5° C. The blocked wells were then washed with wash buffer.

[0204] To selectively bind the samples to the solid support, 100 μL of the following samples were added to the wells: 1) a dilution series of six samples of polysialylated recombinant FVIII standard prepared using a 1 / 10 solution of human plasma diluted in PBS, 3% skim milk and 50 mM benzamide, spanning FVIII concentrations ranging from 1.1 mU / mL to 34.2 mU / mL (representing a dilution factor of 1 / 6,000 to 1 / 192,000); 2) a dilution series of human plasma prepared using a 1 / 10 solution of human plasma diluted in PBS, 3% skim milk and 50 mM benzamide; or 3) a dilution series of test controls prepared using a 1 / 10 solution of human plasma diluted in PBS, 3% skim milk and 50 mM benzamide. Polysialylated recombinant FVIII was prepared by conjugating a 20 kDa polysialic acid reagent containing active aminooxy groups to the oxidized N-glycans of FVIII (see U.S. Patent Application Publication Nos. 2011 / 0027350 and 2011 / 0028693, each of which is incorporated herein by reference in its entirety). The preparation had a FVIII:C activity of 3110 IU FVIII / mL, as measured by a chromogenic method, and contained 483 μg / mL N-acetylneuraminic acid resulting in a degree of polysialylation of about 7-8. Samples were loaded onto the plate and incubated at about 18° C. to about 26° C. for 120±10 minutes. Under these conditions, polysialylated recombinant FVIII was selectively conjugated to the solid support via its polysialic acid (PSA) moiety using an anti-PSA antibody. The plates were then washed six times with wash buffer and then incubated with 200 μL / well of FVIII dilution buffer containing 3.4 g / L imidazole, 5.85 g / L NaCl, 10 mg / mL HSA, pH 7.4, equilibration was performed at about 18° C. to about 26° C. for 5 to 10 minutes.

[0205] After a washing step with washing buffer, the wells were emptied and the FVIII activity was measured by chromogenic assay after standard color development using Immunochrome FVIII Kit (Technoclone, GmbH, Vienna, Austria). The wells were filled with 20 μL of FVIII dilution buffer, after which 20 μL of reagent A and reagent B were added successively. The plate was placed on a mixer set at 500 rpm and incubated for 15 min at about 18° C. to about 26° C. Then 100 μL / well of substrate solution was added and incubated for 45 min at about 18° C. to about 26° C. Finally, the reaction was stopped by adding 40 μL / well of 20% acetic acid. The optical density (OD) was measured in an ELISA reader at 405 nm (reference wavelength 620 nm). The quantitative evaluation is based on a double logarithmic calibration curve. The blank-corrected mean OD of the individual calibration curve standards is related to their FVIII concentration. The resulting calibration curve is used to calculate the FVIII concentrations of samples if their OD is within the OD range covered by the calibration curve. Three different analyses were included in this study to investigate operator-dependent effects.

[0206] Table 6 shows the averages (n=112) of the original data of the calibration curves and their characteristics. In particular, it shows the averages of the blank-corrected ODs measured for the six calibration curve standards D1-D6, with FVIII concentrations ranging from 34.2 to 1.1 mU / mL, and for the corresponding blanks. Besides these direct assay readouts, the characteristic slopes of the calibration curves, the y-intercepts and the correlation coefficients of the calibration curves obtained are shown. Finally, the relative total error (RTE), a combined measure of the accuracy and precision of the curve fit, is shown. The RTE was calculated by backfitting the ODs measured for each calibrator curve standard. The concentrations thus obtained were multiplied by the dilution factor and finally averaged to obtain the average backfit concentration. The RTE is the sum of the absolute differences between the nominal concentration and the average backfit concentration of the assay standards, and is calculated by multiplying the double standard deviation of the backfit average concentration by the nominal concentration.

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[0207] The direct readout of MDAA, the blank-corrected OD of the calibration curve standards, when expressed as the RSD of the average of 112 calibration curves, showed an acceptable variability of 20% or less. The RSDs measured for the individual calibration curve standards varied slightly, ranging from 16.3% to 20.0%. Also, the RSD determined for the assay blank (14.7%) was not appreciably different from these RSDs. Since the entire procedure of the assay was performed at room temperature, these data indicate slight variations in room temperature that could cause variations in the direct readout. However, these slight variations are not expected to adversely affect the assay performance, as shown by the data below. These data yielded very similar calibration curves: their average slopes and y-intercepts had RSDs of 2.6% to 6.7%, respectively, indicating parallelism for these curves constructed on different days by different operators. Furthermore, these curves were linear, as demonstrated by correlation coefficients ranging from 0.9972 to 1.0000 with an average correlation coefficient of r=0.9990. Since the correlation coefficient can be a false indicator of the accuracy of the calibration curve, we also calculated the RTE as a better measure of the performance of the calibration curve. Thus, we obtained an average RTE of 11.2% with individual values ​​ranging from 2.4% to 19.7%. In a more rigorous test, only 12 of the 112 RTEs were higher than 15%, whereas 41 RTEs were lower than 10%. As a further test of the quality of the fit and to validate the selected calibration model, we also calculated the percent agreement of the backfit assay calibration curve standards with their nominal concentrations. Table 7 shows the average results and ranges obtained. [Table 7]

[0208] The average backfit concentrations of the assay calibration standards were in good agreement with their respective nominal concentrations. Assay standard D1 with the highest polysialylated recombinant FVIII concentration showed the highest deviation from the nominal concentration, whereas the lowest assay standard D6 differed little from assay standards D3 and D4. This demonstrated that samples with low polysialylated recombinant FVIII concentrations could also be accurately estimated. However, all individual backfit concentrations were within the range according to current regulations, which require a ±20% agreement rate for at least 70% of the assay calibration standards after backfitting. Figure 13 shows the average calibration curve obtained from 24 trials. The insert shows the agreement rate of the backfit assay calibration standards as a % of the respective nominal concentration.

[0209] Overall, all data confirmed the accuracy, precision and robustness of constructing a calibration curve for MDAA to measure polysialylated FVIII.

[0210] [Example 13] Specificity of MDAA for polysialylated FVIII This example illustrates the specificity of MDAA for polysialylated FVIII using a competitive assay.

[0211] The modified recognition antibody was bound to a solid support as described in Example 12.

[0212] Samples were selectively bound to a solid support and FVIII activity was measured using a chromogenic assay as described in Example 12, with the exception that 1) recombinant PEGylated FVIII samples were diluted to a concentration of 68.4 mU / mL and then mixed 1+1 with 20 kDa polysialic acid (Lipoxen, UK) to achieve final concentrations ranging from 0.06 μg / mL to 1,000 μg / mL.

[0213] Each competing sample was measured twice in one run. The average signal obtained was then related back to that obtained from the sample containing no competitor, and the relative signal was calculated. Figure 14 shows the competition curve obtained by this approach, and in addition, the IC 50 , i.e., semi-competitive, and this IC was run using GraphPad Prism version 5.00 for Windows, GraphPad Software. 50 The corresponding confidence interval CI calculated for 95% The polysialic acid concentrations that provide a 100% fold increase in the capture efficiency are shown in Table 1. The data obtained clearly confirm the specificity of the capture step, since we measured a clear dose-dependent decrease in the signal measured in the presence of polysialic acid. Half-competition was reached under the given assay conditions with a polysialic acid concentration of 60.5 μg / mL.

[0214] [Example 14] Precision of MDAA for polysialylated FVIII This example illustrates the precision of MDAA for polysialylated FVIII.

[0215] The modified recognition antibody was bound to a solid support as described in Example 12.

[0216] Samples were selectively bound to a solid support and FVIII activity was measured using the chromogenic assay described in Example 12, except that 1) six serial 1+1 dilutions of recombinant PEGylated FVIII were prepared starting at a dilution of 1:5,000.

[0217] Results were estimated from only those mean ODs that were within the range covered by the calibration curve, and at least three dilutions were used for that purpose. In most cases, however, the average was calculated for five individual dilutions of the dilution series that yielded an RSD clearly lower than 15%. This confirmed that the dilution series of the assay controls was parallel to that of the assay calibrators and also met the requirement to measure several control samples with high medium and low analyte concentrations. The results represent 39 runs performed by two operators, yielding a total of 113 measurements of polysialylated recombinant FVIII. The mean concentration of 180±11.2 U / mL was converted to an RSD of 6.2%, indicating the precision between runs of the assay. The mean inter-run precision, determined for 2-4 measurements per run, was 3.8%, with values ​​ranging from 0.3% to 7.4%. Figure 15 shows these data and also shows the frequency distribution of the data.

[0218] Considering that this method combines the principles of ligand binding assay and activity assay, the precision of MDAA for polysialylated recombinant FVIII was excellent. Therefore, a relatively high sample dilution, i.e. a dilution series starting at 1 / 5,000 dilution, is required for the selected capture agent. The following chromogenic activity assay involves several reagent replacement and incubation steps. Although the whole procedure is obviously complicated, the RSD, which indicates the precision between runs, was low. The average of 39 trials had an RSD of 6.2%.

[0219] [Example 15] Accuracy and precision of MDAA for polysialylated FVIII in plasma This example illustrates the accuracy and precision of the MDAA for polysialylated FVIII in plasma using a spike recovery assay.

[0220] The modified recognition antibody was bound to a solid support as described in Example 12.

[0221] The samples were selectively bound to a solid support and FVIII activity was measured using the chromogenic assay described in Example 12, except that the samples included plasma from FVIII-deficient mice (E17), rats, and cynomolgus monkeys, measured before and after being spiked with five concentrations of polysialylated recombinant FVIII ranging from 0.05 U / mL to 15 U / mL.

[0222] The intra-run precision (n=6; determined for polysialylated recombinant FVIII at concentrations of 0.05, 0.5 and 15 U / mL) and inter-run precision (n=6; determined for 0.05, 0.25, 0.5, 1 and 15 U / mL) were determined for spiked plasma samples, whereas the precision of the assay was determined by calculating the recovery of the spiked amount. Finally, the total error was calculated as the sum of the inter-run precision and precision. Current EMA guidelines propose that this total error should not exceed 30% or 40% at the lower limit of quantification (LLOQ) of the assay. Table 8 summarizes the precision data obtained for spiked animal plasma and buffer samples, expressed as the corresponding mean RSD. [Table 8]

[0223] We found RSDs below 10% for all but one sample. This particular monkey plasma sample for which we determined an RSD of 13.8% was spiked at 0.05 U / ml, representing the lower limit of quantification of the assay. The mean RSDs, showing the inter- and intra-run precision over the range investigated, were entirely independent of the actual concentration and the respective animal plasma used for spiking. Thus, the precision profile qualified the MDAA for polysialylated recombinant FVIII as a highly precise bioanalytical method suitable for the measurement of polysialylated recombinant FVIII in animal plasma samples, as required, for example, to determine pharmacokinetic parameters. Table 9 shows the determined recovery as a measure of assay precision and finally the total error calculated for individual polysialylated recombinant FVIII concentrations at the limit of quantification of 0.05 U / ml and for polysialylated recombinant FVIII concentrations ranging from 0.25 U / ml to 15 U / ml. [Table 9]

[0224] The recovery of spiked polysialylated recombinant FVIII was excellent in all animal plasma samples tested here, even at the lowest spiked concentration of 0.05 U / mL. Also, the total error was below 15% in all cases investigated. These data, which easily meet the EMA guidelines for bioanalytical assay validation, clearly qualify the MDAA for polysialylated recombinant FVIII to be used for the measurement of polysialylated recombinant FVIII in laboratory animal plasma. Figure 16 finally shows the dose-response curves for all three animal plasma samples diluted 1 / 20 and spiked with polysialylated recombinant FVIII (0.068 U / mL). The minimum dilution of 1 / 20 was defined as the lower limit of quantification of the assay, as these data demonstrated no substantial impact on detectability.

[0225] The linearity of the dose-response curves obtained in animal plasma diluted 1 in 20 was as good as that obtained for each dilution series of the assay standards in buffer alone. Moreover, the slopes were very similar, differing by less than 2% for the spiked E17 and rat plasma samples and by less than 9% for the spiked monkey plasma samples.

[0226] [Example 16] MDAA for PEGylated FIX This example illustrates the MDAA for PEGylated FIX.

[0227] To bind the modified recognition antibody to the solid support, 4 μg / mL of rabbit anti-PEG antibody (B47-2061-1, Epitomics, Inc., Burlingame, CA), 0.1 M NaHCO 3 -Na 2 CO 3 100 μL / well of coating antibody solution containing PBS, 3% skim milk, and 50 mM benzamide was incubated in the wells of an F96 Maxisorp plate overnight at 0±10° C. The plate was then washed with a wash buffer containing PBS and 0.05% Tween 20. The wells of the plate were then blocked by incubation of 200 μL / well of blocking buffer containing PBS, 3% skim milk, and 50 mM benzamide for 60±10 minutes at about 18° C. to about 26° C. The blocked wells were then washed with wash buffer.

[0228] To selectively bind the samples to the solid support, 100 μL of the following samples were added to the wells: 1) a dilution series of PEGylated recombinant FIX standard prepared using PBS containing 10 mg / mL HSA and spanning FIX concentrations ranging from 45.5 mU / mL to 0.28 mU / mL, or 2) a dilution series of unmodified recombinant FIX prepared using PBS containing 10 mg / mL HSA and spanning FIX concentrations ranging from 350 mU / mL to 25,000 mU / mL. PEGylated recombinant FIX with a degree of PEGylation of about 1 was used. The samples were loaded into the plate and incubated at about 18° C. to about 26° C. for 60±10 minutes. Under these conditions, the PEGylated recombinant FIX was selectively bound to the solid support by its PEG moiety using an anti-PEG antibody. Plates were then washed six times with wash buffer and subsequently incubated with 200 μL / well of FIX dilution buffer containing 3.4 g / L imidazole, 5.85 g / L NaCl, 10 mg / mL HSA, pH 7.4. The equilibration was carried out for 3 min at room temperature. After a washing step with wash buffer, wells were emptied and FIX activity was measured using the FIX chromogenic assay according to the manufacturer's instructions (HYPHEN Biomed, Vienna, Austria).

[0229] Figure 17 shows the data obtained for PEGylated and unmodified FIX. PEGylated FIX showed a clear concentration-dependent signal with a linear concentration vs. signal relationship in the range of 5.7-0.28 mU FIX / mL. In contrast, unPEGylated FIX did not show any significant signal even at 1000-fold higher concentrations.

[0230] [Example 17] MDAA Coagulation Assay Format for PEGylated FVIII This example illustrates that MDAA for PEGylated recombinant FVIII can also be performed in a clotting assay format.

[0231] To bind the modified recognition antibody to the solid support, MaxiSorp Startubes (Nunc) were incubated with 0.5 mL of coating antibody solution containing PBS and 20 μg / mL of affinity purified rabbit anti-PEG antibody (#A151) for 18 hours at 0±10° C. The tubes were then washed with a washing buffer containing PBS and 0.05% Twenn 20. The wells of the plate were then blocked by incubation of 1 mL of blocking buffer containing PBS, 3% skim milk, and 50 mM benzamidine for 60±10 minutes at about 18° C. to about 26° C. The blocked wells were then washed with the washing buffer.

[0232] To selectively bind the samples to the solid support, 0.5 mL of 1) a dilution series of five samples containing PEGylated recombinant FVIII standard spanning FVIII concentrations ranging from 0.04 mU / mL to 4 mU / mL, and 2) a dilution series of five samples containing Advate recombinant FVIII standard spanning FVIII concentrations ranging from 0.04 mU / mL to 4 mU / mL. The dilution buffer served as a blank. The samples were loaded into tubes and incubated at about 18°C ​​to about 26°C for 60±10 minutes. The plate was then washed six times with wash buffer, followed by incubation with 0.5 mL of FVIII dilution buffer containing 3.4 g / L imidazole, 5.85 g / L NaCl, 10 mg / mL HSA, pH 7.4. The equilibration was performed at about 18°C ​​to about 26°C for 5 to 10 minutes. The tubes were then emptied and the clotting assay was performed by adding 200 μL of FVIII dilution buffer and 100 μL of FVIII-deficient plasma (#481C00D, Technoclone, GmbH, Vienna, Austria). 2 The mixture was incubated for 3 min at 37±5° C. before adding the 100% glycerol. The tubes were kept in a water bath at 37±5° C. and visually inspected for clot formation.

[0233] Figure 18 shows the results obtained. Clotting occurred only in the tubes containing PEGylated recombinant FVIII, while the tubes containing Advate recombinant FVIII showed no signs of clot formation within 80 minutes. Furthermore, there was a relationship between clotting time and concentration of PEGylated recombinant FVIII. These data demonstrate that techniques other than chromogenic activity measurements can be used with MDAA.

[0234] [Example 18] MDAA Coagulation Assay Format for Polysialylated FVIII This example demonstrates that MDAA for polysialylated recombinant FVIII can also be performed in a clotting assay format.

[0235] To bind the modified recognition antibody to the solid support, MaxiSorp Startubes (Nunc) were incubated with 0.5 mL of coating antibody solution containing PBS and 20 μg / mL of mouse anti-PSA NCAM antibody (MAB5324) for 18 hours at 0±10° C. The tubes were then washed with a washing buffer containing PBS and 0.05% Tween 20. The wells of the plate were then blocked by incubation of 1 mL of blocking buffer containing PBS, 3% skim milk, and 50 mM benzamide for 60±10 minutes at about 18° C. to about 26° C. The blocked wells were then washed with the washing buffer.

[0236] To selectively bind the samples to the solid support, 0.5 mL of 1) a dilution series of five samples containing polysialylated recombinant FVIII standard spanning FVIII concentrations ranging from 0.04 mU / mL to 4 mU / mL, and 2) a dilution series of five samples containing Advate recombinant FVIII standard spanning FVIII concentrations ranging from 0.04 mU / mL to 4 mU / mL. The dilution buffer served as a blank. The samples were loaded into tubes and incubated at about 18°C ​​to about 26°C for 60±10 minutes. The plate was then washed six times with wash buffer, followed by incubation with 0.5 mL of FVIII dilution buffer containing 3.4 g / L imidazole, 5.85 g / L NaCl, 10 mg / mL HSA, pH 7.4. The equilibration was performed at about 18°C ​​to about 26°C for 5 to 10 minutes. The tubes were then emptied and the clotting assay was performed by adding 200 μL of FVIII dilution buffer and 100 μL of FVIII-deficient plasma (#481C00D, Technoclone, GmbH, Vienna, Austria). 2 The mixture was incubated for 3 min at 37±5° C. before adding the 100% glycerol. The tubes were kept in a water bath at 37±5° C. and visually inspected for clot formation.

[0237] The results obtained are shown in Figure 19. Clotting occurred only in the tubes containing polysialylated recombinant FVIII, while the tubes containing Advate showed no signs of clot formation within 80 minutes. Furthermore, there was a relationship between clotting time and the concentration of polysialylated rFVIII. These data demonstrate that techniques other than chromogenic activity measurements can also be used for MDAA.

[0238] In closing, although aspects of the present specification are emphasized by reference to specific embodiments, it is understood that one skilled in the art will readily recognize that these disclosed embodiments are merely illustrative of the principles of the subject matter disclosed herein. Thus, it should be understood that the disclosed subject matter is in no way limited to the specific methods, protocols, and / or reagents, etc. described herein. Thus, various modifications or changes to the disclosed subject matter or alternative configurations of the disclosed subject matter may be made in accordance with the teachings herein without departing from the spirit of the present specification. Finally, the terminology used herein is for the purpose of describing only specific embodiments, and is not intended to limit the scope of the present invention, which is defined only by the claims. Thus, the present invention is not limited to exactly what has been shown and described.

[0239] Certain embodiments of the present invention have been described herein, including the best mode known to the inventors for carrying out the invention. Of course, modifications to these described embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors anticipate that such modifications will be utilized by those skilled in the art as necessary, and the inventors intend for the invention to be practiced in ways other than as specifically described herein. Accordingly, the present invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described embodiments in all possible variations thereof is encompassed by the present invention, unless otherwise indicated herein or otherwise clearly contradicted by context.

[0240] The categorization of alternative embodiments, elements, or steps of the invention should not be construed as limiting. Each group member may be referenced and claimed individually or in any combination with other group members disclosed herein. It is understood that one or more members of a group may be included in or deleted from a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to include the group as modified, thus satisfying all Markush group descriptions used in the appended claims.

[0241] Unless otherwise indicated, all numbers expressing features, items, amounts, parameters, characteristics, periods, etc. used in the specification and claims are understood to be modified in all instances by the term "about". As used herein, the term "about" means that the so specified feature, item, amount, parameter, characteristic, or period encompasses a range above and below the stated feature, item, amount, parameter, characteristic, or period, plus or minus 10 percent of the value. Thus, unless indicated to the contrary, the numerical parameters set forth in the specification and the appended claims are approximations that may vary. At the very least, and not to limit the application of the doctrine of equivalents to the scope of the claims, each numerical indication should be construed at least in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and values ​​setting forth the broad scope of the invention are approximations, the numerical ranges and values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical range or value inherently contains certain necessary errors resulting from the standard deviation found in their respective testing measurements. The recitation of numerical ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and unless otherwise indicated herein, each individual value of the numerical range is incorporated herein as if it were individually recited herein.

[0242] The terms "a," "an," "the," and similar referents used in the context of describing the invention (particularly in the context of the appended claims) are to be construed to include both the singular and the plural, unless otherwise indicated herein or otherwise clearly contradicted by context. All methods described herein may be performed in any suitable order, unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is solely for the purpose of making the invention easier to understand and does not pose a limitation on the scope of the invention or otherwise the claims. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0243] Certain embodiments disclosed herein may be further limited in the claims using the language consisting of or consisting essentially of. When used in a claim, whether filed by amendment or added, the transitional phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. The transitional phrase "consisting essentially of" limits the claim to those specified materials or steps and which do not materially affect the basic and novel characteristic(s). Embodiments of the invention so claimed are essentially or distinctly described and enabled herein.

[0244] All patents, patent publications, and other publications referenced or identified in this specification are individually and specifically incorporated herein by reference in their entirety for the purpose of describing and disclosing, for example, the compositions and methods described in such publications that may be used in connection with the present invention. These publications are provided merely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to such prior disclosure by reason of prior invention or for any other reason. All statements as to the date or representation of the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the correctness of the dates or contents of these documents.

Claims

1. 1. A method for detecting the presence of a recombinant coagulation factor comprising the steps of: incubating a sample containing the recombinant coagulation factor comprising the modification with a capture agent that selectively binds to the modification under conditions that allow selective binding of the capture agent to the modification, thereby forming a factor-agent complex; purifying the factor-agent complex from the sample; Assaying for clotting factor activity. wherein detecting said clotting factor activity indicates the presence of a recombinant clotting factor comprising said modification.

2. The method of claim 1, wherein the sample further comprises coagulation factors that do not have the modification and / or coagulation factors that have different patterns of degree of modification.

3. 3. The method of claim 1 or 2, wherein the recombinant coagulation factor is Factor II, Factor IIa, Factor VII, Factor VIIa, Factor VIII, Factor VIIIa, Factor IX, Factor IXa, Factor X, or Factor Xa.

4. 4. The method of any one of claims 1 to 3, wherein the sample comprises a purified preparation of the recombinant coagulation factor, a partially purified preparation of the recombinant coagulation factor, a non-purified preparation of the recombinant coagulation factor, a formulated preparation of the recombinant coagulation factor, a crude extract of the recombinant coagulation factor, a fractionated extract of the recombinant coagulation factor, a cell lysate comprising the recombinant coagulation factor, or a biological sample.

5. 5. The method of claim 4, wherein the biological sample comprises cells, a tissue sample, a blood sample, a body fluid sample, or an organ sample obtained directly from an individual.

6. 6. The method of claim 5, wherein the bodily fluid is urine, sputum, semen, feces, saliva, bile, cerebrospinal fluid, nasal swab, urogenital swab, nasal aspirate, or spinal fluid.

7. 5. The method of claim 4, wherein the biological sample is a preparation derived from a sample obtained directly from an individual.

8. 8. The method of claim 7, wherein the preparation derived from a sample obtained directly from an individual is a plasma fraction of a blood sample, a serum fraction of a blood sample, or an eluate from a purification process.

9. The method according to any one of claims 1 to 8, wherein the sample is treated to increase the detectability of the activity of the recombinant coagulation factor.

10. 10. The method of claim 9, wherein the treatment comprises dissolving, diluting, purifying, extracting, filtering, distilling, separating, concentrating, inactivating interfering components, adding reagents, or any combination thereof.

11. The capture agent is 1×10 5 M -1 s -1 Super, 1×10 6 M -1 s -1 Super, 1×10 7 M -1 s -1 Over or 1×10 8 M -1 s -1 The method of any one of claims 1 to 10, wherein the coagulation factor has an association rate constant for the coagulation factor comprising the modification of greater than 100%.

12. The capture agent is 1×10 -3 s -1 Less than 1×10 -4 s -1 Less than or 1×10 -5 s -1 The method of any one of claims 1 to 11, wherein the coagulation factor has a dissociation rate constant for said coagulation factor comprising said modification of less than 100 mM.

13. 13. The method of any one of claims 1 to 12, wherein the capture agent has an equilibrium dissociation constant for the coagulation factor comprising the modification of less than 0.500 nM, less than 0.450 nM, less than 0.400 nM, less than 0.350 nM, less than 0.300 nM, less than 0.250 nM, less than 0.200 nM, less than 0.150 nM, less than 0.100 nM, or less than 0.050 nM.

14. The capture agent is 1×10 0 M -1 s -1 Less than 1×10 1 M -1 s -1 Less than 1×10 2 M -1 s -1 Less than 1×10 3 M -1 s -1 Less than or 1×10 4 M -1 s -1 The method of any one of claims 1 to 13, wherein the coagulation factor has an association rate constant for a coagulation factor without the modification or for a coagulation factor with a different pattern or degree of modification of less than 1.

15. The association rate constant (Ka) of the capture agent for a recombinant coagulation factor containing a modification is greater than or equal to 1×10 compared to the association rate constant (Ka) of the capture agent for a recombinant coagulation factor without a modification and / or the association rate constant (Ka) of the capture agent for a recombinant coagulation factor having a different pattern or degree of modification. 0 M -1 s -1 Large, 1×10 1 M -1 s -1 Large, 1×10 2 M -1 s -1 Large, 1×10 3 M -1 s -1 Large or 1×10 4 M -1 s -1 The method according to any one of claims 1 to 14, wherein the

16. 15. The method of any one of claims 1 to 14, wherein the association rate constant (Ka) of the capture agent for a recombinant coagulation factor comprising a modification is at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 100-fold greater, at least 1,000-fold greater, or at least 10,000-fold greater than the association rate constant (Ka) of the capture agent for a recombinant coagulation factor without the modification and / or compared to the association rate constant (Ka) of the capture agent for a recombinant coagulation factor having a different pattern or degree of modification.

17. 15. The method of any one of claims 1 to 14, wherein the ratio of binding specificity of the capture agent to a recombinant coagulation factor comprising a modification to a recombinant coagulation factor without a modification and / or with a different pattern or degree of modification is at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, at least 8:1, at least 9:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 35:1, or at least 40:

1.

18. The method of any one of claims 1 to 17, wherein the capture agent is a multivalent capture agent.

19. The method of any one of claims 1 to 18, wherein the capture agent distinguishes between the recombinant coagulation factor comprising a modification and the same coagulation factor but without the modification.

20. 20. The method of any one of claims 1 to 19, wherein the capture agent distinguishes between the recombinant coagulation factor comprising a modification and the same coagulation factor but with a different pattern or degree of the same modification.

21. The method of any one of claims 1 to 20, wherein the capture agent is an antibody.

22. The antibody may be an anti-acetate antibody, an anti-phosphate antibody, an anti-lipid antibody, or an anti-carbohydrate antibody, an anti-myristate antibody, an anti-palmitate antibody, an anti-farnesol antibody, an anti-geranylgeraniol antibody, or an anti-glycosylphosphatidylinositol (GPI) antibody, an anti-lipoate antibody, an anti-flavin antibody, an anti-heme C antibody, an anti-4'-phosphopantetheinyl antibody, an anti-retinylidene antibody, an anti-diphthamide antibody, an anti-ethanolamine phosphoglycerol antibody, an anti-heme C antibody, an anti-phosphoryl phosphatidyl ... psin antibody, anti-acetyl antibody, anti-formyl antibody, anti-alkyl antibody, anti-methyl antibody, anti-amide antibody, anti-amino acid antibody, anti-butyl antibody, anti-carboxyl antibody, anti-glycosyl antibody, anti-polysialic acid antibody, anti-hydroxyl antibody, anti-malonyl antibody, anti-iodine antibody, anti-phosphate antibody, anti-adenylyl antibody, anti-succinyl antibody, anti-sulfate antibody, anti-selenium antibody, anti-carbohydrate antibody, anti-polysaccharide antibody, anti-starch antibody, anti-hydroxyl-ethyl starch (HES) antibody, anti-saccharide antibody, anti-poly 22. The method of claim 21, wherein the antibody is an ethylene glycol (PEG) antibody, an anti-ubiquitin antibody, an anti-pullulan antibody, an anti-chitosan antibody, an anti-hyaluronic acid antibody, an anti-chondroitin sulfate antibody, an anti-dermatan sulfate antibody, an anti-dextran antibody, an anti-carboxymethyl-dextran antibody, an anti-polyalkylene oxide (PAO) antibody, an anti-polyalkylene glycol (PAG) antibody, an anti-polypropylene glycol (PPG) antibody, an anti-polyoxazoline antibody, an anti-polyacryloylmorpholine antibody, an anti-polyvinyl alcohol (PVA) antibody, an anti-polycarboxylate antibody, an anti-polyvinylpyrrolidone (PVP) antibody, an anti-polyphosphazene antibody, an anti-polyoxazoline antibody, an anti-polyethylene-co-maleic anhydride antibody, an anti-polystyrene-co-maleic anhydride antibody, an anti-poly(1-hydroxymethylethylene hydroxymethylformal) (PHF) antibody, or an anti-2-methacryloyloxy-2'-ethyltrimethylammonium-phosphate (MPC) antibody.

23. 23. The method of any one of claims 1 to 22, wherein the recombinant coagulation factor comprising the modification is PEGylated Factor II, PEGylated Factor IIa, polysialylated Factor II, polysialylated Factor IIa, HES-linked Factor II, HES-linked Factor IIa, silylated Factor II, or silylated Factor IIa.

24. 23. The method of any one of claims 1 to 22, wherein the recombinant coagulation factor comprising said modification is PEGylated Factor VII, PEGylated Factor VIIa, polysialylated Factor VII, polysialylated Factor VIIa, HESylated Factor VII, HESylated Factor VIIa, silylated Factor VII, or silylated Factor VIIa.

25. The method according to any one of claims 1 to 22, wherein the recombinant coagulation factor comprising the modification is PEGylated Factor VIII, PEGylated Factor VIIIa, polysialylated Factor VIII, polysialylated Factor VIIIa, HES-modified Factor VIII, HES-modified Factor VIIIa, silylated Factor VIII, or silylated Factor VIIIa.

26. 23. The method of any one of claims 1 to 22, wherein the recombinant coagulation factor comprising said modification is PEGylated Factor IX, PEGylated Factor IXa, polysialylated Factor IX, polysialylated Factor IXa, HESylated Factor IX, HESylated Factor IXa, silylated Factor IX, or silylated Factor IXa.

27. 23. The method of any one of claims 1 to 22, wherein the recombinant coagulation factor comprising said modification is PEGylated Factor X, PEGylated Factor Xa, polysialylated Factor X, polysialylated Factor Xa, HESylated Factor X, HESylated Factor Xa, silylated Factor X, or silylated Factor Xa.

28. The method of any one of claims 1 to 26, wherein the capture agent is bound to a solid support.

29. 30. The method of claim 28, wherein the solid support is a multiwell plate, a film, a tube, a sheet, a column, or a microparticle.

30. The method of any one of claims 1 to 29, wherein the assaying step is carried out using a qualitative or quantitative assay.

31. The modification may be an acetate group, a phosphate group, a lipid group, a carbohydrate group, a myristate group, a palmitate group, an isoprenoid group, a glycosylphosphatidylinositol (GPI) group, a lipoate group, a flavin group, a heme C group, a 4'-phosphopantetheinyl group, a retinylidene group, a diphthamide group, an ethanolamine phosphoglycerol group, a hypusine group, an acetyl group, a formyl group, an alkyl group, a methyl group, an amide group, an amino acid, a butyl group, a carboxyl group, a glycosyl group, a polysialic acid (PSA) group, a hydroxyl group, a malonyl group, an iodine group, a phosphate group, an adenylyl group, a succinyl group, a sulfate group, a selenium group, a carbohydrate group, a starch group, a hydroxyl-ethyl starch group, a polysaccharide group, a saccharide group, a polyethylene glycol group, a ubiquitin group, 31. The method according to any one of claims 1 to 30, wherein the polysaccharide is a pullulan group, a chitosan group, a hyaluronic acid group, a chondroitin sulfate group, a dermatan sulfate group, a dextran group, a carboxymethyl-dextran group, a polyalkylene oxide group, a polyalkylene glycol group, a polypropylene glycol group, a polyoxazoline group, a polyacryloylmorpholine group, a polyvinyl alcohol group, a polycarboxylate group, a polyvinylpyrrolidone group, a polyphosphazene group, a polyoxazoline group, a polyethylene-co-maleic anhydride group, a polystyrene-co-maleic anhydride group, a poly(1-hydroxymethylethylene hydroxymethylformal) group, or a 2-methacryloyloxy-2'-ethyltrimethylammonium-phosphate group.

32. The recombinant coagulation factor may be modified by any of the following processes: myristoylation, palmitoylation, isoprenylation (prenylation), glypiation, lipoylation, flavinylation, phosphopantetheinylation, retinylidenation, diphthamidylation, ethanolamine phosphoglycerylation, hypusination, acylation, acetylation, formylation, alkylation, amidation, arginylation, polyglutamylation, polyglycylation, butyrylation, gamma-carboxylation, glycosylation, polysialylation, malonylation, hydroxylation, iodination, nucleoylation, glycer ... The method according to any one of claims 1 to 31, wherein the protein is modified by any one of the following: erythroylation, oxidation, phosphoroesterification, phosphoramidation, phosphorylation, adenylylation, propionylation, pyroglutamylation, S-glutathionylation, S-nitrosylation, succinylation, sulfation, selenoylation, glycation, biotinylation, acylation, PEGylation, HESylation, starchylation, citrullination, deamidation, eliminylation, carbamylation, deimination, pupylation, NEDDylation, ubiquitination, SUMOylation, or ISGylation.

33. 33. Use of a kit for carrying out the method according to any one of claims 1 to 32, comprising a capture agent that selectively binds to the modification under conditions allowing selective binding of the capture agent to the modification, thereby forming a coagulation factor-agent complex.

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