Method for detection of a plasma protein

Abstract

The present disclosure relates to a method of determining the concentration of a plasma protein in a sample using fluorescence polarization, wherein the sample is in an assay solution comprising a neutral hydrophilic polymer.

Description

[0001] METHOD FOR DETECTION OF A PLASMA PROTEIN

[0002] RELATED APPLICATION DATA

[0003] The present application claims priority from European Patent Application No. 24209490.2 entitled “Method for detection of a plasma protein” filed on 29 October 2024, the entire contents of which is hereby incorporated by reference.

[0004] FIELD

[0005] The present disclosure relates to a method of determining the concentration of a plasma protein in a sample using fluorescence polarization, wherein the sample is in an assay solution comprising a neutral hydrophilic polymer.

[0006] BACKGROUND

[0007] The therapeutic use of plasma-derived proteins is ever increasing however, protein purification is one of the most costly aspects of therapeutic protein production. Existing methods of protein purification include chromatography (e.g. affinity chromatography, anion exchange chromatography, hydrophobic interaction chromatography, SE-HPLC) and non-chromatography (e.g. precipitation and liquid extraction) purification methods. Major obstacles of existing methods are the high cost and time involved in purification and the need to ensure that the product is of a suitable quality (e.g. purity and stability) for therapeutic use.

[0008] Currently, the levels of plasma proteins are determined at various stages of the manufacturing process to assess protein production or optimisation of conditions. Thus, it is imperative that the concentration of the plasma proteins is determined accurately. There are currently several different means used to quantify plasma proteins including nephelometry, biolayer interferometry, microagglutination assay, proximity assays (such as SPARCL™, HTRF®, and FRET -PINCER®), enzyme linked immunosorbent assay (ELISA) reactions and high-performance liquid chromatography (HPLC). More recently, fluorescence polarisation assays (such as the Valita® Titer Assay) have been used for the rapid, high throughput quantification of plasma immunoglobulin G (IgG). Whilst each of these assays are effective in detecting plasma proteins, to accurately detect plasma proteins (such as IgG) at each step during the manufacturing process each of these techniques requires adjustment of the reaction conditions, such as the sample preparation, dilution linearity, instrumentation requirements and time requirements, resulting in increased running costs and time delays, thus, limiting their usefulness in detecting plasma proteins during the entire manufacturing process.

[0009] It will therefore be apparent to the skilled person that there is a need in the art for improved methods for the detection of plasma proteins in a sample, that can be implemented at any stage during the manufacturing process.

[0010] SUMMARY

[0011] The present disclosure is based on the inventors’ identification of an improved method of determining the concentration of a plasma protein using fluorescence polarization in an assay solution comprising a neutral hydrophilic polymer. The inventors found that using an assay solution comprising a neutral hydrophilic polymer as a substrate provided dilution linearity with consistent and reliable results. In particular, the method allows efficient and accurate determination of the concentration of the plasma protein (such as immunoglobulin G (IgG)) in a plasma sample at any stage of the plasma collection and / or plasma fractionation process using the same conditions, as no sample related interference is observed. Importantly, being able to use a single method to detect plasma proteins at any step along the plasma collection and / or plasma fractionation process reduces processing times and costs compared to existing assays.

[0012] Accordingly, the present disclosure provides an improved method of determining the concentration of a plasma protein in a sample using fluorescence polarization, wherein the sample is in an assay solution comprising a neutral hydrophilic polymer.

[0013] The present disclosure provides a method of determining the concentration of a plasma protein in a sample, the method comprising:

[0014] i. incubating the sample and a plasma protein binding probe conjugated to a fluorescent dye in an assay solution, wherein the assay solution comprises a neutral hydrophilic polymer;

[0015] ii. assaying the incubated solution to detect fluorescence polarization;

[0016] iii. determining a change in polarization; and

[0017] iv. correlating the change in polarization with the concentration of the plasma protein in the sample.

[0018] In one example, the polymer has a hydrodynamic radius of at least about 2 nm. For example, the polymer has a hydrodynamic radius of at least about 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 nm. In one example, the polymer has a hydrodynamic radius of at least about 2.5 nm.

[0019] Methods of determining the hydrodynamic radius of the polymer will be apparent to the skilled person and / or described herein.

[0020] In one example, the polymer is selected from polyethylene glycol, polysucrose, or a derivative thereof. The present disclosure provides a method of determining the concentration of a plasma protein in a sample, the method comprising:

[0021] i. incubating the sample and a plasma protein binding probe conjugated to a fluorescent dye in an assay solution, wherein the assay solution comprises polyethylene glycol, polysucrose, or a derivative thereof;

[0022] ii. assaying the incubated solution to detect fluorescence polarization;

[0023] iii. determining a change in polarization; and

[0024] iv. correlating the change in polarization with the concentration of the plasma protein in the sample.

[0025] In one example, the polymer is polyethylene glycol or a derivative thereof. In one example, the polymer is polyethylene glycol.

[0026] In one example, the polyethylene glycol or derivative thereof has a molecular weight (kDa) between about 0.2 and about 200. For example, between about 0.2 and 150 kDa, or between about 1 and 150 kDa.

[0027] In one example, the polyethylene glycol or derivative thereof has a molecular weight (kDa) between about 1 and about 100. For example, between about 1 and about 90 kDa, or between about 1 and about 80 kDa, or between about 1 and about 70 kDa, or between about 1 and about 60 kDa. In another example, the polyethylene glycol or derivative thereof has a molecular weight (kDa) between about 1 and about 50, or between about 1 and about 40, or between about 1 and about 30, or between about 1 and about 20. In a further example, the polyethylene glycol or derivative thereof has a molecular weight (kDa) between about 1 and about 10.

[0028] In one example, the polyethylene glycol or derivative thereof has a molecular weight (kDa) between about 3 and about 10. For example, the polyethylene glycol or derivative thereof has a molecular weight (kDa) of about 3. In another example, the polyethylene glycol or derivative thereof has a molecular weight (kDa) of about 4. In one example, the polyethylene glycol or derivative thereof has a molecular weight (kDa) between about 5 and about 10. In a further example, the polyethylene glycol or derivative thereof has a molecular weight (kDa) of about 5. In one example, the polyethylene glycol or derivative thereof has a molecular weight (kDa) of about 6. In another example, the polyethylene glycol or derivative thereof has a molecular weight (kDa) of about 7. In a further example, the polyethylene glycol or derivative thereof has a molecular weight (kDa) of about 8. In one example, the polyethylene glycol or derivative thereof has a molecular weight (kDa) of about 9. In another example, the polyethylene glycol or derivative thereof has a molecular weight (kDa) of about 10.

[0029] The present disclosure provides a method of determining the concentration of a plasma protein in a sample, the method comprising:

[0030] i. incubating the sample and a plasma protein binding probe conjugated to a fluorescent dye in an assay solution, wherein the assay solution comprises polyethylene glycol having a molecular weight of about 8 kDa; ii. assaying the incubated solution to detect fluorescence polarization;

[0031] iii. determining a change in polarization; and

[0032] iv. correlating the change in polarization with the concentration of the plasma protein in the sample.

[0033] In one example, the polymer is polysucrose or a derivative thereof. For example, the polymer is polysucrose. In one example, the polysucrose or derivative thereof is a Ficoll®. The skilled person will recognise that Ficoll® refers to a neutral, highly branched, high-mass, hydrophilic polysaccharide. Accordingly, in one example, the polysucrose is a neutral, highly branched, high-mass, hydrophilic polysaccharide.

[0034] In one example, the Ficoll® has a molecular weight (kDa) between about 50 and about 500. For example, the Ficoll® has a molecular weight (kDa) between about 50 and 450. In one example, the Ficoll® has a molecular weight (kDa) of between about 50 and 100. For example, about 50, or about 60, or about 70, or about 80, or about 100. In one example, the Ficoll® has a molecular weight (kDa) of about 70. In another example, the Ficoll® has a molecular weight (kDa) between about 100 and about 450. For example, about 100, or about 150, or about 200, or about 250, or about 300, or about 350, or about 400, or about 450. In one example, the Ficoll® has a molecular weight (kDa) of about 400. In one example, the Ficoll® has a molecular weight (kDa) of about 70 or about 400.

[0035] In one example, the concentration of the polymer in the assay solution is between about 0.5 and 5% (w / v). In another example, the concentration of the polymer in the assay solution is between about 0.5 and 4% (w / v). In a further example, the concentration of the polymer in the assay solution is between about 0.5 and 3% (w / v). In one example, the concentration of the polymer in the assay solution is between about 0.5 and 2.5% (w / v). In another example, the concentration of the polymer in the assay solution is between about 0.5 and 2% (w / v). In a further example, the concentration of the polymer in the assay solution is between about 0.5 and 1.5% (w / v). In one example, the concentration of the polymer in the assay solution is between about 0.75 and 1.25% (w / v).

[0036] In one example, the concentration (in % w / v) of the polymer in the assay solution is about or greater than about 0.1, 0.5 or 1. In one example, the polymer is in the assay solution at a concentration of about 0.1 %(w / v). In another example, the polymer is in the assay solution at a concentration of about 0.5%. In one example, the polymer is in the assay solution at a concentration of about 0.75%. In a further example, the polymer is in the assay solution at a concentration of about 1%. In one example, the polymer is in the assay solution at a concentration of about 1.25%. In another example, the polymer is in the assay solution at a concentration of about 1.5%. In a further example, the polymer is in the assay solution at a concentration of about 2%. In one example, the method further comprises exciting the sample with plane polarized light. For example, the method further comprises exciting the sample with plane polarized light at a wavelength corresponding to an excitation wavelength of the fluorescent dye.

[0037] In one example, the method further comprises exciting the sample with plane polarized light immediately after incubating the sample and a plasma protein binding probe conjugated to a fluorescent dye in the assay solution. For example, the method comprises mixing the sample with the plasma protein binding protein in the assay solution and immediately exciting with plane polarized light.

[0038] In one example, the method further comprises detecting light intensity emitted by the fluorescent dye. For example, the method further comprises detecting light intensity emitted by the fluorescent dye at an appropriate emission wavelength in two planes, one parallel and one perpendicular to the emission plane.

[0039] In one example, the method comprises determining the change in polarisation between excitation and emission light.

[0040] In one example, the plasma protein binding probe specifically binds to the plasma protein.

[0041] In one example, the binding probe is selected from the group consisting of an immunoglobulin binding protein, an aptamer, a Fc receptor or fragment thereof, a glycoprotein, a glycoprotein receptor, a phospholipid, a plasminogen activator protein, a protein kinase domain, a pleckstrin homology domain, a coagulation factor, an anticoagulant, a lysine analogue, a divalent cation, a gamma-carboxyglutamic acid-rich (GLA) domain, a complement system component, a protease, a protease inhibitor, a small molecule, an amphipathic helix, an antibody or antigen binding fragment thereof, a heavy chain immunoglobulin, an antibody mimetic, a cibacronblue F3GA, a L-tryptophan and combinations thereof.

[0042] In one example, the binding probe is an immunoglobulin binding protein. For example, the immunoglobulin binding protein is selected from the group consisting of Protein A, Protein G, Protein L and combinations thereof. In one example, the binding probe is Protein A. In another example, the binding probe is Protein G. In a further example, the binding probe is Protein L.

[0043] In one example, the binding probe is selected from the group consisting of a truncated Protein A having a molecular weight of less than 20kD, a truncated Protein G having a molecular weight of less than 20kD and combinations thereof.

[0044] In one example, the binding probe comprises a truncated Protein A having a molecular weight of less than 20kD. For example, a molecular weight of less than 15kDa. In one example, the truncated Protein A has a molecular weight of less than 10kD. In one example, the truncated Protein A has a molecular weight of less than 8kD. In one example, the truncated Protein A has a molecular weight of less than 5kD. In one example, the truncated Protein A has one, two or three of the four antibody binding regions removed.

[0045] In another example, the binding probe comprises a truncated Protein G having a molecular weight of less than 20kD. For example, a molecular weight of less than 15kDa. In one example, the truncated Protein G has a molecular weight of less than 10kD. In one example, the truncated Protein G has a molecular weight of less than 8kD. In one example, the truncated Protein G has a molecular weight of less than 5kD. In one example, the truncated Protein G has one or two of the three antibody binding regions removed.

[0046] Suitable truncated Protein A and Protein G will be apparent to the skilled person and / or described herein. The present disclosure provides a method of determining the concentration of a plasma protein in a sample, the method comprising: i. incubating the sample and a truncated Protein G probe conjugated to a fluorescent dye in an assay solution, wherein the assay solution comprises polyethylene glycol having a molecular weight of about 8 kDa; ii. assaying the incubated solution to detect fluorescence polarization;

[0047] iii. determining a change in polarization; and

[0048] iv. correlating the change in polarization with the concentration of the plasma protein in the sample.

[0049] In one example, the binding probe is a nucleic acid. For example, the nucleic acid is an aptamer. In one example, the binding probe is an aptamer. For example, the aptamer is a DNA aptamer or an RNA aptamer. In one example, the binding probe is a DNA aptamer. In another example, the binding probe is a RNA aptamer.

[0050] In one example, the binding probe is a Fc receptor or fragment thereof. For example, the Fc receptor or fragment thereof is a Fc-gamma receptor (FcyR), a Fc-alpha receptor (FcaR), a Fc-epsilon receptor (FceR) and / or a polymeric immunoglobulin receptor (plgR). In one example, the binding probe is a Fc-gamma receptor (FcyR). In another example, the binding probe is a Fc-alpha receptor (FcaR). In a further example, the binding probe is a Fc-epsilon receptor (FceR). In one example, the binding probe is a polymeric immunoglobulin receptor (plgR).

[0051] In one example, the binding probe is a glycoprotein. For example, the glycoprotein is a glycoprotein lb (GPIb), a thrombomodulin, von Willebrand factor (vWF) and / or a lactadherin domain. In one example, the glycoprotein is a glycoprotein lb (GPIb). In another example, the glycoprotein is a thrombomodulin. In a further example, the glycoprotein is a vWF. For example, the vWF is a full length or truncated vWF. In one example, the glycoprotein is a lactadherin domain.

[0052] In one example, the binding probe is a glycoprotein receptor. For example, the glycoprotein receptor is an endothelial protein C receptor (EPCR).

[0053] In one example, the binding probe is a phospholipid.

[0054] In one example, the binding probe is a plasminogen activator protein. For example, the plasminogen activator protein is staphylokinase.

[0055] In one example, the binding probe is a protein kinase domain. For example, the protein kinase domain is a protein kinase C domain.

[0056] In one example, the binding probe is a pleckstrin homology domain.

[0057] In one example, the binding probe is a coagulation factor. For example, the coagulation factor is an antihemophilic factor, a Factor Xa (FXa) and / or a Factor la (Fla). In one example, the binding probe is an antihemophilic factor. In another example, the binding probe is a FXa. In a further example, the binding probe is a Fla.

[0058] In one example, the binding probe is an anti-coagulant. For example, the anti-coagulant is warfarin, heparin, Protein S and / or a FXa inhibitor. In one example, the binding probe is warfarin. In another example, the binding probe is heparin. In a further example, the binding probe is Protein S. In one example, the binding probe is a FXa inhibitor.

[0059] In one example, the binding probe is a lysine analogue.

[0060] In one example, the binding probe is a divalent cation. For example, the divalent cation is a calcium (Ca2+) ion and / or a zinc (Zn2+) ion. In one example, the binding probe is calcium (Ca2+) ions. In another example, the binding probe is zinc (Zn2+) ions.

[0061] In one example, the binding probe is a gamma-carboxy glutamic acid-rich (GLA) domain.

[0062] In one example, the binding probe is a complement system component. For example, the complement system component is a complement component 1q (C1q). In one example, the binding probe is a protease. For example, the protease is a serine protease. In one example, the serine protease is a urokinase, a tissue plasminogen activator, an elastase, a kallikrein, a high molecular weight kallikrein and / or a thrombin. In one example, the binding probe is a urokinase. In another example, the binding probe is a tissue plasminogen activator. In a further example, the binding probe is an elastase. In one example, the binding probe is a kallikrein. In another example, the binding probe is a high molecular weight kallikrein. In one example, the binding probe is a thrombin.

[0063] In one example, the binding probe is a protease inhibitor. For example, the protease inhibitor is a serine protease inhibitor or a proteinase inhibitor. In one example, the protease inhibitor is a serine protease inhibitor. For example, the serine protease inhibitor is an elastase inhibitor, a bovine pancreatic trypsin inhibitor (BPTI), an aprotinin, an alpha 2-antiplasmin inhibitor, an alpha 1 -antichymotrypsin, a phenylmethylsulfonyl fluoride and / or a benzamidine. In one example, the binding probe is an elastase inhibitor. In another example, the binding probe is a BPTI. In a further example, the binding probe is aprotinin. In one example, the binding probe is an alpha 2-antiplasmin inhibitor. In one example, the binding probe is an alpha 1 -antichymotrypsin. In another example, the binding probe is a phenylmethylsulfonyl fluoride. In a further example, the binding probe is benzamidine. In one example, the protease inhibitor is a serine, cysteine and threonine protease inhibitor. For example, the serine, cysteine and threonine protease inhibitor is a leupeptin.

[0064] In one example, the binding probe is a small molecule. For example, the small molecule is an anxiolytic benzodiazepine or a salicylate. In one example, the binding probe is an acetylsalicylic acid. In one example, the acetylsalicylic acid is aspirin. In one example, the anxiolytic benzodiazepine is diazepam.

[0065] In one example, the binding probe is an amphipathic helix.

[0066] In one example, the binding probe is protein-based, e.g., a peptide, polypeptide or protein.

[0067] In one example, the binding probe comprises a variable region fragment (Fv). In one example, the binding probe is an antibody or antigen binding fragment thereof.

[0068] In one example, the binding probe is an antibody. Exemplary antibodies are full-length and / or naked (e.g., unconjugated) antibodies.

[0069] In one example, the antibody is an IgG or an IgE or an IgM or an IgD or an IgA or an IgY antibody. For example, the antibody is an IgG antibody.

[0070] In one example, the IgG antibody is an IgGi or an IgG2 or an IgG i or an IgG4.

[0071] In one example, the binding probe is an antigen binding fragment. For example, the antigen binding fragment is a single domain antibody, a minibody, a diabody, a triabody, a tetrabody, a Fv, a single chain Fv (scFv) fragment, a dimeric scFv (di-scFv), a Fab, a F(ab’)2 or a half antibody. In one example, the binding probe is a single domain antibody. In another example, the binding probe is a nanobody. In a further example, the binding probe is a minibody. In one example, the binding probe is a diabody. In another example, the binding probe is a triabody. In a further example, the binding probe is a tetrabody. In one example, the binding probe is a Fv. In another example, the binding probe is a scFv. In a further example, the binding probe is a di-scFv. In one example, the binding probe is a Fab. In another example, the binding probe is a F(ab')2. In one example, the binding probe is a half antibody.

[0072] In one example, the binding probe is a domain antibody (e.g., comprising only a heavy chain variable region or only a light chain variable region) or a heavy chain only antibody or variable region thereof. In one example, the binding probe is a heavy chain immunoglobulin. In one example, the binding probe is an antibody mimetic. In one example, the binding probe is a small antibody -mimetic scaffold protein. For example, the binding probe is a sherpabody. In another example, the binding probe is an adnectin. In a further example, the binding probe is an anticalin. In one example, the binding probe is an affibody. In another example, the binding probe is an avimer. In a further example, the binding probe is a DARPin. In another example, the binding probe is an affitin. In a further example, the binding probe is a fynomer. In one example, the binding probe is a peptide aptamer. In one example, the binding probe is an affimer. In another example, the binding probe is an alphabody. In a further example, the binding probe is a monobody. In another example, the binding probe is a nanoCLAMP. In one example, the binding probe is an optimer. In a further example, the binding probe is a repebody. In another example, the binding probe is a centyrin.

[0073] The skilled person will recognise other small antibody-mimetic scaffold proteins suitable for use in the present disclosure.

[0074] In one example, the binding probe is a cibacronblue F3GA.

[0075] In one example, the binding probe is a L-tryptophan.

[0076] In one example, the assay solution further comprises a buffering agent having a dissociation constant (pKa) between 5.5 and 8.9 at 25°C. In one example, the buffering agent has a pKa of about 5.5 at 25°C. In another example, the buffering agent has a pKa of about 5.6 at 25°C. In a further example, the buffering agent has a pKa of about 5.7 at 25°C. In one example, the buffering agent has a pKa of about 5.8 at 25°C. In another example, the buffering agent has a pKa of about 5.9 at 25°C. In a further example, the buffering agent has a pKa of about 6.0 at 25°C. In one example, the buffering agent has a pKa of about 6.1 at 25°C. In another example, the buffering agent has a pKa of about 6.2 at 25°C. In a further example, the buffering agent has a pKa of about 6.3 at 25°C. In one example, the buffering agent has a pKa of about 6.4 at 25°C. In another example, the buffering agent has a pKa of about 6.5 at 25°C. In a further example, the buffering agent has a pKa of about 6.6 at 25°C. In one example, the buffering agent has a pKa of about 6.7 at 25°C. In another example, the buffering agent has a pKa of about 6.8 at 25°C. In a further example, the buffering agent has a pKa of about 6.9 at 25°C. In one example, the buffering agent has a pKa of about 7.0 at 25°C. In another example, the buffering agent has a pKa of about 7.1 at 25°C. In a further example, the buffering agent has a pKa of about 7.2 at 25°C. In one example, the buffering agent has a pKa of about 7.3 at 25°C. In another example, the buffering agent has a pKa of about 7.4 at 25°C. In a further example, the buffering agent has a pKa of about 7.5 at 25°C. In one example, the buffering agent has a pKa of about 7.6 at 25°C. In another example, the buffering agent has a pKa of about 7.7 at 25°C. In a further example, the buffering agent has a pKa of about 7.8 at 25°C. In one example, the buffering agent has a pKa of about 7.9 at 25°C. In another example, the buffering agent has a pKa of about 8.0 at 25°C. In a further example, the buffering agent has a pKa of about 8.1 at 25°C. In one example, the buffering agent has a pKa of about 8.2 at 25°C. In another example, the buffering agent has a pKa of about 8.3 at 25°C. In a further example, the buffering agent has a pKa of about 8.4 at 25°C. In one example, the buffering agent has a pKa of about 8.5 at 25°C. In another example, the buffering agent has a pKa of about 8.6 at 25°C. In a further example, the buffering agent has a pKa of about 8.7 at 25°C. In one example, the buffering agent has a pKa of about 8.8 at 25°C. In another example, the buffering agent has a pKa of about 8.9 at 25°C.

[0077] The present disclosure provides a method of determining the concentration of a plasma protein in a sample, the method comprising: i. incubating the sample and a truncated Protein G probe conjugated to a fluorescent dye in an assay solution, wherein the assay solution comprises polyethylene glycol having a molecular weight of about 8 kDa and a buffering agent with a dissociation constant (pKa) of between 5.5 and 8.9 at 25°C;

[0078] ii. assaying the incubated solution to detect fluorescence polarization;

[0079] iii. determining a change in polarization; and

[0080] iv. correlating the change in polarization with the concentration of the plasma protein in the sample.

[0081] In one example, the buffering agent is selected from the group consisting of 3-morpholinopropane-l-sulfonic acid (MOPS), 2-(N-morpholino)ethanesulfonic acid (MES), Bis-Tris, N-(2-acetamido)-2 -aminoethanesulfonic acid (ACES), piperazine-N, N'-bis(2-ethanesulfonic acid) (PIPES), N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 2-[(2-Hydroxy-l,l-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid (TES), 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES), 3-(N, N-Bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid (DIPSO), 4-(N-Morpholino)butanesulfonic acid (MOBS), N-[Tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropanesulfonic acid (TAPSO), N-(Hydroxyethyl)piperazine-N'-2 -hydroxypropanesulfonic acid (HEPPSO), Piperazine-N, N'-bis(2-hydroxypropanesulfonic acid) (POPSO), triethanolamine (TEA), 4-(2-Hydroxyethyl)-l-piperazinepropanesulfonic acid (EPPS), Tricine, glycylglycine, bis[(2-hydroxyethyl)amino]acetic acid (Bicine), N-(2-Hydroxyethyl)piperazine-N'-(4-butanesulfonic acid) (HEPBS), tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS), Tris, phosphate buffered saline (PBS), and combinations thereof.

[0082] In one example, the buffering agent is MOPS. In one example, the buffering agent is MES. In one example, the buffering agent is ACES. In one example, the buffering agent is PIPES. In one example, the buffering agent is BES. In one example, the buffering agent is TES. In one example, the buffering agent is HEPES. In one example, the buffering agent is DIPSO. In one example, the buffering agent is MOBS. In one example, the buffering agent is TAPSO. In one example, the buffering agent is HEPPSO. In one example, the buffering agent is POPSO. In one example, the buffering agent is TEA. In one example, the buffering agent is EPPS. In one example, the buffering agent is Bicine. In one example, the buffering agent is HEPBS. In one example, the buffering agent is TAPS. In one example, the buffering agent is PBS.

[0083] In one example, the buffering agent is at a concentration of between 5 mM and 200 mM. For example, the buffering agent is at a concentration of between 10 mM and 150 mM. In one example, the buffering agent is at a concentration of between 25 mM and 125 mM. In another example, the buffering agent is at a concentration of between 25 mM and 100 mM. For example, the buffering agent is at a concentration of about 25 mM, or about 30 mM, or about 40 mM, or about 50 mM, or about 60 mM, or about 70 mM, or about 80 mM, or about 90 mM or about 100 mM. In one example, the buffering agent is at a concentration of 50 mM.

[0084] In one example, the buffering agent is MOPS at a concentration of 50 mM.

[0085] In one example, the assay solution further comprises a salt. For example, the salt is a monovalent and / or a divalent salt.

[0086] In one example, the salt is a monovalent salt. For example, the monovalent salt is selected from the group consisting of sodium chloride, potassium chloride and combinations thereof. In one example, the salt is sodium chloride. In another example, the salt is potassium chloride.

[0087] In one example, the salt is a divalent salt. For example, the divalent salt is selected from a group consisting of magnesium chloride, calcium chloride, barium chloride, copper (II) chloride, nickel chloride, manganese chloride, and combinations thereof. In one example, the salt is magnesium chloride. In another example, the salt is calcium chloride. In a further example, the salt is barium chloride. In one example, the salt is copper (II) chloride. In another example, the salt is nickel chloride. In a further example, the salt is manganese chloride.

[0088] In one example, the salt is in the assay solution at a concentration of between 0 mM and 500 mM. For example, the salt is in the assay solution at a concentration of between 5 mM and 500 mM. In one example, the salt is in the assay solution at a concentration of between 50 mM and 400 mM. In another example, the salt is in the assay solution at a concentration of between 50 mM and 350 mM. In a further example, the salt is in the assay solution at a concentration of between 50 mM and 300 mM. In one example, the salt is in the assay solution at a concentration of between 50 mM and 250 mM. In another example, the salt is in the assay solution at a concentration of between 50 mM and 200 mM. For example, the salt is at a concentration of between 100 mM and 150 mM.

[0089] In one example, the salt is in the assay solution at a concentration of about 5 mM, or about 10 mM, or about 25 mM, or about 50 mM, or about 75 mM, or about 100 mM. In another example, the salt is in the assay solution at a concentration of about 110 mM. In a further example, the salt is in the assay solution at a concentration of about 120 mM. In one example, the salt is in the assay solution at a concentration of about 130 mM. In another example, the salt is in the assay solution at a concentration of about 140 mM. In a further example, the salt is in the assay solution at a concentration of about 150 mM.

[0090] In one example, the assay solution has a pH of between 5 and 10. In a further example, the assay solution has a pH of between 6 and 8. In another example, the assay solution has a pH of between 6.5 and 7.8. In one example, the assay solution has a pH of 6.5. In another example, the assay solution has a pH of 7.1. In one example, the assay solution has a pH of 7.4. In a further example, the assay solution has a pH of 7.5. In another example, the assay solution has a pH of 7.7.

[0091] In one example, the fluorescent dye has a fluorescent signal lifetime of at least 4 nanoseconds (ns). For example, the fluorescent signal lifetime is at least 5 ns. In one example, the fluorescent dye has a fluorescent signal lifetime of at least 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18 or 20 ns. In one example, the fluorescent dye has a fluorescent signal lifetime of between 4-100 ns, or between 4-50 ns, or between 4-40 ns, or between 4-30 ns or between 4-25 ns. In another example, the fluorescent dye has a fluorescent signal lifetime of between 5-100 ns, or between 5-50 ns, or between 5-40 ns, or between 5-30 ns or between 5-25 ns. For example, the fluorescent dye has a fluorescent signal lifetime of between 10-100 ns, or between 10-50 ns, or between 10-40 ns, or between 10-30 ns or between 5-25 ns. In a further example, the fluorescent dye has a fluorescent signal lifetime of between 15-100 ns, or between 15-50 ns, or between 15-40 ns, or between 15-30 ns or between 15-25 ns.

[0092] In one example, the fluorescent dye is an organic dye.

[0093] In one example, the fluorescent dye is an inorganic dye.

[0094] In one example, the fluorescent dye is a quantum dot.

[0095] In one example, the fluorescent dye is a fluorescein, rhodamine, cyanine, dipyrromethene, naphthalene, xanthene, squaraine or triangulenium. In one example, the fluorescent dye is a fluorescein. For example, the fluorescent dye is fluorescein isothiocyanate (FITC). In another example, the fluorescent dye is rhodamine. In a further example, the fluorescent dye is cyanine. In one example, the fluorescent dye is dipyrromethene. In another example, the fluorescent dye is naphthalene. In a further example, the fluorescent dye is xanthene. In another example, the fluorescent dye is squaraine. In a further example, the fluorescent dye is triangulenium.

[0096] In one example, the fluorophore has a peak emission band with a full width at half maximum (FWHM) of less than about 60, 50, 40, 30, 20 or 10 nm. The present disclosure provides a method of determining the concentration of a plasma protein in a sample, the method comprising:

[0097] i. incubating the sample and a truncated Protein G probe conjugated to fluorescein isothiocyanate (FITC) in an assay solution, wherein the assay solution comprises polyethylene glycol having a molecular weight of about 8 kDa and a buffering agent with a dissociation constant (pKa) of between 5.5 and 8.9 at 25°C;

[0098] ii. assaying the incubated solution to detect fluorescence polarization;

[0099] iii. determining a change in polarization; and

[0100] iv. correlating the change in polarization with the concentration of the plasma protein in the sample.

[0101] In one example, the plasma protein is selected from the group consisting of an immunoglobulin (Ig), an apolipoprotein Al, an albumin, a protease, a protease inhibitor, plasminogen, a fibrinogen, a von Willebrand factor, a clotting factor or activated form thereof, a cofactor or activated form thereof, a contact system factor, a prekallikrein activator (PKA), a prothrombin, thrombin, prothrombin complex factor or activated form thereof, a protein C, an antithrombin III, alpha acid glycoprotein, a transport protein, Factor H, a component of the complement pathway, inhibitors of any component of the complement pathway, a highly glycosylated protein Protein S, histidine -rich glycoprotein, mannan binding lectin, C4 -binding protein, fibronectin, GC-globulin, erythropoietin, interferon, tumor factors, tPA, γCSF, an oligomeric form or degradation product of any of the foregoing and combinations thereof.

[0102] In one example, the plasma protein is an Ig. For example, the Ig is an IgG, an IgA, an IgM and / or a RhD immunoglobulin protein product. In one example, the Ig is an IgG. In another example, the Ig is an IgA. In a further example, the Ig is an IgM. In one example, the Ig is a RhD immunoglobulin protein product.

[0103] In one example, the plasma protein is an apolipoprotein Al.

[0104] In one example, the plasma protein is an albumin. For example, a-globulins and / or p-globulins.

[0105] In one example, the plasma protein is a protease. For example, a serine protease or a plasmin, or a kallikrein or a FX / FXa.

[0106] In one example, the plasma protein is a protease inhibitor. For example, a serine protease inhibitor, a Cl esterase inhibitor, an alpha- 1- antitrypsin, an anti-thrombin, a P-antithrombin, an a-antithrombin, an a-2-macroglobulin, a tissue factor pathway inhibitor (TFPI), a heparin cofactor II, a protein C inhibitor (PAI-3), an a-1 esterase inhibitor protein or antiangionetic proteins. In one example, the plasma protein is an alpha-1- antitrypsin. In one example, the plasma protein is a Cl esterase inhibitor. In one example, the plasma protein is an anti-thrombin. For example, p-antithrombin or a-antithrombin or an anti-thrombin III. In one example, the plasma protein is a P-antithrombin. In one example, the plasma protein is an a-antithrombin. In one example, the plasma protein is an a-2-macroglobulin. In one example, the plasma protein is a tissue factor pathway inhibitor (TFPI). In one example, the plasma protein is a heparin cofactor II. In one example, the plasma protein is a protein C inhibitor (PAI-3). In one example, the plasma protein is an a-1 esterase inhibitor protein. In one example, the plasma protein is an antiangiogenic protein. For example, latent-anti-thrombin.

[0107] In one example, the plasma protein is plasminogen.

[0108] In one example, the plasma protein is a fibrinogen.

[0109] In one example, the plasma protein is a von Willebrand factor.

[0110] In one example, the plasma protein is a prekallikrein activator (PKA).

[0111] In one example, the plasma protein is a prothrombin complex factor or activated forms thereof. For example, the plasma protein is a factor Xl / XIa, a factor Il / IIa, a factor Vll / VIIa, a factor IX / IXa, or a X / Xa. In one example, the plasma protein is a factor Xl / XIa. In one example, the plasma protein is a factor II / IIa. In one example, the plasma protein is a factorVII / VIIa. In one example, the plasma protein is a factorIX / IXa. In one example, the plasma protein is a factor X / Xa.

[0112] In one example, the plasma protein is a prothrombin.

[0113] In one example, the plasma protein is a thrombin.

[0114] In one example, the plasma protein is a protein C.

[0115] In one example, the plasma protein is alpha acid glycoprotein.

[0116] In one example, the plasma protein is a transport protein. For example, the plasma protein is haptoglobin. In one example, the plasma protein is hemopexin. In one example, the plasma protein is transferrin. In one example, the plasma protein is a ceruloplasmin. In one example, the plasma protein is a hemoglobulin.

[0117] In one example, the plasma protein is Factor H.

[0118] In one example, the plasma protein is a clotting factor or activated form thereof. For example, the clotting factor is factor X / Xa, factor Vll / VIIa, factor Vlll / VIIIa, a factor IX / IXa, factor Xll / XIIa, factor Xlll / XIIIa and / or factor Xl / XIa. In one example, the coagulation factor is factor X / Xa. In another example, the coagulation factor is factor Vll / VIIa. In a further example, the coagulation factor is factor Vlll / VIIIa. In one example, the coagulation factor is factor IX / IXa. In another example, the coagulation factor is factor Xl / XIa. In a further example, the coagulation factor is factor Xll / XIIa. In one example, the coagulation factor is factor Xlll / XIIa.

[0119] In one example, the plasma protein is a cofactor or activated forms thereof. For example, the plasma protein is factor V / Va, factor Vlll / VIIIa or factor Xlll / XIIIa. In one example, the plasma protein is factor V / Va. In one example, the plasma protein is FVIII / VIIIa. In one example, the plasma protein is factor Xlll / XIIIa.

[0120] In one example, the plasma protein is a contact system factor. For example, the plasma protein is a factor Xl / XIa, factor Xll / XIIa or plasma kallikrein. In one example, the plasma protein is a factor Xl / XIa. In one example, the plasma protein is a factor Xll / XIIa. In one example, the plasma protein is a plasma kallikrein.

[0121] In one example, the plasma protein is a component of the complement system.

[0122] In one example, the plasma protein is a Protein S.

[0123] In one example, the plasma protein is a highly glycosylated protein. For example, a-l-acid glycoprotein, antichymotrypsin, inter-a-trypsin inhibitor, a-2-HS glycoprotein or a C-reactive protein. In one example, the plasma protein is an antichymotrypsin. In one example, the plasma protein is an a-l-acid glycoprotein. In one example, the plasma protein is an inter-a-trypsin inhibitor. In one example, the plasma protein is an a-2-HS glycoprotein. In one example, the plasma protein is a C-reactive protein.

[0124] In one example, the plasma protein is a histidine-rich glycoprotein.

[0125] In one example, the plasma protein is a mannan binding lectin.

[0126] In one example, the plasma protein is a C4-binding protein.

[0127] In one example, the plasma protein is a fibronectin.

[0128] In one example, the plasma protein is a GC-globulin.

[0129] In one example, the plasma protein is a erythropoietin.

[0130] In one example, the plasma protein is a interferon.

[0131] In one example, the plasma protein is a tumor factors.

[0132] In one example, the plasma protein is a tPA.

[0133] In one example, the plasma protein is a yCSF. In one example, the plasma protein is an oligomeric form or degradation product of any of the foregoing plasma proteins.

[0134] It will be apparent to the skilled person from the disclosure herein that reference to a clotting factor (e.g., Factor X or FX) includes reference to the zymogen (or inactive form) as well as the activated form thereof (e.g., Factor Xa or FXa) and vice versa.

[0135] The present disclosure provides a method of determining the concentration of an immunoglobulin (Ig) in a sample, the method comprising:

[0136] i. incubating the sample and a truncated Protein G probe conjugated to a fluorescent dye in an assay solution, wherein the assay solution comprises polyethylene glycol having a molecular weight of about 8 kDa; ii. assaying the incubated solution to detect fluorescence polarization;

[0137] iii. determining a change in polarization; and

[0138] iv. correlating the change in polarization with the concentration of the Ig in the sample.

[0139] The present disclosure provides a method of determining the concentration of an immunoglobulin (Ig) in a sample, the method comprising:

[0140] i. incubating the sample and a truncated Protein G probe conjugated to fluorescein isothiocyanate (FITC) in an assay solution, wherein the assay solution comprises polyethylene glycol having a molecular weight of about 8 kDa;

[0141] ii. assaying the incubated solution to detect fluorescence polarization;

[0142] iii. determining a change in polarization; and

[0143] iv. correlating the change in polarization with the concentration of the Ig in the sample.

[0144] The present disclosure provides a method of determining the concentration of an immunoglobulin (Ig) in a sample, the method comprising:

[0145] i. incubating the sample and a truncated Protein G probe conjugated to fluorescein isothiocyanate (FITC) in an assay solution, wherein the assay solution comprises polyethylene glycol having a molecular weight of about 8 kDa and a buffering agent with a dissociation constant (pKa) of between 5.5 and 8.9 at 25°C;

[0146] ii. assaying the incubated solution to detect fluorescence polarization;

[0147] iii. determining a change in polarization; and

[0148] iv. correlating the change in polarization with the concentration of the Ig in the sample.

[0149] The present disclosure provides a method of determining the concentration of an immunoglobulin G (IgG) in a sample, the method comprising:

[0150] i. incubating the sample and a truncated Protein G probe conjugated to a fluorescent dye in an assay solution, wherein the assay solution comprises polyethylene glycol having a molecular weight of about 8 kDa; ii. assaying the incubated solution to detect fluorescence polarization;

[0151] iii. determining a change in polarization; and

[0152] iv. correlating the change in polarization with the concentration of the IgG in the sample.

[0153] The present disclosure provides a method of determining the concentration of an immunoglobulin G (IgG) in a sample, the method comprising:

[0154] i. incubating the sample and a truncated Protein G probe conjugated to fluorescein isothiocyanate (FITC) in an assay solution, wherein the assay solution comprises polyethylene glycol having a molecular weight of about 8 kDa; ii. assaying the incubated solution to detect fluorescence polarization;

[0155] iii. determining a change in polarization; and

[0156] iv. correlating the change in polarization with the concentration of the IgG in the sample.

[0157] The present disclosure provides a method of determining the concentration of an immunoglobulin G (IgG) in a sample, the method comprising:

[0158] i. incubating the sample and a truncated Protein G probe conjugated to fluorescein isothiocyanate (FITC) in an assay solution, wherein the assay solution comprises polyethylene glycol having a molecular weight of about 8 kDa and a buffering agent with a dissociation constant (pKa) of between 5.5 and 8.9 at 25°C;

[0159] ii. assaying the incubated solution to detect fluorescence polarization;

[0160] iii. determining a change in polarization; and

[0161] iv. correlating the change in polarization with the concentration of the IgG in the sample.

[0162] The skilled person will recognise suitable sources of plasma proteins for use in a method of the disclosure. Exemplary protein sources (i.e., samples) include plasma or serum of human or animal origin, fermentation broth, cell culture, protein suspension, milk or other original sources. Immunoglobulin containing material or solutions may contain monoclonal or polyclonal immunoglobulin(s). In some embodiments, the immunoglobulin-containing starting material is a solution comprising polyclonal antibodies. In other embodiments the starting material comprises a monoclonal antibody or a fragment thereof. It is therefore within the knowledge of a skilled person that the term “immunoglobulin” as used herein can also be identified as antibody including monoclonal antibody or polyclonal antibody, either natural or recombinant.

[0163] In one example, the plasma protein is a natural or recombinant plasma protein. In one example, the plasma protein is a naturally occurring plasma protein. In another example, the plasma protein is a recombinantly produced plasma protein.

[0164] In one example, the plasma protein is derived from serum, plasma, a fermentation broth, a cell culture harvest or a protein suspension.

[0165] In one example, the sample is serum, plasma, a plasma fraction, or a purified or partially purified plasma or plasma fraction thereof, a fermentation broth ora purified or partially purified fermentation broth thereof, a cell culture harvest or a purified or partially purified cell culture harvest thereof or a protein suspension.

[0166] In one example, the plasma sample is obtained by a finger-prick test. In another example, the plasma sample is obtained during plasmapheresis.

[0167] In one example, the sample is a protein mixture. For example, the protein mixture comprises plasma proteins, peptide hormones, growth factors, cytokines and polyclonal immunoglobulins proteins, plasma proteins selected from human and animal blood clotting factors including fibrinogen, prothrombin, thrombin, prothrombin complex, FX, FXa, FIX, FIXa, FVII, FVIIa, FXI, FXIa, FXII, FXIIa, FXIII and FXIIIa, von Willebrand factor, transport proteins including albumin, transferrin, ceruloplasmin, haptoglobin, hemoglobulin and hemopexin, protease inhibitors including -antithrombin, a-antithrombin, a-2-macroglobulin, Cl-inhibitor, tissue factor pathway inhibitor (TFPI), heparin cofactor II, protein C inhibitor (PAI-3), Protein C and Protein S, a-1 esterase inhibitor proteins, a-1 antitrypsin, antiangionetic proteins including latent-antithrombin, highly glycosylated proteins including a-1 -acid glycoprotein, antichymotrypsin, inter-a-trypsin inhibitor, a-2-HS glycoprotein and C-reactive protein and other proteins including histidine -rich glycoprotein, mannan binding lectin, C4-binding protein, fibronectin, GC -globulin, plasminogen, blood factors such as erythropoietin, interferon, tumor factors, tPA and / or yCSF. In one example, the plasma or plasma fraction is selected the group consisting of a human blood plasma sample, an IgG intermediate product, a cryo-rich plasma, a cryo-poor plasma, a Supernatant I (SN I), a Cohn Fraction II (Fr II), a Cohn Fraction 11+111 (Fr II+III), a Cohn Fraction I+II+III (FrI+II+III), a Kistler / Nitschmann Precipitate A (KN A), a Kistler / Nitschmann Precipitate B (KN B), a Kistler / Nitschmann Precipitate of Supernatant B (KN B+l), formulated plasma fractionation product and combinations thereof. In one example, the plasma or plasma fraction is a human blood plasma sample. In another example, the plasma fraction is an IgG intermediate product. In one example, the plasma fraction is cryo-rich plasma. In another example, the plasma fraction is cryo-poor plasma. In a further example, the plasma fraction is Supernatant I (SN I). In one example, the plasma fraction is Cohn Fraction II (Fr II). In another example, the plasma faction is Cohn Fraction II+III (Fr II+III). In a further example, the plasma fraction is Cohn Fraction I+II+III (FrI+II+III). In one example, the plasma fraction is Kistler / Nitschmann Precipitate A (KN A). In another example, the plasma fraction is Kistler / Nitschmann Precipitate B (KN B). In a further example, the plasma fraction is Kistler / Nitschmann Precipitate of Supernatant B (KN B+l). In one example, the plasma fraction is a formulated plasma fractionation product.

[0168] In one example, the plasma fraction is a suspended paste. For example, the suspended paste is selected from a group consisting of Cohn Fraction II (Fr II), Cohn Fraction II+III (Fr II+III), Cohn Fraction I+II+III (FrI+II+III), Kistler / Nitschmann Precipitate A (KN A), Kistler / Nitschmann Precipitate B (KN B), Kistler / Nitschmann Precipitate of Supernatant B (KN B+l), and combinations thereof. In one example, the suspended paste is a Cohn Fraction II (Fr II) paste. In one example, the suspended paste is a Cohn Fraction II+III (Fr II+III) paste. In another example, the suspended paste is a Cohn Fraction I+II+III (FrI+II+III) paste. In another example, the suspended paste is a Kistler / Nitschmann Precipitate A (KN A) paste. In another example, the suspended paste is a Kistler / Nitschmann Precipitate B (KN B) paste. In a further example, the suspended paste is a Kistler / Nitschmann Precipitate of Supernatant B (KN B+l) paste.

[0169] In one example, the plasma fraction is selected from the group consisting of a mammalian plasma fraction, a human plasma fraction, an equine plasma fraction, and a bovine plasma fraction. In one example, the plasma fraction is a mammalian plasma fraction. In one example, the plasma fraction is a human plasma fraction. In one example, the plasma fraction is an equine plasma fraction. In one example the plasma fraction is a bovine plasma fraction. In one example the plasma fraction is a bovine plasma fraction comprising human polyclonal antibodies.

[0170] In one example, the plasma has not been subjected to any purification steps. For example, the plasma is an unpurified plasma sample.

[0171] In one example, the method of the disclosure is performed at the point of plasma collection. For example, the method of the disclosure is performed during plasmapheresis. In another example, the method of the disclosure is performed on a sample obtained from the subject during plasma donation. In a further example, the method of the disclosure is performed on a plasma sample immediately following collection from the subject. For example, the method is performed on the plasma sample collected from the subject.

[0172] In one example, the plasma or plasma fraction has been purified or partially purified.

[0173] In one example, the purified or partially purified plasma or plasma fraction thereof has been subjected to one or more steps selected from the group consisting of clarification, ethanol precipitation, octanoic acid fractionation, ammonium sulphate precipitation, affinity chromatography, ion exchange chromatography, viral inactivation, viral filtration, isoagglutinin affinity chromatography, ultrafiltration / diafiltration, bulk formulation, final formulation and combinations thereof. In one example, the purified or partially purified plasma or plasma fraction thereof has been subjected to clarification. In one example, the purified or partially purified plasma or plasma fraction thereof has been subjected to ethanol precipitation. In one example, the purified or partially purified plasma or plasma fraction thereof has been subjected to octanoic acid fractionation. In one example, the purified or partially purified plasma or plasma fraction thereof has been subjected to ammonium sulphate precipitation. In one example, the purified or partially purified plasma or plasma fraction thereof has been subjected to affinity chromatography. In one example, the purified or partially purified plasma or plasma fraction thereof has been subjected to ion exchange chromatography. In one example, the purified or partially purified plasma or plasma fraction thereof has been subjected to ion exchange chromatography. In one example, the purified or partially purified plasma or plasma fraction thereof has been subjected to viral inactivation. In one example, the purified or partially purified plasma or plasma fraction thereof has been subjected to viral filtration. In one example, the purified or partially purified plasma or plasma fraction thereof has been subjected to isoagglutinin affinity chromatography. In one example, the purified or partially purified plasma or plasma fraction thereof has been subjected to ultrafiltration / diafiltration. In one example, the purified or partially purified plasma or plasma fraction thereof has been subjected to bulk formulation. In one example, the purified or partially purified plasma or plasma fraction thereof has been subjected to final formulation.

[0174] In one example, the method of the disclosure is performed before and / or after one or more purification steps selected from the group consisting of clarification, ethanol precipitation, octanoic acid fractionation, ammonium sulphate precipitation, affinity chromatography, ion exchange chromatography, viral inactivation, viral filtration, isoagglutinin affinity chromatography, ultrafiltration / diafiltration, bulk formulation, final formulation and combinations thereof.

[0175] In one example, the method of the disclosure is performed before and / or after clarification. In one example, the method of the disclosure is performed before clarification. In one example, the method of the disclosure is performed after clarification. In one example, the method of the disclosure is performed before and after clarification.

[0176] In one example, the method of the disclosure is performed before and / or after ethanol precipitation. In one example, the method of the disclosure is performed before ethanol precipitation. In one example, the method of the disclosure is performed after ethanol precipitation. In one example, the method of the disclosure is performed before and after ethanol precipitation.

[0177] In one example, the method of the disclosure is performed before and / or after octanoic acid fractionation. In one example, the method of the disclosure is performed before octanoic acid fractionation. In one example, the method of the disclosure is performed after octanoic acid fractionation. In one example, the method of the disclosure is performed before and after octanoic acid fractionation.

[0178] In one example, the method of the disclosure is performed before and / or after ammonium sulphate precipitation. In one example, the method of the disclosure is performed before ammonium sulphate precipitation. In one example, the method of the disclosure is performed after ammonium sulphate precipitation. In one example, the method of the disclosure is performed before and after ammonium sulphate precipitation.

[0179] In one example, the method of the disclosure is performed before and / or after affinity chromatography. In one example, the method of the disclosure is performed before affinity chromatography. In one example, the method of the disclosure is performed after affinity chromatography. In one example, the method of the disclosure is performed before and after affinity chromatography. In one example, the method of the disclosure is performed before and / or after ion exchange chromatography. In one example, the method of the disclosure is performed before ion exchange chromatography. In one example, the method of the disclosure is performed after ion exchange chromatography. In one example, the method of the disclosure is performed before and after ion exchange chromatography.

[0180] In one example, the method of the disclosure is performed before and / or after viral inactivation. In one example, the method of the disclosure is performed before viral inactivation. In one example, the method of the disclosure is performed after viral inactivation. In one example, the method of the disclosure is performed before and after viral inactivation.

[0181] In one example, the method of the disclosure is performed before and / or after viral filtration. In one example, the method of the disclosure is performed before viral filtration. In one example, the method of the disclosure is performed after viral filtration. In one example, the method of the disclosure is performed before and after viral filtration.

[0182] In one example, the method of the disclosure is performed before and / or after isoagglutinin affinity chromatography. In one example, the method of the disclosure is performed before isoagglutinin affinity chromatography. In one example, the method of the disclosure is performed after isoagglutinin affinity chromatography. In one example, the method of the disclosure is performed before and after isoagglutinin affinity chromatography.

[0183] In one example, the method of the disclosure is performed before and / or after ultrafiltration / diafiltration. In one example, the method of the disclosure is performed before ultrafiltration / diafiltration. In one example, the method of the disclosure is performed after ultrafiltration / diafiltration. In one example, the method of the disclosure is performed before and after ultrafiltration / diafiltration.

[0184] In one example, the method of the disclosure is performed before and / or after bulk formulation. In one example, the method of the disclosure is performed before bulk formulation. In one example, the method of the disclosure is performed after bulk formulation. In one example, the method of the disclosure is performed before and after bulk formulation.

[0185] In one example, the method of the disclosure is performed before and / or after final formulation. In one example, the method of the disclosure is performed before final formulation. In one example, the method of the disclosure is performed after final formulation. In one example, the method of the disclosure is performed before and after final formulation.

[0186] In one example, the method comprises diluting the sample and / or the probe in the assay solution prior to incubating. In one example, the method comprises diluting the sample probe in the assay solution prior to incubating. In another example, the method comprises diluting the probe in the assay solution prior to incubating. In a further example, the method comprises diluting the sample and the probe in the assay solution prior to incubating.

[0187] Methods of serially diluting the sample and / or probe in the assay solution will be apparent to the skilled person and / or described herein.

[0188] In one example, the sample and / or probe is serially diluted in the assay solution to a dilution factor of between 1:10 to 1:60 before incubating.

[0189] In one example, the sample is serially diluted in the assay solution to a dilution factor of between 1: 10 to 1:60 before incubating. In one example, the sample is serially diluted in the assay solution at a dilution factor of 1:10, or 1:12.5, or 1:20, or 1:24, or 1:25, or 1:40, or 1:50, or 1:60 before incubating. In another example, the probe is serially diluted in the assay solution to a dilution factor of between 1: 10 to 1:60 before incubating. In one example, the probe is serially diluted in the assay solution at a dilution factor of 1:10, or 1:12.5, or 1:20, or 1:24, or 1:25, or 1:40, or 1:50, or 1:60 before incubating.

[0190] In one example, the method comprises adding the diluted sample to the diluted probe before incubating, wherein the total dilution factor of the sample and the probe in the assay solution is between 1:100 to 1:3000. For example, the total dilution factor of the sample and the probe in the assay solution is 1:100, or 1:250, or 1:600, or 1:2000, or 1:3000.

[0191] In one example, the method determines the concentration of the plasma protein in the sample with a coefficient of variation of < 3.0%. For example, the method determines the concentration of the plasma protein in the sample with a coefficient of variation of 3.0%, or 2.9%, or 2.8%, or 2.7%, or 2.6%, or 2.5%. In one example, the method determines the concentration of the plasma protein in the sample with a coefficient of variation of < 2.5%. For example, the method determines the concentration of the plasma protein in the sample with a coefficient of variation of 2.5%, or 2.4%, or 2.3%, or 2.2%, or 2.1%, or 2.0%. In one example, the method determines the concentration of the plasma protein in the sample with a coefficient of variation of < 2.0%. For example, the method determines the concentration of the plasma protein in the sample with a coefficient of variation of 2.0%, or 1.9%, or 1.8%, or 1.7%, or 1.6%, or 1.5%. In one example, the method determines the concentration of the plasma protein in the sample with a coefficient of variation of < 1.5%. For example, the method determines the concentration of the plasma protein in the sample with a coefficient of variation of 1.5%, or 1.4%, or 1.3%, or 1.2%, or 1.1%, or 1.0%. In one example, the method determines the concentration of the plasma protein in the sample with a coefficient of variation of < 1.0%.

[0192] In one example, the concentration of the plasma protein in the sample is between 2 to 250 g / L plasma protein. For example, the concentration of the plasma protein in the sample is about 2 g / L plasma protein, or about 5 g / L plasma protein, or about 10 g / L plasma protein, or about 15 g / L plasma protein, or about 20 g / L plasma protein, or about 25 g / L plasma protein. In another example, the concentration of the plasma protein in the sample is about 30 g / L plasma protein, or about 40 g / L plasma protein, or about 50 g / L plasma protein, or about 60 g / L plasma protein, or about 70 g / L plasma protein, or about 80 g / L plasma protein, or about 90 g / L plasma protein. In a further example, the concentration of the plasma protein in the sample is about 100 g / L plasma protein, or about 110 g / L plasma protein, or about 120 g / L plasma protein, or about 130 g / L plasma protein, or about 140 g / L plasma protein, or about 150 g / L plasma protein. In one example, the concentration of the plasma protein in the sample is about 160 g / L plasma protein, or about 170 g / L plasma protein, or about 180 g / L plasma protein, or about 190 g / L plasma protein, or about 200 g / L plasma protein. In another example, the concentration of the plasma protein in the sample is about 210 g / L plasma protein, or about 220 g / L plasma protein, or about 230 g / L plasma protein, or about 240 g / L plasma protein, or about 250 g / L plasma protein.

[0193] In one example, the method of the disclosure detects the plasma protein in the sample in a concentration range of 2 to 250 g / L plasma protein. For example, the method of the disclosure detects IgG in the sample in a concentration range of 2 to 250 g / L IgG.

[0194] In one example, the method is performed at a temperature in the range of 18 to 25 °C. For example, the method is performed at room temperature.

[0195] In one example, the method is carried out in a well of a multi-well plate. Suitable multi-well plates for use in the present disclosure will be apparent to the skilled person and / or described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0196] Figure 1 is a graphical representation showing the effect of different assay solutions on the coefficient of variation (CV).

[0197] DETAILED DESCRIPTION

[0198] General

[0199] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter.

[0200] Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0201] The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the present disclosure.

[0202] Any example of the present disclosure herein shall be taken to apply mutatis mutandis to any other example of the disclosure unless specifically stated otherwise. Stated another way, any specific example of the present disclosure may be combined with any other specific example of the disclosure (except where mutually exclusive).

[0203] Any example of the present disclosure disclosing a specific feature or group of features or method or method steps will be taken to provide explicit support for disclaiming the specific feature or group of features or method or method steps.

[0204] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (for example, in cell culture, immunology, molecular biology, protein chemistry, and biochemistry).

[0205] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0206] Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0207] As used herein the term "derived from" shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source.

[0208] Furthermore, as used herein the singular forms of “a”, “and” and “the” include plural references unless the context clearly dictates otherwise.

[0209] All publications cited herein are hereby incorporated by reference in their entirety. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

[0210] Any discussion of documents, acts, materials, devices, articles or the like that has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

[0211] Selected Definitions

[0212] As used herein, the term “determine” or “determining” in reference to the concentration of a plasma protein in a sample shall be understood to mean to ascertain or obtain, qualitatively or quantitatively, the concentration of plasma protein in the sample.

[0213] The terms “concentration” or “titer” or “titre” as used herein in refers to the amount of the plasma protein present in the sample at a given time point. The concentration may be quantified in absolute or relative terms. Generally, the concentration is defined as weight of plasma protein per volume of assay solution, or grams per litre (g / L).

[0214] The term “incubate” or “incubating” as used herein refers to combining or mixing the sample and plasma protein binding probe in the assay solution under conditions suitable for the plasma protein binding probe to bind to the plasma protein in the sample, i.e., form a complex.

[0215] As used herein, the phrase “assaying the incubated solution to detect fluorescence polarization” shall be understood to mean exciting the sample with plane polarized light at a wavelength corresponding to an excitation wavelength of the fluorescent dye, and detecting light intensity emitted by the fluorescent dye at an appropriate emission wavelength both in two planes, one of which is parallel to the plane of the excitation plane and one of which is perpendicular to the plane of the excitation light. For example, the excitation plane is vertical or horizontal and the emitted light is detected in vertical and horizontal planes. The change in polarization between the excitation and emission light (i.e., the degree to which the emission intensity moves from the excitation plan (i.e., vertical) to a perpendicular plane (i.e., horizontal) is a function of the degree of rotation of the fluorescent dye. A change (i.e., an increase) in polarisation of emitted light is observed when the plasma protein binding probe is bound to the plasma protein, compared to the plasma protein binding probe alone, as the complex will rotate slower.

[0216] The phrase “correlating the change in polarization with the concentration of the plasma protein in the sample” as used herein shall be understood to mean calculating the plasma protein concentration in the sample based on the change in polarization of light emitted by the fluorescent dye. Methods for calculating the plasma protein concentration are known in the art and include inferring the protein concentration from a standard curve of known concentrations, or by calculating dissociation constants and inferring concentration from first principles.

[0217] The term “protein” shall be taken to include a single polypeptide chain, i.e., a series of contiguous amino acids linked by peptide bonds or a series of polypeptide chains covalently or non-covalently linked to one another (i.e., a polypeptide complex). For example, the series of polypeptide chains can be covalently linked using a suitable chemical or a disulfide bond. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions.

[0218] The term “polypeptide” or “polypeptide chain” will be understood from the foregoing paragraph to mean a series of contiguous amino acids linked by peptide bonds. The term “plasma” shall refer to the straw -coloured / pale yellow component of blood obtained from one or more blood donor(s). Methods of obtaining plasma from a donor will be apparent to a skilled person and / or described herein. For example, plasma is obtained by removing red blood cells from donated blood. For example, plasma is obtained by plasmapheresis.

[0219] The term “plasma fraction” or “fraction thereof’ shall refer to plasma which has been fractionated to isolate one or more desirable protein components from the plasma. For example, plasma may be fractionated to isolate cryoprecipitates (proteins that precipitate out of solution when a unit of fresh frozen plasma is slowly thawed in the cold) and cryosupematant (also known as cryo-poor plasma). For example, plasma may be fractionated by ethanol precipitation to produce IgG-containing Oncley fractions, Cohn fractions, ammonium sulphate precipitates, or Precipitates A (KN A), B (KN B), and the Precipitate of Supernatant B (KN B+l) from plasma as described in US patent 3,301,842. Plasma fractions include 11+111 precipitate produced according to Cohn methods such as Method 6, Cohn et. al. J. Am; Chem. Soc., 68 (3), 459-475 (1946), Method 9, Oncley et al. J. Am; Chem. Soc., 71, 541-550 (1946), or the I+II+III precipitate, Method 10, Cohn et.al. J. Am; Chem. Soc., 72, 465-474 (1950); as well as the method of Deutsch et.al. J. Biol. Chem. 164, 109-118 (1946) or the Precipitate-A, B and the Precipitate of Supernatant B of Nitschmann and Kistler Vox Sang. 7, 414-424 (1962); Helv. Chim. Acta 37, 866-873 (1954). For example, the plasma may be fractionated by octanoic acid fractionation as described in European application 893450. Typically, Cohn Fractions, and Kistler / Nitschmann Precipitate’s A (KN A), B (KN B) and the Precipitate of Supernatant B (KN B+l) exist as a suspended paste. Other purification techniques including chromatography may be used.

[0220] The term “immunoglobulin G (IgG)”, also known as “gamma globulin” or “immune globulin”, shall be taken to mean antibody of isotype G. There are several subclasses of IgG, for example, IgGl, IgG2, IgG3 and IgG4.

[0221] The term “cryo-precipitate” or “cryo-precipitates” refers to proteins in plasma that precipitate out of solution when a unit of fresh frozen plasma is slowly thawed in the cold. Cryo-precipitates include factor VIII, fibrinogen, von Willebrand factor, factor XIII and platelet membrane microparticles.

[0222] The term “cryo-poor plasma” shall be taken to mean plasma removed of cryo-precipitates.

[0223] The term “cryo-rich plasma” shall be taken to mean plasma comprising components typically found in cryoprecipitates.

[0224] The term “partially purified plasma or plasma fraction thereof’ shall be understood to mean any fraction or solution from any stage of plasma purification.

[0225] The term “partially purified fermentation broth thereof’ or “partially purified cell culture harvest thereof’ shall be understood to mean any fraction or solution from any stage of fermentation or cell culture harvest.

[0226] The term “industrial or commercial scale” or “large scale” or “manufacturing scale” shall refer to the amount of product that would be produced in a batch that was designed for clinical testing, formulation, sale and / or distribution to the public. For example, industrial scale refers to large scale purification of IgG from the plasma or fraction thereof to produce the plasma protein product.

[0227] As used herein, the term “polymer” takes its usual definition in the art and so refers to a homopolymer or a co-polymer formed from the polymerisation of one or more monomers.

[0228] As used herein, the term “homopolymef ’ takes its usual definition in the art and so refers to a polymer whose polymer chains comprise one type of monomer. As used herein, the term “co-polymer” takes its usual definition in the art and so refers to a polymer whose polymer chains comprise two or more different types of monomers. As used herein the term “monomer” takes its usual definition in the art, and so refers to a molecular compound that may chemically bind to another monomer to form a polymer.

[0229] As used herein, the term “hydrophilic polymer” refers to a polymer that, in the absence of chemical or physical crosslinking between the polymer chains, has a solubility in water about or greater than about 10 percent by weight (wt %) at 25 °C. A hydrophilic polymer may include some monomer units that are hydrophilic and some monomer units that are hydrophobic, while still exhibiting overall hydrophilic properties. In some embodiments, the solubility is greater than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more, percent by weight (wt %) at 25 °C.

[0230] As used herein, the term “neutral polymer” refers to a polymer that is substantially non-ionisable in aqueous solution.

[0231] As used herein, the term “neutral hydrophilic polymer” refers to a polymer that is both a neutral polymer and a hydrophilic polymer.

[0232] As used herein, the term “radius of gyration” (also known as Rg) refers to the mean radius of a polymer and is calculated as the root mean square distance of the component units (e.g. monomer) of the polymer molecule from the centre of mass of the polymer molecule (see Stepto et al. Pure Appl Chem. 87(1): 71, the entire contents of which is incorporated herein by reference). The radius of gyration may be given in units of, e.g., Angstrom or nanometer (nm). The person skilled in the art will appreciate that there are a variety of methods for determining the radius of gyration of a polymer. The "z average radius of gyration" of a polymer as used herein refers to the R of a polymer measured using light scattering (eg, SLS, DLS, MALS). Accordingly, methods for measuring z-average R are known and can be used herein. For example, z-average R can be measured as disclosed inUS Patent No. 7531073, US Patent Application Publication Nos. 2010 / 0003515 and 2009 / 0046274, Wyatt (Anal. Chim. Acta 272 :1-40), and Mori and Barth (Size Exclusion Chromatography, Springer-Verlag, Berlin, 1999). Further example standard methods for determining Rginclude, but are not limited to, ASTM D4001-20, ASTM D2857-22, ASTM D5296-19, ASTM D6474-20, or ASTM D6579-11. In some embodiments, the radius of gyration is determined according to any one of the methods described in Linegar et al., Hydrodynamic radius of polyethylene glycol in solution obtained by dynamic light scattering. Colloid J 72, 279-281 (2010), Kim et al., Polymer Dynamics in PEG-Silica Nanocomposites: Effects of Polymer Molecular Weight, Temperature and Solvent Dilution, Macromolecules 2012, 45, 10, 4225-4237, Zhao et al., Viscoelasticity of Poly (ethylene glycol) Solutions on Supported Lipid Bilayers via Quartz Crystal Microbalance with Dissipation, Macromolecules 2015, 48, 6, 1824-1831, Georgalis et al., Light scattering studies onFicoll PM70 solutions reveal two distinct diffusive modes, Journal of Colloid and Interface Science, 2012, 386, 141-147, or Yunhong Rong, Probing the structure of Dextran Systems and their Organization, Thesis (2008), Rutgers, The State University of New Jersey, the entire contents (including in particular, any and all methods for determining radius of gyration disclosed therein) of each of which is hereby incorporated herein by reference for all purposes. Other techniques include, but are not limited to, neutron or x-ray scattering (eg SANS), gel chromatography, analytical centrifugation, and nuclear magnetic resonance (NMR). It will be appreciated the radius of gyration may depend upon the environment in which the polymer is situated, and may for example, depend in part upon factors such as solvent, temperature, and pH. Radii of gyration may be stated for a polymer as determined in aqueous solution, neutral pH, and at standard atmospheric temperature. In some embodiments, the radius of gyration is given for the neutral hydrophilic polymer, as determined in the assay solution (according to the method described herein, i.e. Example 4). In some embodiments, the radius of gyration is as determined by DLS for the neutral hydrophilic polymer when present at a concentration of 10 g / L in a solution of 50 mM MOPS buffer, 130 mM NaCl, pH 7.1 at 25 °C.

[0233] As used herein, the term “hydrodynamic radius” (also known as “Stokes radius”) refers to the effective radius of a molecule (such as polymer) measured by assuming that the molecule in the solution moves through the solution and experiences resistance to the viscosity of the solution) Rh. In other words, the Stokes radius is a measure of the effective radius of a molecule in solution as it diffuses in a fluid medium. Typically, the radius may be seen to represent the radius of a hypothetical hard sphere that diffuses at the same rate as the actual molecule or particle. The hydrodynamic radius of a molecule affects its rate of diffusion in aqueous solution as well as its ability to move within a macromolecular gel. The hydrodynamic radius of a molecule is determined not only by its molecular weight, but also by its structure, including shape and compactness. Methods for determining hydrodynamic radii are well known in the art and include, for example, dynamic light scattering (DLS), or size exclusion chromatography (SEC) as described in US Pat. Nos. 6,406,632 and 7,294,513). In some embodiments, the hydrodynamic radius is given for the neutral hydrophilic polymer, as determined in the assay solution (according to the method described herein, i.e. Example 4). In some embodiments, the hydrodynamic radius is as determined by DLS for the neutral hydrophilic polymer when present at a concentration of 10 g / L in a solution of 50 mM MOPS buffer, 130 mM NaCl, pH 7.1 at 25 °C.

[0234] As used herein, the term “coefficient of variation” refers to the ratio of the standard deviation to the mean. The skilled person will appreciate that the higher the coefficient of variation, the greater the level of dispersion around the mean. The lower the value of the coefficient of variation, the more precise the estimate. As used herein, the coefficient of variation is expressed as a percentage.

[0235] Determining the concentration of a plasma protein

[0236] The present disclosure provides a method of determining the concentration of a plasma protein in a sample, the method comprising:

[0237] i. incubating the sample and a plasma protein binding probe conjugated to a fluorescent dye in an assay solution, wherein the assay solution comprises a neutral hydrophilic polymer;

[0238] ii. assaying the incubated solution to detect fluorescence polarization;

[0239] iii. determining a change in polarization; and

[0240] iv. correlating the change in polarization with the concentration of the plasma protein in the sample.

[0241] Assay Solution

[0242] The present disclosure provides a method of determining the concentration of a plasma protein in a sample comprising incubating the sample and a plasma protein binding probe conjugated to a fluorescent dye in an assay solution, wherein the assay solution comprises a neutral hydrophilic polymer.

[0243] Neutral Hydrophilic Polymer

[0244] According to the method described herein, incubation is performed in an assay solution comprising a neutral hydrophilic polymer. There is no particular limitation as to suitable neutral hydrophilic polymers and the skilled person will be able to readily determine suitable neutral hydrophilic polymers for use in the present disclosure. Suitable examples of neutral hydrophilic polymers include, but are not limited to, poly(olefinic alcohol) such as poly(vinyl alcohol), poly(hydroxyethyl acrylate), poly(hydroxyalkyl methacrylate) such as poly(hydroxyethyl methacrylate) (pHEMA), poly(N-vinyl pyrrolidone) (PVP), polyalkylene glycols and polyalkylene oxides such as polyethylene oxide) (PEO), polyethylene glycol) (PEG), polypropylene oxide), and poly(methyl vinyl ether) (PMVE). Further examples of neutral hydrophilic polymers include, but are not limited to, polysaccharides including cellulose and ethers and esters of cellulose, such as hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), hydroxyethyl methyl cellulose (HEMC), hydroxypropyl methyl cellulose (HPMC), dextran, dextrin, maltodextrin, agarose, guar gum, amylose, polysucrose, and Ficoll, hydrophilic polypeptides and poly(amino acids) such as poly-L-serine, and derivatives and combinations thereof.

[0245] In one example, the polymer is present in the assay solution at any concentration. In some embodiments, the concentration (in % w / v) of the polymer in the assay solution is about or greater than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 or more. In some embodiments, the concentration (in % w / v) of the polymer in the assay solution is about or greater than about 0.1, 0.5 or 1. In some embodiments, the concentration (in % w / v) of the polymer in the assay solution is between about 0.1 and about 1.5, between 0.1 and about 1.

[0246] The person skilled in the art will appreciate that neutral hydrophilic polymers will adopt particular conformation(s) and or size(s) in solution. Various properties for describing such conformations and / or sizes are at the disposal of the person skilled in the art. One such descriptor is the radius of gyration. Without intending to limit the scope of the present disclosure, it is believed that it may be advantageous for the neutral hydrophilic polymer to be included in the assay solution for use in the method described herein, to have a radius of gyration above a particular length. In some embodiments, the neutral hydrophilic polymer has a radius of gyration of at least about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.0 nm. In some embodiments, the neutral hydrophilic polymer has a radius of gyration of at least about 2.0 nm. In some embodiments, the neutral hydrophilic polymer has a radius of gyration of at least about 2.5 nm. In some embodiments, the neutral hydrophilic polymer has a radius of gyration of at least about 3.0 nm. In some embodiments, the neutral hydrophilic polymer has a radius of gyration of at least about 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0 nm. In some embodiments the radius of gyration is between about 3.0 and about 200 nm, between about 3.0 and about 150 nm, between about 3.0 and about 100 nm, between about 3.0 and about 75 nm, between about 3.0 and about 50 nm, between about 3.0 and about 40 nm, between about 3.0 and about 30 nm, between about 3.0 and about 20 nm, between about 2.0 and about 10 nm, between about 2.5 and about 10 nm, between about 2.0 and about 15 nm, between about 2.5 and about 15 nm, between about 3.0 and about 15 nm, between about 5.0 and about 15 nm, or between about 3.0 and about 10 nm.

[0247] For any of the aforementioned embodiments describing the radius of gyration of the neutral hydrophilic polymer, the radius of gyration may be that of the neutral hydrophilic polymer in the assay solution. For any of the aforementioned embodiments describing the radius of gyration of the neutral hydrophilic polymer, the radius of gyration may be that of the neutral hydrophilic polymer as determined according to the method described in Example 4, viz. in some embodiments, the neutral hydrophilic polymer has a radius of gyration (determined by DLS) when present at a concentration of 10 g / L in a solution of 50 mM MOPS buffer, 130 mM NaCl, pH 7.1 at 25 °C.

[0248] In some embodiments, the neutral hydrophilic polymer has a hydrodynamic radius of at least about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.0 nm. In some embodiments, the neutral hydrophilic polymer has a hydrodynamic radius of at least about 2.0 nm. In some embodiments, the neutral hydrophilic polymer has a hydrodynamic radius of at least about 2.5 nm. In some embodiments, the neutral hydrophilic polymer has a hydrodynamic radius of at least about 3.0 nm. In some embodiments, the neutral hydrophilic polymer has a hydrodynamic radius of at least about 2.0, 2.5, 3.5, 4.0, 4.5 or 5.0 nm. In some embodiments the hydrodynamic radius is between about 0.5 and about 200 nm, between about 0.5 and about 150 nm, between about 0.5 and about 100 nm, between about 0.5 and about 75 nm, between about 0.5and about 50 nm, between about 0.5 and about 40 nm, between about 0.5 and about 30 nm, between about 0.5and about 20 nm or between about 0.5 and about 10 nm, 3.0 and about 200 nm, between about 3.0 and about 150 nm, between about 3.0 and about 100 nm, between about 3.0 and about 75 nm, between about 3.0 and about 50 nm, between about 3.0 and about 40 nm, between about 3.0 and about 30 nm, between about 3.0 and about 20 nm, between about 2.0 and about 15 nm, between about 2.5 and about 15 nm, between about 2.0 and about 10 nm, between about 2.5 and about 10 nm, between about 3.0 and about 15 nm, between about 5.0 and about 15 nm, or between about 3.0 and about 10 nm. For any of the aforementioned embodiments describing the hydrodynamic radius of the neutral hydrophilic polymer, the hydrodynamic radius may be that of the neutral hydrophilic polymer in the assay solution. For any of the aforementioned embodiments describing the hydrodynamic radius of the neutral hydrophilic polymer, the hydrodynamic radius may be that of the neutral hydrophilic polymer as determined according to the method described in Example 4, viz. in some embodiments, the neutral hydrophilic polymer has a hydrodynamic radius (determined by DLS) when present at a concentration of 10 g / L in a solution of 50 mM MOPS buffer, 130 mM NaCl, pH 7.1 at 25 °C.

[0249] In some embodiments, the neutral hydrophilic polymer is branched. In some embodiments, the neutral hydrophilic polymer is linear. In one example, the neutral hydrophilic polymer has an average molecular weight (in kDa) of about or greater than about 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195 or 200. In one example, the neutral hydrophilic polymer has an average molecular weight (in kDa) of about or less than about 200, 195, 190, 185, 180, 175, 170, 165, 160, 155, 150, 145, 140, 135, 130, 125, 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.8, 0.6, 0.4, 0.2.

[0250] In one example, the neutral hydrophilic polymer is selected from polyethylene glycol, polysucrose, or a derivative or combination thereof. In some embodiments, the neutral hydrophilic polymer is a polyethylene glycol or a derivative thereof. In some embodiments, the neutral hydrophilic polymer is a polysucrose or a derivative thereof.

[0251] Polyethylene glycol (PEG) is also known as polyethylene oxide (PEO) or poly (oxy ethylene) (POE). Typically, polyethylene glycol refers to oligomers or polymers with a molecular weight below 20 kDa, polyethylene oxide to polymers with a molecular weight above 20 kDa, and poly (oxy ethylene) to a polymer of any molecular weight having the general formula HO-(CH2CH2O)n-H (which may be for example, either a polyethylene oxide or polyethylene glycol). As used herein, all three terms are used are interchangeably to refer to polymers of the formula HO-(CH2CH2O)n-H, wherein n is any integer, for example, an integer between 2 and 23000 (molar mass of a given polyethylene glycol being approximately equal to 44.05 * n + 18.02 g / mol). As such, it will be understood that a reference to polyethylene glycol will be understood to also constitute a reference to polyethylene oxide and poly(oxyethylene). Polyethylene glycols are known to persons skilled in the art and are widely commercially available. There is no particular limitation as to suitable polyethylene glycols or derivatives thereof for the purposes of the present disclosure. The polyethylene glycol may be branched or unbranched. Further, the polyethylene glycol may be substituted or unsubstituted. For example, some polyethylene glycols may be substituted at one or both ends with an alkyl group, such as a C1-C5 alkyl group, or a phenyl group, which may be optionally substituted, e.g., with an alkyl group, such as a C1-C5 alkyl group. Other forms of derivatisation that essentially retain the properties of the polyethylene glycol moiety that make them suitable for use according to the present disclosure will be known and understood to persons skilled in the art.

[0252] As used herein, polyethylene glycol derivative includes any compound comprising a polyethylene glycol moiety. Suitable polyethylene glycol moieties include, but are not limited to, polyethylene glycol laurates, polyethylene glycol lauryl ethers, polyethylene glycol monostearates, polyethylene glycol dioleates, polyethylene glycol monooleates, polyethylene glycol isostearates, polyethylene glycol.

[0253] In one example, the polyethylene glycol or the derivative thereof has an average molecular weight (in kDa) of about or greater than about 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195 or 200. In one example, the polyethylene glycol or the derivative thereof has an average molecular weight (in kDa) of about or less than about 200, 195, 190, 185, 180, 175, 170, 165, 160, 155, 150, 145, 140, 135, 130, 125, 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.8, 0.6, 0.4, 0.2.

[0254] In one example, the polyethylene glycol or the derivative thereof has an average molecular weight (in kDa) in a range provided by any two of the previously described upper and / or lower amounts, for example, in one example, the polyethylene glycol or the derivative thereof has an average molecular weight (in kDa) between about 0.2 and about 200, between about 1 and about 100, between about 3 and about 10. In one example, the polyethylene glycol or the derivative thereof has an average molecular weight (in kDa) between about 0.2 and about 200. In one example, the polyethylene glycol or the derivative thereof has an average molecular weight (in kDa) between about 1 and about 100. In one example, the polyethylene glycol or the derivative thereof has an average molecular weight (in kDa) between about 3 and about 10. In one example, the polyethylene glycol or the derivative thereof has an average molecular weight (in kDa) of about 8. In one example, the polyethylene glycol or the derivative thereof has an average molecular weight (in kDa) about or greater than about 4.

[0255] In one example, the polyethylene glycol moiety of the polyethylene glycol derivative has an average molecular weight (in kDa) of about or greater than about 0.2, 0.4, 0.6, 0.8, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195 or 200. In one example, the polyethylene glycol moiety of the polyethylene glycol derivative has an average molecular weight (in kDa) of about or less than about 200, 195, 190, 185, 180, 175, 170, 165, 160, 155, 150, 145, 140, 135, 130, 125, 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.8, 0.6, 0.4, 0.2.

[0256] In one example, the polyethylene glycol moiety of the polyethylene glycol derivative has an average molecular weight (in kDa) in a range provided by any two of the previously described upper and / or lower amounts, for example, in one example, the polyethylene glycol moiety of the polyethylene glycol derivative has an average molecular weight (in kDa) between about 0.2 and about 200, between about 1 and about 100, between about 3 and about 10. In one example, the polyethylene glycol moiety of the polyethylene glycol derivative has an average molecular weight (in kDa) between about 0.2 and about 200. In one example the polyethylene glycol moiety of the polyethylene glycol derivative has an average molecular weight (in kDa) between about 1 and about 100. In one example, the polyethylene glycol moiety of the polyethylene glycol derivative has an average molecular weight (in kDa) between about 3 and about 10. In one example, the polyethylene glycol moiety of the polyethylene glycol derivative has an average molecular weight (in kDa) of about 8.

[0257] In one example, the polyethylene glycol or the derivative thereof has a melting point (in °C) of or greater than 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80. In one example, the polyethylene glycol or the derivative thereof has a melting point (in °C) of or less than 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46 or 45. In one example, the polyethylene glycol or the derivative thereof has a melting point (in °C) in a range provided by any two of the previously described upper and / or lower amounts, for example, in one example, the polyethylene glycol or the derivative thereof has a melting point (in °C) between 45 and 80, between 50 and 75, between 55 and 75, between 55 and 70, between 45 and 75, between 50 and 70, or between 55 and 65, between 55 and 80, between 60 and 80, or between 65 and 75. The above melting points refer to the melting point of the polyethylene glycol or the derivative thereof, at standard atmospheric pressure (1 atm).

[0258] In some embodiments, the neutral hydrophilic polymer is a polysucrose or a derivative thereof. Polysucrose will be understood to encompass any polymer derived from sucrose, including, but not limited to, Ficoll. In some embodiments, the polysucrose has a molecular weight (in kDa) between about 50 and about 500. In some embodiments, the polysucrose has a molecular weight (in kDa) between about 70 and about 400. In some embodiments, the polysucrose has a molecular weight (in kDa) of at least about 50. In some embodiments, the polysucrose has a molecular weight (in kDa) of at least about 70. In some embodiments, the polysucrose has a molecular weight (in kDa) of about 70 or about 400. In some embodiments, the polysucrose has a molecular weight (in kDa) of about 70. In some embodiments, the polysucrose has a molecular weight of about 400 kDa.

[0259] In some embodiments, the neutral hydrophilic polymer is a Ficoll or derivative thereof. As used herein, “Ficoll” refers to a hydrophilic water soluble polymer formed from the copolymerization of (poly)sucrose and epichlorohydrin. The Ficoll may be linear, although is typically branched, or highly -branched. A reference to Ficoll will be understood to encompass copolymers according to the following structural formula (wherein a, b, c and n are positive integers):

[0260]

[0261] The person skilled in the art will appreciate that Ficoll derivatives may be formed, e.g. by modifying one or more functional groups comprised by the Ficoll (e.g. the hydroxyl groups or sucrose moiety), or conjugating the Ficoll to some other moiety. Ficoll polymers are commercially available, and are typically named according to the average molecule weight of the Ficoll molecules, e.g. Ficoll® 400 refers to an average molecule weight of 400 kDa. The actual molecular weight of a Ficoll sample as determined by e.g. intrinsic viscosity, may vary by as much as ±25% relative to the value stated by the provider. In some embodiments, the Ficoll has a molecular weight (in kDa) between about 50 and about 500. In some embodiments, the Ficoll has a molecular weight (in kDa) between about 70 and about 400. In some embodiments, the Ficoll has a molecular weight (in kDa) of at least about 50. In some embodiments, the Ficoll has a molecular weight (in kDa) of at least about 70. In some embodiments, the Ficoll has a molecular weight (in kDa) of about 70 or about 400. In some embodiments, the Ficoll has a molecular weight (in kDa) of about 70. In some embodiments, the Ficoll has a molecular weight of about 400 kDa.

[0262] Plasma protein binding probes

[0263] The present disclosure provides a method for determining the concentration of a plasma protein in a sample, comprising incubating the sample and a plasma protein binding probe conjugated to a fluorescent dye in an assay solution.

[0264] In one example, the plasma protein binding probe specifically binds to the plasma protein.

[0265] As used herein, the term “specifically binds” shall be taken to mean that the binding interaction between the plasma protein binding probe and plasma protein is dependent on the presence of the antigenic determinant or epitope. The binding probe preferentially binds or recognizes a specific antigenic determinant or epitope even when present in a mixture of other molecules or organisms. In one example, the binding probe reacts or associates more frequently, more rapidly, with greater duration and / or with greater affinity with the specific component or cell expressing same than it does with alternative antigens or cells. It is also understood by reading this definition that, for example, a binding probe the specifically binds to a particular component may or may not specifically bind to a second antigen. As such, “specific binding” does not necessarily require exclusive binding or non-detectable binding of another antigen. The term “specifically binds” can be used interchangeably with “selectively binds” herein. Generally, reference herein to binding means specific binding, and each term shall be understood to provide explicit support for the other term. Methods for determining specific binding will be apparent to the skilled person. For example, a binding protein comprising the binding region of the disclosure is contacted with the plasma protein or a cell expressing same or a mutant form thereof or an alternative antigen. The binding to the component or mutant form or alternative antigen is then determined and a binding region that binds as set out above is considered to specifically bind to the plasma protein.

[0266] In one example, the plasma protein binding probe of the present disclosure can take various forms (including natural or synthetic proteins) and bind one or more plasma proteins. Exemplary plasma protein binding probes are described herein and include a nucleic acid (e.g., an aptamer), a polypeptide, a peptide, a small molecule, an antibody or an antigen binding fragment of an antibody.

[0267] Immunoglobulin binding proteins

[0268] In one example, the plasma protein binding probe comprises an immunoglobulin binding protein. For example, the immunoglobulin binding protein comprises a bacterial protein. For example, the bacterial protein is an immunoglobulin-binding bacterial protein.

[0269] In one example, the immunoglobulin binding protein is selected from the group consisting of Protein A, Protein G, Protein L and combinations thereof.

[0270] It will be apparent to the skilled person that Protein A, Protein G and Protein L bind to the Fc portion of an immunoglobulin. In one example, the immunoglobulin binding protein is a truncated immunoglobulin binding protein. For example, the truncated binding protein is modified to have at least one immunoglobulin binding domain removed. For example, the immunoglobulin binding protein is a truncated immunoglobulin binding protein having a molecular weight of less than 20kDa. For example, the truncated binding protein has a molecular weight of less than 15kDa. In one example, the truncated binding protein has a molecular weight of less than 10kDa. In one example, the binding protein has a molecular weight of less than 8kD. In one example, the binding protein has a molecular weight of less than 5kD.

[0271] In one example, the plasma protein binding probe comprises Protein A. For example, the Protein A is a truncated Protein A. In another example, the Protein A is a truncated Protein A having a molecular weight of less than 20kD.

[0272] In one example, the plasma protein binding probe comprises Protein G. For example, the Protein G is a truncated Protein G. In another example, the Protein G is a truncated Protein G having a molecular weight of less than 20kD.

[0273] In one example, the plasma protein binding probe comprises Protein L. For example, the Protein L is a truncated Protein L.

[0274] In one example, the immunoglobulin binding protein (i.e., Protein A, Protein G and / or Protein L) specifically binds IgG. For example, the plasma protein binding probe is Protein A and specifically binds to IgG. In another example, the plasma protein binding probe is Protein G and specifically binds to IgG. In a further example, the plasma protein binding probe is Protein L and specifically binds to IgG.

[0275] In another example, the immunoglobulin binding protein (i.e., Protein A, Protein G and / or Protein L) specifically binds IgM. For example, the plasma protein binding probe is Protein L and specifically binds to IgM.

[0276] In another example, the immunoglobulin binding protein (i.e., Protein A, Protein G and / or Protein L) specifically binds IgA. For example, the plasma protein binding probe is Protein L and specifically binds to IgA.

[0277] Aptamer

[0278] In one example, the plasma protein binding probe comprises a nucleic acid aptamer (adaptable oligomer). In one example, the aptamer is a DNA aptamer. In another example, the aptamer is an RNA aptamer.

[0279] Aptamers are single stranded oligonucleotides or oligonucleotide analogs that are capable of forming a secondary and / or tertiary structure that provides the ability to bind to a particular target molecule, such as a plasma protein. Thus, aptamers are the oligonucleotide analogy to antibodies. In general, aptamers comprise about 15 to about 100 nucleotides, such as about 15 to about 40 nucleotides, for example about 20 to about 40 nucleotides, since oligonucleotides of a length that falls within these ranges can be prepared by conventional techniques.

[0280] An aptamer can be isolated from or identified from a library of aptamers. An aptamer library is produced, for example, by cloning random oligonucleotides into a vector (or an expression vector in the case of an RNA aptamer), wherein the random sequence is flanked by known sequences that provide the site of binding for PCR primers. An aptamer that provides the desired biological activity (e.g., binds specifically to a plasma protein) is selected. An aptamer with increased activity is selected, for example, using SELEX (Sytematic Evolution of Ligands by Exponential enrichment). Suitable methods for producing and / or screening an aptamer library are described, for example, in Elloington and Szostak, Nature 546:818-22, 1990; US 5270163; and / or US 5475096.

[0281] In one example, the plasma protein binding probe is an aptamer and specifically binds to IgA. Fc receptor and fragments thereof

[0282] In one example, the plasma protein binding probe of the present disclosure comprises a fragment crystallizable (Fc) receptor (FcR).

[0283] As discussed herein, generally a FcR binds a Fc domain of a human immunoglobulin. However, the FcR may bind an immunoglobulin of any other mammalian species, including for example, a Camelid species, a rodent (e.g. a mouse, rat, rabbit, guinea pig) or non-human primate (e.g. chimpanzee, macaque) species. It will be appreciated that the scope of the present disclosure encompasses alleles, variants and mutations of FcR.

[0284] The skilled person will appreciate that there are several different types of FcRs based on the type of antibody they recognise. For example, those that bind IgG, are called Fc-gamma receptors (FcyR), those that bind IgA are called Fc-alpha receptors (FcaR) and those that bind IgE are called Fc-epsilon receptors (FceR).

[0285] In one example, the FcR is a Fc-gamma receptor (FcyR). For example, the FcyR is a FcyRI (CD64), a FcyRIIA (CD32), a FcyRIIB (CD32), a FcyRIIIA (CD 16a), and / or a FcyRIIIB (CD 16b)

[0286] In one example, the FcR is a Fc-alpha receptor (FcaR). For example, the FcaR is a FcaRI (CD89).

[0287] In one example, the FcR is a Fc-epsilon receptor (FceR). For example, the FceR is a FceRI or a FceRII (CD23).

[0288] In one example, the FcR is a polymeric immunoglobulin receptor (plgR). The skilled person will be aware that a plgR recognized the J chain region of polymerized IgA and IgM.

[0289] In one example, the plasma protein binding probe is a FcR and specifically binds to IgG. In one example, the plasma protein binding probe is a plgR and specifically binds to IgA.

[0290] Glycoproteins and receptors thereof

[0291] In one example, the plasma protein binding probe comprises a glycoprotein.

[0292] In one example, the glycoprotein is glycoprotein lb (GPIb). In one example, the plasma protein binding probe is GPIb and specifically binds to vWF.

[0293] GPIb, also known as CD42, is a platelet surface membrane glycoprotein composed of a heterodimer, an alpha chain and a beta chain, that is linked by disulfide bonds. The GPIb functions as a receptor for von Willebrand factor (vWF).

[0294] In one example, the glycoprotein is a thrombomodulin (also known as CD141 or BDCA-3). In one example, the plasma protein binding protein is thrombomodulin and specifically binds to Protein C.

[0295] In one example, the glycoprotein is von Willebrand factor (vWF). In one example, the plasma protein binding probe is vWF and specifically binds factor VIII (FVIII).

[0296] In one example, the plasma protein binding probe comprises a glycoprotein receptor. For example, a transmembrane glycoprotein receptor. In one example, the glycoprotein receptor is an N-glycosylated type I membrane protein.

[0297] In one example, the transmembrane glycoprotein receptor is an endothelial protein C receptor (EPCR; also known as activated protein C receptor (APC receptor)). In one example, the plasma protein binding probe is EPCR and specifically binds to Protein C.

[0298] In one example, the plasma protein binding probe comprises a lactadherin domain. Lactadherin is a glycoprotein secreted by a variety of cell types and contains two EGF domains and two C domains (C1C2 and C2) with sequence homology to the Cl and C2 domains of blood coagulation factors V and VIII. Similar to these coagulation factors, lactadherin binds to phosphatidylserine (PS)-containing membranes with high affinity.

[0299] In one example, the lactadherin domain is a C1C2 domain. In another example, the lactadherin domain is a C2 domain.

[0300] Phospholipids

[0301] In one example, the plasma protein binding probe comprises a phospholipid.

[0302] The skilled person will recognise that phospholipids are a class of lipids that have a hydrophilic head containing a phosphate group and two hydrophobic tails derived from fatty acids, joined by an alcohol residue (usually a glycerol).

[0303] Exemplary phospholipids suitable for use in the present disclosure will be apparent to the skilled person and include phosphatidic acid (phosphatidate); phosphatidylethanolamine (cephalin); phosphatidylcholine (lecithin); phosphatidylserine; phosphoinositides such as phosphatidylinositol, phosphatidylinositol phosphate, phosphatidylinositol bisphosphate and phosphatidylinositol trisphosphate; and phosphosphingolipids such as ceramide phosphorylcholine (Sphingomyelin), ceramide phosphorylethanolamine (Sphingomyelin) and ceramide phosphoryllipid.

[0304] In one example, the plasma protein binding probe is a phospholipid and specifically binds to apolipoprotein Al.

[0305] Plasminogen Activator Proteins

[0306] In one example, the plasma protein binding probe comprises a plasminogen activator protein.

[0307] In one example, the plasminogen activator protein is staphylokinase.

[0308] The skilled person will recognise that staphylokinase is a 136 aa long bacteriophage encoded protein expressed by lysogenic strains of Staphylococcus aureus. Staphylokinase forms a complex with human plasmin catalysing the activation of plasminogen.

[0309] In one example, the plasma protein binding probe is staphylokinase and specifically binds to plasminogen.

[0310] Protein Kinase Domains

[0311] In one example, the plasma protein binding probe comprises a protein kinase C domain.

[0312] Protein kinase C (PKC) is a family of protein kinase enzymes that are involved in controlling the function of other proteins through the phosphorylation of hydroxyl groups of serine and threonine amino acid residues on these proteins, or a member of this family.

[0313] The structure of PKC is known in the art and consists of a regulatory domain and a catalytic domain tethered together by a hinge region. The regulatory domain comprises a Cl and a C2 domain which bind to DAG and Ca2+respectively to recruit PKC to the plasma membrane.

[0314] In one example, the protein kinase C domain is the Cl domain. In another example, the protein kinase C domain is the C2 domain. Pleckstrin Homology Domain

[0315] In one example, the plasma protein binding probe comprises a pleckstrin homology (PH) domain.

[0316] The PH domain is known in the art and is a small modular domain that occurs in a wide range of proteins involved in intracellular signalling or as a constituent of the cytoskeleton. The PH domain comprises approximately 120 amino acids. The domains can bind phosphatidylinositol within biological membranes and proteins such as the beta / gamma subunits of heterotrimeric G proteins. Through these interactions, PH domains play a role in recruiting proteins to different membranes, thus targeting them to appropriate cellular compartments or enabling them to interact with other components of the signal transduction pathways.

[0317] Coagulation Factors

[0318] In one example, the plasma protein binding probe comprises a coagulation factor.

[0319] In one example, the coagulation factor is an antihemophilic factor (also known as Factor VIII). In one example, the plasma protein binding probe is antihemophilic factor and specifically binds to FVIII.

[0320] In one example, the coagulation factor is Factor Xa (FXa). In one example, the plasma protein binding probe is FXa and specifically binds to Antithrombin III (ATIII).

[0321] In one example, the coagulation factor is Factor la (Fla; also known as fibrin). In one example, the plasma protein binding probe is fibrin and specifically binds to FXIII. In one example, the plasma protein binding probe is fibrin and specifically binds to fibrinogen.

[0322] Anti-coagulants

[0323] In one example, the plasma protein binding probe comprises an anti-coagulant.

[0324] In one example, the anti-coagulant is warfarin. In another example, the anti-coagulant is heparin. In a further example, the anti-coagulant is Protein S.

[0325] In one example, the plasma protein binding probe is Protein S and specifically binds to Protein C. In one example, the plasma protein binding probe is warfarin and specifically binds to albumin. In one example, the plasma protein binding probe is heparin and specifically binds to vWF. In one example, the plasma protein binding probe is heparin and specifically binds to ATIII.

[0326] In one example, the anti-coagulant is a Factor Xa (FXa) inhibitor. For example, the FXa inhibitor is apixaban or edoxaban. In one example, the FXa inhibitor is apixaban. In another example, the FXa inhibitor is edoxaban.

[0327] In one example, the plasma protein binding probe is apixaban and specifically binds to FXa. In one example, the plasma protein binding probe is edoxaban and specifically binds to FXa.

[0328] Lysine Analogues

[0329] In one example, the plasma protein binding probe comprises a lysine analogue.

[0330] In one example, the lysine analogue is an anti-fibrinolytic. For example, tranexamic acid (TXA) or epsilon aminocaproic acid (EACA).

[0331] In one example, the lysine analogue is a cyclopropyllysine (KCP).

[0332] In one example, the lysine analogue is a benzylamine (Kba).

[0333] In one example, the lysine analogue is a meto-aminophenylalanine (F3a).

[0334] In on example, the lysine analogue is a para-aminophenylalanine (F4a) In one example, the lysine analogue is a pyridylalanine (AP).

[0335] In one example, the lysine analogue is tyrosine (Y).

[0336] In one example, the plasma protein binding probe is a lysine analogue and specifically binds to plasmin. In one example, the plasma protein binding probe is a lysine analogue and specifically binds to plasminogen.

[0337] Divalent cations

[0338] In one example, the plasma protein binding probe comprises a divalent cation.

[0339] In one example, the divalent cation is calcium (Ca2+) ions. In one example, the plasma protein binding probe is Ca2+ and specifically binds to FXIII.

[0340] In one example, the divalent cation is zinc (Zn2+) ions. In one example, the plasma protein binding probe is ZN2+ and specifically binds to prekallikrein activator.

[0341] Gamma-carb oxy glutamic acid-rich (GLA) Domains

[0342] In one example, the plasma protein binding probe comprises a gamma-carboxyglutamic acid-rich (GLA) domain or variant thereof.

[0343] The GLA domain contains glutamate residues that have been post-translationally modified by vitamin K-dependent carboxylation to form gamma-carboxy glutamate (Gia).

[0344] Proteins known to comprise a GLA domain are known in the art and include, but are not limited to, vitamin K-dependent proteins S and Z, prothrombin, transthyretin, osteocalcin, matrix GLA protein, inter-alpha-trypsin inhibitor heavy chain H2 and growth arrest-specific protein 6.

[0345] Complement System Components

[0346] In one example, the plasma protein binding probe comprises a complement system component.

[0347] In one example, the complement system component is complement component 1q (C1q). In one example, the plasma protein binding probe is C1q and specifically binds to IgM.

[0348] Proteases

[0349] In one example, the plasma protein binding probe is a protease. For example, the protease is an aspartic protease, a glutamic protease, a metalloprotease, a cysteine protease, a serine protease, and / or a threonine protease.

[0350] In one example, the protease is a serine protease. In one example, the serine protease is a urokinase (also known as a urokinase -type plasminogen activator). In another example, the serine protease is a tissue plasminogen activator (also known as a tissue-type plasminogen activator or tPA). In a further example, the serine protease is an elastase. In one example, the serine protease is a kallikrein. For example, the kallikrein is a high molecular weight kallikrein. In another example, the serine protease is a thrombin.

[0351] In one example, the plasma protein binding probe is urokinase and specifically binds to plasminogen. In one example, the plasma protein binding probe is urokinase and specifically binds to tPA. In one example, the plasma protein binding probe is elastase and specifically binds to alpha- 1 antitrypsin. In one example, the plasma protein binding probe is kallikrein and specifically binds to prekallikrein activator. In one example, the plasma protein binding probe is high molecular weight kallikrein and specifically binds to prekallikrein activator. In one example, the plasma protein binding probe is thrombin and specifically binds to fibrinogen. In one example, the plasma protein binding probe is thrombin and specifically binds to ATIII.

[0352] Protease Inhibitors

[0353] In one example, the plasma protein binding probe is a protease inhibitor. For example, the protease inhibitor is an aspartic protease inhibitor, a cysteine protease inhibitor, a metalloprotease inhibitor, a serine protease inhibitor and / or a threonine protease inhibitor.

[0354] In one example, the protease inhibitor is a serine protease inhibitor or serpin.

[0355] In one example, the serine protease inhibitor is an elastase inhibitor. In another example, the serine protease inhibitor is a bovine pancreatic trypsin inhibitor (BPTI). For example, the BPTI is aprotinin. In a further example, the serine protease inhibitor is an alpha 2-antiplasmin inhibitor. In one example, the serine protease inhibitor is an alpha 1 -antichymotrypsin. In another example, the serine protease inhibitor is a phenylmethylsulfonyl fluoride (or benzylsulfonyl fluoride). In a further example, the serine protease inhibitor is a benzamidine. For example, a benzamidine hydrochloride.

[0356] In one example, the plasma protein binding probe is aprotinin and specifically binds to a serine protease. In one example, the plasma protein binding probe is alpha 2-antiplasmin inhibitor and specifically binds to a serine protease. In one example, the plasma protein binding probe is alpha 1 -antichymotrypsin and specifically binds to alpha- 1 antitrypsin. In one example, the plasma protein binding probe is benzamidine and specifically binds to a serine protease.

[0357] In one example, the protease inhibitor is a serine, cysteine and threonine protease inhibitor. For example, the serine, cysteine and threonine protease inhibitor is a leupeptin.

[0358] In one example, the plasma protein binding probe is leupeptin and specifically binds to a serine protease. In one example, the protease inhibitor is a proteinase inhibitor.

[0359] In one example, the proteinase inhibitor is a serine proteinase inhibitor.

[0360] In one example, the binding probe is a kunitz domain of a protease inhibitor. For example, a kunitz domain peptide. The skilled person will recognize that kunitz domain peptides comprise a disulfide rich alpha and beta fold and are relatively small with a length of about 50 to 60 amino acids and a molecular weight of about 6 kDa. Exemplary kunitz domain proteins include aprotinin (bovine pancreatic trypsin inhibitor, BPTI), Alzheimer's amyloid precursor protein (APP), and tissue factor pathway inhibitor (TFPI).

[0361] Small molecules

[0362] In one example, the plasma protein binding probe is a small molecule.

[0363] In one example, the small molecule is an anxiolytic benzodiazepine. For example, a diazepam.

[0364] In one example, the small molecule is a salicylate. For example an acetylsalicylic acid. In one example, the acetylsalicylic acid is aspirin.

[0365] In one example, the plasma protein binding probe is diazepam and specifically binds to albumin. In one example, the plasma protein binding probe is aspirin and specifically binds to albumin.

[0366] Amphipathic helix

[0367] In one example, the plasma protein binding probe is an amphipathic helix. The skilled person will recognise that an amphipathic (or amphiphilic) helix is a helix with both hydrophobic and hydrophilic amino acid residues arranged in such a way as to create two faces on opposite sides of the helix, one face being hydrophobic.

[0368] In one example, the amphipathic helix is an amphipathic alpha helix.

[0369] In one example, the plasma protein binding probe is an amphipathic alpha helix and specifically binds to an apolipoprotein Al.

[0370] Antibodies and antigen binding fragment thereof

[0371] In one example, the plasma protein binding probe of the present disclosure comprises an antigen binding domain of an antibody.

[0372] As used herein, the term “antigen binding domain” shall be taken to mean a region of an antibody that is capable of specifically binding to an antigen, i.e., a VH or a VL or an Fv comprising both a VH and a VL. The antigen binding domain need not be in the context of an entire antibody, e.g., it can be in isolation (e.g., a domain antibody) or in another form, e.g., as described herein, such as a scFv.

[0373] As used herein, the term “Fv” shall be taken to mean any protein, whether comprised of multiple polypeptides or a single polypeptide, in which a VLand a VHassociate and form a complex having an antigen binding domain, i.e., capable of specifically binding to an antigen. The VHand the VLwhich form the antigen binding domain can be in a single polypeptide chain or in different polypeptide chains. Furthermore, an Fv of the disclosure (as well as any protein of the disclosure) may have multiple antigen binding domains which may or may not bind the same antigen. This term shall be understood to encompass fragments directly derived from an antibody as well as proteins corresponding to such a fragment produced using recombinant means. In some examples, the VH is not linked to a heavy chain constant domain (CH) 1 and / or the VL is not linked to a light chain constant domain (CL). Exemplary Fv containing polypeptides or proteins include a Fab fragment, a Fab’ fragment, a F(ab’) fragment, a scFv, a diabody, a triabody, a tetrabody or higher order complex, or any of the foregoing linked to a constant region or domain thereof, e.g., CH2 or CH3 domain, e.g., a minibody. A "Fab fragment" consists of a monovalent antigen-binding fragment of an immunoglobulin, and can be produced by digestion of a whole antibody with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain or can be produced using recombinant means. A "Fab' fragment" of an antibody can be obtained by treating a whole antibody with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of a heavy chain comprising a VH and a single constant domain. Two Fab' fragments are obtained per antibody treated in this manner. A Fab’ fragment can also be produced by recombinant means. A "F(ab')2 fragment” of an antibody consists of a dimer of two Fab' fragments held together by two disulfide bonds, and is obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction. A “Fab2” fragment is a recombinant fragment comprising two Fab fragments linked using, for example a leucine zipper or a CH3 domain.

[0374] In one example, the binding probe is an antibody.

[0375] For the purposes for the present disclosure, the term “antibody” includes a protein capable of specifically binding to one or a few closely related antigens by virtue of an antigen binding domain contained within a Fv. This term includes four chain antibodies (e.g., two light chains and two heavy chains), recombinant or modified antibodies (e.g., chimeric antibodies, humanized antibodies, human antibodies, CDR-grafted antibodies, primatized antibodies, de-immunized antibodies, synhumanized antibodies, half-antibodies, bispecific antibodies). An antibody generally comprises constant domains, which can be arranged into a constant region or constant fragment or fragment crystallizable (Fc). Exemplary forms of antibodies comprise a four-chain structure as their basic unit. Full-length antibodies comprise two heavy chains (~50 to 70 kD) covalently linked and two light chains (~23 kDa each). A light chain generally comprises a variable region (if present) and a constant domain and in mammals is either a K light chain or a λ light chain. A heavy chain generally comprises a variable region and one or two constant domain(s) linked by a hinge region to additional constant domain(s). Heavy chains of mammals are of one of the following types a, 5, e, y, or p. Each light chain is also covalently linked to one of the heavy chains. For example, the two heavy chains and the heavy and light chains are held together by inter-chain disulfide bonds and by non-covalent interactions. The number of inter-chain disulfide bonds can vary among different types of antibodies. Each chain has an N-terminal variable region (VH or VL wherein each are ~110 amino acids in length) and one or more constant domains at the C-terminus. The constant domain of the light chain (CL which is ~110 amino acids in length) is aligned with and disulfide bonded to the first constant domain of the heavy chain (CHI which is 330 to 440 amino acids in length). The light chain variable region is aligned with the variable region of the heavy chain. The antibody heavy chain can comprise 2 or more additional CHdomains (such as, CH2, CH3 and the like) and can comprise a hinge region between the CH1 and CH2 constant domains. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGi, IgG2, IgG3, IgG4, IgAi and IgA2) or subclass. In one example, the antibody is a murine (mouse or rat) antibody or a primate (such as, human) antibody. In one example the antibody heavy chain is missing a C-terminal lysine residue. In one example, the antibody is humanized, synhumanized, chimeric, CDR-grafted or deimmunized.

[0376] Methods for generating antibodies are known in the art and / or described in Harlow and Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988). Generally, in such methods the soluble molecule or a region thereof (e.g., an extracellular domain) or immunogenic fragment or epitope thereof or a cell expressing and displaying same (i.e., an immunogen), optionally formulated with any suitable or desired carrier, adjuvant, or pharmaceutically acceptable excipient, is administered to a non-human animal, for example, a mouse, chicken, rat, rabbit, guinea pig, dog, horse, cow, goat or pig. The immunogen may be administered intranasally, intramuscularly, subcutaneously, intravenously, intradermally, intraperitoneally, or by other known routes.

[0377] Monoclonal antibodies are one exemplary form of antibody contemplated by the present disclosure. The term “monoclonal antibody" or “mAb” refers to a homogeneous antibody population capable of binding to the same antigen(s), for example, to the same epitope within the antigen. This term is not intended to be limited as regards to the source of the antibody or the manner in which it is made.

[0378] For the production of mAbs any one of a number of known techniques may be used, such as, for example, the procedure exemplified in US4196265 or Harlow and Lane (1988), supra.

[0379] Alternatively, ABL-MYC technology (NeoClone, Madison WI 53713, USA) is used to produce cell lines secreting MAbs (e.g., as described in Largaespada et al, J. Immunol. Methods. 197: 85-95, 1996).

[0380] The antibody of the present disclosure may be a synthetic antibody. For example, the antibody is a chimeric antibody, a humanized antibody, a human antibody or a de-immunized antibody.

[0381] In one example, an antibody described herein is a chimeric antibody. The term “chimeric antibody” refers to antibodies in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species (e.g., murine, such as mouse) or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species (e.g., primate, such as human) or belonging to another antibody class or subclass. Methods for producing chimeric antibodies are described in, e.g., US4816567; and US5807715.

[0382] The antibodies of the present disclosure may be humanized or human.

[0383] The term "humanized antibody” shall be understood to refer to a subclass of chimeric antibodies having an antigen binding site or variable region derived from an antibody from a non-human species and the remaining antibody structure based upon the structure and / or sequence of a human antibody. In a humanized antibody, the antigen-binding site generally comprises the complementarity determining regions (CDRs) from the non-human antibody grafted onto appropriate FRs in the variable regions of a human antibody and the remaining regions from a human antibody. Antigen binding sites may be wild-type (i.e., identical to those of the non-human antibody) or modified by one or more amino acid substitutions. In some instances, FR residues of the human antibody are replaced by corresponding non-human residues.

[0384] Methods for humanizing non-human antibodies or parts thereof (e.g., variable regions) are known in the art. Humanization can be performed following the method of US5225539, or US5585089. Other methods for humanizing an antibody are not excluded.

[0385] The term "human antibody" as used herein refers to antibodies having variable regions (e.g. VH, VL) and, optionally constant regions derived from or corresponding to sequences found in humans, e.g. in the human germline or somatic cells.

[0386] The "human" antibodies can include amino acid residues not encoded by human sequences, e.g. mutations introduced by random or site directed mutations in vitro (in particular mutations which involve conservative substitutions or mutations in a small number of residues of the antibody, e.g. in 1, 2, 3, 4, 5 or 6 of the residues of the antibody, e.g. in 1, 2, 3, 4, 5 or 6 of the residues making up one or more of the CDRs of the antibody). These “human antibodies” do not actually need to be produced by a human, rather, they can be produced using recombinant means and / or isolated from a transgenic animal (e.g., mouse) comprising nucleic acid encoding human antibody constant and / or variable regions (e.g., as described above). Human antibodies can be produced using various techniques known in the art, including phage display libraries (e.g., as described in US5885793).

[0387] Human antibodies which recognize a selected epitope can also be generated using a technique referred to as "guided selection." In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope (e.g., as described in US5565332).

[0388] Single domain antibodies

[0389] In some examples, an antigen binding fragment of an antibody of the disclosure is or comprises a singledomain antibody (which is used interchangeably with the term “domain antibody” or “dAb” or “nanobody”). A singledomain antibody is a single polypeptide chain comprising all or a portion of the heavy chain variable domain of an antibody.

[0390] Diabodies, Triabodies and Tetrabodies

[0391] In some examples, an antigen binding fragment of the disclosure is or comprises a diabody, triabody, tetrabody or higher order protein complex such as those described in WO98 / 044001 and / or WO94 / 007921.

[0392] For example, a diabody is a protein comprising two associated polypeptide chains, each polypeptide chain comprising the structure VL-X-VH or VH-X-VL, wherein X is a linker comprising insufficient residues to permit the VH and VL in a single polypeptide chain to associate (or form an Fv) or is absent, and wherein the VH of one polypeptide chain binds to a VL of the other polypeptide chain to form an antigen binding site, i.e., to form a Fv molecule capable of specifically binding to one or more antigens. The VL and VH can be the same in each polypeptide chain or the VL and VH can be different in each polypeptide chain so as to form a bispecific diabody (i.e., comprising two Fvs having different specificity).

[0393] Single Chain Fv (scFv) Fragments

[0394] The binding probe of the disclosure can be a scFv The skilled artisan will be aware that scFvs comprise VH and VL regions in a single polypeptide chain and a polypeptide linker between the VH and VL which enables the scFv to form the desired structure for antigen binding (i.e., for the VH and VL of the single polypeptide chain to associate with one another to form a Fv). For example, the linker comprises in excess of 12 amino acid residues with (Gly4Ser)3being one of the more favored linkers for a scFv.

[0395] The present disclosure also contemplates a disulfide stabilized Fv (or diFv or dsFv), in which a single cysteine residue is introduced into a FR of VHand a FR of VLand the cysteine residues linked by a disulfide bond to yield a stable Fv.

[0396] Alternatively, or in addition, the present disclosure encompasses a dimeric scFv, i.e., a protein comprising two scFv molecules linked by a non-covalent or covalent linkage, e.g., by a leucine zipper domain (e.g., derived from Fos or Jun). Alternatively, two scFvs are linked by a peptide linker of sufficient length to permit both scFvs to form and to bind to an antigen, e.g., as described in US20060263367.

[0397] Half-antibodies

[0398] In some examples, the antigen binding fragment of the present disclosure is a half-antibody or a halfmolecule. The skilled artisan will be aware that a half antibody refers to a protein comprising a single heavy chain and a single light chain. In one example, a half antibody forms when an antibody dissociates to form two molecules each containing a single heavy chain and a single light chain.

[0399] Methods for generating half antibodies are known in the art and exemplary methods are described herein. In one example, the half antibody can be secreted by introducing into cells genes of the single heavy chain and single light chain that constitute the IgG of interest for expression. In one example, a constant region (e.g., an IgG4constant region) comprises a “key or hole” (or “knob or hole”) mutation to prevent heterodimer formation. In one example, a constant region (e.g., an IgG i constant region) comprises a T366W mutation (or knob). In another example, a constant region (e.g., an IgG i constant region) comprises a T366S, L368A and Y407V mutation (or hole). In another example, the constant region comprises T350V, T366L, K392L and T394W mutations (knob). In another example, the constant region comprises T350V, L351Y, F405A and Y407V mutations (hole). Exemplary constant region amino acid substitutions are numbered according to the EU numbering system.

[0400] Other antibody fragments

[0401] The present disclosure also contemplates other antibodies and antibody fragments, such as:

[0402] (i) minibodies, e.g., as described inUS5837821;

[0403] (ii) heteroconjugate proteins, e.g., as described in US4676980;

[0404] (iii) heteroconjugate proteins produced using a chemical cross-linker, e.g., as described in US4676980; and (iv) Faba (e.g., as described in EP19930302894).

[0405] Heavy chain immunoglobulins

[0406] Heavy chain immunoglobulins differ structurally from many other forms of immunoglobulin (e.g., antibodies), in so far as they comprise a heavy chain, but do not comprise a light chain. Accordingly, these immunoglobulins are also referred to as “heavy chain only antibodies”. Heavy chain immunoglobulins are found in, for example, camelids and cartilaginous fish (also called IgNAR).

[0407] The variable regions present in naturally occurring heavy chain immunoglobulins are generally referred to as "VHH domains" in camelid Ig and V-NAR in IgNAR, in order to distinguish them from the heavy chain variable regions that are present in conventional 4-chain antibodies (which are referred to as "VH domains") and from the light chain variable regions that are present in conventional 4-chain antibodies (which are referred to as "VL domains").

[0408] Heavy chain immunoglobulins do not require the presence of light chains to bind with high affinity and with high specificity to a relevant antigen. This means that single domain binding fragments can be derived from heavy chain immunoglobulins, which are easy to express and are generally stable and soluble.

[0409] A general description of heavy chain immunoglobulins from camelids and the variable regions thereof and methods for their production and / or isolation and / or use is found inter alia in the following references WO94 / 04678, WO97 / 49805 and WO 97 / 49805.

[0410] A general description of heavy chain immunoglobulins from cartilaginous fish and the variable regions thereof and methods for their production and / or isolation and / or use is found inter alia in W02005118629.

[0411] Antibody mimetics

[0412] Adnectins

[0413] In one example, a binding probe of the present disclosure comprises an adnectin. Adnectins are based on the tenth fibronectin type III (10Fn3) domain of human fibronectin in which the loop regions are altered to confer antigen binding. For example, three loops at one end of the p-sandwich of the10Fn3 domain can be engineered to enable an Adnectin to specifically recognize an antigen. For further details see US20080139791 or W02005056764.

[0414] Anticalins

[0415] In a further example, a binding probe of the disclosure comprises an anticalin. Anticalins are derived from lipocalins, which are a family of extracellular proteins which transport small hydrophobic molecules such as steroids, bilins, retinoids and lipids. Lipocalins have a rigid -sheet secondary structure with a plurality of loops at the open end of the conical structure which can be engineered to bind to an antigen. Such engineered lipocalins are known as anticalins. For further description of anticalins see US7250297 or US20070224633.

[0416] Affibodies

[0417] In a further example, a binding probe of the disclosure comprises an affibody. An affibody is a scaffold derived from the Z domain (antigen binding domain) of Protein A of Staphylococcus aureus which can be engineered to bind to antigen. The Z domain consists of a three-helical bundle of approximately 58 amino acids. Libraries have been generated by randomization of surface residues. For further details see EP1641818. Avimers

[0418] In a further example, a binding probe of the disclosure comprises an Avimer. Avimers are multidomain proteins derived from the A-domain scaffold family. The native domains of approximately 35 amino acids adopt a defined disulphide bonded structure. Diversity is generated by shuffling of the natural variation exhibited by the family of A-domains. For further details see W02002088171.

[0419] DARPins

[0420] In a further example, a binding probe of the disclosure comprises a Designed Ankyrin Repeat Protein (DARPin). DARPins are derived from Ankyrin which is a family of proteins that mediate attachment of integral membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33 residue motif consisting of two a-helices and a P-tum. They can be engineered to bind different target antigens by randomizing residues in the first a-helix and a P-tum of each repeat. Their binding interface can be increased by increasing the number of modules (a method of affinity maturation). For further details see US20040132028.

[0421] Sherpabodies

[0422] In one example, a binding probe of the disclosure comprises a sherpabody. The skilled person will recognize that sherpabodies are small antibody-mimetic scaffold proteins around 60 amino acids or less and comprise an improved SH3 -domain fold derived from the human ciliary adaptor protein nephrocystin. Sherpabodies can be engineered to bind to different targets and selected for by phage affinity selection processes using standard solid phase sorting.

[0423] Affitins

[0424] In one example, the binding probe of the disclosure comprises an affitin. The skilled person will recognize that affitins, also known as nanofitins, consist of 66 amino acids and have a molecular mass of about 7 kDa. Affitins are structurally derived from the DNA binding protein Sac7d, found in Sulfolobus acidocaldarius, and by randomizing the amino acids on the binding surface of Sac7d and subjecting the resulting protein to rounds of ribosome display, the affitin has the ability to selectively bind various target antigens.

[0425] Fynomers

[0426] In one example, the binding probe of the disclosure comprises a fynomer. The skilled person will recognize that fynomers are small globular binding proteins with a molecular weight of 7 kDa, composed of amino acids 83 to 145 of the Src homology 3 (SH3) domain of the human tyrosine-protein kinase Fyn18. Fynomers can be engineered to yield specific and high-affinity binding domains to target specific proteins.

[0427] Affimer

[0428] In one example, the binding probe is an affimer. The skilled person will recognize that affimers are small proteins that bind to target proteins with affinity in the nanomolar range. Affimers are 12-14 kDa proteins derived from the cysteine protease inhibitor family of cystatins and share a common tertiary structure of an alpha-helix lying on top of an anti-parallel beta sheet. Affimer proteins display two peptide loops that can be randomized to bind to desired target proteins. The skilled person will recognize that phage display libraries can be used to screen for affimers that exhibit high-specificity binding.

[0429] Alphabodies

[0430] In one example, the binding probe is an alphabody. The skilled person will recognize that alphabodies are also known as cell-penetrating alphabodies or CP AB and are small 10 kDa proteins. Alphabodies are a single peptide chain containing three alpha-helices, composed of four heptad repeats, connected by glycine / serine rich linker regions. The three alpha-helices (A, B and C) were designed to maintain correct folding and thermostability even when modified. Residues in the groove between helices A and C can be modified to bind convex targets, or on the outside of helix C to bind concave protein targets.

[0431] Monobodies

[0432] In one example, the binding probe is a monobody. Monobodies are synthetic binding proteins constructed using a fibronectin type III domain (FN3) as a molecular scaffold. Specifically, this class of binding proteins are built upon a diversified library of the 10th FN3 domain of human fibronectin. The native FN3 scaffold consists of 94 amino acids and has a molecular mass of about 10 kDa. The FN3 domain has a structure similar to antibody variable domains, with seven beta sheets forming a beta-sandwich and three exposed loops on each side corresponding to the three complementarity -determining regions. The skilled person will recognize that high affinity and specificity for different target molecules can be generated from combinatorial libraries in which portions of the FN3 scaffold are diversified.

[0433] NanoCLAMPs

[0434] In one example, the binding probe is a nanoCLAMP. The skilled person will recognize that nanoCLAMPs (Clostridial Antibody Mimetic Proteins) are recombinant 15 kDa antibody mimetic proteins selected for tight, selective and gently reversible binding. The nanoCLAMP scaffold is based on an IgG-like, thermostable carbohydrate binding module family 32 (CBM32) from a Clostridium perfringens hyaluronidase (Mu toxin). nanoCLAMPs to specific targets are generated by varying the amino acid sequences and sometimes the length of three solvent exposed, adjacent loops that connect the beta strands making up the beta-sandwich fold, conferring binding affinity and specificity for the target.

[0435] Optimers

[0436] In one example, the binding probe is an optimer. The skilled person will recognize that optimers are short synthetic oligonucleotides composed of RNA or DNA that bind to a specific target molecule. These nucleic acid molecules can exhibit cognate base-pairing to produce sections of double-stranded DNA or RNA within the Optimer molecules. The skilled person will understand that as not all of the bases in the Optimer sequence will be compatible for internal double-stranded pairing, single-stranded loop and bulge regions will remain in the secondary and tertiary structures, where hydrogen bond acceptor and donor groups are exposed and available to interact with the selected target for target engagement and target binding. Optimer ligands can be produced via solid-phase synthesis and are selected via an automated, high throughput in vitro screening process. Following selection of the appropriate sequence the identified Optimer undergoes a process to determine the minimum oligonucleotide fragment within this sequence that possesses the correct target-binding characteristics. The Optimer is trimmed to contain only this sequence, removing additional free non-target binding nucleotide bases. This reduces the molecular weight of the Optimer from 29 kDa to ~5 kDa and increases the stability of the molecule through a reduction in entropy, as additional motion of the free nucleotides is removed.

[0437] Repebodies

[0438] In one example, the binding probe is a repebody. The skilled person will recognize that repebodies are composed of leucine-rich repeat modules, each of which has a p-strand-tum-a helix structure, in a horseshoe-shaped solenoid fold. Repebodies are based on variable lymphocyte receptors (VLRs), which are non-immunoglobulin antibodies in jawless vertebrates.

[0439] Centyrins

[0440] In one example, the binding probe is a centyrin. The skilled person will recognize that centyrins are small, engineer proteins comprising a consensus fibronectin type III (FN3) domain sequence based on human Tenascin C. Amino acids within the centyrin framework can be varied to bind antigens of interest with high specificity and affinity.

[0441] Obody

[0442] In one example, the binding probe is an obody. The skilled person will recognize that an obody is small, versatile single-domain protein binding module that binds protein, carbohydrate, nucleic acid and small-molecule ligands. In one example, the obody is an OB-fold anticodon recognition domain from aspartyl tRNA synthetase taken from the thermophile Pyrobaculum aerophilum.

[0443] Knottin

[0444] In one example, the binding probe is an inhibitor cystine knot (or knottin). The skilled person will recognize that knottin is a protein structural motif containing three disulfide bridges. It will also be apparent to the skilled person that knottin’ s are present in a number of different proteins including, but not limited to, agouti related peptide, agouti signalling peptide, albumin I, covalitoxin-II, DkTx, grammotoxin, GsMTx-4, guangxitoxin, hainantoxin, hanatoxin, heteroscodratoxin-1, huwentoxin, maurocalcine, theraphosa leblondi toxin, 5-palutoxin, phrixotoxin, psalmotoxin, robustoxin, stromatoxin, tachystatin, vanillotoxin and vejocalcin.

[0445] Atrimer

[0446] In one example, the binding probe is an atrimer. The skilled person will recognize that atrimers are trivalent adjustable scaffolds comprising three binding domains containing five amino-acid loops that can be modified to bind the antigen of interest with high specificity.

[0447] Fluorescent Dye

[0448] As described herein, the plasma protein binding probe as used herein is conjugated to a fluorescent dye. There is no particular limitation on fluorescent dyes to which the plasma protein binding probe is conjugated, that may be used in the methods described herein.

[0449] As used herein, the term "fluorescent dye" (or "fluorescent probe" or "fluorophore") should be understood to mean a fluorescent chemical, probe or moiety that can re-emit light upon light excitation. Fluorescent dyes will be known to those skilled in the art. Examples of fluorescent dyes include, but are not limited to, organic dyes, organometallic compounds, metal chelates, quantum dots or nanoparticles, and combinations of the above. Further examples include, but are not limited to, fluorescent proteins such as GFP, YFP and RFP, and non-protein organic fluorophores including tetrapyrrole derivatives, pyrene derivatives, xanthene derivatives, and cyanine derivatives.

[0450] Further examples of suitable fluorescent dyes include, but are not limited to, ATTO 565, ATTO 655, Acridine Orange, Acridine Yellow, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, BODIPY 500 / 510, BODIPY 530 / 550, BODIPY FL, BODIPY TR-X, Cascade Blue, Coumarin 6, CY2, CY3B, CY3, CY3.5, CY5, CY5.5, Dansyl, DAPI, DPH, Erythrosin, Ethidium Bromide, Fluorescein, FITC, FURA-2, GFP, Hoechst 33258, Hoechst 33342, HPTS, Indocyanine Green, KU530, KU560, Laurdan, Lucifer Yellow, Nile Red, Oregon Green, Prodan, Pyrene, Rhodamine 101, Rhodamine 110, Rhodamine 123, Rhodamine 6G, Rhodamine B, Ru(bpy)3[PF6]2, Ru(bpy)2(dcpby)[PF6]2, SeTau dyes such as SeTau-380, SeTau-404, SeTau-405, SeTau-647, SeTau-425-NHS, SITS, SNART, Stilbene SITS, SITA, Texas Red, TOTO-1, YOYO-1, and YOYO-3. Such dyes are commercially available, typically in a form comprising a reactive moiety to facilitate labelling (e.g. of the plasma protein binding probe to be used according to the methods described herein). For example, dyes may be commercially available in a form comprising e.g. an amine -reactive handle such as an NHS group, or a cysteine-reactive handle such as a maleimide. Where not commercially available, it is well within the skills of a competent synthetic chemist to modify a dye to contain a reactive moiety to facilitate labelling to e.g. a plasma protein binding probe. Likewise, attachment or conjugation of a fluorescent dye (comprising a reactive moiety) to a plasma protein binding probe will be well within the ability of the skilled person. Commercially available dyes comprising reactive moieties are typically accompanied with manufacturer’s instructions for conjugating the fluorescent dye to various substrates, e.g. proteins.

[0451] In some embodiments, the fluorescent dye is an organic dye. As used herein “organic dye” refers to all nonparticulate, compounds that do not contain mineral or inorganic components and are capable of fluorescence.

[0452] In some embodiments, the fluorescent dye is a fluorescein, rhodamine, cyanine, dipyrromethene, naphthalene, xanthene, anthracene, squaraine or triangulenium, or derivative thereof. Such classes of compounds are known in the art. In some embodiments, the fluorescent dye is a fluorescein, rhodamine, cyanine, xanthene, or derivative thereof. In some embodiments, the fluorescent dye is a fluorescein or derivative thereof. In some embodiments, the fluorescent dye is selected from the group consisting of: Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 633, Alexa Fluor 647, and Alexa Fluor 680. In some embodiments, the fluorescent dye is fluorescein isothiocyanate (FITC).

[0453] In some embodiments, the fluorescent dye is an inorganic dye. Inorganic dyes refer to fluorescent dyes that are not organic dyes. In some embodiments, the fluorescent dye is a quantum dot. As the skilled person will be aware, a quantum dot is a semiconductor composed of atoms from groups II-VI or III-V elements of the periodic table (e.g., CdSe, CdTe, InP). The optical properties of quantum dots can be manipulated by synthesizing a (usually stabilizing) shell. Such quantum dots are known as core-shell quantum dots (e.g., CdSe / ZnS, InP / ZnS, InP / CdSe).

[0454] In some embodiments, the quantum dot is selected from the group consisting of CdSe / ZnS core / shell quantum dots, CdTe / CdSe core / shell quantum dots, CdSe / ZnTe core / shell quantum dots, and alloyed semiconductor quantum dots (e.g., CdSeTe). In some embodiments, the quantum dots are less than about 10 nm in diameter. In some embodiments, the quantum dots are between about 2 nm to about 5.5 nm in diameter. In some embodiments, the quantum dots are between about 1.5 nm to about 4.5 nm in diameter. If different colour emission is needed for creating multiple sensors (multiplex detection), this can be achieved by changing the size of the quantum dot core yielding different emission wavelengths.

[0455] Fluorescence lifetime (or fluorescence signal lifetime) is the time during which the fluorescent dye remains in an excited state, and is typically reported in nanoseconds (ns). As used herein, the term "long life fluorescent dye" should be understood to mean a fluorescent dye having a fluorescence lifetime (in ns) of about or at least about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18 or 20 or more. In some embodiments, the fluorescent dye has a fluorescence lifetime of at least 4 ns. In some embodiments, the fluorescent dye has a fluorescence lifetime of at least 5 ns. In some embodiments, the fluorescent dye has a fluorescence lifetime (in ns) of between about 4 and about 100, between about 5 and about 50, between about 5 and about 40, between about 5 and about 30, between about 5 and about 25, between about 10 and about 100, between about 10 and about 50, between about 10 and about 40, between about 10 and about 30, between about 10 and about 25, between about 15 and about 100, between about 15 and about 50, between about 15 and about 40, between about 15 and about 30, between about 15 and about 25.

[0456] As used herein, the term “full width at half maximum” (or “FWHM”) refers to the width (in nanometers) of the spectral distribution at about half of its maximum value. Accordingly, a full width at half maximum of a peak emission band, refers to the full width at half maximum of the peak emission band (or wavelength) of a fluorescent dye, the peak emission band (or wavelength) referring to the wavelength at which the fluorescence of the fluorescent dye exhibits maximum intensity across the entire fluorescence spectrum of the fluorescent dye.

[0457] In some embodiments, the fluorescent dye has a peak emission band with a full width at half maximum (FWHM) of about or less than about less than about 60, 50, 40, 30, 20 or 10 nm. In some embodiments, the fluorescent dye has a peak emission band with a full width at half maximum (FWHM) of less than about 20. In some embodiments, the fluorescent dye has a peak emission band with a full width at half maximum (FWHM) between about 10 and about 60. In some embodiments, the fluorescent dye has a peak emission band with a full width at half maximum (FWHM) between about 10 and about 40. In some embodiments, the fluorescent dye has a peak emission band with a full width at half maximum (FWHM) between about 10 and about 30.

[0458] Detecting fluorescence polarization

[0459] Methods of detecting fluorescence polarization will be apparent to the skilled person and / or are described herein.

[0460] The skilled person will understand that smaller molecules rotate faster than larger molecules in solution, and that the rotation rate can be determined by fluorescence polarization. When a plasma protein in a sample is incubated with a binding protein that is labelled with a fluorescent probe, the presence of protein-probe complex in the sample can be detected by means of fluorescence polarization.

[0461] As described herein, the method of the disclosure determines the concentration, qualitatively and quantitatively, of a plasma protein in a sample. The sample (comprising the plasma protein) is incubated with the plasma protein binding probe (which is conjugated to a fluorescent dye) and the sample is excited with plane polarized light at a wavelength corresponding to an excitation wavelength of a fluorescent dye. The light intensity emitted by the dye is used to detect the level of fluorescent polarization. The level of fluorescent polarization is then correlated with antibody titer in the sample, In one example, the sample is excited with plane polarized light. For example, the sample is excited with plane polarized light at a wavelength corresponding to an excitation wavelength of the fluorescent dye.

[0462] In one example, the sample is excited with plane polarized light immediately after incubating the sample and a plasma protein binding probe conjugated to a fluorescent dye in an assay solution. For example, the sample is mixed with the plasma protein binding protein in the assay solution and immediately excited with plane polarized light.

[0463] In one example, the light intensity emitted by the fluorescent dye is detected. For example, the light intensity emitted by the fluorescent dye is detected at an appropriate emission wavelength in two planes, one parallel and one perpendicular to the emission plane.

[0464] In one example, the method comprises determining the change in polarisation between excitation and emission light.

[0465] The skilled person will be aware of fluorescence polarization analysers that are suitable for use in the present disclosure. Examples of fluorescence polarization analysers include BMG PheraStar, BMG ClarioStar and the Tecan Infinite F500.

[0466] As used herein, the term "fluorescence polarisation analyser" shall be understood to refer to a machine capable of receiving a sample and exciting the sample with plane polarised light at a wavelength corresponding to an excitation wavelength of a fluorescent dye, detecting light intensity emitted by the fluorescent dye at an appropriate emission wavelength in two planes, one parallel and one perpendicular to the emission plane, and typically determining the change in polarisation between excitation and emission light. Typically, the machine is capable of receiving and reading a multiplicity of sample contained in a multi-well plate, typically in a high throughput manner.

[0467] In one example, the method is performed in a multi-well plate. Suitable multi-well plates will be apparent to the skilled person and include, for example, at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 96, 100 or 384 wells.

[0468] In one example, the method is performed at high throughput. In one example, the method is performed during large scale or commercial manufacturing.

[0469] Additional purification steps

[0470] Additional purification steps may be performed before and / or after the method of the disclosure. For example, the method is performed on a purified or partially purified sample. In one example, the sample is serum, plasma or plasma fraction thereof, a fermentation broth or a purified or partially purified fermentation broth thereof, a cell culture harvest or a purified or partially purified cell culture harvest thereof or a protein suspension.

[0471] In one example, the purified or partially purified sample comprising the plasma protein has been subjected to one or more steps selected from a group consisting of: precipitation, such as ethanol, ammonium sulphate and octanoic acid fractionation; affinity chromatography; ion exchange chromatography; viral inactivation; viral filtration; isoagglutinin affinity chromatography, ultrafiltration / diafiltration, bulk formulation, final formulation and combinations thereof. Additional purification steps will be apparent to the skilled person and / or described herein.

[0472] In one example, the method of the disclosure is performed before and / or after one or more purification steps selected from a group consisting of: precipitation, such as ethanol, ammonium sulphate and octanoic acid fractionation; affinity chromatography; ion exchange chromatography; viral inactivation; viral filtration; isoagglutinin affinity chromatography, ultrafiltration / diafiltration, bulk formulation, final formulation and combinations thereof.

[0473] In one example, the method of the disclosure is performed before one or more purification steps selected from a group consisting of: precipitation, such as ethanol, ammonium sulphate and octanoic acid fractionation; affinity chromatography; ion exchange chromatography; viral inactivation; viral filtration; isoagglutinin affinity chromatography, ultrafiltration / diafiltration, bulk formulation, final formulation and combinations thereof.

[0474] In one example, the method of the disclosure is performed after one or more purification steps selected from a group consisting of: precipitation, such as ethanol, ammonium sulphate and octanoic acid fractionation; affinity chromatography; ion exchange chromatography; viral inactivation; viral filtration; isoagglutinin affinity chromatography, ultrafiltration / diafiltration, bulk formulation, final formulation and combinations thereof.

[0475] In one example, the method of the disclosure is performed before and / or after the sample comprising the plasma protein is subjected to a precipitation step. In one example, the method of the disclosure is performed before the sample comprising the plasma protein is subjected to a precipitation step. In one example, the method of the disclosure is performed after the sample comprising the plasma protein is subjected to a precipitation step.

[0476] In one example, the precipitation step is ethanol precipitation. For example, cold ethanol may be used to isolate and enrich IgG by removing albumin and a- and P-globulins from the plasma or fractions thereof. For example, as described in WO2011 / 149472.

[0477] In one example, the precipitation step is octanoic acid fractionation. Octanoic acid may be used to remove of plasma lipids and plasma proteins (other than IgG). For example, as described in WO2011 / 131787.

[0478] In one example, the precipitation step is ammonium sulphate precipitation.

[0479] In one example, the method of the disclosure is performed before and / or after the sample comprising the plasma protein is subjected to an affinity chromatography step. In one example, the method of the disclosure is performed before the sample comprising the plasma protein is subjected to an affinity chromatography step. In one example, the method of the disclosure is performed after the sample comprising the plasma protein is subjected to an affinity chromatography step.

[0480] In one example, the method of the disclosure is performed before and / or after the sample comprising the plasma protein is subjected to an ion exchange chromatography step. In one example, the method of the disclosure is performed before the sample comprising the plasma protein is subjected to an ion exchange chromatography step. In one example, the method of the disclosure is performed after the sample comprising the plasma protein is subjected to an ion exchange chromatography step.

[0481] In one example, the ion exchange chromatography is anion exchange chromatography. For example, anion exchange chromatography may be used to remove IgA, remaining IgM and other plasma components (other than IgG).

[0482] The anion exchanger can be a resin-based anion exchanger, an anion exchange membrane adsorber, or any other format of anion exchanger with a positively charged substrate for capturing negatively charged particles. In one example, the anion exchanger is an anion exchange membrane adsorber. In another example, the anion exchanger is a resin-based anion exchanger. In a further example, the anion exchanger is a monolithic anion exchanger.

[0483] In one example, the sample has been subjected to anion exchange chromatography using a resin-based anion exchanger. For example, the anion exchange chromatography resin is a strong anion exchanger. In one example, the strong anion exchange resin comprises a matrix consisting of a poly(styrene-divinylbenzene) matrix. In one example, the strong anion exchanger comprises a quatemized polyethyleneimine functional group. Suitable resin-based anion exchanges will be apparent to the skilled person and include, for example, POROS™ HQ 50.

[0484] In one example, the anion exchange chromatography step is performed in flow through mode. In another example, the anion exchange chromatography step is performed in bind-and-elute mode. In one example, the anion exchange chromatography step comprises a buffer selected from the group consisting of sodium citrate, 2-(N-morpholino)ethanesulfonic acid (MES) buffer, sodium dihydrogen phosphate, BisTris, phosphate, L-histidine and combinations thereof. In one example, the anion exchange chromatography step comprises a buffer comprising MES buffer. In another example, the anion exchange chromatography step comprises phosphate buffer.

[0485] In one example, the method of the disclosure is performed before and / or after the sample comprising the plasma protein is subjected to a viral inactivation step. In one example, the method of the disclosure is performed before the sample comprising the plasma protein is subjected to a viral inactivation step. In one example, the method of the disclosure is performed after the sample comprising the plasma protein is subjected to a viral inactivation step.

[0486] In one example, the viral inactivation may be effected by adjusting the solution to low pH. Low pH may be a pH of between 2 to 4. In one example, low pH viral inactivation is performed in the presence of caprylate. In another example, viral inactivation may be effected by contacting the plasma or fraction thereof, or an IgG-enriched preparation or IgG-containing pharmaceutical composition with n-Octyl-P-D-Glucopyranoside (OG), thereby forming an OG-IgG mixture. In a further example, low pH viral inactivation is performed in the presence of N, N-Dimethylmyristylamine V-oxidc (TDAO).

[0487] In a further example, viral inactivation may be effected by exposing the protein mixture, plasma or plasma fraction, a protein depleted preparation or composition (e.g., a plasma protein depleted preparation e.g., an IgG-depleted preparation) to a solvent-detergent inactivation step. Suitable solvent-detergent treatments would be apparent to the skilled person and include, for example environmentally friendly detergents. Exemplary environmentally friendly detergents suitable for use in the present disclosure and in particular for use in inactivating lipid enveloped viruses include \, \-Dimcthylmyristylaminc V-oxidc (TDAO), polysorbate 80 (PS80), polyoxyethylene (10) isooctylcyclohexyl ether (TRITON® X-100-reduced), and a non-ionic surfactant prepared from glucose and alcohol (e.g., Simulsol™ formulations). In one example, the detergent is \, \ -Dimcthylmyristylaminc N-oxide (TDAO). In one example, the detergent is polysorbate 80. In another example, the detergent is polyoxyethylene (10) isooctylcyclohexyl ether (TRITON® X-100-reduced). In a further example, the detergent is a non-ionic surfactant prepared from glucose and alcohol.

[0488] In one example, the method of the disclosure is performed before and / or after the sample comprising the plasma protein is subjected to a viral filtration step. In one example, the method of the disclosure is performed before the sample comprising the plasma protein is subjected to a viral filtration step. In one example, the method of the disclosure is performed after the sample comprising the plasma protein is subjected to a viral filtration step.

[0489] In one example, the viral filtration is performed with membranes of pore sizes from 15-20 nm to remove microbes and viruses from a solution or eluate or pharmaceutical composition. Exemplary nanofilters include Planova S20N (Asahi), Virosart HC (Sartorius) and Planova 20N (Asahi).

[0490] In one example, the method of the disclosure is performed before and / or after the sample comprising the plasma protein is subjected to an immunoaffinity chromatography step. In one example, the method of the disclosure is performed before the sample comprising the plasma protein is subjected to an immunoaffinity chromatography step. In one example, the method of the disclosure is performed after the sample comprising the plasma protein is subjected to an immunoaffinity chromatography step. In one example, the immunoaffinity chromatography step is isoagglutinin affinity chromatography. For example, the isoagglutinin affinity chromatography is performed using Eshmuno anti-A and anti-B resin. For example, isoagglutinin affinity chromatography may be used to remove isoagglutinins A and B.

[0491] In one example, the method of the disclosure is performed before and / or after the sample comprising the plasma protein is subjected to an ultrafiltration / diafiltration step. In one example, the method of the disclosure is performed before the sample comprising the plasma protein is subjected to an ultrafiltration / diafiltration chromatography step. In one example, the method of the disclosure is performed after the sample comprising the plasma protein is subjected to an ultrafiltration / diafiltration chromatography step.

[0492] An exemplary ultrafiltration / diafiltration membrane is Pellicon 2 Cassettes (Millipore) or Polyethersulfone orHydrosart cassettes (Sartorius).

[0493] In one example, the method of the disclosure is performed before and / or after the sample comprising the plasma protein is subjected to a bulk formulation step. In one example, the method of the disclosure is performed before the sample comprising the plasma protein is subjected to a bulk formulation step. In one example, the method of the disclosure is performed after the sample comprising the plasma protein is subjected to a bulk formulation step.

[0494] In one example, the method of the disclosure is performed before and / or after the sample comprising the plasma protein is subjected to a final formulation step. In one example, the method of the disclosure is performed before the sample comprising the plasma protein is subjected to a final formulation step. In one example, the method of the disclosure is performed after the sample comprising the plasma protein is subjected to a final formulation step. For example, the sample is formulated in the final commercially available formulation.

[0495] The present disclosure is described in the following numbered paragraphs:

[0496] 1. A method of determining the concentration of a plasma protein in a sample, the method comprising: i. incubating the sample and a plasma protein binding probe conjugated to a fluorescent dye in an assay solution, wherein the assay solution comprises a neutral hydrophilic polymer;

[0497] ii. assaying the incubated solution to detect fluorescence polarization;

[0498] iii. determining a change in polarization; and

[0499] iv. correlating the change in polarization with the concentration of the plasma protein in the sample.

[0500] 2. The method of paragraph 1, wherein the polymer has a hydrodynamic radius of at least about 2.0 nm.

[0501] 3. The method of paragraph 2, wherein the polymer has a radius of gyration of at least about 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0 nm.

[0502] 4. The method of paragraph 1, wherein the polymer is selected from polyethylene glycol, polysucrose, or a derivative thereof.

[0503] 5. The method of paragraph 4, wherein the polymer is polyethylene glycol or a derivative thereof. 6. The method of paragraphs 4 or 5, wherein the polyethylene glycol or derivative thereof has a molecular weight (kDa) between about 0.2 and about 200.

[0504] 7. The method of any one of paragraphs 4 to 7, wherein the polyethylene glycol or derivative thereof has a molecular weight (kDa) between about 1 and about 100.

[0505] 8. The method of any one of paragraphs 4 to 7, wherein the polyethylene glycol or derivative thereof has a molecular weight (kDa) between about 3 and about 10.

[0506] 9. The method of any one of paragraphs 4 to 8, wherein the polyethylene glycol or derivative thereof has a molecular weight (kDa) of about 8.

[0507] 10. The method of paragraph 4, wherein the polymer is poly sucrose or a derivative thereof.

[0508] 11. The method of paragraph 10, wherein the poly sucrose or derivative thereof is a Ficoll.

[0509] 12. The method of any one of paragraphs 10 or 11, wherein the Ficoll has a molecular weight (kDa) between about 50 and about 500.

[0510] 13. The method of any one of paragraphs 10 to 12, wherein the Ficoll has a molecular weight (kDa) of about 70 or about 400.

[0511] 14. The method of any one of paragraphs 1 to 13, wherein the concentration (in % w / v) of the polymer in the assay solutionis about or greater than about 0.1, 0.5 or 1.

[0512] 15. The method of any one of paragraph 1 to 14, wherein the plasma protein binding probe specifically binds to the plasma protein and wherein the binding probe is selected from the group consisting of an immunoglobulin binding protein, an aptamer, a Fc receptor or fragment thereof, a glycoprotein, glycoprotein receptor, a phospholipid, a plasminogen activator protein, a protein kinase domain, a pleckstrin homology domain, a coagulation factor, an anticoagulant, a lysine analogue, a divalent cation, a gamma-carboxyglutamic acid-rich (GLA) domain, a complement system component, a protease, a protease inhibitor, a small molecule, an amphipathic helix, an antibody or antigen binding fragment thereof, a heavy chain immunoglobulin, an antibody mimetic, a cibacronblue F3GA, a L-tryptophan and combinations thereof.

[0513] 16. The method of paragraph 15, wherein:

[0514] (i) the immunoglobulin binding protein is selected from the group consisting of Protein A, Protein G, Protein L and combinations thereof;

[0515] (ii) the aptamer is a DNA aptamer or an RNA aptamer;

[0516] (iii) the Fc receptor or fragment thereof is a Fc-gamma receptor (FcyR), a Fc-alpha receptor (FcaR), a Fc- epsilon receptor (FceR) and / or a polymeric immunoglobulin receptor (plgR); (iv) the glycoprotein is a glycoprotein lb (GPIb), a thrombomodulin, von Willebrand factor (vWF) and / or a lactadherin domain;

[0517] (v) the glycoprotein receptor is an endothelial protein C receptor (EPCR);

[0518] (vi) the plasminogen activator protein is staphylokinase;

[0519] (vii) the protein kinase domain is a protein kinase C domain;

[0520] (viii) the coagulation factor is an antihemophilic factor, a Factor Xa (FXa) and / or a Factor la;

[0521] (ix) the anti-coagulant is warfarin, heparin, Protein S and / or a FXa inhibitor;

[0522] (x) the divalent cation is calcium (Ca2+) ions and / or zinc (Zn2+) ions;

[0523] (xi) the complement system component is complement component 1q (C1q);

[0524] (xii) the protease is a serine protease;

[0525] (xiii) the protease inhibitor is a serine protease inhibitor or a proteinase inhibitor;

[0526] (xiv) the small molecule is an anxiolytic benzodiazepine or a salicylate;

[0527] (xv) the antibody mimetic is a sherpabody, an adnectin, an anticalin, an affibody, an avimer, a DARPin, an affitin, a fynomer, a peptide aptamer, an affimer, an alphabody, a monobody, a nanoCLAMP, an optimer, a repebody, a centyrin, an obody, a kunitz domain peptide, a knottin and / or an atrimer; and / or (xvi) a single domain antibody, a minibody, a diabody, a triabody, a tetrabody, a Fv, a single chain Fv (scFv) fragment, a dimeric scFv (di-scFv), a Fab, a F(ab’)2or a half antibody.

[0528] 17. The method of paragraph 16, wherein

[0529] (i) the serine protease is a urokinase, a tissue plasminogen activator, an elastase, a kallikrein, a high molecular weight kallikrein and / or a thrombin; or

[0530] (ii) the serine protease inhibitor is an elastase inhibitor, a bovine pancreatic trypsin inhibitor (BPTI), an aprotinin, an alpha 2-antiplasmin inhibitor, an alpha 1 -antichymotrypsin, a phenylmethylsulfonyl fluoride and / or a benzamidine.

[0531] 18. The method of any one of paragraphs 1 to 17, wherein the plasma protein binding probe comprises a binding region that specifically binds to the plasma protein, wherein the binding probe is selected from the group consisting of a truncated Protein A having a molecular weight of less than 20kD, a tmncated Protein G having a molecular weight of less than 20kD and combinations thereof.

[0532] 19. The method of any one of paragraphs 1 to 18, wherein the assay solution further comprises a buffering agent having a dissociation constant (pKa) between 5.5 and 8.9 at 25°C.

[0533] 20. The method of paragraph 19, wherein the buffering agent is selected from the group consisting of 3-morpholinopropane-1 -sulfonic acid (MOPS), 2-(N-morpholino)ethanesulfonic acid (MES), Bis-Tris, N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), piperazine-N, N'-bis(2-ethanesulfonic acid) (PIPES), N-Bis(2-hydroxyethyl)-2 -aminoethanesulfonic acid (BES), 2-[(2 -Hydroxy-1, 1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid (TES), 4-(2 -hydroxy ethyl)- 1 -piperazineethanesulfonic acid (HEPES), 3-(N, N-Bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid (DIPSO), 4-(N-Morpholino)butanesulfonic acid (MOBS), N-[Tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropanesulfonic acid (TAPSO), N-(Hydroxyethyl)piperazine-N'-2-hydroxypropanesulfonic acid (HEPPSO), Piperazine-N, N'-bis(2-hydroxypropanesulfonic acid) (POPSO), triethanolamine (TEA), 4-(2 -Hydroxy ethyl)- 1 -piperazinepropanesulfonic acid (EPPS), Tricine, glycylglycine, bis[(2-hydroxyethyl)amino]acetic acid (Bicine), N-(2-Hydroxyethyl)piperazine-N'-(4-butanesulfonic acid) (HEPBS), tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS), Tris, phosphate buffered saline (PBS), and combinations thereof.

[0534] 21. The method of paragraph 20, wherein the buffering agent is MOPS.

[0535] 22. The method of any one of paragraphs 19 to 21, wherein the buffering agent is at a concentration of between 5 mM and 200 mM.

[0536] 23. The method of paragraph 22, wherein the buffering agent is at a concentration of between 25 m and 100 mM.

[0537] 24. The method of paragraph 22 or 23, wherein the buffering agent is at a concentration of 50 mM.

[0538] 25. The method of any one of paragraphs 1 to 24, wherein the assay solution further comprises a salt.

[0539] 26. The method of paragraph 25, wherein the salt is a monovalent and / or a divalent salt.

[0540] 27. The method of paragraphs 26, wherein the monovalent salt is selected from the group consisting of sodium chloride, potassium chloride and combinations thereof.

[0541] 28. The method of any one of paragraphs 25 to 27, wherein the salt is sodium chloride.

[0542] 29. The method of paragraph 26, wherein the divalent salt is selected from a group consisting of magnesium chloride, calcium chloride, barium chloride, copper (II) chloride, nickel chloride, manganese chloride, and combinations thereof.

[0543] 30. The method of any one of paragraph 25 to 29, wherein the salt is in the assay solution at a concentration of between 0 mM and 500 mM.

[0544] 31. The method of paragraph 30, wherein the salt is at a concentration of between 100 mM and 150 mM.

[0545] 32. The method of any one of paragraphs 1 to 31, wherein the assay solution has a pH of between 5 and 10.

[0546] 33. The method of paragraph 32, wherein the assay solution has a pH of between 6 and 8.

[0547] 34. The method of any one of paragraphs 1 to 33, wherein the fluorescent dye has a fluorescent signal lifetime of at least 4 ns. 35. The method of paragraph 34, wherein the fluorescent signal lifetime is at least 5 ns.

[0548] 36. The method of any one of paragraphs 1 to 35, wherein the fluorescent dye is an organic dye.

[0549] 37. The method of any one of paragraphs 1 to 35, wherein the fluorescent dye is an inorganic dye.

[0550] 38. The method of any one of paragraphs 1 to 35, wherein the fluorescent dye is a quantum dot.

[0551] 39. The method of any one of paragraphs 1 to 38, wherein the fluorescent dye is a fluorescein, rhodamine, cyanine, dipyrromethene, naphthalene, xanthene, squaraine or triangulenium.

[0552] 40. The method of any one of paragraphs 1 to 35or 39, wherein the fluorescent dye is a fluorescein.

[0553] 41. The method of any one of paragraphs 1 to 35, wherein the fluorescent dye is fluorescein isothiocyanate (FITC).

[0554] 42. The method of any one of paragraphs 1 to 41, wherein the fluorophore has a peak emission band with a full width at half maximum (FWHM) of less than about 60, 50, 40, 30, 20 or 10 nm.

[0555] 43. The method of any one of paragraphs 1 to 42, wherein the plasma protein is a natural or recombinant plasma protein.

[0556] 44. The method of any one of paragraphs 1 to 43, wherein the plasma protein is selected from the group consisting of immunoglobulin (Ig), an apolipoprotein Al, an albumin, a protease, a protease inhibitor, plasminogen, a fibrinogen, a von Willebrand factor, a clotting factor or activated form thereof, a cofactor or activated form thereof, a contact system factor, a prekallikrein activator (PKA), a prothrombin, thrombin, prothrombin complex factor or activated form thereof, a protein C, an anti-thrombin III, alpha acid glycoprotein, a transport protein, Factor H, a component of the complement pathway, inhibitors of any component of the complement pathway, a highly glycosylated protein Protein S, histidine-rich glycoprotein, mannan binding lectin, C4-binding protein, fibronectin, GC-globulin, erythropoietin, interferon, tumor factors, tPA, γCSF, an oligomeric form or degradation product of any of the foregoing and combinations thereof.

[0557] 45. The method of paragraph 44, wherein:

[0558] (i) the Ig is an IgG, an IgA, an IgM and / or a RhD immunoglobulin protein product; and / or

[0559] (ii) the complement component is complement component 1q (C1q), Clr and / or Cis; and / or

[0560] (iii)the coagulation factor is factor Xa, factor VII, factor VIII, a factor IX, factor XII, factor XIII and / or factor XI; and / or

[0561] (iv) the protease is a serine protease or a plasmin. 46. The method of paragraph 45, wherein the plasma protein is IgG.

[0562] 47. The method of any one of paragraphs 1 to 46, wherein the sample is serum, plasma, a plasma fraction, or a purified or partially purified plasma or plasma fraction thereof, a fermentation broth or a purified or partially purified fermentation broth thereof, a cell culture harvest or a purified or partially purified cell culture harvest thereof or a protein suspension.

[0563] 48. The method of any one of paragraphs 1 to 47, wherein the plasma or plasma fraction is selected the group consisting of a human blood plasma sample, an IgG intermediate product, a cryo-rich plasma, a cryo-poor plasma, a Supernatant I (SN I), a Cohn Fraction II (Fr II), a Cohn Fraction 11+111 (Fr II+III), a Cohn Fraction I+II+III (FrI+II+III), a Kistler / Nitschmann Precipitate A (KN A), a Kistler / Nitschmann Precipitate B (KN B), a Kistler / Nitschmann Precipitate of Supernatant B (KN B+l), formulated plasma fractionation product and combinations thereof.

[0564] 49. The method of paragraphs 47or 48, wherein the purified or partially purified plasma or plasma fraction thereof has been subjected to one or more steps selected from the group consisting of clarification, ethanol precipitation, octanoic acid fractionation, ammonium sulphate precipitation, affinity chromatography, ion exchange chromatography, viral inactivation, viral filtration, isoagglutinin affinity chromatography, ultrafiltration / diafiltration, bulk formulation, final formulation and combinations thereof.

[0565] 50. The method of any one of paragraphs 1 to 49, wherein the method comprises diluting the sample and / or the probe in the assay solution before incubating.

[0566] 51. The method of paragraph 50, wherein the method comprises serially diluting the sample and / or probe in the assay solution before incubating, wherein the sample and / or probe is serially diluted in the assay solution to a dilution factor of between 1:10 to 1:60 before incubating.

[0567] 52. The method of paragraph 51, wherein the method comprises adding the diluted sample to the diluted probe before incubating, wherein the total dilution factor of the sample and the probe in the assay solution is between 1:100 to 1:3000.

[0568] 53. The method of any one of paragraphs 1 to 52, wherein the method determines the concentration of the plasma protein in the sample with a coefficient of variation of < 3.0%.

[0569] 54. The method of any one of paragraph 53, wherein the method determines the concentration of the plasma protein in the sample with a coefficient of variation of < 2.0%.

[0570] 55. The method of any one of paragraphs 1 to 54, wherein the method is performed at a temperature in the range of 18 to 25 °C. 56. The method of any one of paragraphs 1 to 55, wherein the method is carried out in a well of a multi-well plate.

[0571] The present disclosure includes the following non-limiting Examples.

[0572] EXAMPLES

[0573] Example 1: Quantitation of Immunoglobulin G (IgG) using the fluorescence polarization assay

[0574] A fluorescence polarization assay was used to quantitate the IgG content in a series of samples. The assay was a plate-based, mix and measure assay. The standard curve comprised seven standard points and ranges from 10 to 100 mg / L. First the assay standard, control and the samples were diluted to the target concentrations / range using the assay solution. Subsequently, the pre-diluted standards, control and test samples were mixed with assay solution in the assay plate. The microtiter plate wells were pre-coated with a fluorescein-labeled protein G derivative which allows quantitation of the IgG content by measuring the fluorescence polarization (FP).

[0575] Initial testing was performed using 50 m MOPS, 130 mM NaCl, pH 7.1.

[0576] Cryo-poor plasma (CPP) and purified Privigen samples were used to assess the buffers. The results are shown in Table 1. Testing was off-set against two different standards, Siemens Protein Standard or Privigen.

[0577] Table 1: IgG detection using initial assay buffer

[0578]

[0579]

[0580] As shown in Table 1, the coefficient of variation (CV) across the two samples was inconsistent dependent on the sample and the standard used.

[0581] Additive Screen for Assay Solution

[0582] To reduce the hands-on time for IgG quantification and enable high throughput analysis of samples at all stages of the plasma purification process, a buffer screen was undertaken to assess the impact on IgG quantification.

[0583] The following additives were tested in the assay solution comprising 50 mM MOPS buffer, 130 mM NaCl, pH7.1:

[0584] 1. Neutral hydrophilic polymers:

[0585] a. 1% PEG (200, 4000, 8000, 20000)

[0586] b. 1% Ficoll 70 c. 1% PVP10000

[0587] 2. Charged polymers:

[0588] a. 1% Poly(natrium-4-styrolsulfonat) (PSS70000)

[0589] 3. Inert branched polymers:

[0590] a. 1% Dextran (10,000; 20,000)

[0591] 4. Inert dimer:

[0592] a. 1% Iodixanol

[0593] 5. Surfactants:

[0594] a. 0.05% PS20 (Tween)

[0595] b. 0.1% Poloxamer

[0596] 6. Proteins:

[0597] a. 0.1%, 0.5%, 1%, 2% Human serum albumin

[0598] b. IgG depleted plasma (50% v / v)

[0599] 7. Inert monomers:

[0600] a. 1% Sucrose

[0601] b. 1% Glycerol

[0602] The following samples were assessed during the testing process:

[0603] 1. 2 lots: Privigen

[0604] 2. 1 lot: Affinity chromatography eluate (1stpurification step of cryo-poor plasma)

[0605] 3. 2 lots: Cryo-poor plasma (CPP)

[0606] 4. 1 lot: Siemens N Protein Standard S / L (close to plasma)

[0607] After predilution to ~100 mg / L all samples were diluted with the same dilution factors in the assay solution comprising the relevant additive. Each sample dilution consisted of two dilution steps, whereby no dilution step was greater than 1:60. An example of the sample dilutions is provided in Table 2.

[0608] Table 2: Sample dilutions

[0609]

[0610] A 7-point standard curve was prepared in the assay solution (with the relevant additive) using Privigen as the standard ranging from 10-100 mg / L. An example of the preparation of the standard curve is provided in Table 3. Table 3: Preparation of standard curve

[0611]

[0612] A control (N Protein Control SL / L) was also used as a quality control using a 1:10 and 1:12.5 dilution, resulting in a 1:125 dilution.

[0613] A Waters Andrew+ liquid handler and the OneLab software were used for sample preparation and the transfer to the assay plate. The standard, control, and samples are in Matrix tubes pre-diluted with assay buffer (with the relevant additive) in a deep well plate. The fluorescein labeled probe (FITC labelled truncated protein G) was reconstituted using 70 µL assay buffer (with the relevant additive) per well. Subsequently, 50 µL of the diluted standards, control, and the samples were added to the plate in technical duplicates. Finally, a CLARIOstar Plus plate reader (BMG Labtech) was used to read the plate and obtain the fluorescence polarization measurement.

[0614] The polarization values, fitting of the standard curve and determining IgG concentrations in the diluted samples was performed automatically using the “SMART control Data Analysis MARS” software. Results below 2.00 g / L were automatically indicated as < 2.00 g / L. Results above 250.00 g / L were automatically indicated as > 250.00 g / L.

[0615] Coefficients of variation (CVs) were assessed, with the following acceptance criteria:

[0616] • CV of technical duplicates of the standards < 5.0%.

[0617] • CV of technical duplicates of the control < 3.0%.

[0618] • CV of technical duplicates of the samples < 3.0%.

[0619] Results

[0620] The results of the buffer screen are shown in Tables 4-6

[0621] As shown in Table 4, the neutral hydrophilic polymers PEG, Ficoll and PVP showed overall good dilution consistency in all samples. PEG8000 was subsequently tested over the concentrations of 0.1% to 3% and the results are shown in Table 7.

[0622] As shown in Table 5, the inert charged linear charged polymer PSS showed good dilution consistency, however higher variability (CV2-7%) between samples. Inert branched polymers (Dextran), the inert dimer (Iodixanol) and the inert monomers (glycerol and sucrose) all showed no acceptable dilution consistency.

[0623] As shown in Table 6, the addition of PS20 into the assay solution showed highly variable results between sample types. Whilst improved dilution linearity was observed a higher variability (CVl-12%) was observed in technical duplicates. Use of poloxamer 188 did not generate a signal as there was strong interference with the assay. HSA showed high interference when used in the final dilution step on the assay plate and was re-tested in the predilutions only at 0.1%, 0.5%, 1% and 2% and displayed and overall acceptable dilutional linearity.

[0624] IgG depleted plasma (50% v / v) also showed strong interference when used in the final dilution step on the assay plate and was re-tested in pre-dilutions at 100% and 50%. 100% pre-dilution showed high variability, whilst 50% pre-dilution showed an acceptable dilution linearity.

[0625] Assay results using the neutral hydrophilic polymers in the assay solution were shown to be comparable to existing HPLC and nephelometry techniques for calculation of IgG concentration.

[0626] Table 4: Assay solution comprising neutral hydrophilic polymers

[0627]

[0628]

[0629] Table 5: Assay solution comprising inert charged linear polymers, inert branched polymers, inert dimers & inert monomers

[0630]

[0631]

[0632] Table 6: Assay solution comprising 0.05% PS20 (Surfactants)

[0633]

[0634]

[0635]

[0636] Example 2: Effect of assay solution pH and conductivity on IgG detection

[0637] The impact of buffer pH and conductivity on the ability of the assay to detect IgG at different stages of the plasma purification process was assessed.

[0638] The assay was run according to Example 1 using an assay buffer comprising 50 mM MOPS, 130 mM NaCl, 10g / l PEG8000 with the pH tested at pH6.5, 7.1, 7.4, 7.5 and 7.7. The standard curve was generated using the Siemens N Protein Standard SL. As shown in Table 8, buffer pH had no effect on quantitation of IgG in the samples.

[0639] The assay was run according to Example 1 using an assay buffer comprising 50 mM MOPS, 130 10g / l PEG8000 pH 7.1 with the NaCl concentration tested at 0 mM, 110 mM, 130 mM and 150 mM. The standard curve was generated using the Siemens N Protein Standard SL. As shown in Table 9, conductivity of the assay solution (i.e., concentration of NaCl) had no effect on quantitation of IgG in the samples.

[0640] Example 3: Quantitation of IgG derived from cell culture harvest

[0641] To assess whether the assay solution comprising the neutral hydrophilic polymer could also be used to determine IgG concentration from cell culture harvest material, the assay was performed according to Example 1 using cell culture harvest from recombinant IgG production. The standard curve was generated using Privigen.

[0642] The results are shown in Table 10 and Figure 1. The effect of improved CV and dilution consistency is less pronounced than in plasma samples. Overall, the assay solution comprising PEG showed a better or similar good CV and dilution consistency. This indicates that the improved assay solution comprising MOPS / PEG buffer can be used not only for plasma samples but also for recombinant samples.

[0643] S

[0644]

[0645] S9

[0646] Table 9: Assay solution comprising 50 mM MOPS, 130 10g / L PEG8000, pH7.1 and variable NaCl concentration

[0647]

[0648]

[0649] Table 10: IgG detection using CD-CHO media or assay solution with and without 1% PEG.

[0650]

[0651] Example 4: Measurement of hydrodynamic diameter of polymer additives using the DLS method The hydrodynamic diameter (2 x hydrodynamic radius) of several of the polymers tested as additives in the assay solution comprising 50 mM MOPS buffer, 130 mM NaCl, pH 7.1 was tested using dynamic light scattering (DLS).

[0652] The Zetasizer Pro instrument (Malvern Panalytical Ltd., UK) was used for the DLS measurements with a back scatter signal acquisition setting and measurement setting at optimal position. The attenuation and measurement process were set at automatic. The sample was applied at room temperature (25 °C) to a 1 mL sample volume in an assay buffer comprising the relevant additive using the 1 ml cuvette (DTS0012). The data acquisition was started following a 180 s equilibration time in the instrument cuvette holder. Data were obtained and processed using ZS XPLORER v3.2.0.84 software. A General model as defined in the ZS XPLORER (v 3.2.0.84) software was used to analyze the intensity -based size distribution data. Parameters such as hydrodynamic diameter, diffusion coefficient, d90, and span were obtained. All measurements were repeated in duplicate.

[0653] The mean results of the duplicates are shown in Table 11. The results show that PS20 has a larger size consistent with that of the polymer additives which showed good dilution consistency, thereby suggesting its highly variable results in the assay are due to it being a charged polymer. Additionally, the DLS measurements show that PEG 200, which also demonstrated high assay variability, has a significantly smaller hydrodynamic diameter than those polymers which show good dilution consistency.

[0654] Table 11: Hydrodynamic diameter of various polymer additives using DLS

[0655]

Claims

CLAIMS1. A method of determining the concentration of a plasma protein in a sample, the method comprising: i. incubating the sample and a plasma protein binding probe conjugated to a fluorescent dye in an assay solution, wherein the assay solution comprises a neutral hydrophilic polymer;ii. assaying the incubated solution to detect fluorescence polarization;iii. determining a change in polarization; andiv. correlating the change in polarization with the concentration of the plasma protein in the sample.

2. The method of claim 1, wherein the polymer has a hydrodynamic radius of at least about 2.0 nm or at least about 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0 nm.

3. The method of claim 1 or claim 2, wherein the polymer is:(i) selected from polyethylene glycol, polysucrose, or a derivative thereof;(ii) polyethylene glycol or a derivative thereof; or(iii) polysucrose or a derivative thereof;optionally wherein the polyethylene glycol or derivative thereof has a molecular weight (kDa):(a) between about 0.2 and about 200;(b) between about 1 and about 100;(c) between about 3 and about 10; and / or(d) about 8; oroptionally wherein the poly sucrose or derivative thereof is a Ficoll, wherein the Ficoll has a molecular weight (kDa) between about 50 and about 500, or about 70, or about 400.

4. The method of any one of claims 1 to 3, wherein the concentration (in % w / v) of the polymer in the assay solution is about or greater than about 0.1, 0.5 or 1.

5. The method of any one of claims 1 to 4, wherein the plasma protein binding probe specifically binds to the plasma protein and wherein the binding probe is selected from the group consisting of an immunoglobulin binding protein, an aptamer, a Fc receptor or fragment thereof, a glycoprotein, glycoprotein receptor, a phospholipid, a plasminogen activator protein, a protein kinase domain, a pleckstrin homology domain, a coagulation factor, an anticoagulant, a lysine analogue, a divalent cation, a gamma-carboxyglutamic acid-rich (GLA) domain, a complement system component, a protease, a protease inhibitor, a small molecule, an amphipathic helix, an antibody or antigen binding fragment thereof, a heavy chain immunoglobulin, an antibody mimetic, a cibacron blue F3 GA, a L-tryptophan and combinations thereof, wherein optionally:(i) the immunoglobulin binding protein is selected from the group consisting of Protein A, Protein G, Protein L and combinations thereof;(ii) the aptamer is a DNA aptamer or an RNA aptamer;(iii) the Fc receptor or fragment thereof is a Fc- gamma receptor (FcyR), a Fc-alpha receptor (FcaR), a Fc-epsilon receptor (FceR) and / or a polymeric immunoglobulin receptor (plgR);(iv) the glycoprotein is a glycoprotein lb (GPIb), a thrombomodulin, von Willebrand factor (vWF) and / or a lactadherin domain;(v) the glycoprotein receptor is an endothelial protein C receptor (EPCR);(vi) the plasminogen activator protein is staphylokinase;(vii) the protein kinase domain is a protein kinase C domain;(viii) the coagulation factor is an antihemophilic factor, a Factor Xa (FXa) and / or a Factor la;(ix) the anti-coagulant is warfarin, heparin, Protein S and / or a FXa inhibitor;(x) the divalent cation is calcium (Ca2+) ions and / or zinc (Zn2+) ions;(xi) the complement system component is complement component 1q (C1q);(xii) the protease is a serine protease;(xiii) the protease inhibitor is a serine protease inhibitor or a proteinase inhibitor;(xiv) the small molecule is an anxiolytic benzodiazepine or a salicylate;(xv) the antibody mimetic is a sherpabody, an adnectin, an anticalin, an affibody, an avimer, a DARPin, an affitin, a fynomer, a peptide aptamer, an affimer, an alphabody, a monobody, a nanoCLAMP, an optimer, a repebody, a centyrin, an obody, a kunitz domain peptide, a knottin and / or an atrimer; and / or (xvi) a single domain antibody, a minibody, a diabody, a triabody, a tetrabody, a Fv, a single chain Fv (scFv) fragment, a dimeric scFv (di-scFv), a Fab, a F(ab’)2 or a half antibody; and wherein optionally:(i) the serine protease is a urokinase, a tissue plasminogen activator, an elastase, a kallikrein, a high molecular weight kallikrein and / or a thrombin; or(ii) the serine protease inhibitor is an elastase inhibitor, a bovine pancreatic trypsin inhibitor (BPTI), an aprotinin, an alpha 2-antiplasmin inhibitor, an alpha 1 -antichymotrypsin, a phenylmethylsulfonyl fluoride and / or a benzamidine.

6. The method of any one of claims 1 to 5, wherein the plasma protein binding probe comprises a binding region that specifically binds to the plasma protein, wherein the binding probe is selected from the group consisting of a truncated Protein A having a molecular weight of less than 20kD, a truncated Protein G having a molecular weight of less than 20kD and combinations thereof.

7. The method of any one of claims 1 to 6, wherein the assay solution further comprises:(i) a buffering agent having a dissociation constant (pKa) between 5.5 and 8.9 at 25°C, optionally wherein the buffering agent is selected from the group consisting of 3 -morpholinopropane- 1 -sulfonic acid (MOPS), 2-(N- morpholino)ethanesulfonic acid (MES), Bis-Tris, N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), piperazine-N, N'-bis(2-ethanesulfonic acid) (PIPES), N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 2-[(2-Hydroxy-l,l-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid (TES), 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid (HEPES), 3-(N, N-Bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid (DIPSO), 4-(N-Morpholino)butanesulfonic acid (MOBS), N-[Tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropanesulfonic acid (TAPSO), N- (Hydroxyethyl)piperazine-N'-2-hydroxypropanesulfonic acid (HEPPSO), Piperazine-N, N'-bis(2 -hydroxypropanesulfonic acid) (POPSO), triethanolamine (TEA), 4-(2- Hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS), Tricine, glycylglycine, bis[(2- hydroxyethyl)amino] acetic acid (Bicine), N-(2-Hydroxyethyl)piperazine-N'-(4-butanesulfonic acid) (HEPBS), tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS), Tris, phosphate buffered saline (PBS), and combinations thereof; and / or(ii) a salt, optionally wherein the salt is a monovalent and / or a divalent salt.

8. The method of claim 7, wherein the buffering agent is at a concentration of between 5 mM and 200 mM and / or between 25 mM and 100 m, and / or at 50 mM.

9. The method of claim 7 or 8, wherein:(i) the monovalent salt is selected from the group consisting of sodium chloride, potassium chloride and combinations thereof; or(ii) the divalent salt is selected from a group consisting of magnesium chloride, calcium chloride, barium chloride, copper (II) chloride, nickel chloride, manganese chloride, and combinations thereof.

10. The method of any one of claims 7 to 9, wherein the salt is in the assay solution at a concentration of between 0 mM and 500 mM and / or between 100 mM and 150 mM.

11. The method of any one of claims 1 to 10, wherein the assay solution has a pH of between 5 and 10 and / or between 6 and 8.

12. The method of any one of claims 1 to 11, wherein:(i) the fluorescent dye has a fluorescent signal lifetime of at least 4 ns and / or at least 5 ns; and / or(ii) the fluorescent dye is an organic dye, an inorganic dye, a quantum dot, a fluorescein, rhodamine, cyanine, dipyrromethene, naphthalene, xanthene, squaraine, triangulenium, and / or fluorescein isothiocyanate (FITC); and / or(iii) the fluorophore has a peak emission band with a full width at half maximum (FWHM) of less than about 60, 50, 40, 30, 20 or 10 nm.

13. The method of any one of claims 1 to 12, wherein the plasma protein is a natural or recombinant plasma protein, wherein the plasma protein is selected from the group consisting of immunoglobulin (Ig), an apolipoprotein Al, an albumin, a protease, a protease inhibitor, plasminogen, a fibrinogen, a von Willebrand factor, a clotting factor or activated form thereof, a cofactor or activated form thereof, a contact system factor, a prekallikrein activator (PKA), a prothrombin, thrombin, prothrombin complex factor or activated form thereof, a protein C, an anti-thrombin III, alpha acid glycoprotein, a transport protein, Factor H, a component of the complement pathway, inhibitors of any component of the complement pathway, a highly glycosylated protein Protein S, histidine -rich glycoprotein, mannanbinding lectin, C4-binding protein, fibronectin, GC- globulin, erythropoietin, interferon, tumor factors, tPA, γCSF, an oligomeric form or degradation product of any of the foregoing and combinations thereof, wherein:(i) the Ig is an IgG, an IgA, an IgM and / or a RhD immunoglobulin protein product; and / or(ii) the complement component is complement component 1q (C1q), Clr and / or Cis; and / or(iii) the coagulation factor is factor Xa, factor VII, factor VIII, a factor IX, factor XII, factor XIII and / or factor XI; and / or(iv) the protease is a serine protease or a plasmin.

14. The method of any one of claims 1 to 13, wherein:(i) the sample is serum, plasma, a plasma fraction, or a purified or partially purified plasma or plasma fraction thereof, a fermentation broth or a purified or partially purified fermentation broth thereof, a cell culture harvest or a purified or partially purified cell culture harvest thereof or a protein suspension; and / or (ii) the plasma or plasma fraction is selected the group consisting of a human blood plasma sample, an IgG intermediate product, a cryo-rich plasma, a cryo-poor plasma, a Supernatant I (SN I), a Cohn Fraction II (Fr II), a Cohn Fraction 11+111 (Fr II+III), a Cohn Fraction I+II+III (FrI+II+III), a Kistler / Nitschmann Precipitate A (KN A), a Kistler / Nitschmann Precipitate B (KN B), a Kistler / Nitschmann Precipitate of Supernatant B (KN B+l), formulated plasma fractionation product and combinations thereof, optionally wherein the purified or partially purified plasma or plasma fraction thereof has been subjected to one or more steps selected from the group consisting of clarification, ethanol precipitation, octanoic acid fractionation, ammonium sulphate precipitation, affinity chromatography, ion exchange chromatography, viral inactivation, viral filtration, isoagglutinin affinity chromatography, ultrafiltration / diafiltration, bulk formulation, final formulation and combinations thereof.

15. The method of any one of claims 1 to 14, wherein the method comprises diluting the sample and / or the probe in the assay solution before incubating, optionally wherein the method comprises serially diluting the sample and / or probe in the assay solution before incubating, wherein the sample and / or probe is serially diluted in the assay solution to a dilution factor of between 1:10 to 1:60 before incubating and optionally wherein the method comprises adding the diluted sample to the diluted probe before incubating, wherein the total dilution factor of the sample and the probe in the assay solution is between 1: 100 to 1:3000.

16. The method of any one of claims 1 to 15, wherein the method determines the concentration of the plasma protein in the sample with a coefficient of variation of < 3.0% or with a coefficient of variation of < 2.0%.

17. The method of any one of claims 1 to 16, wherein the method is performed at a temperature in the range of 18 to 25 °C and / or is carried out in a well of a multi-well plate.

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