EA040036B1 - VACUUM BLOOD COLLECTION TUBES CONTAINING PROTEASE INHIBITORS FOR EVALUATION OF CONTACT SYSTEM ACTIVATION - Google Patents

Description

Cross-reference to related applications

This application claims priority on the filing date of U.S. Application No. 62/204644, filed August 2015, and U.S. Provisional Application No. 62/214308, filed September 4, 2015. The entire contents of each of these referenced applications are incorporated herein by reference.

State of the art

Accurate measurement of the activation level of the contact system in vivo using patient plasma is complicated by the property of ex vivo activation during blood collection. The blood of patients with certain diseases associated with the contact system, such as hereditary angioedema, is particularly susceptible to activation of the contact system ex vivo, because it lacks the C1 inhibitor, a natural inhibitor of this pathway. Therefore, measurement of specific pathway biomarkers (eg, 2-chain high molecular weight kininogen) may overestimate the degree of contact system activation that a patient has if blood is not carefully collected and processed.

Brief description of the invention

The present disclosure is based at least in part on the development of non-glass vacuum blood collection tubes containing mixtures (cocktails) of protease inhibitors in a liquid formulation that prevents ex vivo activation of the contact system during blood collection. Thus, the vacuum blood collection tubes disclosed herein provide an accurate measurement of the endogenous level of contact system activation in patients, especially in patients deficient in a natural inhibitor (eg, C1 inhibitor) of this pathway.

Accordingly, in one aspect, the description relates to a vacuum blood collection tube containing a liquid formulation that contains a mixture of protease inhibitors, which is substantially free of protease inhibitors that are unstable in aqueous solution. The vial may not be a glass vial. In some embodiments, the vial is plastic. In some embodiments, the vacuum blood collection tube contains 0.5 ml of any of the liquid formulations described herein, which can be diluted 10-fold upon use.

In some embodiments, the vacuum tube protease inhibitor mixture described herein comprises at least one serine protease inhibitor (eg, plasma kallikrein inhibitor) and at least one cysteine protease inhibitor. In one example, a mixture of protease inhibitors includes EPI-KAL2, which can be biotinylated, and leupeptin. The amount of EPI-KAL2 in the liquid composition containing it can vary from 5 to 15 μM. Alternatively or additionally, the amount of leupeptin in the liquid formulation may vary from 200 to 300 μM.

In some embodiments, the mixture of protease inhibitors described herein may contain at least two serine protease inhibitors, at least one of which is a trypsin inhibitor, such as a soybean trypsin inhibitor. In some examples, the mixture of protease inhibitors includes benzamidine, soy trypsin inhibitor, leupeptin and AEBSF. In some examples, the liquid composition in a vacuum blood collection tube may contain 80-120 mM benzamidine, 1-3 mg/ml soybean trypsin inhibitor, 200-300 μM leupeptin, and/or 10-30 mM AEBSF.

The liquid formulation in any of the vacuum blood collection tubes described herein may additionally contain polybrene and EDTA. In some embodiments, any liquid formulation described herein may have a pH of 4-6 (eg, 4.5).

In another aspect, the present description provides a method for assessing the endogenous level of activation of the contact system in an individual. The method includes: (i) collecting blood from an individual into any of the vacuum blood collection tubes described herein; (ii) processing the blood to obtain a plasma sample; and (iii) measuring the activation level of the contact system in the plasma sample. In some embodiments, the measurement step (step (iii)) can be performed by measuring the level of one or more biomarkers indicative of activation of the contact system. Such biomarkers may include prekallikrein, plasma active kallikrein (pKal), a2M-pKal, active factor XII, active factor XI, high molecular weight kininogen (HMWK), and/or a bradykinin metabolite. In one example, one or more biomarkers include cleaved HMWK and/or intact HMWK.

In another aspect, the present description provides a method for assessing the level of a drug with a directed effect on the contact system in an individual. The method includes: (i) collecting blood from an individual into a vacuum blood collection tube as described herein, wherein the individual receives a drug targeted to a contact system component; (ii) processing the blood to obtain a plasma sample; and (iii) measuring the individual's plasma drug level.

Also provided herein is a method for assessing the immunogenicity of a contact-targeted drug, wherein the method comprises: (i) collecting blood from an individual in a vacuum blood collection tube as described herein, wherein the individual receives the targeted drug. action on the component of the contact system; (ii) processing the blood to obtain a plasma sample; and (iii) measuring the level of antibodies that bind to the drug in the plasma sample. Such a method may further comprise before step (iii)

- 1 040036 isolation of antibodies that bind to a drug from a plasma sample. In some examples, anti-drug antibodies (ADA) can be isolated by solid phase extraction and acid dissociation (SPEAD).

In any of the methods described herein, the subject may be a diseased human who, in some cases, may be receiving treatment with a drug targeting a component (e.g., plasma kallikrein) of the contact system, e.g., a drug (e.g., an antibody), specifically directed to plasma kallikrein (for example, to the active form of plasma kallikrein). In some examples, blood is taken from a patient with a disease associated with the contact system, such as hereditary angioedema (HAE) or idiopathic angioedema. In some cases, the patient suffers from NAE with a normal level of C1-inhibitor (C1-INH).

In any of the methods described herein, the vacuum blood collection tube may not be the first tube filled with the individual's blood. Alternatively or additionally, the method step [step (ii)] can be performed within one hour of the blood collection step [step (i)].

A detailed description of one or more embodiments of the invention are set forth in the description below. Other features and advantages of the present invention will be apparent from the following drawings and the detailed description of several embodiments, as well as from the claims.

Brief description of the drawings

The following drawings are part of the present disclosure and are included to further disclose certain aspects of the present disclosure, which may be better understood with reference to one or more of these drawings in conjunction with a detailed description of specific embodiments provided herein.

In FIG. 1 is a photograph demonstrating that the SCAT169 and SCAT153 tubes prevented activation of the contact system as measured by 2 strand Western blot. When 10% ellagic acid is added to plasma, the contact system is activated, resulting in the conversion of 1-chain HMWK to 2-chain HMWK (see sodium citrate plasma). In contrast, the plasma of SCAT169 and SCAT153 contains the same amount of 1-strand HMWK before and after the addition of ellagic acid;

in fig. 2 is a graph showing the change in kininogen breakdown (in percent 2HMWK/cHMWK) based on plasma sampling methods from healthy individuals. The clinical collection center is indicated, as well as the type of collection tubes used, including K2EDTA (EDTA), sodium citrate, SCAT169, or P100. The grouping of the measuring points corresponds, from left to right: center 1: EDTA, center 2: citrate; center 3: citrate, center 4: citrate, center 1: SCAT169, center 2: SCAT169, center 4: SCAT169, center 5: SCAT169 and center 4: P100;

in fig. 3 is a graph showing kininogen breakdown (percent 2HMWK/cHMWK) in plasma collected in SCAT169 tubes from healthy individuals from different clinical centers. The grouping of the measuring points corresponds, from left to right: center 1: salesman, center 4, center 5 and centers 4 and 5;

in fig. 4 is a graph showing kininogen breakdown (percent 2-HMWK/cHMWK) in healthy patients compared to patients with HAE type I or II, HAE nC1-INH, and idiopathic angioedema (AE). The grouping of measurement points corresponds, from left to right: healthy individuals, basal HAE I/II, attack HAE I/II, basal HAE (nC1-INH), attack HAE (nC1-INH), basal (level) idiopathic AE and attack idiopathic AE;

in fig. 5 is a graph showing plasma levels of 2-chain HMWK in healthy individuals and NAE patients. A: Shows the percent levels of 2-chain HMWK in the plasma of healthy individuals. As shown in the graph, Clinical Center C did not implement removal of the primary tube prior to collection of SCAT169 plasma. B: Shows the percentage of 2-chain HMWK in the plasma of NAE patients.

Detailed description of the invention

The present disclosure is based at least in part on the development of vacuum blood collection tubes containing protease inhibitor cocktails that prevent contact system activation. In order to accurately assess the endogenous level of contact system activation in a patient or healthy volunteer, it is important that blood is carefully collected and processed. One or more of the following precautions may be taken to obtain an accurate assessment of the characteristics associated with the contact system, as described in this document:

(i) preferably, the vacuum blood collection tubes that have been described herein are not primary blood-filled tubes in which increased activation of the contact system due to local trauma after puncture of the vessel with a needle can be observed;

(ii) blood must not come into contact with glass (use plastic tubes or catheters);

(iii) the blood must be processed to obtain plasma within a short period of time (on-

- 2 040036 example, approximately 1 h) after collection; and/or (iv) the use of protease inhibitors in the collection tube should stabilize the plasma against activation of the ex vivo contact system that prevents accurate determination of the patient's endogenous state.

Advantages of the vacuum blood collection tubes described herein include at least: (1) the use of vacuum non-glass (eg, plastic) tubes for standardized and simplified blood collection; (2) using a liquid formulation containing protease inhibitor cocktails to minimize hydrolysis as much as possible; (3) the optional absence of protease inhibitors that are unstable in aqueous solutions (eg, PPACK II, H-D-Phe-Phe-Arg-chloromethyl ketone is also known); and (4) administering, in some embodiments, a plasma kallikrein inhibitor such as EPI-KAL2 (may be biotinylated), which allows the tubes to contain a reagent that allows detection of activated plasma kallikrein using an immunoassay. See, for example, WO 95/21601, the corresponding descriptions of which are incorporated herein by reference.

An unexpected observation in this study was that the use of a liquid protease inhibitor cocktail prevented or reduced hemolysis. If blood was collected in vacuum tubes containing a lyophilized preparation of protease inhibitors, it was highly hemolyzed, which may affect the measurement of some analytes. However, if blood was collected in a vacuum tube containing a solution of a mixture of the same protease inhibitors, it did not hemolyze.

Therefore, measurements aimed at evaluating the degree of activation of the contact system in plasma samples obtained by processing blood samples collected in vacuum tubes described herein gave more accurate levels of assessment of patients with various diseases. Obtaining an accurate assessment of the activation of the contact system may allow the identification of diseases or subsets of patients with various diseases that are possibly mediated by this pathway and, therefore, can be treated with an inhibitor of the contact system.

In addition, the use of the vacuum blood collection tubes described herein may assist in the accurate determination of drug levels and/or evaluation of the immunogenicity of a therapeutic molecule directed against activated forms of proteins in the contact system (e.g., plasma kallikrein, FXIIa, and 2-chain kininogen). ). Tubes may offer a similar advantage to therapeutic molecules that target activated proteins in the downstream contact system activation pathway that do not require calcium to produce an activated target (eg, FXIa and FIXa). The advantage of these tubes is mainly applicable to biological therapeutic molecules, since the PK assays and immunogenicity assays used are usually immunoassays in which the binding site (eg, idiotype, in the case of a therapeutic antibody) is recognized. If the therapeutic target is activated ex vivo, it may bind to the plasma biological agent and thereby interfere with detection of PK and immunogenicity in immunoassays. In vitro protease inhibitors can prevent target activation. The use of a liquid formulation prevents hemolysis, which may interfere with some laboratory tests.

Vacuum blood collection tubes containing a cocktail of protease inhibitors in a liquid formulation.

Vacuum blood collection tubes are commonly used in medical practice to collect blood samples for various applications. The tubes described herein may be non-glass tubes containing a liquid formulation that contains a mixture of protease inhibitors (protease inhibitor cocktail). In some embodiments, the protease inhibitor cocktail may include at least one serine protease inhibitor and at least one cysteine protease inhibitor. The at least one serine protease inhibitor may be a plasma kallikrein inhibitor. Such protease inhibitor cocktails may contain multiple (eg, 2, 3, 4, or 5) serine protease inhibitors, at least one of which may be a human trypsin or plasmin inhibitor. Preferably, the protease inhibitor cocktails described herein are substantially free of a protease inhibitor that is unstable in aqueous solution, i.e., the activity of the protease inhibitor that is unstable in aqueous solution is insignificant compared to the overall inhibitory activity of the protease cocktail. In some instances, the amount of protease inhibitor that is unstable in aqueous solution may be less than 5% (w/w) of the total amount of protease inhibitors in the cocktail, such as less than 2%, less than 1%, or less than 0.5%. In some instances, the protease inhibitor cocktail is completely free of a protease inhibitor that is unstable in aqueous solution (eg, in aqueous solution at pH 4-6). One example of a protease inhibitor that is unstable in aqueous solution is PPACK II, also known as H-D-Phe-Phe-Arg-chloromethyl ketone.

In table. 1 below are examples of serine protease inhibitors, cysteine protease inhibitors and trypsin protease inhibitors that can be used to prepare the described

- 3 040036 herein Protease Inhibitor Cocktails

Categories Examples of inhibitors Serine protease inhibitors * Benzamidine * 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF); * * Himostatin; * N alpha-Tosyl-Lys Chloromethyl Ketone (TLCK); * Tos-Phe-CH2C1; N-p-Tosyl-P-phenylalanine chloromethyl ketone (TRNK) * 1-({(6R,7S)-3-[(acetyloxy)methyl]-7-methoxy- 5,5-dioxido-8-oxo-5-thia-1-azabicyclo[4.2.0]oct2-en-2-yl}carbonyl)-L-proline * Patamostat mesylate; * Gabeksat mesylate; * Msaapvck (Meosuc-aapv-cmk; MeOSuc-Ala-Ala-ProVal-CMK) * Nafamostat mesylate; * Rosmarinic acid; * Purpurogallin; * 2-(4-( (1-Acetimidoyl-3- pyrrolidinyl)oxy)phenyl)-3-(7-amidino-2naphthyl)propionic acid hydrochloride pentahydrate * 4-(4-Bromophenylsulfonylcarbomayl)benzoyl-Lvalyl-b-proline-1(RS)-(1-trifluoroacetyl-2methylprolyl)amide * L-658758; CHEMBL446371; L658758 * Sivelestat; * Patamostat; * Cholesterol sulfate; * Elastase III inhibitor; * Gabeksat; * 4',6-diamidino-2-phenylindole; * 4-aminobenzamidine; * 3,4-dichloroisocoumarin; * Bivalirudin Trichloroacetate

- 4 040036

* Pradaksa; * HIRUDINE; * Ximelagatran; * Lepirudin; Refludan; hbw 023 * Bivalirudin; * Letaxaban; * Eribaxaban; * Dabigatran etexilate mesylate; * Apixaban; * Tanexaban; * Rivaroxaban; Xarelto; 366789-02-8 * Plasma kallikrein inhibitors such as EPL- KAL2, DX-88, DX-2930 and the like The following examples are human trypsin and/or plasmin inhibitors: * Soy trypsin inhibitor * 4-(2-aminoethyl)benzenesulfonylfluoride * 4-aminobenzamidine * alpha 1-antitrypsin * Aprotinin * Camostat * Eco-protein (E coli) * inter-alpha inhibitor * Nafamostat * NCO 650 * Ovomucin * Somatomedin B * Trypsin inhibitor (Bowman-Birk, soy) * Trypsin inhibitor (Kunitz, soy) * Urinastatin Cysteine protease inhibitor * Geldanamycin; 30562-34-6; AKOS022185390 * Calpastatin; * L-Proline, N-[[(2S,3S)-3- [(propylamino)carbonyl]-2-oxiranyl]carbonyl]-Lisoleucyl-;

- 5 040036

* Protease I inhibitor; * (L-3-trans-(Propylcarbamoyl)oxirane-2carbonyl)-L-isoleucyl-P-proline; * Calpain III inhibitor; * [L-3-trans-(Propylcarbamoyl)oxirane-2carbonyl]-L-isoleucyl-L-proline; * Omuralid; * (S)-MG132; * Lactacystin; * Z-Phe-ala-diazomethane; * Leipeptin; * 4-Hydroxynonenal; * trans-Epoxysuccinyl-P-leucylamido(4guanidino)butane; * Loxistatin; * Clasto-lactacystinbeta-lactone; * L-Proline, *Z-FA-FMK; * N-acetyleucyl-leucyl-methioninal;(?) * nitroaspirin; * Allnal; * Aloxystatin; * ethyl 3-({4-methyl-1-[(3-methylbutyl)amino]-1oxopentan-2-yl}carbamoyl)oxirane-2-carboxylate; * (+/-) 4-HYDROXINONE-2-ENAL;

In some examples, a protease inhibitor cocktail for use in preparing a vacuum blood collection tube contains at least one serine protease inhibitor (e.g., 1, 2, or 3), which may include at least one trypsin/plasmin inhibitor (e.g., 1, 2 or 3) and at least one cysteine protease inhibitor (eg 1, 2 or 3). Such a cocktail of protease inhibitors may include three serine protease inhibitors (eg, benzamidine, AEBSF, and a trypsin/plasmin inhibitor such as soybean trypsin inhibitor) and one cysteine protease inhibitor (eg, leupeptin).

In other examples, the protease inhibitor cocktail may contain at least one serine protease inhibitor (eg, a plasma kallikrein inhibitor) and at least one cysteine protease inhibitor (eg, leupeptin).

Plasma kallikrein inhibitor can be EPI-KAL2 (Met His Ser Phe Cys Ala Phe Lys Ala Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp; SEQ ID NO: 1), which is a specific plasma kallikrein, a recombinant protease inhibitor, due to which the tubes can contain a reagent that allows the detection of activated plasma kallikrein, using, for example, immunoassays.

Any of the protease inhibitor cocktails may be dissolved in a suitable solution to form a liquid formulation. A suitable solution may be an acid-citrate-dextrose solution, which may contain trisodium citrate, citric acid and dextrose. The solution may have a pH of about 4-6, 4-5, 4.5-5.0, or 4.2-4.7, such as 4.5. In some embodiments, the solution has a pH of approximately 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 , 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 or 6.0. In some embodiments, the implementation of the solution has a pH value of approximately 4.5. The liquid formulation may further comprise a cationic polymer such as a hexadimethyl bromide (Polybrene®) molecule, which can reduce contact system activation by interacting with negatively charged surfaces, and a chelating agent (eg, EDTA) that can inhibit metalloproteases.

The concentration of each protease inhibitor in the cocktail can be 5x or 10x the final concentration of that inhibitor for use in inhibiting the respective protease, depending on the dilution factor in practice. Final concentration of specific com

- 6 040036 commercial protease inhibitor is known in the art and can be obtained according to the manufacturer's protocol. In some examples, the concentration of EPI-KAL2 may vary from 5-15 μM (eg, 510, 7-12 μM, or 10-15 μM). In some embodiments, the concentration of EPI-KAL2 is about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or about 15 µM. In some examples, the concentration of leupeptin may be from 200 to 300 μM (eg, 200-250, 240-270, or 250-300 μM). In some embodiments, the concentration of leupeptin is about 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or about 300 µM. In some examples, the concentration of soybean trypsin inhibitor may vary from 1 to 3 mg/ml (eg, 1-2 or 2-3 mg/ml). In some embodiments, the soybean trypsin inhibitor concentration is about 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 or about 3.0 mg/mL. In some examples, the concentration of benzamidine may vary from 80 to 120 mm (for example, 80-100 or 100-120 mm). In some embodiments, the benzamidine concentration is about 80, 85, 90, 95, 100, 105, 110, 115, or about 120 mM. In some examples, the concentration of AEBSF may be from 10 to 30 mm (for example, 10-20 or 20-30 mm). In some embodiments, the AEBSF concentration is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or about 30 mm.

If a peptide protease inhibitor (eg, EPI-KAL2) is used, it can be biotinylated according to conventional procedures. For example, a peptide inhibitor can be biotinylated as follows. Briefly, the peptide inhibitor can be dissolved in a suitable solution such as phosphate buffered saline (PBS). Freshly prepared SulfoNHS-LC-Biotin can be added to the peptide inhibitor solution and incubated on ice for a suitable period of time. Excess unreacted and hydrolyzed biotin can be removed using a column to desalt protein solutions. The labeling of the peptide inhibitor can be confirmed by ELISA and the protein concentration can be determined, for example, by the Bradford assay.

Any of the liquid formulations described herein may be prepared by conventional means, for example by dissolving the respective components in a suitable solution and placing in vacuum blood collection tubes, which are preferably non-glass. The tubes can be stored at -20°C and can be thawed on ice or at a refrigerated temperature (eg, about 4°C), such as in a refrigerator, for a suitable period of time before use.

The use of vacuum tubes for blood collection containing cocktails of protease inhibitors in liquid form.

Any vacuum tube for blood collection described herein can be used to collect blood samples from individuals for use in the analysis of endogenous signs associated with the contact system, including, but not limited to, the level of activation of the contact system, the level of drug that targets the component of the contact system, in serum and/or the immunogenicity of such a drug. To reduce ex vivo activation of the contact system (e.g., local trauma following needle puncture), the vacuum blood collection tube described herein may not be the primary blood-filled tube when blood is drawn from an individual. For example, an individual's first blood sample can be collected in a primary tube that can be removed, and then vacuum tubes are used to collect subsequent blood samples that can be used for analysis. The primary tube may be a conventional blood collection tube used in normal practice.

After blood collection, blood samples can be processed to obtain plasma samples within a suitable period of time (eg, no more than an hour). Plasma samples can be further analyzed to evaluate signs associated with the contact system of the individual from whom the primary blood sample was taken.

Blood samples may be collected from the individual, if necessary, analyzed as described herein. In some instances, the subject is a patient who may have, suspected, or at risk of developing a contact system related disease. For example, the patient may have had previous cases of NAE or may be at risk of developing NAE. The patient may have type I or type II HAE, either deficient in C1-INH or producing atypical C1-INH. Alternatively, the patient may have type III HAE that is not associated with C1-INH deficiency. In other examples, the patient has a history of idiopathic angioedema or may be at risk for idiopathic angioedema. Such a patient may have been previously treated or is now being treated with a drug that targets a component of the contact system (eg, pKal or FXIIa or high molecular weight kininogen).

i. Evaluation of the endogenous level of activation of the contact system.

In one aspect, plasma samples as described herein can be assayed for the endogenous level of contact system activation of an individual, which may be a patient suffering from a contact system related disease (e.g., NAE or idiopathic neurotic edema) who is suspected of having or is at risk of developing this disease. Such a patient may be treated for the disease, such as treatment with an inhibitor

- 7 040036 pKal (for example, anti-pKal antibodies). In other cases, such a patient may not receive such treatment.

Alternatively, the patient may be a healthy individual free of such diseases.

The level of contact system activation in a plasma sample can be determined by measuring one or more biomarkers indicative of contact system activation.

Plasma kallikrein (pKal) is the major bradykinin-producing enzyme in the bloodstream. Activation of pKal can occur via the contact system or via factor XIIa, both associated with the pathology associated with hereditary angioedema (HAE). Plasma kallikrein circulates as an inactive zymogen called prekallikrein, which mainly binds to its substrate, high molecular weight kininogen (HMWK). In response to stimulation, prekallikrein is cleaved to form active plasma kallikrein. Such activation of kallikrein can be mediated, for example, by factor XIIa after activation by FXII to FXIIa, or by a contact cascade effector. Approximately 75-90% of circulating prekallikrein is bound to HMWK through interaction with an inactive site with HMWK domain 6, which hydrolyzes additional HMWK molecules to form cleaved HMWK and bradykinin. Active plasma kallikrein cleaves HMWK at two sites, resulting in the release of bradykinin, a key mediator of pain, inflammation, edema, and angiogenesis. Another cleavage product, cleaved kininogen, contains amino acid chains linked together via a disulfide bridge. Cugno et al., Blood (1997) 89:3213-3218.

Examples of biomarkers that can be used to assess the level of contact system activation in a patient's blood sample (thereby determining whether the patient has an increased level and/or activity of the contact system, for example, an increased level of pKal), are presented in table. 2 below.

Table 2. Biomarker contact system

Biomarker Analysis Baseline in a patient with NAE relative to normal Δ indication or in acute edema Prekallikrein ELISA or analysis enzymatic activity Not changed (~500 nM) or slightly reduced Further decline Active pKal ELISA or enzymatic activity Not changed or slightly raised Promoted complex a2MrKa! ELISA Raised if the attack was recent Promoted Complex ClINH-pKal ELISA Raised if the attack was recent Promoted FXIIa ELISA or enzymatic activity Unchanged or slightly elevated Promoted FXIa ELISA or enzymatic activity Not changed or slightly raised Promoted Intact HMWK ELISA Not changed or slightly downgraded Downgraded Split HMWK ELISA Not changed or slightly raised Promoted Metabolite of bradykinin ELISA or LC MS Not changed or slightly raised Promoted

One or more biomarkers indicating activation of the contact system can be analyzed using standard methods. One particularly suitable type of assay for detection, qualitatively, semi-quantitatively or quantitatively, is an immunoassay. An immunoassay is any assay in which a target molecule (eg, a biomarker molecule associated with activation of a contact system) is detected and/or quantified using a binding agent, as described herein, that specifically binds to the molecule target. The binding agent may be an antibody, which may be a full-length antibody or an antigen-binding fragment thereof. The immunoassay may be a competitive or non-competitive immunoassay and may be a homogeneous or heterogeneous immunoassay. For example, an immunoassay for detecting a contact system biomarker can be enzyme immunoassay (EIA), radioimmunoassay (RIA), fluoroimmunoassay (FIA), chemiluminescent immunoassay (CLIA), particle counting immunoassay (CIA), quantitative enzyme immunoassay (IEMA), enzyme immunosorbent assay. assay (ELISA), sidestream immunoassay, sandwich immunoassay, immuno-PCR assay, close ligation method, Western blot assay, or immunoprecipitation assay. Additional suitable biomarker detection immunoassays provided herein will be apparent to those skilled in the art. Those skilled in the art will appreciate that the present disclosure is not limited to immunoassays, however, and that detection assays that are not based on an antibody or antigen-binding antibody fragment, such as mass spectrometry, are also effective for detecting and/or quantifying biomarkers of the contact system, as proposed in this document.

The type of detection assay used to detect and/or quantify a contact system biomarker, such as proposed herein, will depend on the particular situation in which the assay is to be used (e.g., clinical or research use) and on the type and amount of detectable biomarkers, and the type and number of patient samples run in parallel to indicate several parameters. For example, elevated levels of cleaved kininogen (2-chain kininogen) can be detected in plasma samples collected from patients with NAE or from healthy individuals during acute NAE using Western blot analysis. Although Western blot analyzes allow simultaneous analysis of biomarkers of the contact system in many samples, they are limited by the number of biomarkers that can be assessed in parallel. Accordingly, in some embodiments, if a plurality of the contact system biomarkers provided herein are analyzed in a single sample or multiple samples, then assays suitable for such multiple assays are preferred. Examples of such assays include, without limitation, peptide microarrays and chip-based laboratory assays that have been designed for high throughput, multiplex alternatives for smaller scale immunoassays, such as Western blots.

In some examples, a plasma sample can be placed in a multiwell microplate in the presence or absence of a pKal inhibitor and/or contact system activator. The mixture can be incubated on ice in the presence of the labeled peptide substrate pKal for a suitable period of time (eg 2 minutes) and a corn trypsin inhibitor (CTI) can be added to the mixture to stop the activation reaction. The mixture can be diluted if necessary and proteolytic activity can be determined by measuring the level of the fluorescent peptide substrate. The results of such an analysis can be based on the determination of the endogenous level of activation of the contact system of the individual from whom the plasma sample was taken. They can also be used to determine the inhibitory activity of a pKal inhibitor, if used.

ii. Evaluation of endogenous levels of drugs targeting the contact system.

In yet another embodiment, the present invention relates to the use of the vacuum blood collection tube described herein to determine levels of a drug targeted to a contact system component. Drug levels are needed to evaluate pharmacokinetic parameters. For example, if the contact system is activated ex vivo in plasma samples collected to quantify a plasma kallikrein inhibitor (e.g., DX-2930) in plasma, then excess activated plasma kallikrein may bind to the drug and thereby protect its detection in the assay. Any of the vacuum blood collection tubes described herein can be used to more accurately assess drug levels, such as in a sample collected from an individual.

To put this method into practice, plasma samples obtained from an individual (e.g., human) receiving a drug that is directed to a component of the contact system (e.g., pKal) can be obtained from blood samples collected in vacuum blood collection tubes described herein, following the methods described herein. The level of the drug in the plasma sample can be measured in accordance with normal practice. In some cases, drug levels can be measured using an immunoassay, such as those described herein.

iii. Evaluation of the immunogenicity of a drug targeting the contact system.

In yet another embodiment, the present invention relates to the use of a vacuum

- 9 040036 blood collection tubes for determining the immunogenicity of a biological inhibitor against a component of the contact system (eg pKal). For example, it is common practice to develop immunogenicity assays that are capable of measuring anti-drug antibodies (ADA) in the presence of excess drug in the blood plasma. Such a need for an immunogenicity assay that can measure ADA certainly applies to therapeutic monoclonal antibodies, which can have multi-week half-lives and high circulating drug levels. To overcome the effect of excess drug in the sample, methods are used to separate anti-drug antibodies from the drug. Such anti-drug antibodies can be isolated by solid phase extraction and acid dissociation (SPEAD), which involves incubating the biotinylated form of the drug with a plasma sample for an extended period of time (usually overnight) followed by isolating the biotinylated drug, which may be bound anti-drug antibodies using a streptavidin-coated plate. The plate is then treated with acid to release the anti-drug antibody. The released antibodies can be applied directly to another assay plate for detection. In the absence of protease inhibitors in the collection tubes, the contact system can be activated ex vivo, resulting in the production of active plasma kallikrein. After the acid release and recoating steps above, both active pKal and anti-drug antibodies will bind to the surface of the plate. Anti-drug antibody is usually detected with a labeled drug, which can also bind to active pKal if present, resulting in a false positive signal in the ADA assay.

The use of vacuum blood collection tubes that contain the protease inhibitor cocktails described herein can prevent this false positive signal.

General methods.

In the practice of the present invention will be used, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are known in practice in this field. Such techniques are explained in detail in the literature, for example Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel, et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995).

Without going into details, it is believed that a person skilled in the art can, based on the above description, apply the present invention to its fullest extent. Therefore, the following specific embodiments are to be construed as merely illustrative and not limiting the remainder of the description in any way. All publications cited in this document are incorporated by reference for the purposes or subject matter indicated herein.

Example 1 Preparation of Protease Inhibitor Cocktails that Prevent Contact System Activation.

Cocktails of protease inhibitors that prevent the activation of the contact system.

Vacuum plastic tubes were used for standardized and simplified blood collection.

The following two protease inhibitor cocktails were used in a liquid formulation to prevent hydrolysis:

1) 10x cocktail of protease inhibitors A: SCAT169.

Vacuum plastic tubes, total volume 5 ml, containing (0.5 ml): 100 mM benzamidine, 400 µg/ml polybrene, 2 mg/ml soy trypsin inhibitor, 20 mM EDTA, 263 µM leupeptin and 20 mM AEBSF (4-( 2-aminoethyl)benzenesulfonyl fluoride hydrochloride) dissolved in acid-citrate dextrose (100 mM trisodium citrate, 67 mM citric acid and 2% dextrose, pH 4.5).

2) 10x cocktail of protease B inhibitors: SCAT153.

Vacuum plastic tubes, 5 ml total volume, containing (0.5 ml): 10 µM biotinized EPI-KAL2, 400 µg/ml polybrene, 20 mM EDTA and 2 63 µM leupeptin dissolved in the mixture

- 10 040036 acid-citrate-dextrose (100 mM trisodium citrate, 67 mM citric acid and 2% dextrose, pH 4.5).

A cocktail of protease B inhibitors (SCAT153) was loaded with biotinylated EPI-KAL2. SCAT169 (Specialized coagulation test tubes, formulation 169) and SCAT153 (Specialized coagulation test tubes, formulation 153) were stored at 2-8°C.

Example 2 SCAT169 and SCAT153 tubes prevent contact system activation as shown by 2-strand Western blot analysis.

Plasma collected in SCAT169 or SCAT153 tubes blocked ex vivo contact system activation induced by the addition of ellagic acid (FIG. 1), a known contact system activator, as measured by conversion of 1-strand to 2-strand HMWK by Western blot analysis.

The observed activation of the contact system induced by ellagic acid was compared in plasma samples collected in 3 different blood collection tubes: sodium citrate tubes (standard tubes used in clinical chemistry laboratories to measure coagulation), SCAT169 tubes, and SCAT153 tubes.

1-strand HWMK was substantially completely converted in samples activated with ellagic acid in sodium citrate tubes and the appearance of 2-strand HMWK was detected.

In contrast, 1-strand HMWK was maintained in ellagic acid-activated plasma in SCAT169 and SCAT153 tubes.

These results indicate that the SCAT169 and SCAT153 tubes are effective in preventing ex vivo contact system activation that can occur during plasma collection and processing.

Example 3 Protease inhibitor cocktails SCAT169 and SCAT153 increased plasma clotting time.

Plasma clotting time was measured in samples processed in 3 different blood collection tubes: sodium citrate tubes, SCAT169 tubes, and SCAT153 tubes, for three separate donor samples (Table 1).

As shown in prothrombin and activated partial thromboplastin times, clotting time was increased in samples with SCAT159 and SCAT153 compared to sodium citrate. The results are shown in table. 3 below.

Table 3 Effect of protease inhibitors on plasma clotting time

Number donor: Prothrombin time (s) Activated partial thromboplastin time (C) Citrate SCAT169 SCAT 153 Citrate SCAT169 SCAT 153 1 12.4 214.55 55.3 42.8 > 240 > 240 2 12.8 159.6 27.85 37.6 > 240 > 240 3 11.4 >300 87.1 34.6 > 240 > 240

Example 4 Use of Vacuum Blood Collection Tubes for Endogenous Contact System Activation Levels in Individuals.

The study of biomarkers of contact system activation in plasma is hampered by inadvertent activation during blood collection and processing. In this study, the use of specialized blood collection tubes (SCAT159 and SCAT153) to assess plasma levels of cleaved high molecular weight kininogen (cHMWK) in healthy individuals and patients with type I/II hereditary angioedema (HAE), idiopathic angioedema, or HAE with normal C1INH (HAEnC1, also referred to as nC1-INH) was analyzed as follows.

To avoid artificial activation of the contact pathway during blood sampling, standardized blood collection methods and commonly used tubes containing protease inhibitors were used in the study. Blood samples were collected from healthy individuals and individuals with the disease, as described above (during the period of attenuation and outbreak of the disease) to assess the percentage of cHMWK using Western blot analysis. Blood samples were placed into SCAT159 or SCAT153 tubes (5 ml total volume, 0.5 ml of 10x protease inhibitor cocktail) using a catheter with a butterfly needle system. The blood samples were then processed to obtain plasma samples within 1 hour after blood collection.

Plasma samples in the SCAT tube were analyzed by Simple Western (SBHD) and Western Blot (TGA) to determine the level of cleaved kininogen (2-chain kininogen) in plasma samples, following the methods described, for example, in WO 2015/061183.

Plasma was collected from healthy individuals at Clinical Centers 1-5 in plastic collection tubes containing various anticoagulants, protease inhibitors, or protein stabilizers before being processed into plasma. In particular, the tubes were of the type K2EDTA, sodium citrate, SCAT 169 or P100

- 11 040036 (BD Biosciences). As shown in FIG. 2, the tubes found to marginally reduce ex vivo contact system activation contained protease inhibitors and were either P100 tubes or SCAT169 tubes, which were 5 ml vacuum plastic blood collection tubes containing 0.5 ml 10x concentrated mixture of 100 mM benzamidine, 400 µg/ml polybrene, 2 mg/ml soy trypsin inhibitor, 20 mM EDTA, 263 µM leupeptin, and 20 mM AEBSF dissolved in acid-citrate-dextrose (100 mM trisodium citrate, 67 mM citric acid and 2% dextrose, pH 4.5). Collection methods using tubes containing protease inhibitor cocktails that prevent an increase in cHMWK in the analysis of plasma from healthy donors.

The results obtained in this study showed that in healthy individuals, cHMWK levels were stable (<5%) at room temperature (RT) for at least 24 h after blood collection in commonly used tubes, but cHMWK levels were elevated (12 %) in plasma samples obtained from a commercial supplier, indicating the importance of optimizing blood collection techniques when studying contact pathway activation.

The effects of cleavage of kininogen in plasma collected in SCAT169 tubes from healthy individuals were also evaluated at two different clinical centers (centers 4 and 5) and from a commercial supplier (center 1) (FIG. 3). The commercial supplier collected blood samples directly into SCAT 169 tubes without removing the first tube.

The cleavage of kininogen was also assessed in blood samples collected in SCAT169 tubes from individuals with a type of angioedema (type I/II HAE, nC1-INH HAE and idiopathic AE). Plasma was collected from healthy individuals or individuals with various types of angioedema both at rest (baseline) and during an attack (attack) to measure the amount of cHMWK in various diseases. The percentage of cHMWK was clearly elevated at baseline in patients with HAE (n=21) compared to healthy controls (n=26), but not in plasma in patients with idiopathic angioedema (n=4) or HAEnC1 (n =5) (Fig. 4), which indicates a limited activity of kallikrein in plasma, although it does not exclude the role of activation of the contact pathway during acute attacks in these diseases.

Plasma evaluated for detection of cHMWK, sensitive to contact system activation stimulated by collection methods and tubes. Overall, this assay provides an improved way to collect plasma samples to evaluate the activation of the contact system, which prevents ex vivo degradation of HMWK.

Example 5 Use of Vacuum Blood Collection Tubes for Endogenous Contact System Activation Levels in Individuals.

Dedicated blood collection tubes (SCAT159 and SCAT153) were evaluated for efficacy in assessing plasma levels of cleaved high molecular weight kininogen (cHMWK) in healthy individuals and patients with type I/II hereditary angioedema (HAE).

Briefly, blood samples were collected from healthy individuals and from patients with NAE, during periods of attenuation (basal) and outbreaks (attacks) of the disease. The percentage of 2-chain HMWK in plasma was detected using Western blot analysis. Plasma samples were collected in a phase 1b double-blind study in randomized patients with type I/II NAE from different centers who received 2 subcutaneous doses of anti-pKal antibody (DX-2930) on days 0 and 15 in close groups of 30 , 100, 300 or 400 mg or placebo. Blood samples were obtained before and after administration of the anti-pKal antibody (DX-2930) on days 1, 8, 22, 64, 92 and 120.

As shown in FIG. 5A, the percentage of plasma levels of 2-chain HMWK in samples from healthy individuals collected from three different clinical centers (A, B, and C) varied depending on the collection method and the type of tube used. Samples using sodium citrate tubes had higher levels of 2-chain HMWK compared to samples in SCAT169 tubes. Notably, samples collected at Clinical Center C that were collected directly into SCAT169 tubes without tube removal prior to the tube containing the protease inhibitor cocktail had higher levels of 2-chain HMWK compared to the tube removal samples.

Similarly, as shown in FIG. 5B, the percentage of plasma levels of 2-chain HMWK in HAE samples was higher in sodium citrate tubes compared to plasma collected in SCAT169 tubes, which is likely due to exogenous activation of the contact system associated with plasma assembly and processing.

This study highlights the benefits of using blood collection tubes containing protease inhibitor cocktails as described herein and proposes an improved method for collecting plasma samples to evaluate contact system activation, which prevents ex vivo activation of abnormal contact system (e.g. HMWK cleavage).

Example 6 Use of Vacuum Blood Collection Devices to Assess Plasma Drug Levels.

Blood was taken from patients with NAE treated with DX-2930 and from healthy individuals as controls in the usual way and placed in collection tubes. After placing the samples of the first blood draw into one or more collection tubes, 5 ml of the sample of the subsequent blood draw were placed into the SCAT159 or SCAT153 tubes. The blood samples were then processed to obtain plasma samples within 1 hour after blood collection.

The amount of DX-2930 in SCAT plasma samples was measured by standard immunoassays such as ELISA. Briefly, an anti-idiotypic anti-DX-2930 monoclonal Fab variant was applied to the surface of a 96-well plate overnight, and unbound anti-idiotypic Fab molecules were removed by washing the plate multiple times. Then, SCAT plasma samples were added to the plate and incubated at room temperature for 2-3 hours. The plate was washed several times, and a biotinylated anti-idiotypic monoclonal antibody IgG variant against DX-2930 was added to the plate, followed by an incubation step and a washing step. Then, streptavidin conjugated with horseradish peroxidase was added to the tablet. After 30 min incubation, it was washed again and analyzed for the signal released by the dye. The signal intensity corresponds to the amount of DX-2930 in the plasma sample.

Example 7 Use of Vacuum Blood Collection Devices to Evaluate the Immunogenicity of a Drug.

Plasma samples from patients with NAE treated with DX-2930 were obtained from blood samples collected in SCAT159 or SCAT153 tubes as described above. Ahtu-DX-2930 antibodies in the plasma sample were isolated by solid phase and acid dissociation (SPEAD).

Briefly, plasma samples were incubated with biotinylated DX-2930 and incubated overnight. The mixture was then placed in a streptavidin-coated plate to capture biotinylated DX-2930, which binds to anti-DX-2930 antibodies in the plasma sample, if any. Anti-DX-2930 antibodies were then released upon acid treatment and formed a layer on a Meso Scale Discovery (MSD) plate. Ruthenium labeled DX-2 93 O was added to the MSD plate and the electrochemiluminescent signal was measured to detect the presence of anti-DX-2930 antibodies.

Other embodiments of the invention

All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is merely an example of a general series of equivalent or similar features.

From the foregoing description, a person skilled in the art can easily determine the main characteristics of the present invention, and without departing from its essence and scope, can make various changes and modifications to the invention in order to adapt it to various applications and conditions of use. Thus, other embodiments are also within the scope of the claims.

Equivalents and area.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments of the present description disclosed herein. The scope of the present disclosure is not limited to the above description, but rather is set forth in the appended claims.

In the claims, the singular means one or more than one, unless otherwise indicated or obvious from the context. Claims and descriptions that include or between one or more members of a group are deemed to be satisfied if one, more than one, or all of the members of the group are present, used, or related to a particular product or method, unless otherwise indicated or evident from the context. The present description includes embodiments that use, apply, or otherwise relate to a given product or method, only one member of this group. The present description includes embodiments that use, apply, or otherwise relate to a given product or method by more than one member or all members of the group.

In addition, the present description covers all variations, combinations and permutations in which one or more restrictions, elements, sentences and descriptive terms from one or more of the listed claims are introduced into another claim. For example, any clause that depends on another clause can be modified to include one or more of the restrictions found in any other clause that depends on the same main clause. If the elements are presented as a list, such as in the Markush group format, then each subgroup of elements is also expanded, and any element(s) can be removed from the group. It should be understood that in general, if the present disclosure or aspects of the present disclosure are indicated as containing specific elements and/or features, then some embodiments of the present disclosure or aspects of the present disclosure consist or essentially consist of such elements and/or features. For simplicity, these options have not been haec verba specifically set forth in the present description. It should also be noted that the terms containing and including are intended to be open-ended and allow the inclusion of additional elements or steps. When a range is specified, the end values are included. In addition, unless otherwise indicated or obvious from the context and for the understanding of a person skilled in

- 13 040036 of this area, then the values that are expressed in ranges can take on any particular value or subrange in the specified ranges in various embodiments of the present disclosure, a tenth of the unit of the lower limit of the range, unless the context clearly indicates otherwise.

This application relates to various issued patents, published patent applications, journal articles and other publications, each of which is incorporated herein by reference. If there is a conflict between any of the included references and the original description, then the description should be used as a template. In addition, any particular embodiment of the present disclosure that falls within the scope of the prior art may simply be excluded from one or more of the claims. Since such embodiments are considered to be known to those of ordinary skill in the art, they may be excluded, even if the exclusion is not explicitly stated herein. Any particular embodiment of the present invention may be excluded from any claim for any reason, whether or not it is related to its existence in the prior art.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments of the present disclosure disclosed herein. The scope of the present embodiments described herein is rather limited to what is set forth in the appended claims than is intended to be limited by the foregoing description. Those skilled in the art will appreciate that various changes and modifications to this disclosure may be made without departing from the spirit or outside the scope of the invention as defined in the following claims.

Claims (35)

CLAIM 1. Vacuum blood collection tube containing a liquid formulation that contains a mixture of protease inhibitors, polybrene and EDTA, where the mixture of protease inhibitors consists of 80-120 μM benzamidine, 1-3 mg/ml soybean trypsin inhibitor, 200-300 μM leupeptin and 10-30 μM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF). 2. The vacuum blood collection tube of claim 1, wherein the tube is not a glass tube. 3. The vacuum blood collection tube of claim 2, wherein the tube is a plastic tube. 4. Vacuum blood collection tube according to any one of claims 1 to 3, wherein the mixture of protease inhibitors consists of 100 mM benzamidine, 2 mg/ml soybean trypsin inhibitor, 263 μM leupeptin and 20 mM AEBSF. 5. A vacuum blood collection tube according to any one of claims 1 to 4, wherein the liquid composition further comprises trisodium citrate, citric acid, and dextrose. 6. The vacuum blood collection tube of claim 5, wherein the liquid formulation contains 100 mM trisodium citrate, 67 mM citric acid, and 2% dextrose. 7. Vacuum blood collection tube according to any one of claims 1 to 6, where: a) the liquid formulation contains 400 μg/ml polybrene and 20 mM EDTA; b) the liquid composition has a pH of 4-6; c) the tube contains 0.5 ml of the liquid composition; and/or d) the protease inhibitor mixture is substantially free of protease inhibitors that are unstable in aqueous solution. 8. The vacuum blood collection tube of claim 7, wherein the liquid formulation has a pH value of 4.5. 9. A method for assessing the endogenous level of activation of the contact system in a blood sample from an individual, where the method includes: (i) collecting blood from the individual in a vacuum blood collection tube according to any one of claims 1 to 8; (ii) processing the blood to obtain a plasma sample; and (iii) measuring the activation level of the contact system in the plasma sample. 10. The method of claim 9, wherein in step (i) the vacuum blood collection tube is not a primary tube filled with the individual's blood, wherein step (ii) is performed within one hour of step (i); and/or where stage (iii) is carried out by measuring the level of one or more biomarkers indicating the activation of the contact system. 11. The method according to any one of claims 9 or 10, wherein the subject is a human. 12. The method of claim 11, wherein the person is a patient with a disease associated with the contact system. 13. The method of claim 12 wherein the disease is hereditary angioedema (HAE) or idiopathic angioedema. 14. The method of claim 13 wherein the patient suffers from HAE with normal C1-INH. 15. The method according to any one of claims 9-14, in which the person receives treatment with a drug directed to a component of the contact system. 16. The method of claim 15 wherein the contact system component is plasma kallikrein. - 14 040036 17. The method of claim 15 or 16 wherein the drug inhibits active plasma kallikrein. 18. The method according to any one of claims 15-17, wherein the drug is an antibody that binds to active plasma kallikrein. 19. The method according to any one of claims 10-18, wherein the one or more biomarkers are selected from the group consisting of prekallikrein, active plasma kallikrein (pKal), a2M-pKal, active factor XII, active factor XI, high molecular weight kininogen (HMWK) and a metabolite of bradykinin. 20. The method of claim 19 wherein the one or more biomarkers comprise cleaved HMWK and/or intact HMWK. 21. A method for changing the level of a drug targeting a contact system in an individual, wherein the method includes: (i) collecting blood from the individual in a vacuum blood collection tube according to any one of claims 1 to 8, where the individual receives a drug that targets a component of the contact system; (ii) processing the blood to obtain a plasma sample; and (iii) measuring the level of the drug in the plasma sample. 22. The method of claim 21, wherein in step (i) the vacuum blood collection tube is not a primary tube filled with the individual's blood; and/or wherein step (ii) is carried out within one hour of step (i). 23. The method according to any one of claims 21 or 22, wherein the subject is a human. 24. The method of claim 23, wherein the person is a patient suffering from a disease associated with the contact system. 25. The method of claim 24 wherein the disease is hereditary angioedema (HAE). 26. The method of any one of claims 21-25, wherein the drug inhibits active plasma kallikrein. 27. The method of claim 26 wherein the drug is an antibody that binds to active plasma kallikrein. 28. A method for assessing the immunogenicity of a drug targeted to a contact, where the method includes: (i) collecting blood from the individual in a vacuum blood collection tube according to any one of claims 1 to 8, where the individual receives a drug that targets a component of the contact system; (ii) processing the blood to obtain a plasma sample; and (iii) measuring the level of antibodies that bind to the drug in the plasma sample. 29. The method of claim 28, wherein in step (i) the vacuum blood collection tube is not a primary tube filled with the individual's blood; and/or wherein step (ii) is carried out within one hour of step (i). 30. The method according to any one of claims 28 or 29, wherein the subject is a human. 31. The method of claim 30, wherein the human is a patient suffering from a disease associated with the contact system. 32. The method of claim 31 wherein the disease is hereditary angioedema (HAE). 33. The method of any one of claims 28-32, wherein the drug inhibits active plasma kallikrein. 34. The method according to any one of claims 28-33, wherein the drug is an antibody that binds to active plasma kallikrein. 35. The method according to any one of claims 28-34, wherein the method further comprises, prior to step (iii), isolating antibodies that bind to the drug from the plasma sample.