Vacutainer with protease inhibitor for assessing activation of contact system

Abstract

This article discloses vacuum blood collection tubes comprising a mixture of protease inhibitors in liquid form and their use for assessing characteristics related to a subject's contact system, including endogenous levels of contact system activation, endogenous levels of a drug targeting the components of the contact system during treatment, and / or the immunogenicity of such a drug.

Description

[0001] This application is a divisional application of Chinese patent application No. 2016800472546, filed on August 12, 2016, entitled "Vacuum blood collection tube containing protease inhibitor for evaluating contact system activation".

[0002] Cross-reference to related applications

[0003] This application claims the benefit of the filing dates of U.S. Provisional Application No. 62 / 204,644, filed August 13, 2015, and U.S. Provisional Application No. 62 / 214,308, filed September 4, 2015. The entire contents of each of these cited applications are incorporated herein by reference. Technical Field

[0004] This application belongs to the technical field of blood collection tubes for evaluating drug levels in targeted contact activation systems. Background Technology

[0005] Accurately measuring in vivo levels of contact system activation using patient plasma presents challenges due to the tendency for activation to occur ex vivo during blood collection. Blood from patients with certain contact system-related diseases (e.g., hereditary angioedema) is particularly prone to ex vivo contact system activation because it lacks natural inhibitors of this pathway—C1 inhibitors. Therefore, measuring pathway-specific biomarkers (e.g., 2-chain high molecular weight kininogen) may overestimate the degree of contact system activation present in patients unless the blood is carefully collected and processed. Summary of the Invention

[0006] This disclosure is based, at least in part, on the development of non-glass vacuum blood collection tubes containing a mixture (cocktail) of protease inhibitors in a liquid formulation that prevents ex vivo activation of the contact system during blood collection. Therefore, the vacuum blood collection tubes described herein allow for precise measurement of the endogenous level of contact system activation in patients, particularly those lacking natural inhibitors of this pathway (e.g., C1 inhibitors).

[0007] Therefore, one aspect of this disclosure describes a vacuum blood collection tube comprising a liquid formulation containing a mixture of protease inhibitors, said liquid formulation being substantially free of protease inhibitors unstable in aqueous solutions. The tube may be a non-glass tube. In some embodiments, the tube is plastic. In some embodiments, the vacuum blood collection tube contains 0.5 ml of any of the liquid formulations described herein, which may be diluted 10-fold before use.

[0008] In some embodiments, the mixture of protease inhibitors in the vacuum blood collection tubes described herein comprises at least one serine protease inhibitor (e.g., a plasma kallikrein inhibitor) and at least one cysteine ​​protease inhibitor. In one embodiment, the mixture of protease inhibitors comprises EPI-KAL2 (which may be biotinylated) and leucopeptide. The content of EPI-KAL2 may range from 5 to 15 μM in the liquid formulation containing it. Optionally or additionally, the amount of leucopeptide in the liquid formulation may range from 200 to 300 μM.

[0009] In some embodiments, the mixture of protease inhibitors described herein may comprise at least two serine protease inhibitors, at least one of which is a trypsin inhibitor, such as soybean trypsin inhibitor. In some embodiments, the mixture of protease inhibitors comprises benzalkonium chloride, soybean trypsin inhibitor, leucopeptide, and AEBSF. In some embodiments, the liquid formulation in the vacuum blood collection tube may comprise 80-120 mM benzalkonium chloride, 1-3 mg / ml soybean trypsin inhibitor, 200-300 μM leucopeptide, and / or 10-30 mM AEBSF.

[0010] The liquid formulation in any vacuum blood collection tube described herein may further comprise polybrene and EDTA. In some embodiments, the pH of any liquid formulation described herein may be 4-6 (e.g., 4.5).

[0011] In another aspect, this disclosure provides a method for assessing the endogenous level of contact system activation in a subject. The method includes: (i) collecting blood from the subject into any vacuum blood collection tube described herein; (ii) processing the blood to produce a plasma sample; and (iii) measuring the level of contact system activation in the plasma sample. In some embodiments, the measurement step (step (iii)) can be performed by measuring the level of one or more biomarkers indicating contact system activation. Such biomarkers may include prokallikrein, active plasma kallikrein (pKal), α2M-pKal complex, active factor XII, active factor XI, high molecular weight kininogen (HMWK), and / or bradykinin metabolites. In one embodiment, one or more biomarkers include cleaved HMWK and / or intact HMWK.

[0012] In another aspect, this disclosure provides a method for assessing the level of a drug in a subject using a targeted contact system. The method includes: (i) collecting blood from a subject into a vacuum blood collection tube as described herein, wherein the subject is administered a component of the drug from the targeted contact system; (ii) processing the blood to produce a plasma sample; and (iii) measuring the level of the drug in the plasma sample.

[0013] Furthermore, this disclosure provides a method for evaluating the immunogenicity of a drug for a targeted contact system, the method comprising: (i) collecting blood from a subject into a vacuum blood collection tube as described herein, wherein the subject is administered a component of the drug for the targeted contact system; (ii) processing the blood to produce a plasma sample; and (iii) measuring the level of antibodies bound to the drug in the plasma sample. Such a method may further comprise: prior to step (iii), separating the drug-bound antibodies from the plasma sample. In some embodiments, the separated antidrug antibodies (ADA) may be determined by solid-phase extraction and acid dissociation (SPEAD).

[0014] In any of the methods described herein, the subject may be a human patient, and in some cases, the human patient may be treated with a drug that targets a component of the contact system (e.g., plasma kallikrein), such as a drug that specifically targets plasma kallikrein (e.g., the active form of plasma kallikrein) (e.g., an antibody). In some embodiments, the blood is from a human patient suffering from a disease associated with the contact system (e.g., hereditary angioedema (HAE) or idiopathic angioedema). In some cases, the human patient has HAE with a normal C1-inhibitor (C1-INH).

[0015] In any of the methods described herein, the vacuum blood collection tube may not be the first tube containing blood from the subject. Optionally or additionally, the processing step [step (ii)] may be performed within one hour after the blood collection step [step (i)].

[0016] Details of one or more embodiments of the invention are set forth in the following description. Other features or advantages of the invention will become apparent from the following drawings and detailed descriptions of several embodiments, as well as from the appended claims. Attached Figure Description

[0017] The following figures form part of and are included in this specification to further illustrate certain aspects of this disclosure, which will be better understood by referring to one or more of these figures in conjunction with the detailed description of the specific embodiments presented herein.

[0018] Figure 1 These are photographs showing the SCAT169 and SCAT153 tubes preventing contact activation, as measured in a 2-chain protein blot assay. When 10% ellagic acid was added to the plasma, the contact system was activated, resulting in the conversion of 1-chain HMWK to 2-chain HMWK (see sodium citrate plasma). In contrast, SCAT169 and SCAT153 plasma contained the same amount of 1-chain HMWK before and after the addition of ellagic acid.

[0019] Figure 2This is a graph showing changes in kininogen cleavage (2-HMWK / cHMWK percentage), based on plasma sample collection methods performed from healthy subjects. The clinical sites of collection and the types of tubes used for collection are shown, including K2EDTA (EDTA), sodium citrate, SCAT169, or P100. The data points are grouped from left to right to correspond to: Site 1: EDTA; Site 2: Citrate; Site 3: Citrate; Site 4: Citrate; Site 1: SCAT169; Site 2: SCAT169; Site 4: SCAT169; Site 5: SCAT169; and Site 4: P100.

[0020] Figure 3 This is a graph showing the percentage of kininogen cleavage (2-HMWK / cHMWK) in plasma collected from different clinical sites in SCAT 169 tubes from healthy subjects. The data points are grouped from left to right to correspond to site 1: commercial vendor, site 4, site 5, and sites 4 and 5.

[0021] Figure 4 This is a graph showing the percentage of kininogen cleavage (2-HMWK / cHMWK) in healthy subjects compared to subjects with type I or II HAE, nC1-INH HAE, and idiopathic angioedema (AE). The data points are grouped from left to right to correspond to healthy subjects, basal HAE I / II, attack HAE I / II, basal HAE (nC1-INH), attack HAE (nC1-INH), basal idiopathic AE, and attack idiopathic AE.

[0022] Figure 5A and Figure 5B These are graphs showing the levels of 2-chain HMWK in the plasma of healthy subjects and patients with HAE. Figure 5A This shows the percentage of plasma 2-chain HMWK levels from healthy subjects. As shown in the figure, clinical site C did not use the initially discarded tube prior to SCAT169 plasma collection. Figure 5B This indicates the percentage of plasma 2-chain HMWK levels from patients with HAE. Invention Details

[0023] This disclosure is based, at least in part, on the development of vacuum blood collection tubes containing a mixture of protease inhibitors to prevent contact system activation. Careful blood collection and processing are essential for accurately assessing the endogenous levels of contact system activation in patients or healthy volunteers. One or more of the following precautions can be taken to ensure accurate assessment of contact system-related characteristics as described herein:

[0024] (i) The vacuum blood collection tubes described herein are preferably not the first tube containing blood, which may exhibit increased contact system activation due to local trauma following needle puncture of the blood vessel;

[0025] (ii) Blood may not come into contact with glass (using plastic tubing or catheters);

[0026] (iii) After collection, the blood can be processed into plasma within a short period of time (e.g., ~1 hour); and / or

[0027] (iv) The use of protease inhibitors in the collection tube can stabilize the plasma and prevent ex vivo contact activation, which hinders the accurate measurement of the endogenous patient condition.

[0028] The advantages of the vacuum blood collection tubes described herein include at least: (1) the standardization and simplification of blood collection using vacuum non-glass (e.g., plastic) tubes; (2) the minimization of hydrolysis using liquid formulations containing a mixture of protease inhibitors; (3) the optional omission of protease inhibitors (e.g., PPACK II, also known as HD-Phe-Phe-Arg-chloromethyl ketone) that are unstable in aqueous solutions; and (4) the inclusion of a plasma kallikrein inhibitor, such as EPI-KAL2 (which may be biotinylated), in some embodiments, which enables the tube to contain reagents that allow for the detection of activated plasma kallikrein using immunoassays. See, for example, WO95 / 21601, the relevant disclosure of which is incorporated herein by reference.

[0029] This study unexpectedly observed that the use of liquid form of the protease inhibitor mixture prevented or reduced hemolysis. When blood was collected into vacuum tubes containing a lyophilized formulation of the protease inhibitor, significant hemolysis occurred, which could interfere with the measurement of certain analytes. However, when blood was collected into vacuum tubes containing a solution of the same protease inhibitor mixture, no hemolysis occurred.

[0030] Therefore, measurements of the degree of contact system activation in plasma samples processed from blood samples collected into the vacuum tubes described herein provide a more precise level of assessment for patients with various diseases. Obtaining accurate assessments of contact activation allows for the identification of diseases or subsets of patients with various conditions potentially mediated by this pathway, thus making them suitable for treatment with inhibitors of the contact system.

[0031] Furthermore, the use of vacuum blood collection tubes described herein facilitates the accurate determination of drug levels and / or the immunogenicity assessment of therapeutic molecules that resist the activated form of proteins in the contact system (e.g., plasma kallikrein, FXIIa, and 2-chain kallikreinogen). Tubes offer similar advantages for therapeutic molecules that target downstream activating proteins of the contact system without requiring calcium to produce activated targets (e.g., FXIIa and FIXa). The advantages of these tubes are primarily applicable to biological therapeutic molecules, as PK and the immunogenicity assays used are typically immunoassays that recognize binding sites (e.g., individual genotype in the case of therapeutic antibodies). If the therapeutic target is activated in vitro, it can bind to biologic components present in plasma, thus interfering with detection in PK and immunogenicity immunoassays. Protease inhibitors in the tubes prevent target activation. The use of liquid formulations prevents hemolysis, which can interfere with certain laboratory assays.

[0032] Vacuum blood collection tubes containing a mixture of protease inhibitors in a liquid formulation

[0033] Vacuum blood collection tubes are commonly used in medical practice for collecting blood samples for various purposes. The tubes described herein can be non-glass tubes containing a liquid formulation comprising a mixture of protease inhibitors (protease inhibitor mixture). In some embodiments, the protease inhibitor mixture may contain 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 a protease inhibitor mixture may contain multiple (e.g., 2, 3, 4, or 5) serine protease inhibitors, at least one of which may be a trypsin or human plasmin inhibitor. Preferably, the protease inhibitor mixture described herein is substantially free of protease inhibitors unstable in aqueous solution, i.e., the activity of protease inhibitors unstable in aqueous solution is very small relative to the total inhibitory activity of the protease mixture. In some cases, the amount of protease inhibitors unstable in aqueous solution may be less than 5% (w / w) of the total protease inhibitors in the mixture, for example, less than 2%, less than 1%, or less than 0.5%. In some cases, the protease inhibitor mixture is completely free of protease inhibitors unstable in aqueous solution (e.g., aqueous solution with pH 4-6). An example of a protease inhibitor that is unstable in aqueous solution is PPACK II, also known as HD-Phe-Phe-Arg-chloromethyl ketone.

[0034] Table 1 below lists exemplary serine protease inhibitors, cysteine ​​protease inhibitors, and trypsin protease inhibitors that can be used to prepare the protease inhibitor mixtures described herein.

[0035]

[0036]

[0037]

[0038] In some embodiments, the protease inhibitor mixture for manufacturing vacuum blood collection tubes comprises at least one serine protease inhibitor (e.g., 1, 2, or 3) and at least one cysteine ​​protease inhibitor (e.g., 1, 2, or 3), wherein the serine protease inhibitor may include at least one trypsin / plasmin inhibitor (e.g., 1, 2, or 3). Such a protease inhibitor mixture may comprise three serine protease inhibitors (e.g., benzalkonium chloride, AEBSF, and trypsin / plasmin inhibitors, such as soybean trypsin inhibitor) and one cysteine ​​protease inhibitor (e.g., leucopeptide).

[0039] In other embodiments, the protease inhibitor mixture may comprise at least one serine protease inhibitor (e.g., a plasma kallikrein inhibitor) and at least one cysteine ​​protease inhibitor (e.g., leucopeptide). The plasma kallikrein inhibitor may be EPI-KAL2 (Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp GlyPro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys GluGlu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu GluCys Lys Lys Met Cys Thr Arg Asp; SEQ ID NO: 1), which is a specific plasma kallikrein recombinant protease inhibitor that enables the tube to contain reagents that allow for the detection of activated plasma kallikrein using, for example, immunoassays.

[0040] Any mixture of protease inhibitors is soluble in a suitable solution to form a liquid formulation. The suitable solution may be an acid-citrate-dextrose solution, which may contain trisodium citrate, citric acid, and dextrose. The pH of the solution may be approximately 4-6, 4-5, 4.5-5.0, or 4.2-4.7, for example, 4.5. In some embodiments, the pH of the solution is 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 pH of the solution is approximately 4.5. The liquid formulation may further contain a cationic polymer, such as hexadimethrine bromide molecules (polybrene). ® It can reduce the activation of the contact system by interacting with negatively charged surfaces and chelating agents (e.g., EDTA), which can inhibit metalloproteinases.

[0041] The concentration of each protease inhibitor in the mixture can be 5 or 10 times the final concentration of that inhibitor used to inhibit the corresponding protease, depending on the actual dilution factor. Specific final concentrations of commercially available protease inhibitors are known in the art and are available from the manufacturer's formulations. In some embodiments, the concentration range of EPI-KAL2 can be 5-15 μM (e.g., 5-10, 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 embodiments, the concentration range of leucopeptide can be 200-300 μM (e.g., 200-250, 240-270, or 250-300 μM). In some embodiments, the concentration of leucopeptide is about 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or about 300 μM. In some embodiments, the concentration range of soybean trypsin inhibitor may be 1-3 mg / mL (e.g., 1-2 or 2-3 mg / mL). In some embodiments, the concentration of soybean trypsin inhibitor 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 embodiments, the concentration range of benzalkonium chloride may be 80-120 mM (e.g., 80-100 or 100-120 mM). In some embodiments, the concentration of benzalkonium chloride is about 80, 85, 90, 95, 100, 105, 110, 115, or about 120 mM. In some embodiments, the concentration range of AEBSF can be 10-30 mM (e.g., 10-20 or 20-30 mM). In some embodiments, the concentration of AEBSF 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.

[0042] When using peptide-based protease inhibitors (e.g., EPI-KAL2), they can be biotinylated using standard methods. For example, peptide inhibitors can be biotinylated as follows: In short, the peptide inhibitor can be dissolved in a suitable solution, such as phosphate-buffered saline (PBS). Freshly prepared Sulfo-NHS-LC-biotin can be added to the peptide inhibitor solution and incubated on ice for an appropriate time. Excess unreacted, hydrolyzed biotin can be removed using a spin-desalting column. The labeling of the peptide inhibitor can be confirmed by ELISA, and the protein concentration can be determined using, for example, a Bradford assay.

[0043] Any of the liquid formulations described herein can be prepared by conventional methods, such as dissolving the appropriate components in a suitable solution and placing them in a vacuum blood collection tube, which is preferably non-glass. The tube can be stored at -20°C and can be thawed on ice or at a refrigerated temperature (e.g., about 4°C), such as in a refrigerator, for an appropriate period of time before use.

[0044] Applications of vacuum blood collection tubes containing a mixture of liquid protease inhibitors

[0045] Any vacuum blood collection tube described herein may be used to collect blood samples from subjects for analysis of endogenous characteristics associated with the contact system, including but not limited to: the level of contact system activation, serum levels of drugs targeting components of the contact system, and / or the immunogenicity of such drugs. To reduce ex vivo activation of the contact system (e.g., due to local trauma following needle puncture), the vacuum blood collection tube described herein may not be the first tube containing blood when drawing blood from a subject. For example, initial blood from the subject may be collected in a disposable first tube, and subsequent blood samples may be collected using a vacuum blood collection tube for analysis. The first tube may be a conventional blood collection tube used in routine practice.

[0046] Following blood collection, the blood sample can be processed to produce a plasma sample within an appropriate timeframe (e.g., no more than one hour). The plasma sample can then be further analyzed to assess characteristics related to the contact system from which the subject (from whom the initial blood sample was obtained) was derived.

[0047] Blood samples may be collected from subjects who require the analyses described herein. In some cases, the subject is a human patient who may have a systemic exposure disorder (HAE), be suspected of having a systemic exposure disorder, or be at risk of having a systemic exposure disorder. For example, a human patient may have previously experienced a HAE or may be at risk of having a HAE. A human patient may have type I or type II HAE, which is characterized by a lack of C1-INH or the production of atypical C1-INH. Alternatively, a human patient may have type III HAE that is not associated with a C1-INH deficiency. In other embodiments, a human patient may have previously experienced idiopathic angioedema or may be at risk of having idiopathic angioedema. Such a human patient may have previously been treated with a drug that targets a component of the systemic exposure disorder (e.g., pKal or FXIIa or high molecular weight kininogen) or is currently being treated with a drug that targets a component of the systemic exposure disorder (e.g., pKal or FXIIa or high molecular weight kininogen).

[0048] i. Assess the intrinsic level of activation of the contact system.

[0049] In one aspect, plasma samples described herein may be analyzed to assess endogenous levels of exposure system activation in subjects who may have an exposure system-related disease (e.g., HAE or idiopathic angioedema), are suspected of having an exposure system-related disease (e.g., HAE or idiopathic angioedema), or are at risk of an exposure system-related disease (e.g., HAE or idiopathic angioedema). Such subjects may be receiving treatment for their disease, such as treatment involving pKal inhibitors (e.g., anti-pKal antibodies). In other cases, such subjects may not be receiving such treatment. Alternatively, subjects may be healthy subjects without the disease.

[0050] The level of contact system activation in plasma samples can be determined by measuring one or more biomarkers that indicate contact system activation.

[0051] Plasma kallikrein (pKal) is the primary circulating enzyme that produces bradykinin. pKal can be activated via the contact system or via factor XIIa, both of which are associated with the pathology of hereditary angioedema (HAE). Plasma kallikrein circulates as an inactive proenzyme called prokallikrein, which typically binds its substrate, high molecular weight kininogen (HMWK). In response to a stimulus, prokallikrein is cleaved to form active plasma kallikrein. This activation of kallikrein can be mediated, for example, by factor XIIa following activation of FXII to FXIIa, or by effectors in the contact cascade. Approximately 75–90% of circulating prokallikrein binds to HMWK by interacting with the inactive site of domain 6 of HMWK, which hydrolyzes additional molecules of HMWK to produce 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. The other cleavage product (cleaved kininogen) contains an amino acid chain linked together by disulfide bonds. (Cugno et al., Blood (1997) 89:3213-3218.)

[0052] Exemplary biomarkers that can be used to assess the level of contact system activation in a patient's blood sample (thus determining whether the patient has elevated levels of contact system and / or contact system activity, such as elevated levels of pKal or pKal activity) are provided in Table 2 below:

[0053] Table 2. Biomarkers of the contact system

[0054]

[0055] One or more biomarkers indicating activation of the contact system can be analyzed using conventional methods. A particularly suitable type of assay for qualitative, semi-quantitative, or quantitative detection is an immunoassay. An immunoassay is any assay in which a target molecule (e.g., a biomarker molecule associated with activation of the contact system) is detected and / or quantified by using a binding agent that specifically binds to the target molecule as described herein. The binding agent can be an antibody, which can be a full-length antibody or its antigen-binding fragment. Immunoassays can be competitive or non-competitive and can be homogeneous or heterogeneous. For example, immunoassays for detecting contact system biomarkers can be enzyme immunoassays (EIA), radioimmunoassays (RIA), fluorescence immunoassays (FIA), chemiluminescent immunoassays (CLIA), counting immunoassays (CIA), immunoenzyme assays (IEMA), enzyme-linked immunosorbent assays (ELISA), lateral flow immunoassays, sandwich immunoassays, immuno-PCR assays, adjacent linkage assays, Western blot assays, or immunoprecipitation assays. Other suitable immunoassays for detecting the biomarkers provided herein will be apparent to those skilled in the art. However, it will be apparent to those skilled in the art that this disclosure is not limited to immunoassays, and that detection assays (such as mass spectrometry) not based on antibodies or antibody fragments binding to antigens can also be used for the detection and / or quantification of contact system biomarkers as provided herein.

[0056] The type of assay used to detect and / or quantify contact system biomarkers (such as those provided herein) will depend on the specific application of the assay (e.g., clinical or research application), and on the type and number of biomarkers to be detected, as well as the type and number of patient samples to be analyzed in parallel, such as parameters. For example, using Western blotting assays, elevated levels of cleaved kininogen (2-chain kininogen) can be detected in plasma samples collected from HAE patients or healthy subjects during an acute HAE episode. While Western blotting assays allow for the simultaneous analysis of contact system biomarkers in multiple samples, the number of biomarkers that can be evaluated in parallel is limited. Therefore, in some embodiments analyzing the multiple contact system biomarkers provided herein in a single or multiple samples, assays suitable for this type of multiplex analysis are preferred. Examples of such assays include, but are not limited to, peptide microarray and lab-on-a-chip assays, which have been designed to provide a high-throughput, multiplex-ready alternative to less scalable immunoassays such as Western blotting.

[0057] In some embodiments, plasma samples may be placed in porous microplates with or without pKal inhibitors and / or contact system activators. In the presence of a pKal-labeled peptide substrate, the mixture may be incubated on ice for an appropriate period (e.g., 2 minutes), and a corn trypsin inhibitor (CTI) may be added to the mixture to terminate the activation reaction. If necessary, the mixture may be diluted, and proteolytic activity may be determined by measuring the level of the fluorescent peptide substrate. Results obtained from such assays can be used to determine the endogenous level of contact system activation in the subject from whom the plasma sample was obtained. If used, they can also be used to determine the inhibitory activity of pKal inhibitors.

[0058] ii. Assess the endogenous levels of the drug in the targeted contact system.

[0059] Another aspect of this disclosure relates to the use of the vacuum blood collection tubes described herein to determine the level of a drug in a targeted contact system. Drug levels are required to assess pharmacokinetic parameters. For example, if the contact system is activated in vitro in a plasma sample collected to determine the amount of a plasma kallikrein inhibitor (e.g., DX-2930) in plasma, excessive activation of the plasma kallikrein can bind the drug, thus hindering its detection in the assay. Any vacuum blood collection tube described herein can be used (e.g., in samples collected from a subject) to more accurately assess drug levels.

[0060] To implement this method, a plasma sample derived from a blood sample collected in a vacuum blood collection tube as described herein can be prepared from a blood sample belonging to a subject (e.g., a human patient) receiving drug treatment with a component of a targeted contact system (e.g., pKal). Drug levels in the plasma sample can be measured according to standard practice. In some cases, drug levels can be measured by immunoassays, such as those described herein.

[0061] iii. Evaluate the immunogenicity of drugs targeting contact systems.

[0062] Another aspect of this disclosure relates to the use of vacuum blood collection tubes for determining the immunogenicity of bioinhibitors against components of the contact system (e.g., pKal). For example, in cases where excessive drug is present in circulating plasma, it is routine practice to develop an immunogenicity assay capable of measuring antibodies against the drug (“ADA”). This requirement for an immunogenicity assay capable of measuring ADA is indeed present for therapeutic monoclonal antibodies that can have a multi-week half-life and high drug levels in circulation. To overcome interference from excessive drug in the sample, techniques are implemented to separate the antidrug antibody from the drug. Such antidrug antibodies can be separated by solid-phase extraction and acid dissociation (SPEAD), which involves incubating the biotinylated form of the drug with the plasma sample for an extended period (typically overnight), followed by separation of the biotinylated drug that binds to the antidrug antibody using a strepto-antibiotin-coated plate. The plate is then acid-treated to release the antidrug antibody. The released antibody can be directly coated onto another assay plate for detection. In the absence of protease inhibitors in the collection tube, the contact system can be in vitro activated, leading to the production of active plasma kinin-releasing enzymes. Following the acid release and recoating steps described above, both the active pKal and the antidrug antibody bind to the surface of the plate. Antidrug antibodies are typically detected using labeled drugs, which can also bind to the active pKal (if present), leading to false positive signals in ADA assays.

[0063] The use of vacuum blood collection tubes containing a mixture of protease inhibitors, as described in this article, can prevent such false positive signals.

[0064] General technology

[0065] Unless otherwise indicated, the implementation of this invention will employ conventional techniques within the scope of the art, including molecular biology (including recombinant technology), microbiology, cell biology, biochemistry, and immunology. These techniques are well illustrated in the following literature, such as: *Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989)*, Cold Spring Harbor Press; *Oligonucleotide Synthesis* (edited by MJ Gait, 1984); *Methods in Molecular Biology*, Humana Press; *Cell Biology: A Laboratory Notebook* (edited by JE Cellis, 1998) Academic Press; *Animal Cell Culture* (edited by RIFreshney, 1987); *Introduction to Cell and Tissue Culture* (JP Mather and PE Roberts, 1998) Plenum Press; *Cell and Tissue Culture: Laboratory Procedures* (edited by A. Doyle, JB Griffiths, and DG Newell, 1993-8) J. Wiley and Sons; *Methods in Enzymology* (Academic Press, Inc.); *Handbook of Experimental Immunology* (edited by DM Weir and CC Blackwell); *Gene Transfer Vectors for Mammalian*. Cells (edited by JM Miller and MP Calos, 1987); Current Protocols in Molecular Biology (edited by FM Ausubel et al., 1987); PCR: The Polymerase Chain Reaction (edited by Mullis et al., 1994); Current Protocols in Immunology (JEColigan et al., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach approach (edited by P. Shepherd and C. Dean, Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999)); The Antibodies (edited by M. Zanetti and JD Capra, Harwood Academic Publishers, 1995).

[0066] Further detailed explanation is unnecessary, and those skilled in the art will be able to utilize the invention to its fullest extent based on the foregoing description. Therefore, the specific embodiments described below should be interpreted merely as exemplary and in no way limit the remainder of this disclosure. All publications cited herein for the purposes or subject matter mentioned herein are incorporated herein by reference.

[0067] Example 1 Preparation of a mixture of protease inhibitors to prevent activation of the contact system

[0068] A mixture of protease inhibitors was developed to prevent activation by the contact system. Vacuum-sealed plastic tubing was used to standardize and simplify blood collection.

[0069] To prevent hydrolysis, a mixture of the following two protease inhibitors is used in liquid formulations:

[0070] 1) 10X Protease Inhibitor Mixture A: SCAT169

[0071] Vacuum 5 mL total volume plastic tube containing (0.5 mL): 100 mM benzylamidin, 400 µg / mL polybrene, 2 mg / mL soybean trypsin inhibitor, 20 mM EDTA, 263 µM leucopeptide 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).

[0072] 2) 10X Protease Inhibitor Mixture B: SCAT153

[0073] Vacuum-sealed 5 ml total volume plastic tube containing (0.5 ml): 10 µM biotinylated EPI-KAL2, 400 µg / mL polygluconine, 20 mM EDTA and 263 µM leucinogen, dissolved in acid-citrate-dextrose (100 mM trisodium citrate, 67 mM citric acid and 2% dextrose, pH 4.5).

[0074] Biotinylated EPI-KAL2 is contained in protease inhibitor mixture B (SCAT153). Store SCAT169 (dedicated coagulation assay tube, formulation 169) and SCAT153 (dedicated coagulation assay tube, formulation 153) tubes at 2–8°C.

[0075] Example 2 SCAT169 and SCAT153 tubes prevent activation of the contact system, as demonstrated by 2-strand protein blotting assays.

[0076] Plasma collected from SCAT169 or SCAT153 tubes blocked contact system activation induced by in vitro addition of ellagic acid, a well-known contact system activator. Figure 1 ), such as as measured by Western blot analysis via the conversion of 1-chain to 2-chain HMWK.

[0077] Ellagic acid-induced activation of the contact system was observed in comparisons of plasma samples collected from three different blood collection tubes: sodium citrate tubes (standard tubes used in clinical chemistry laboratories for coagulation measurements), SCAT169 tubes, and SCAT153 tubes.

[0078] In the ellagic acid-activated sample in a sodium citrate tube, the 1-chain HMWK was almost completely consumed, and the 2-chain HMWK was detected.

[0079] In contrast, 1-chain HMWK was retained in ellagic acid-activated plasma from SCAT169 and SCAT153 tubes.

[0080] These results provide evidence that SCAT169 and SCAT153 tubes effectively prevent ex vivo contact system activation that may occur during plasma sample collection and processing.

[0081] Example 3 A mixture of SCAT169 and SCAT153 protease inhibitors prolongs plasma clotting time.

[0082] For three separate donor samples, plasma clotting time was measured in samples processed in three different blood collection tubes: sodium citrate tubes, SCAT169 tubes, and SCAT153 tubes (Table 1).

[0083] As demonstrated by thrombin and activated partial prothrombin kinase, clotting time was increased in samples of SCAT159 and SCAT153 compared to sodium citrate. The results are presented in Table 3 below.

[0084] Table 3. Effects of protease inhibitors on plasma clotting time

[0085]

[0086] Example 4 Use of vacuum blood collection tubes to assess the intrinsic level of contact system activation in human subjects.

[0087] The detection of plasma biomarkers activated by contact systems faces challenges due to unintentional activation during blood collection and processing. In this study, the use of dedicated blood collection tubes (SCAT159 and SCAT153) in assessing cleaved high molecular weight kininogen (cHMWK) levels in the plasma of healthy subjects and those with type I / II hereditary angioedema (HAE), idiopathic angioedema, or HAE with normal C1-INH (HAEnC1, also known as nC1-INH) was investigated.

[0088] To avoid artificial activation of the contact pathway during blood sampling, this study used standardized blood collection techniques and custom-made tubes containing protease inhibitors. Blood samples were collected from healthy subjects and the aforementioned diseased subjects (during disease quiescence and flare-ups) to assess the percentage of cHMWK using Western blotting. Blood samples were placed into SCAT159 or SCAT153 tubes (5 mL total volume, 0.5 mL 10X protease inhibitor mixture) using catheters with a butterfly needle system. The blood samples were then processed to produce plasma samples within 1 hour of blood collection.

[0089] The level of cleaved kininogen (2-chain kininogen) in plasma samples was determined by analyzing SCAT tube plasma samples using methods disclosed, for example, in WO 2015 / 061183, by simple immunoblotting (SBHD) and protein blotting (TGA).

[0090] Plasma is collected from clinical sites 1-5 of healthy subjects into plastic collection tubes containing various anticoagulants, protease inhibitors, or protein stabilizers, and then processed into plasma. Specifically, the tube types include K2EDTA, sodium citrate, SCAT169, or P100 (BD Biosciences). Figure 2 The study revealed that tubes containing protease inhibitors were found to minimize activation of the ex vivo contact system. These were P100 or SCAT169 tubes, 5 mL plastic vacuum blood collection tubes containing 0.5 mL of a 10X concentrated mixture of the following components: 100 mM benzalkonium chloride, 400 µg / mL polybrene, 2 mg / mL soybean trypsin inhibitor, 20 mM EDTA, 263 µM leucopeptide, and 20 mM AEBSF, dissolved in acid-citrate-dextrose (100 mM trisodium citrate, 67 mM citric acid, and 2% dextrose, pH 4.5). When analyzing plasma from healthy volunteers, the collection method using tubes containing the protease inhibitor mixture prevented increases in cHMWK.

[0091] The results of this study showed that in healthy subjects, cHMWK levels remained stable (<5%) at room temperature (RT) for at least 24 hours after blood was collected into custom tubes. However, cHMWK levels were elevated (12%) in plasma samples obtained from commercial vendors, highlighting the importance of optimizing blood collection techniques when examining exposure pathway activation.

[0092] The role of kininogen cleavage in plasma collected from two different clinical sites (sites 4 and 5) in healthy subjects and from SCAT169 tubes collected from a commercial vendor (site 1) was also evaluated. Figure 3 Commercial vendors collect blood samples directly into SCAT 169 tubes instead of using initial discard tubes.

[0093] Kininogen cleavage was also evaluated in blood samples collected from subjects with various types of angioedema (type I / II HAE, nC1-INH HAE, and idiopathic AE) into SCAT169 tubes. Plasma was collected from healthy subjects or from subjects with various types of angioedema at rest (basal) and during exacerbations (episodes) to measure levels of cHMWK in various disease states. Compared with healthy controls (n=26), the percentage of cHMWK was significantly elevated at baseline in subjects with HAE (n=21), but not significantly elevated in plasma from subjects with idiopathic angioedema (n=4) or HAEnC1 (n=5). Figure 4 The results indicate limited plasma kallikrein activity, but the role of exposure pathway activation during acute exacerbations of these conditions cannot be ruled out.

[0094] Plasma samples evaluated for the detection of cHMWK are susceptible to contact system activation by the collection method and tube stimulation. In summary, this study provides an improved method for collecting plasma samples to assess contact system activation, which avoids ex vivo cleavage of HMWK.

[0095] Example 5: Use of vacuum blood collection tubes to assess the intrinsic level of contact system activation in human subjects

[0096] The use of specialized blood collection tubes (SCAT159 and SCAT153) in assessing the levels of cleaved high molecular weight kininogen (cHMWK) in the plasma of healthy subjects and those with type I / II hereditary angioedema (HAE).

[0097] In short, blood samples were collected from healthy subjects and from patients with HAE during disease quiescence (basal) and flare-ups (episodes). The percentage of 2-chain HMWK in plasma was determined using Western blotting. Plasma samples were collected from randomized patients with type I / II HAE in a phase 1b, multicenter, double-blind study who received two subcutaneous doses of anti-pKal antibody (DX-2930) or placebo at 30, 100, 300, or 400 mg on days 0 and 15 in closed groups. Blood samples were obtained on days 1, 8, 22, 64, 92, and 120, either before or after anti-pKal antibody (DX-2930) administration.

[0098] like Figure 5AThe study showed that the percentage of plasma 2-chain HMWK levels in samples collected from three different clinical sites (A, B, and C) from healthy subjects varied depending on the collection method and tube type used. Samples collected using sodium citrate tubes had higher levels of 2-chain HMWK compared to samples in SCAT169 tubes. Notably, samples collected directly into SCAT169 tubes and without using discard tubes before the tube containing the protease inhibitor mixture, collected at clinical site C, had higher levels of 2-chain HMWK compared to samples collected using discard tubes.

[0099] Similarly, such as Figure 5B The results showed that the percentage of plasma 2-chain HMWK levels in samples from HAE was higher in sodium citrate tubes compared to plasma collected in SCAT169 tubes, which may be due to exogenous activation of the contact system associated with plasma collection and processing.

[0100] This study demonstrates the advantages of using blood collection tubes containing a mixture of protease inhibitors, as described herein, and provides an improved method for collecting plasma samples to evaluate contact system activation, which avoids aberrant ex vivo contact system activation (e.g., as demonstrated by HMWK cleavage).

[0101] Example 6 Uses of vacuum blood collection tubes to assess plasma drug levels

[0102] Following standard practice, blood is drawn from HAE patients receiving DX-2930 treatment and healthy controls and placed into collection tubes. After the initial blood sample is placed into one or more collection tubes, subsequent blood samples (5 ml each) are placed into SCAT159 or SCAT153 tubes. The blood samples are then processed to produce plasma samples within one hour of collection.

[0103] The amount of DX-2930 in SCAT plasma samples was measured using a standard immunoassay (e.g., ELISA). Briefly, an anti-idiotype monoclonal antibody in the Fab form against DX-2930 was coated onto the surface of a 96-well plate and incubated overnight, followed by multiple washes to remove unbound anti-idiotype Fab molecules. The SCAT plasma sample was then added to the plate, and the plate was incubated at room temperature for 2–3 hours. The plate was washed several times, and an anti-idiotype monoclonal antibody in the biotinylated IgG form against DX-2930 was added, followed by incubation and washing. Horseradish peroxidase-conjugated streptavidin was then added to the plate. After incubation for 30 minutes, the plate was washed again, and the signal of dye release was examined. The signal intensity corresponds to the amount of DX-2930 in the plasma sample.

[0104] Example 7Applications of vacuum blood collection tubes in assessing drug immunogenicity

[0105] As described above, plasma samples from HAE patients receiving DX-2930 treatment were prepared from blood samples collected in SCAT159 or SCAT153 tubes. Anti-DX-2930 antibodies in the plasma samples were separated by solid-phase extraction and acid dissociation (SPEAD).

[0106] In short, plasma samples were incubated overnight with biotinylated DX-2930. The mixture was then placed into a strepto-biotin-coated plate to capture biotinylated DX-2930 bound to the plasma sample (if present). The anti-DX-2930 antibody was then released by acid treatment and directly coated onto a Meso Scale Discovery (MSD) plate. Ruthenium-labeled DX-2930 was added to the MSD plate, and the electrochemiluminescence signal was measured to detect the presence of the anti-DX-2930 antibody.

[0107] Other implementation methods

[0108] All features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification can be replaced by an alternative feature for the same, equivalent, or similar purpose. Therefore, unless otherwise expressly stated, each disclosed feature is merely an example of a general series of equivalent or similar features.

[0109] From the above description, those skilled in the art can readily identify the basic features of the present invention, and various changes and modifications can be made to adapt it to various uses and conditions without departing from the spirit and scope of the invention. Therefore, other embodiments are also within the scope of the claims.

[0110] Equivalence principle and scope

[0111] Using only conventional experiments, those skilled in the art will recognize or be able to identify many equivalent embodiments of the specific implementations of this disclosure described herein. The scope of this disclosure is not intended to be limited to the foregoing description, but rather as set forth in the appended claims.

[0112] In the claims, articles such as “a,” “an,” and “the” may refer to one or more, unless otherwise indicated or otherwise apparent from the context. Unless otherwise indicated or otherwise apparent from the context, a claim or description including “or” among one or more members of the group is considered satisfied if one, more than one, or all of the group members appear, employ, or otherwise relate to the given product or method. This disclosure includes embodiments in which an exact member of the group appears, employs, or otherwise relates to the given product or method. This disclosure includes embodiments in which more than one or all of the group members appear, employ, or otherwise relate to the given product or method.

[0113] Furthermore, this disclosure includes all variations, combinations, and arrangements in which one or more limitations, elements, clauses, and descriptive terms from one or more of the enumerated claims are introduced into another claim. For example, any claim dependent on another claim may be modified to include one or more limitations appearing in any other claim dependent on the same basic claim. Where elements appear as a list, for example in Markush groups, individual subgroups of elements are also disclosed, and any element (one or more) may be removed from the group. Generally, it should be understood that certain embodiments of this disclosure or aspects of this disclosure, where referred to as including specific elements and / or features, consist of or are substantially composed of such elements and / or features. For simplicity, those embodiments are not explicitly illustrated herein. It is also noted that the terms “comprising” and “including” are intended to be open-ended and allow for the inclusion of additional elements or steps. Where a range is given, endpoints are included. Furthermore, unless otherwise indicated or otherwise apparent from the context and understood by one of ordinary skill in the art, values ​​expressed as ranges may assume any specific value or subrange within that range in different embodiments of this disclosure, up to one-tenth of the lower limit of that range, unless the context explicitly indicates otherwise.

[0114] This application references various published patents, disclosed patent applications, journal articles, and other publications, all of which are incorporated herein by reference. In the event of any conflict between any incorporated references and this description, this description shall prevail. Furthermore, any specific embodiments of this disclosure falling within the prior art may be expressly excluded from any one or more of the claims. Because such embodiments are considered to be known to one of ordinary skill in the art, they may be excluded, even if such exclusion is not expressly stated herein. Any specific embodiment of this disclosure may be excluded from any claim for any reason, regardless of when, in relation to the existence of prior art.

[0115] Using only conventional experiments, those skilled in the art will recognize or be able to identify many equivalent ways of the specific embodiments described herein. The scope of the embodiments described herein is not intended to be limited to the above description, but rather as set forth in the appended claims. Those skilled in the art will recognize that various changes and modifications can be made to this description without departing from the spirit and scope of this disclosure as defined in the following claims.

Claims

1. Use of a liquid formulation comprising a mixture of protease inhibitors in the preparation of a vacuum blood collection tube for evaluating the level of a drug in a targeted contact system for a subject, wherein the mixture of protease inhibitors comprises EPI-KAL2 and leucosterol, wherein the vacuum blood collection tube comprises a liquid formulation.

2. The use according to claim 1, wherein the mixture of protease inhibitors comprises 5-15 μM EPI-KAL2 and 200-300 μM leucopeptide.

3. The use according to claim 2, wherein the mixture of protease inhibitors comprises 10 μMEPI-KAL2 and 263 μM leucopeptide.

4. The use according to claim 2, wherein EPI-KAL2 is biotinylated.

5. The use according to claim 3, wherein EPI-KAL2 is biotinylated.

6. The use according to claim 1, wherein the vacuum blood collection tube is a non-glass tube.

7. The use according to claim 1, wherein the vacuum blood collection tube is a plastic tube.

8. The use according to claim 1, wherein the liquid formulation further comprises polybrene and EDTA.

9. The use according to claim 8, wherein the liquid formulation comprises 400 μg / mL polybrene and 20 mM EDTA.

10. The use according to claim 1, wherein the pH of the liquid formulation is 4-6.

11. The use according to claim 10, wherein the pH of the liquid formulation is 4.

5.

12. The use according to claim 1, wherein the vacuum blood collection tube contains 0.5 mL of liquid preparation.

13. The use according to claim 1, wherein the mixture of protease inhibitors is substantially free of protease inhibitors that are unstable in aqueous solution.

14. The use according to any one of claims 1-13, wherein the subject is a human subject.

15. The use according to claim 14, wherein the human subject is a human patient suffering from a disease related to the contact system.

16. The use according to claim 15, wherein the disease is hereditary angioedema (HAE) or idiopathic angioedema.

17. The use according to any one of claims 1-13, wherein the drug inhibits active plasma kallikrein.

18. The use according to claim 17, wherein the drug is an antibody that binds to active plasma kallikrein.

19. Use of a liquid formulation comprising a mixture of protease inhibitors in the preparation of a vacuum blood collection tube for evaluating the immunogenicity of a drug for a targeted contact system in a subject, wherein the mixture of protease inhibitors comprises EPI-KAL2 and leucopeptide, wherein the vacuum blood collection tube comprises the liquid formulation.

20. The use according to claim 19, wherein the mixture of protease inhibitors comprises 5-15 μM EPI-KAL2 and 200-300 μM leucopeptide.

21. The use according to claim 20, wherein the mixture of protease inhibitors comprises 10 μM EPI-KAL2 and 263 μM leucopeptide.

22. The use according to claim 20, wherein EPI-KAL2 is biotinylated.

23. The use according to claim 21, wherein EPI-KAL2 is biotinylated.

24. The use according to claim 19, wherein the vacuum blood collection tube is a non-glass tube.

25. The use according to claim 24, wherein the vacuum blood collection tube is a plastic tube.

26. The use according to claim 19, wherein the liquid formulation further comprises polybrene and EDTA.

27. The use according to claim 26, wherein the liquid formulation comprises 400 μg / mL polybrene and 20 mM EDTA.

28. The use according to claim 19, wherein the pH of the liquid formulation is 4-6.

29. The use according to claim 28, wherein the pH of the liquid formulation is 4.

5.

30. The use according to claim 19, wherein the vacuum blood collection tube comprises 0.5 mL of liquid preparation.

31. The use according to claim 19, wherein the mixture of protease inhibitors is substantially free of protease inhibitors that are unstable in aqueous solution.

32. The use according to any one of claims 19-31, wherein the subject is a human subject.

33. The use according to claim 32, wherein the human subject is a human patient suffering from a disease related to the contact system.

34. The use according to claim 33, wherein the disease is hereditary angioedema (HAE).

35. The use according to any one of claims 19-31, wherein the drug inhibits active plasma kallikrein.

36. The use according to claim 35, wherein the drug is an antibody that binds to active plasma kallikrein.

37. The use according to any one of claims 19-31, wherein the immunogenicity of the drug targeting the contact system is evaluated by isolating antibodies binding to the drug from a plasma sample.

38. The use according to claim 37, wherein the antibody is separated by solid-phase extraction and acid dissociation SPEAD assay.

39. Use of a liquid formulation comprising a mixture of protease inhibitors in the preparation of a vacuum blood collection tube for assessing endogenous levels of contact system activation in a subject, wherein the mixture of protease inhibitors comprises EPI-KAL2 and leucopeptide, wherein the vacuum blood collection tube comprises the liquid formulation.

40. The use according to claim 39, wherein the mixture of protease inhibitors comprises 5-15 μM EPI-KAL2 and 200-300 μM leucopeptide.

41. The use according to claim 40, wherein the mixture of protease inhibitors comprises 10 μM EPI-KAL2 and 263 μM leucopeptide.

42. The use according to claim 40, wherein EPI-KAL2 is biotinylated.

43. The use according to claim 41, wherein EPI-KAL2 is biotinylated.

44. The use according to claim 39, wherein the vacuum blood collection tube is a non-glass tube.

45. The use according to claim 44, wherein the vacuum blood collection tube is a plastic tube.

46. ​​The use according to claim 39, wherein the liquid formulation further comprises polybrene and EDTA.

47. The use according to claim 46, wherein the liquid formulation comprises 400 μg / mL polybrene and 20 mM EDTA.

48. The use according to claim 39, wherein the pH of the liquid formulation is 4-6.

49. The use according to claim 48, wherein the pH of the liquid formulation is 4.

5.

50. The use according to claim 39, wherein the vacuum blood collection tube comprises 0.5 mL of liquid preparation.

51. The use according to claim 39, wherein the mixture of protease inhibitors is substantially free of protease inhibitors that are unstable in aqueous solution.

52. The use according to any one of claims 39-51, wherein the subject is a human subject.

53. The use according to claim 52, wherein the human subject is a human patient suffering from a disease related to the contact system.

54. The use according to claim 53, wherein the disease is hereditary angioedema (HAE).

55. The use according to claim 39, wherein the intrinsic level of contact system activation in a subject is assessed by measuring the level of one or more biomarkers indicating contact system activation.

56. The use according to claim 55, wherein one or more biomarkers are selected from the group consisting of prokallikrein, active plasma kallikrein pKal, α2M-pKal complex, active factor XII, active factor XI, high molecular weight kininogen (HMWK), and bradykinin metabolites.

57. The use according to claim 56, wherein one or more biomarkers comprise cleaved HMWK and / or whole HMWK.

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