Method for testing the efficacy of Andexanet

Abstract

Providing a method for testing the efficacy of andexanet. [Solution] This disclosure relates to a kit and method for measuring the potency of an andexanet sample in neutralizing a factor Xa inhibitor and restoring the activity of wild-type factor Xa. This disclosure provides a method for determining the activity of an andexanet sample, comprising: mixing a test sample containing an andexanet with a mixture containing human factor Xa (HFXa) and a direct factor Xa (fXa) inhibitor, wherein the molar ratio of FXa to Xa inhibitor is 0.2:1 to 0.3:1; adding a chromogenic fXa substrate to the mixture, wherein the chromogenic fXa substrate can release a chromophore upon reaction with HFXa; detecting the amount of chromophore released; and calculating the activity of the andexanet sample in releasing HFXa from a direct fXa inhibitor from the amount of chromophore released.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 039,795, filed on June 16, 2020, which is incorporated herein by reference for all purposes.

Background Art

[0002] Andexanet alfa (Factor Xa [recombinant] Inactivated - zhzo) is a modified recombinant inactive form of Factor Xa that is specifically designed to bind to and capture Factor Xa inhibitors. Factor Xa inhibitors, such as apixaban, rivaroxaban, edoxaban, betrixaban, and enoxaparin, are commonly used anticoagulants. However, the use of such anticoagulants is associated with acute massive bleeding episodes. Andexanet alfa or andexanet is an effective antidote to prevent and stop such bleeding.

[0003] It is important to evaluate each batch of andexanet for its activity and potency prior to use.

Summary of the Invention

Means for Solving the Problems

[0004] The present disclosure provides compositions, kits, and methods for measuring the potency (activity) of andexanet alfa in test samples. Potency can be quantified based on the ability of a test sample to restore the activity of human wild - type fXa in a mixture with an fXa inhibitor. The activity of human fXa can be further calibrated with an fXa protein, such as one derived from bovine or human, for which an international standard has been established.

[0005] One embodiment of the present disclosure provides a method for determining the activity of an andexanet sample, comprising: mixing a test sample containing an andexanet with a mixture containing human factor Xa (HFXa) and a direct factor Xa (fXa) inhibitor, wherein the molar ratio of FXa to the Xa inhibitor is 0.2:1 to 0.3:1; adding a chromogenic fXa substrate to the mixture, wherein the chromogenic fXa substrate can release a chromophore upon reaction with HFXa; detecting the amount of released chromophore; and calculating the activity of the andexanet sample in releasing HFXa from the direct fXa inhibitor from the amount of released chromophore.

[0006] Another embodiment provides a method for determining the activity of an andexanet sample, comprising: mixing a test sample containing an andexanet with a mixture containing bovine factor Xa (BFXa) and a direct factor Xa (fXa) inhibitor, wherein the molar ratio of BFXa to the fXa inhibitor is 0.15:1 to 0.25:1; adding a chromogenic fXa substrate to the mixture, wherein the chromogenic fXa substrate can release a chromophore upon reaction with BFXa; detecting the amount of chromophore released; and calculating the activity of the andexanet sample in releasing BFXa from the direct fXa inhibitor from the amount of chromophore released.

[0007] Exemplary, non-limiting drawings are provided as embodiments of the present disclosure. [Brief explanation of the drawing]

[0008] [Figure 1]The enzymatic activity of human fXa was compared to that of bovine fXa (Hyphen BFXa) for cleavage of Spectrozyme-fXa substrates. Human fXa (○) or BFXa (□) was serially diluted in 1×TBS (pH 7.4, 0.1% BSA) at concentrations of 250, 125, 62.5, 31.25, 15.625, 7.8125, 3.90625, and 0 ng / mL. 100 μL of the serially diluted fXa was mixed with 50 μL of Spectrozyme-fXa (2 mM). The reaction was quenched by adding 50 μL of acetic acid after incubation at room temperature for 10 minutes. Based on the gradient (OD405 relative to concentration), bovine fXa exhibited approximately 31% lower activity than HFXa. [Figure 2A] Bovine fXa and human fXa were compared by matching enzyme concentrations in a direct efficacy assay. 2A. BFXa (1×) / vetrixaban (1×); 2B. BFXa (1×) / vetrixaban (1.5×); 2C. BFXa (1×) / vetrixaban (2×). For HFXa, BFXa, and betrixaban, the stock solutions were first prepared separately. FXa / vetrixaban mixtures were prepared by mixing different volumes of stock solutions of FXa and betrixaban to obtain HFXa / vetrixaban or BFXa / vetrixaban. FXa / vetrixaban mixture (50 μL) was added to the reaction mixture as described in the direct efficacy assay. The HFXa (1×) / vetrixaban (1×) mixture (○) was used as a control. BFXa / vetrixaban mixtures (□) were tested with different concentrations of betrixaban while maintaining the same concentration of BFXa (1×). Panel a): BFXa (1×) / vetrixaban (1×); Panel b: BFXa (1×) / vetrixaban (1.5×); Panel c): BFXa (1×) / vetrixaban (2×). [Figure 2B-C]Bovine fXa and human fXa were compared by matching enzyme concentrations in a direct efficacy assay. 2A. BFXa (1×) / vetrixaban (1×); 2B. BFXa (1×) / vetrixaban (1.5×); 2C. BFXa (1×) / vetrixaban (2×). For HFXa, BFXa, and betrixaban, the stock solutions were first prepared separately. FXa / vetrixaban mixtures were prepared by mixing different volumes of stock solutions of FXa and betrixaban to obtain HFXa / vetrixaban or BFXa / vetrixaban. FXa / vetrixaban mixture (50 μL) was added to the reaction mixture as described in the direct efficacy assay. The HFXa (1×) / vetrixaban (1×) mixture (○) was used as a control. BFXa / vetrixaban mixtures (□) were tested with different concentrations of betrixaban while maintaining the same concentration of BFXa (1×). Panel a): BFXa (1×) / vetrixaban (1×); Panel b: BFXa (1×) / vetrixaban (1.5×); Panel c): BFXa (1×) / vetrixaban (2×). [Figure 3A-B] The results of optimizing bovine FXa and betrixaban concentrations by matching enzyme activity are shown. 3A. BFXa (1.64×) / betrixaban (2×); 3B. BFXa (1.45×) / betrixaban (2×). Stock solutions for HFXa, BFXa, and betrixaban were first prepared separately. FXa / betrixaban mixtures were prepared by mixing different volumes of stock solutions of FXa and betrixaban to obtain HFXa / betrixaban or BFXa / betrixaban. FXa / betrixaban mixtures (50 μL) were added to the reaction mixture as described in the direct efficacy assay. HFXa (1×) / betrixaban (1×) mixture (○) was used as a control. BFXa / betrixaban mixtures (□) were tested with different concentrations of BFXa while maintaining the same concentration of betrixaban (2×). Panel a): BFXa (1.64×) / vetrixaban (2×); Panel b: BFXa (1.45×) / vetrixaban (2×). [Modes for carrying out the invention]

[0009] I. Definition All numerical specifications, such as pH, temperature, time, concentration, and molecular weight, including ranges, are approximate values ​​that change (+) or (-) in increments of 0.1. It should be understood that the term "approximately" is used with all numerical specifications, although this is not always explicitly stated. Furthermore, it should be understood that reagents described herein are merely illustrative, and their equivalents are known in the art, although this is not always explicitly indicated.

[0010] "Composition" means a combination of an active agent and another compound or composition that is inactive (e.g., a detectable agent or label) or active.

[0011] Factor Xa, or fXa, or fXa protein, is a serine protease in the blood coagulation pathway and is produced from inactive factor X (fX). The nucleotide sequence encoding human factor X ("fX") can be found in GenBank under acceptance number "NM_000504". Upon catalytic cleavage of the first 52 residues of the heavy chain, fX is activated to fXa (SEQ ID NO: 1, Table 1). FXa contains a light chain and a heavy chain (as shown in Table 1). The first 45 amino acid residues of the light chain (residues 1-45 in SEQ ID NO: 1) are called the Gla domain because it contains 11 post-translationally modified γ-carboxyglutamic acid (Gla) residues. It also contains a short (6 amino acid residues) aromatic stack sequence (residues 40-45 in SEQ ID NO: 1). Chymotrypsin digestion selectively removes 1-44 residues, resulting in Gla domain-less fXa. The serine protease catalytic domain of fXa is located in the C-terminal heavy chain. The heavy chain of fXa is highly homologous to other serine proteases such as thrombin, trypsin, and activated protein C.

[0012] "Natural fXa" or "wild-type fXa" refers to fXa that is naturally occurring in plasma or isolated in its original, unmodified form, possessing the biological activity to activate prothrombin and thereby promote blood clot formation. This term includes naturally occurring polypeptides isolated from tissue samples and recombinantly produced fXa. "Active fXa" refers to fXa that possesses the biological activity to activate prothrombin. "Active fXa" may be natural or modified fXa that retains coagulant activity.

[0013] As used herein, “fXa derivative” refers to a modified fXa protein having alterations or significant deletions of the Gla-domain (e.g., deletion of at least 50%, 60%, 70%, 80%, or 90% of amino acid residues 6–39 of the light chain) and alterations to the active site, such that the fXa derivative has reduced ability to assemble into the prothrombinase complex and reduced or no catalytic activity compared to the wild-type protein. Furthermore, like the wild-type protein, the fXa derivative can bind to and / or substantially neutralize fXa inhibitors. Examples of fXa derivatives are provided in U.S. Patent No. 8,153,590 and No. 8,268,783 and their biological equivalents.

[0014] The term "active site" refers to a portion of an enzyme or antibody that undergoes a chemical reaction. A "modified active site" is an active site that has been structurally modified to provide an active site with increased or decreased chemical reactivity or specificity. Examples of active sites include, but are not limited to, the catalytic domain of human factor X containing amino acid residues 235-488 and the catalytic domain of human factor Xa containing amino acid residues 195-448. An example of a modified active site is, but is not limited to, the catalytic domain of human factor Xa containing amino acid residues 195-448 of SEQ ID NO: 2, which has at least one amino acid substitution at position Arg306, Glu310, Arg347, Lys351, Lys414, or Arg424.

[0015] Examples of fXa derivatives include "Gla-domain-less fXa" or "des-Gla fXa," which refers to fXa or fXa derivatives that lack a Gla-domain and have other modifications in addition to the removal of the Gla-domain. Examples of Gla-domain-less fXa in the present invention include, but are not limited to, fXa derivatives lacking all or part of amino acid residues 1 to 39 (or 6 to 39) of SEQ ID NO: 1.

[0016] Another example of an fXa derivative is "Gla-deficient fXa," which refers to fXa or an fXa derivative with a reduced number of free side-chain γ-carboxyl groups in its Gla-domain. Like Gla-domain-less fXa, Gla-deficient fXa can also have other modifications. Examples of Gla-deficient fXa include uncarboxylated fXa, low-carboxylated fXa, and decarboxylated fXa. "Uncarboxylated fXa" or "decarboxylated fXa" refers to an fXa derivative that lacks γ-carboxyl groups in the γ-carboxyglutamic acid residue of the Gla-domain, such as fXa in which all of its Gla-domain γ-carboxyglutamic acid residues are replaced by different amino acids, or fXa in which all of its side-chain γ-carboxyls are removed or masked by means of amination, esterification, etc. With respect to recombinantly expressed proteins, uncarboxylated fXa is sometimes called non-carboxylated fXa. "Low-carboxylated fXa" refers to fXa derivatives in which the number of γ-carboxyl groups in the Gla domain is reduced compared to wild-type fXa, such as fXa in which one or more but not all of its Gla-domain γ-carboxyglutamic acid is replaced by one or more different amino acids, or fXa in which at least one but not all of its side-chain γ-carboxyl groups are removed or masked by means of amination and esterification.

[0017] In some embodiments, the fXa derivative has a deletion of at least amino acid residues 6-39 of the light chain of wild-type fXa (see the light chain of SEQ ID NO: 1). In some embodiments, the fXa derivative also has a deletion of the EGF1 domain (amino acids 46-84) and / or the EGF2 domain (amino acids 85-128 of the light chain). In some embodiments, the light chain of the fXa derivative contains at least amino acids 129-139 of the wild-type light chain (or 95-105 of SEQ ID NO: 3).

[0018] Sequence ID No. 2 (Table 2), also known as "andexanet alpha" or simply "andexanet," contains two mutations compared to wild-type fXa. The first mutation is the deletion of amino acid residues 6-39 in the Gla-domain of fX. The second mutation is the substitution of active site residue S379 with an Ala residue. These amino acid substitutions correspond to amino acid 296 of Sequence ID No. 1, respectively. Andexanet contains two chains, referred to as Sequence ID No. 3 (light chain) and No. 4 (heavy chain), respectively.

[0019] fXa derivatives also include those that involve mutations, post-translational modifications, and / or alterations to the protein during the manufacturing process. For example, in another embodiment, fXa derivatives with or without the Ser379 modification may include modifications to the His (to Ala) and / or Asp (to Ala / Asn) residues in the three catalytic residues, as well as deletion or modification of the Gla domain. These modifications result in fXa derivatives with reduced enzyme activity but that do not compete with fXa when assembling into the prothrombinase complex.

[0020] In some embodiments, the fXa derivative has a heavy chain with one or two O-linked glycosylations. In some embodiments, the heavy chain may have C-terminal shortening of amino acid residues or up to 13, 14, or 15 amino acid residues. In some embodiments, the light chain may have N-terminal shortening.

[0021] The present disclosure provides various biological equivalents of the disclosed sequences of fXa derivatives or, alternatively, polypeptides having a specific sequence identity to these fXa derivatives. In one aspect, such biological equivalents retain the structural features of these fXa derivatives, namely a modified active site or heavy chain and a deleted or modified Gla domain. In another aspect, such biological equivalents retain the functional features of these fXa derivatives, namely not competing with fXa upon assembly into the prothrombinase complex and having a reduced catalytic activity. In another aspect, such biological equivalents can bind to and / or substantially neutralize an fXa inhibitor.

[0022] [Table 1]

[0023] [Table 2]

[0024] The term "factor Xa inhibitor" or "inhibitor of factor Xa" refers to a compound that can directly or indirectly inhibit the activity of the coagulation factor Xa that catalyzes the conversion of prothrombin to thrombin in vitro and / or in vivo.

[0025] A "direct factor Xa inhibitor" or "direct fXa inhibitor" binds directly to fXa and inhibits its activity. Non-limiting examples include rivaroxaban (Xarelto), apixaban (Eliquis), betrixaban (Bevyxxa), darexaban (YM150), edoxaban (Lixiana), otamixaban, letaxaban (TAK-442), eliquixaban (PD0348292) and their pharmaceutically acceptable salts and combinations thereof.

[0026] An "indirect factor Xa inhibitor" or "indirect fXa inhibitor" inhibits fXa activity through one or more other factors. Non-exclusive examples of indirect factor Xa inhibitors include enoxaparin, fondaparinux, hydraparinux, biotinylated hydraparinux, fragmin, tinzaparin, low molecular weight heparin ("LMWH"), and combinations thereof.

[0027] II. Efficacy Assay Anticoagulants fulfill a market need for the treatment or prevention of unwanted thrombosis in patients prone to blood clot formation, such as those with coagulation disorders, those confined during periods of immobility, or those undergoing surgery. However, one of the major limitations of anticoagulant therapy is the risk of treatment-related bleeding and the limited ability to rapidly reverse anticoagulant activity in cases of overdose or when emergency surgery is required.

[0028] Andexanet Alpha (Andexanet) is a recombinant human factor Xa decoy protein that can reverse the inhibition of factor Xa, thereby serving as an effective antidote to factor Xa-based anticoagulant therapy. Andexanet is manufactured as a recombinant protein produced from cultured cells. It is important to measure the potency of each production batch before use.

[0029] The efficacy assay involves adding an andexanet sample to an fXa / inhibitor mixture in which fXa is at least partially inhibited by the inhibitor. Upon addition of the andexanet sample, the inhibitor is captured and releases all or part of the bound fXa, which can be measured by its ability to cleave the fXa-specific chromogenic substrate and release the chromophore.

[0030] Interestingly, it has been found that a suitable fXa / inhibitor mixture requires a much larger number of inhibitors than fXa. For example, when human fXa was used with a specific fXa inhibitor, betrixaban maleate, the preferred molar ratio was 0.25:1.

[0031] In certain situations, the efficacy of the measured andexanet is preferably expressed together with fXa calibrated to an international standard. Unfortunately, until recently, there was no suitable international standard for human fXa. For this purpose, Example 2 investigated the use of bovine fXa.

[0032] Interestingly, bovine fXa exhibited significantly lower chromogenic activity. This presented a challenge. If bovine fXa is matched to human fXa by mass or concentration, bovine fXa will have lower activity. On the other hand, if bovine fXa has matched activity, it will have an excess molar quantity compared to human fXa, thus affecting the fXa / inhibitor ratio and the degree of inhibition in the assay. Another surprising finding was that betrixaban was less potent in inhibiting bovine fXa than human fXa.

[0033] Based on these observations, the betrixaban concentration in the mixture was increased (1.5×, 2.0×) to reduce the background activity of BFXa in the absence of andexanet, while the same concentration was maintained for BFXa (1×), as shown in Figures 2(b-c). A doubling of the betrixaban concentration reduced the BFXa background to a level similar to that of HFXa in the absence of andexanet. However, the maximum absorbance for BFXa (1×) was lower than that of the control with HFXa (1×) / vetrixaban (1×) in the presence of the highest andexanet concentration.

[0034] The remarkable finding of this disclosure is that, despite differences in chromogenic activity and the ability to be inhibited by fXa inhibitors, the maximum and minimum absorbance, EC 50 Furthermore, equivalent profiles between bovine fXa and human fXa controls, when converted to the assay range, could be achieved by adjusting the bovine fXa concentration in the mixture. In some embodiments, the bovine fXa / inhibitor molar ratio may be around 0.18:1. In some embodiments, the bovine fXa concentration may be around 16.2 nM.

[0035] Accordingly, according to one embodiment of the present disclosure, a method is provided for demonstrating the activity of an andexanet sample. In some embodiments, the method involves mixing a test sample containing an andexanet with a mixture containing human factor Xa (HFXa) and a direct factor Xa (fXa) inhibitor. Next, a chromogenic fXa substrate is added to the mixture, and the chromogenic fXa substrate can release a chromophore upon reaction with HFXa. Once the amount of released chromophore is detected, the activity of the andexanet sample can be calculated in relation to the release of HFXa from the direct fXa inhibitor.

[0036] As used herein, "andexanet sample" refers to a sample containing one or more andexanet molecules. An andexanet sample may be a sample of a specific volume or mass obtained from a larger volume or mass prepared through pharmaceutical manufacturing or before clinical use.

[0037] In some embodiments, the molar ratio of fXa to fXa inhibitor in the mixture is 0.2:1 to 0.3:1. In some embodiments, the molar ratio of fXa to fXa inhibitor in the mixture is 0.1:1 to 0.5:1, 0.12:1 to 0.5:1, 0.14:1 to 0.5:1, 0.15:1 to 0.5:1, 0.16:1 to 0.5:1, 0.18:1 to 0.5:1, 0.2:1 to 0.5:1, 0.22:1 to 0.5:1, 0.24:1 to 0.5:1, 0.25:1 to 0.5:1, 0.26:1 to 0.5:1, 0.28:1 to 0. 0.5:1, 0.3:1~0.5:1, 0.1:1~0.48:1, 0.12:1~0.48:1, 0.14:1~0.48:1, 0.15:1~0.48:1, 0.16:1~0.48:1, 0.18:1~0.48:1, 0.2:1~0.48:1, 0.22:1~0.48:1, 0.24:1~0.48:1, 0.25:1~0.48:1, 0.26:1~0.48:1, 0.28:1~0.48:1, 0.3 :1~0.48:1, 0.1:1~0.46:1, 0.12:1~0.46:1, 0.14:1~0.46:1, 0.15:1~0.46:1, 0.16:1~0.46:1, 0.18:1~0.46:1, 0.2:1~0.46:1, 0.22:1~0.46:1, 0.24:1~0.46:1, 0.25:1~0.46:1, 0.26:1~0.46:1, 0.28:1~0.46:1, 0.3:1~0.46 :1, 0.1:1~0.45:1, 0.12:1~0.45:1, 0.14:1~0.45:1, 0.15:1~0.45:1, 0.16:1~0.45:1, 0.18:1~0.45:1, 0.2:1~0.45:1, 0.22:1~0.45:1, 0.24:1~0.45:1, 0.25:1~0.45:1, 0.26:1~0.45:1, 0.28:1~0.45:1 or 0.3:1~0.45:1.

[0038] In some embodiments, the molar ratio of fXa to fXa inhibitor in the mixture is 0.1:1~0.44:1, 0.12:1~0.44:1, 0.14:1~0.44:1, 0.15:1~0.44:1, 0.16:1~0.44:1, 0.18:1~0.44:1, 0.2:1~0.44:1, 0.22:1~0.44:1, 0.24:1~0.44:1, 0.25:1~0.44:1, 0.26:1~0.44 :1, 0.28:1~0.44:1, 0.3:1~0.44:1, 0.1:1~0.42:1, 0.12:1~0.42:1, 0.14:1~0.42:1, 0.15:1~0.42:1, 0.16:1~0.42:1, 0.18:1~0.42:1, 0.2:1~0.42:1, 0.22:1~0.42:1, 0.24:1~0.42:1, 0.25:1~0.42:1, 0.26:1~0.42:1, 0.28 :1~0.42:1, 0.3:1~0.42:1, 0.1:1~0.4:1, 0.12:1~0.4:1, 0.14:1~0.4:1, 0.15:1~0.4:1, 0.16:1~0.4:1, 0.18:1~0.4:1, 0.2:1~0.4:1, 0.22:1~0.4:1, 0.24:1~0.4:1, 0.25:1~0.4:1, 0.26:1~0.4:1, 0.28:1~0.4:1, 0.3:1~0.4 :1, 0.1:1~0.38:1, 0.12:1~0.38:1, 0.14:1~0.38:1, 0.15:1~0.38:1, 0.16:1~0.38:1, 0.18:1~0.38:1, 0.2:1~0.38:1, 0.22:1~0.38:1, 0.24:1~0.38:1, 0.25:1~0.38:1, 0.26:1~0.38:1, 0.28:1~0.38:1 or 0.3:1~0.38:1.

[0039] In some embodiments, the molar ratio of fXa to fXa inhibitor in the mixture is 0.1:1~0.36:1, 0.12:1~0.36:1, 0.14:1~0.36:1, 0.15:1~0.36:1, 0.16:1~0.36:1, 0.18:1~0.36:1, 0.2:1~0.36:1, 0.22:1~0.36:1, 0.24:1~0.36:1, 0.25:1~0.36:1, 0.26:1~0.36:1, 0 0.28:1~0.36:1, 0.3:1~0.36:1, 0.1:1~0.35:1, 0.12:1~0.35:1, 0.14:1~0.35:1, 0.15:1~0.35:1, 0.16:1~0.35:1, 0.18:1~0.35:1, 0.2:1~0.35:1, 0.22:1~0.35:1, 0.24:1~0.35:1, 0.25:1~0.35:1, 0.26:1~0.35:1, 0.28:1~0.35 :1, 0.3:1~0.35:1, 0.1:1~0.34:1, 0.12:1~0.34:1, 0.14:1~0.34:1, 0.15:1~0.34:1, 0.16:1~0.34:1, 0.18:1~0.34:1, 0.2:1~0.34:1, 0.22:1~0.34:1, 0.24:1~0.34:1, 0.25:1~0.34:1, 0.26:1~0.34:1, 0.28:1~0.34:1, 0.3:1~0 These are 0.34:1, 0.1:1~0.32:1, 0.12:1~0.32:1, 0.14:1~0.32:1, 0.15:1~0.32:1, 0.16:1~0.32:1, 0.18:1~0.32:1, 0.2:1~0.32:1, 0.22:1~0.32:1, 0.24:1~0.32:1, 0.25:1~0.32:1, 0.26:1~0.32:1, 0.28:1~0.32:1, or 0.3:1~0.32:1.

[0040] In some embodiments, the molar ratio of fXa to fXa inhibitor in the mixture is 0.1:1~0.3:1, 0.12:1~0.3:1, 0.14:1~0.3:1, 0.15:1~0.3:1, 0.16:1~0.3:1, 0.18:1~0.3:1, 0.2:1~0.3:1, 0.22:1~0.3:1, 0.24:1~0.3:1, 0.25:1~0.3:1, 0.26:1~0.3:1, 0.28:1~0.3:1, 0.1:1~0.28:1, 0.12:1~0.28:1, 0.14: 1~0.28:1, 0.15:1~0.28:1, 0.16:1~0.28:1, 0.18:1~0.28:1, 0.2:1~0.28:1, 0.22:1~0.28:1, 0.24:1~0.28:1, 0.25:1~0.28:1, 0.26:1~0.28:1, 0.1:1~0.26:1, 0.12:1~0.26:1, 0.14:1~0.26:1, 0.15:1~0.26:1, 0.16:1~0.26:1, 0.18:1~0.26:1, 0.2:1~0.26:1, 0.22:1~ 0.26:1, 0.24:1~0.26:1, 0.25:1~0.26:1, 0.1:1~0.25:1, 0.12:1~0.25:1, 0.14:1~0.25:1, 0.15:1~0.25:1, 0.16:1~0.25:1, 0.18:1~0.25:1, 0.2:1~0.25:1, 0.22:1~0.25:1, 0.24:1~0.25:1, 0.1:1~0.24:1, 0.12:1~0.24:1, 0.14:1~0.24:1, 0.15:1~0.24:1, 0.16:1~0. The ratios are 24:1, 0.18:1~0.24:1, 0.2:1~0.24:1, 0.22:1~0.24:1, 0.1:1~0.22:1, 0.12:1~0.22:1, 0.14:1~0.22:1, 0.15:1~0.22:1, 0.16:1~0.22:1, 0.18:1~0.22:1, 0.2:1~0.22:1, 0.1:1~0.2:1, 0.12:1~0.2:1, 0.14:1~0.2:1, 0.15:1~0.2:1, 0.16:1~0.2:1, or 0.18:1~0.2:1.

[0041] In some embodiments, the molar ratio of fXa to the fXa inhibitor in the mixture is, but is not limited, 0.21–0.29, 0.22–0.28, 0.23–0.27, 0.24–0.26, 0.245–0.255, 0.248–0.252, or about 0.25.

[0042] In some embodiments, the concentration of HFXa is about 8-14 nM after the test sample is added. In some embodiments, the concentration is 5-20 nM, 6-20 nM, 7-20 nM, 8-20 nM, 9-20 nM, 10-20 nM, 11-20 nM, 12-20 nM, 5-19 nM, 6-19 nM, 7-19 nM, 8-19 nM, 9-19 nM, 10-19 nM, 11-19 nM, 12-19 nM, 5-18 nM, 6-18 nM, 7-18 nM, 8~18nM, 9~18nM, 10~18nM, 11~18nM, 12~18nM, 5~17nM, 6~17nM, 7~17nM, 8~17nM, 9~17nM, 10~1 7nM, 11~17nM, 12~17nM, 5~16nM, 6~16nM, 7~16nM, 8~16nM, 9~16nM, 10~16nM, 11~16nM, 12~16nM , 5~15nM, 6~15nM, 7~15nM, 8~15nM, 9~15nM, 10~15nM, 11~15nM, 12~15nM, 5~14nM, 6~14nM, 7~1 4nM, 8~14nM, 9~14nM, 10~14nM, 11~14nM, 12~14nM, 5~13nM, 6~13nM, 7~13nM, 8~13nM, 9~13nM, 1 These ranges from 0 to 13 nM, 11 to 13 nM, 12 to 13 nM, 5 to 12 nM, 6 to 12 nM, 7 to 12 nM, 8 to 12 nM, 9 to 12 nM, 10 to 12 nM, 11 to 12 nM, 5 to 11 nM, 6 to 11 nM, 7 to 11 nM, 8 to 11 nM, 9 to 11 nM, 10 to 11 nM, 5 to 10 nM, 6 to 10 nM, 7 to 10 nM, 8 to 10 nM, or 9 to 10 nM.

[0043] Any of the direct fXa inhibitors may be used for the assay. In some embodiments, the direct fXa inhibitor is selected from the group consisting of betrixaban, apixaban, rivaroxaban, edoxaban, otamixaban, retaxaban, and eribaxaban. In some embodiments, the direct fXa inhibitor is betrixaban. In some embodiments, the direct fXa inhibitor is apixaban. In some embodiments, the direct fXa inhibitor is rivaroxaban.

[0044] In some embodiments, the method may be useful to further involve verifying the activity of the andexanet sample using a standard curve prepared with a reference andexanet sample.

[0045] Another embodiment of the present disclosure provides a method for determining the activity of an undexanet sample using an fXa protein for which an established international standard exists. In some embodiments, such fXa is bovine fXa (BFXa). In some embodiments, such fXa is human fXa (HFXa). Human fXa (HFXa) is known to have a molecular weight of about 46 kDa, and bovine fXa (BFXa) is known to have a molecular weight of about 45.3 kDa.

[0046] Therefore, in some embodiments, this method involves mixing a test sample containing andexanet with a mixture containing bovine factor Xa (BFXa) and a direct factor Xa (fXa) inhibitor. Subsequently, a chromogenic fXa substrate is added to the mixture, and the chromogenic fXa substrate can release a chromophore upon reaction with BFXa. The amount of released chromophore can be detected and used to calculate the activity of the andexanet sample when releasing BFXa from the direct fXa inhibitor.

[0047] In some embodiments, the molar ratio of BFXa to fXa inhibitor is approximately 0.15:1 to 0.25:1. In some embodiments, the molar ratio of BFXa to fXa inhibitor is approximately 0.1:1 to 0.5:1, 0.12:1 to 0.5:1, 0.14:1 to 0.5:1, 0.15:1 to 0.5:1, 0.16:1 to 0.5:1, 0.18:1 to 0.5:1, 0.2:1 to 0.5:1, 0.22:1 to 0.5:1, 0.24:1 to 0.5:1, 0.25:1 to 0.5:1, 0.1:1 to 0.48:1, and 0.12:1. ~0.48:1, 0.14:1~0.48:1, 0.15:1~0.48:1, 0.16:1~0.48:1, 0.18:1~0.48:1, 0.2:1~0.48:1, 0.22:1~0.48:1, 0.24:1~0.48:1, 0.25:1~0.48:1, 0.1:1~0.46:1, 0.12:1~0.46:1, 0.14:1~0.46:1, 0.15:1~0.46:1, 0.16: 1~0.46:1, 0.18:1~0.46:1, 0.2:1~0.46:1, 0.22:1~0.46:1, 0.24:1~0.46:1, 0.25:1~0.46:1, 0.1:1~0.45:1, 0.12:1~0.45:1, 0.14:1~0.45:1, 0.15:1~0.45:1, 0.16:1~0.45:1, 0.18:1~0.45:1, 0.2:1~0.45:1, 0.22: The ratios are 1-0.45:1, 0.24:1-0.45:1, 0.25:1-0.45:1, 0.1:1-0.44:1, 0.12:1-0.44:1, 0.14:1-0.44:1, 0.15:1-0.44:1, 0.16:1-0.44:1, 0.18:1-0.44:1, 0.2:1-0.44:1, 0.22:1-0.44:1, 0.24:1-0.44:1, or 0.25:1-0.44:1.

[0048] In some embodiments, the molar ratio of BFXa to fXa inhibitor is approximately 0.1:1~0.42:1, 0.12:1~0.42:1, 0.14:1~0.42:1, 0.15:1~0.42:1, 0.16:1~0.42:1, 0.18:1~0.42:1, 0.2:1~0.42:1, 0.22:1~0.42:1, 0.24:1~0.42:1, 0.25:1~0.42:1, 0.1:1~0.4:1, 0.12:1~0.4:1, 0.14:1~0.4:1, 0.15:1~0.4:1, 0.16:1~0.4:1, 0.18:1~0.4:1, 0.2 :1~0.4:1, 0.22:1~0.4:1, 0.24:1~0.4:1, 0.25:1~0.4:1, 0.1:1~0.38:1, 0.12:1~0.38:1, 0.14:1~0.38:1, 0.15:1~0.38:1, 0.16:1~0.38:1, 0.18:1~ 0.38:1, 0.2:1~0.38:1, 0.22:1~0.38:1, 0.24:1~0.38:1, 0.25:1~0.38:1, 0.1:1~0.36:1, 0.12:1~0.36:1, 0.14:1~0.36:1, 0.15:1~0.36:1, 0.16:1~0 0.36:1, 0.18:1~0.36:1, 0.2:1~0.36:1, 0.22:1~0.36:1, 0.24:1~0.36:1, 0.25:1~0.36:1, 0.1:1~0.35:1, 0.12:1~0.35:1, 0.14:1~0.35:1, 0.15:1~0 0.35:1, 0.16:1~0.35:1, 0.18:1~0.35:1, 0.2:1~0.35:1, 0.22:1~0.35:1, 0.24:1~0.35:1, 0.25:1~0.35:1, 0.1:1~0.34:1, 0.12:1~0.34:1, 0.14:1~0. 34:1, 0.15:1~0.34:1, 0.16:1~0.34:1, 0.18:1~0.34:1, 0.2:1~0.34:1, 0.22:1~0.34:1, 0.24:1~0.34:1, 0.25:1~0.34:1, 0.1:1~0.32:1, 0.12:1~0. 32:1, 0.14:1~0.32:1, 0.15:1~0.32:1, 0.16:1~0.32:1, 0.18:1~0.32:1, 0.2:1~0.32:1, 0.22:1~0.32:1, 0.24:1~0.32:1 or 0.25:1~0.32:1, 0.1:1~0.3:1, 0.12:1~0.3:1, 0.14:1~0.3:1, 0.15:1~0.3:1, 0.16:1~0.3:1, 0.18:1~0.3:1, 0.2:1~0.3:1, 0.22:1~0.3:1, 0.24:1~0.3:1, 0.25:1~0.3:1, 0.1:1~0.28:1, 0.12:1~0.28:1, 0.14:1~0.28:1, 0.15:1~0.28:1, 0.16:1~0.28:1, 0.18:1~0.28: 1, 0.2:1~0.28:1, 0.22:1~0.28:1, 0.24:1~0.28:1, 0.25:1~0.28:1, 0.1:1~0.26:1, 0.12:1~0.26:1, 0.14:1~0.26:1, 0.15:1~0.26:1, 0.16:1~0.26:1, 0.18:1~0.26:1, 0.2:1~0.26:1, 0.22:1~0.26:1, 0.24:1~0.26:1, 0.25:1~0.26:1, 0.1:1~0 0.25:1, 0.12:1~0.25:1, 0.14:1~0.25:1, 0.15:1~0.25:1, 0.16:1~0.25:1, 0.18:1~0.25:1, 0.2:1~0.25:1, 0.22:1~0.25:1, 0.24:1~0.25:1, 0.1:1~0.24:1, 0.12:1~0.24:1, 0.14:1~0.24:1, 0.15:1~0.24:1, 0.16:1~0.24:1, 0.18:1~0.24:1, 0 The ratios are 0.2:1~0.24:1, 0.22:1~0.24:1, 0.1:1~0.22:1, 0.12:1~0.22:1, 0.14:1~0.22:1, 0.15:1~0.22:1, 0.16:1~0.22:1, 0.18:1~0.22:1, 0.2:1~0.22:1, 0.1:1~0.2:1, 0.12:1~0.2:1, 0.14:1~0.2:1, 0.15:1~0.2:1, 0.16:1~0.2:1, and 0.18:1~0.2:1.

[0049] In some embodiments, the molar ratio of BFXa to the fXa inhibitor is approximately 0.15:1 to 0.25:1, 0.16:1 to 0.23:1, 0.16:1 to 0.23:1, 0.16:1 to 0.20:1, 0.16:1 to 0.19:1, 0.17:1 to 0.19:1, or approximately 0.18:1.

[0050] In some embodiments, the concentration of BFXa is about 10-20 nM after the test sample is added. In some embodiments, the concentration of BFXa is about 10-20 nM, 11-20 nM, 12-20 nM, 13-20 nM, 14-20 nM, 15-20 nM, 16-20 nM, 17-20 nM, 10-19 nM, 11-19 nM, 12-19 nM, 13-19 nM, 14-19 nM, 15-19 nM, 16-19 nM, 17-19 nM, 10-18 nM, 11-18 nM, 12-18 nM, 1 These ranges from 3 to 18 nM, 14 to 18 nM, 15 to 18 nM, 16 to 18 nM, 17 to 18 nM, 10 to 17 nM, 11 to 17 nM, 12 to 17 nM, 13 to 17 nM, 14 to 17 nM, 15 to 17 nM, 16 to 17 nM, 10 to 16 nM, 11 to 16 nM, 12 to 16 nM, 13 to 16 nM, 14 to 16 nM, 15 to 16 nM, 10 to 15 nM, 11 to 15 nM, 12 to 15 nM, 13 to 15 nM, or 14 to 15 nM.

[0051] In some embodiments, the concentration of BFXa is approximately 14–18 nM, 15–17 nM, or 15.5–16.5 nM after the test sample is added.

[0052] Any of the direct fXa inhibitors may be used for the assay. In some embodiments, the direct fXa inhibitor is selected from the group consisting of betrixaban, apixaban, rivaroxaban, edoxaban, otamixaban, retaxaban, and eribaxaban. In some embodiments, the direct fXa inhibitor is betrixaban. In some embodiments, the direct fXa inhibitor is apixaban. In some embodiments, the direct fXa inhibitor is rivaroxaban.

[0053] In some embodiments, the method may be useful to further involve verifying the activity of the andexanet sample using a standard curve prepared with a reference andexanet sample.

[0054] Another embodiment of this disclosure provides a method for determining the activity of an undexanet sample using an fXa protein for which an established international standard exists. In some embodiments, such fXa is bovine fXa (BFXa). Human fXa (HFXa) is known to have a molecular weight of about 46 kDa, and bovine fXa (BFXa) has a molecular weight of about 45.3 kDa.

[0055] Therefore, in some embodiments, this method involves mixing a test sample containing andexanet with a mixture containing bovine factor Xa (BFXa) and a direct factor Xa (fXa) inhibitor. Subsequently, a chromogenic fXa substrate is added to the mixture, and the chromogenic fXa substrate can release a chromophore upon reaction with BFXa. The amount of released chromophore can be detected and used to calculate the activity of the andexanet sample when releasing BFXa from the direct fXa inhibitor.

[0056] In some embodiments, the chromogenic fXa substrate is Spectrozyme-Xa.

[0057] In some embodiments, methods are provided to calibrate the activity of human fXa (HFXa) protein by testing a reference undexanet with both HFXa and bovine fXa (BFXa). The test results can then be used to express HFXa activity along with BFXa activity. [Examples]

[0058] This disclosure will be further understood by reference to the following embodiments, which are intended purely to illustrate this disclosure. This disclosure is not limited in scope by the embodiments described herein, but is intended only as an example of one aspect of this disclosure. Any functionally equivalent method is within the scope of this disclosure. In addition to those described herein, various modifications of this disclosure will be apparent to those skilled in the art from the above and the accompanying drawings. Such modifications are included within the scope of the accompanying claims.

[0059] Example 1. Testing the efficacy of the andexanet sample. This example describes an assay for determining the efficacy of an andexanet alpha sample in neutralizing a factor Xa inhibitor.

[0060] This assay measures the identity and potency of undexanet alpha based on its ability to bind to the direct fXa inhibitor, betrixaban maleate, and reverse the inhibition of human fXa in an assay mixture composed of human fXa and betrixaban. The recovered human fXa activity is measured with an fXa-specific chromogenic substrate that releases a chromophore upon cleavage by fXa. The potency of the test sample is determined by comparing the response of the test sample to the response obtained for the standard in a software program using a homogenization test, measured by the difference in the slope of a 4-parameter curve fit. The potency is calculated by the ratio of the EC50 of the sample to the reference.

[0061] The process is as follows: 1. Take out a fixed amount of human fXa and fXa inhibitor (e.g., betrixaban, stock solution 226 μg / mL) and thaw at room temperature. 2. Preparation of fXa standards (RS), assay controls, and test samples (TS) a) Prepare two dilutions of the standard and each 0.6 mg / mL test sample in assay buffer. Unless otherwise specified, use the concentration of the standard actually measured at release, and use the indicated (target) concentration of all test samples. For the 10.0 mg / mL final bulk, see the example below. Diluted solution of the example Required amount of TS = (0.6 mg / mL × 1000 μL) / 10.0 mg / mL = 60.0 μL Required amount of assay buffer = 1000 μL - 60 μL = 940 μL b) Serially diluted solutions of the standard (RS), assay control (CTRL), and test sample (TS) are prepared in a diluted microplate by performing 2.4-fold serial dilution. See below for the dilution scheme of the example.

[0062] [Table 3]

[0063] 3. Preparation of fXa and betrixaban (fXa inhibitor) a) Dilute fXa to 50 μg / mL, then further dilute it to 1.0 μg / mL in assay buffer. b) Dilute the fXa inhibitor (226 μg / mL betrixaban stock solution) to 40 ng / mL in the assay buffer as shown in the table below. c) Combine 6 mL of 1.0 μg / mL fXa and 6 mL of 40 ng / mL fXa inhibitor to add equivolume diluted fXa and fXa inhibitor (vetrixaban) to a suitable container. Mix the wells. 4. Preparation of fXa and fXa inhibitor controls a) Prepare an fXa control by mixing 100 μL of diluted fXa with 300 μL of assay buffer. b) Prepare an fXa inhibitor (vetrixaban) control by mixing 200 μL of the fXa inhibitor mixture with 200 μL of the assay buffer. Transfer 5.50 μL / well of the standard (RS), assay control (CTRL), and test sample (TS) to the assay plate. Add 6.50 μL of the human fXa / inhibitor mixture (prepared in step 3(c)) to all RS, CTRL, TS-1, and TS-2 wells. 7. Transfer 100 μL / well of the fXa control and fXa / inhibitor control to the assay plate. Add 100 μL of assay buffer to each blank well. 8. To mix, shake the plate for approximately 1 minute on an orbital plate shaker set to approximately 300 rpm. 9. Cover the plate with an adhesive plate sealer and incubate in the dark for 60 ± 5 minutes. 10. While the sample is incubating, thaw the Spectrozyme fXa substrate and prepare it at a concentration of 2.0 mM. Add 4.5 mL of assay buffer to 3.0 mL of a disposable, fixed volume of thawed Spectrozyme fXa substrate and mix the wells. Prepare this reagent immediately before use. This volume is sufficient for one assay plate. Add 11.50 μL of the prepared Spectrozyme FXa substrate to all wells, and immediately transfer the plate to a plate shaker set to approximately 300 rpm for about 1 minute. 12. Incubate the plate in the dark for 10 ± 1 minutes, then stop the reaction by adding 50 μL of stop solution. 13. Read the microplate at 405 nm as the test wavelength and 490 nm as the reference wavelength. 14. Calculate the efficacy of the test andexanet sample.

[0064] Example 2. Calibration of the effective unit The efficacy assay tested in Example 1 was developed based on the ability of andexanet to bind to a direct fXa inhibitor (betrixaban) in an assay mixture consisting of andexanet, HFXa, and betrixaban, and to reverse its inhibition of human fXa (HFXa) activity. The recovered HFXa activity is measured with an fXa-specific chromogenic substrate that releases a chromophore upon cleavage by fXa. The reversal activity of the test sample is compared to an andexanet standard to obtain specific activity (expressed as either % or mg / mg). The standard used in Example 1 was HFXa, but its efficacy could not be traced from an independent international standard because an international standard for HFXa (e.g., WHO) had not been established until recently.

[0065] This example describes the development of a direct potency unit assay using bovine BFXa (Hyphen-BioMed) instead of HFXa. BFXa activity was calibrated against the international standard National Institute for Biological Standard and Control (NIBSC) (UK) [NIBSC code: 75 / 595].

[0066] Materials and methods Human FXa (HFXa) was purchased from Haematologic Technologies, Inc. (Essex Junction, VT) (catalog number HCXA-0060). A 1.0 μg / mL 2× working stock was freshly prepared in Tris-buffered saline (1× TBS, pH 7.4, 0.1% BSA).

[0067] BFXa standards were purchased from Hyphen BioMed (France) (catalog numbers: BE1010 / BE101K). Each vial of Hyphen BFXa contained 50 μg of purified bovine FXa or 111 units / vial (i.e., 2.2 units / μg or 0.45 μg / unit) calibrated against NIBSC BFXa (75 / 595), which is not a WHO international standard. Each vial was reconstituted with 1 mL of H2O as recommended to obtain 50 μg / mL or 111 units / mL. Working stocks were prepared at 0.9 μg / mL (2.0 U / mL) in 1 × TBS (pH 7.4, 0.1% BSA).

[0068] The FXa inhibitor (vetrixaban) was provided as a 1 mM (452 ​​μg / mL, mw: 425 free bases) DMSO stock solution. A 2× working stock of 40 ng / mL was prepared in 1× TBS (pH 7.4, 0.1% BSA) and mixed with HFXa. When the betrixaban concentration in the mixture was varied, alternative working stocks of 400, 300, 200, and 40 ng / mL were prepared and mixed with BFXa.

[0069] The FXa substrate Spectrozyme-Xa substrate was purchased from Sekisui Diagnostics (Lexington, MA) (catalog number 222L). One vial of Spectrozyme-FXa (50 μmol / vial) was reconstituted with 10 mL of H2O to obtain a 5 mM stock solution. A 2 mM working stock was prepared in 1 × TBS (pH 7.4, 0.1% BSA).

[0070] Andexanet Alpha (50 mg / vial) was reconstituted with 4.7 mL of H2O as recommended, and the absorbance (A) was measured. 280 A protein concentration of 9.7 mg / mL was obtained by ). A small, fixed amount was stored at -80°C. A working stock was prepared at 0.6 mg / mL in 1×TBS (pH 7.4, 0.1% BSA), and this was used to prepare standards ranging from 0 to 600 μg / mL.

[0071] result Existing direct efficacy assays used for release and stability testing of andexanet samples are endpoint methods involving the mixing of equivolumes (50 μL) of each reagent. Human FXa and betrixaban (inhibitors) are first prepared as 2× stock solutions at 1.0 μg / mL (HFXa) and 40 ng / mL (betrixaban), respectively, and then combined in equivolumes (mixed in a 1:1 ratio) to obtain 0.5 μg / mL of HFXa and 20 ng / mL of betrixaban, respectively, in the mixture. Next, the HFXa / inhibitor mixture (50 μL) is added to the reaction mixture.

[0072] The specific activity Hyphen BFXa was calibrated against the manufacturer's NIBSC BFXa(75 / 595) standard. Reconstituted stock concentrations (50 μg / mL, 111 units / mL, 0.45 μg / unit) were used as working standards in the development of direct potency unit assays.

[0073] Comparison of color development activity between HFXa and Hyphen BFXa Under the same conditions, Hyphen BFXa exhibits approximately 31% lower chromogenic activity than HFXa based on μg / mL concentration (Figure 1). This observation indicates that the direct efficacy assay needs to be optimized for BFXa, as matching the mass or concentration with HFXa results in BFXa having lower activity, while matching the activity results in a molar excess of BFXa compared to HFXa, thus affecting the FXa / inhibitor ratio and degree of inhibition in the assay. In addition, betrixaban was found to have lower efficacy in inhibiting BFXa. Therefore, the assay conditions will require further optimization for the concentrations of both BFXa and betrixaban (inhibitor).

[0074] Comparison of HFXa and Hyphen BFXa in direct efficacy assays For the initial setup of the direct potency unit assay, the same concentrations of BFXa and HFXa as those used in the direct potency assay were employed. For the HFXa mixture, a 2× stock solution was prepared with 1.0 μg / mL of HFXa and 40 ng / mL of betrixaban. A control mixture of HFXa (1×) / vetrixaban (1×) was prepared by mixing equal volumes (1:1) to obtain 0.5 μg / mL of HFXa and 20 ng / mL of betrixaban. 50 μL of the FXa / vetrixaban mixture was added to the reaction in the direct potency assay.

[0075] For the BFXa mixture, stock solutions were prepared at 0.9 μg / mL (2.0 units / mL) for BFXa and 200-400 ng / mL for betrixaban. Higher betrixaban stock solution concentrations were prepared to meet the volume requirements for preparing the BFXa / betrixaban mixture. The BFXa(1×) / betrixaban(1×) mixture was prepared by mixing appropriate volumes of BFXa and betrixaban to obtain 0.5 μg / mL of BFXa and 20 ng / mL of betrixaban in the mixture. 50 μL of the fXa / betrixaban mixture was added to the reaction in the direct potency assay.

[0076] As shown in Figure 2(a), BFXa exhibited lower absorbance compared to HFXa at the highest anddexanet concentration, consistent with the observation of lower chromogenic activity of BFXa. However, in the absence of anddexanet, BFXa exhibited higher background in the presence of betrixaban, indicating lower inhibition of BFXa activity.

[0077] Based on these observations, the betrixaban concentration in the mixture was increased (1.5×, 2.0×) to reduce the background activity of BFXa in the absence of andexanet, while the same concentration was maintained for BFXa (1×), as shown in Figures 2(b-c). A doubling of the betrixaban concentration reduced the BFXa background to a level similar to that of HFXa in the absence of andexanet. However, the maximum absorbance for BFXa (1×) was lower than that of the control with HFXa (1×) / vetrixaban (1×) in the presence of the highest andexanet concentration.

[0078] Optimization of BFXa concentration in direct efficacy assays Figure 3 shows the effect of varying the BFXa concentration on maximum absorbance while maintaining the same concentration of betrixaban (2×). The mixture with BFXa(1.64×) / vetrixaban(2×) exhibited the maximum acceptable absorbance, but slightly higher than the HFXa control (Figure 3a). Further adjustment of the BFXa concentration with BFXa(1.45×) / vetrixaban(2×) yielded the final optimized conditions with comparable profiles between BFXa and the HFXa control (Figure 3b).

[0079] The optimized conditions shown in Figure 3b satisfy the intended assay settings for replacing HFXa with BFXa in the original direct efficacy assay, with maximum and minimum absorbance, EC 50Furthermore, the BFXa and HFXa controls exhibited comparable profiles in terms of assay range. Similar assay procedures and criteria used in direct potency assays can be used in direct potency unit assays.

[0080] Therefore, any modifications for the preparation of the BFXa / betrixaban mixture must be adapted to the changes made to the BFXa and betrixaban concentrations in the mixture. The sample protocol is as follows: 1) Preparation of BFXa (2.0 U / mL) - Reconstitution of BFXa (Hyphen BE101K, 50 μg / vial) by adding 1.0 mL of purified water to the Hyphen BFXa vial. This will result in 50 μg / mL of BFXa (111 units / mL). Mix the contents of the vial and keep the vial on ice. - Dilute the reconstituted BFXa to 0.92 μg / mL (2.0 units / mL) in assay buffer. 2) Preparation of betrixaban stock solution (200 ng / mL) - Dilute the betrixaban stock solution to 200 ng / mL in assay buffer. 3) Preparation of BFXa / vetrixaban mixture - For example, 8 volumes of BFXa (0.92 μg / mL) and 2.0 mL of betrixaban (200 μg / mL) are combined to mix 8 volumes of BFXa with 2 volumes of betrixaban. - Add the BFXa / vetrixaban mixture (50 μL) to the direct potency unit assay mixture.

[0081] The BFXa concentrations / mass in 50 μL of the mixture are 1.6 units / mL and 0.08 units, respectively. The betrixaban concentrations / mass in 50 μL of the mixture are 40 ng / mL and 2 ng, respectively.

[0082] Since the Hyphen BFXa used in the assay is calibrated against NIBSC BFXa(75 / 595), the direct potency units for the undexanet alpha standard are traceable from an independent international standard. Hyphen BFXa can also be used to calibrate HFXa reference samples.

[0083] The recently made-to-date HFXa international standard [NIBSC code: 15 / 102] can be used in the same way as the BFXa standard for HFXa calibration, or it can be used directly in traceable potency unit assays from an independent international standard.

[0084] The content of all papers, patents and patent applications, as well as all other documents and electronically available information referred to or cited herein is incorporated herein by reference to the same extent as each individual publication is incorporated by reference specifically and individually.

[0085] The applicant reserves the right to physically incorporate into this application any material and information from any such paper, patent, patent application or other physical and electronic document.

[0086] This disclosure is described extensively and generally herein. Each of the narrowly defined species and subgenera classifications within the general disclosure also forms part of this disclosure. This includes the general descriptions of this disclosure with any proviso or negative limitation that remove any subject from the genus, whether or not the extracted subject is specifically enumerated herein. Other embodiments are within the scope of the following claims. In addition, where any feature or aspect of this disclosure is described in terms of the Markush group, a person skilled in the art will recognize that this disclosure is also described in terms of any individual member or subgroup of any member of the Markush group. The present invention provides, for example, the following items: (Item 1) A method for determining the activity of an andexanet sample, The test sample containing andexanet is mixed with a mixture containing human factor Xa (HFXa) and a direct factor Xa (fXa) inhibitor, wherein the molar ratio of fXa to the fXa inhibitor is 0.2:1 to 0.3:1. Adding a chromogenic fXa substrate to the mixture, wherein the chromogenic fXa substrate is capable of releasing a chromophore upon reaction with the HFXa; To detect the amount of chromophore released; and The activity of the andexanet sample when releasing HFXa from the direct fXa inhibitor is calculated from the amount of chromophore released. A method that includes this. (Item 2) The method according to item 1, wherein the molar ratio of HFXa to the fXa inhibitor is 0.22 to 0.28. (Item 3) The method according to item 1, wherein the molar ratio of HFXa to the fXa inhibitor is 0.24 to 0.26. (Item 4) The HFXa is the method according to any one of items 1 to 3, having a concentration of approximately 8 to 14 nM after the test sample is added. (Item 5) The HFXa is the method described in item 4, having a concentration of approximately 10-12 nM after the test sample is added. (Item 6) The method according to any one of items 1 to 5, wherein the direct fXa inhibitor is selected from the group consisting of betrixaban, apixaban, rivaroxaban, edoxaban, otamixaban, retaxaban, and eribaxaban. (Item 7) The method according to item 6, wherein the direct fXa inhibitor is betrixaban. (Item 8) The chromogenic fXa substrate is spectrozyme-Xa, one of items 1 to 7. The method described in item 1. (Item 9) The method according to any one of items 1 to 8, further comprising verifying the activity of the andexanet sample using a standard curve prepared using a reference andexanet sample. (Item 10) A method for determining the activity of an andexanet sample, The test sample containing andexanet is mixed with a mixture containing bovine factor Xa (BFXa) and a direct factor Xa (fXa) inhibitor, wherein the molar ratio of BFXa to the fXa inhibitor is 0.15:1 to 0.25:1. Adding a chromogenic fXa substrate to the mixture, wherein the chromogenic fXa substrate is capable of releasing a chromophore upon reaction with the BFXa; To detect the amount of chromophore released; and The activity of the andexanet sample when releasing BFXa from the direct fXa inhibitor is calculated from the amount of chromophore released. A method that includes this. (Item 11) The method according to item 10, wherein the molar ratio of BFXa to the fXa inhibitor is 0.16 to 0.20. (Item 12) The method according to item 10, wherein the molar ratio of BFXa to the fXa inhibitor is 0.17 to 0.19. (Item 13) The BFXa is obtained by adding the test sample and having a concentration of approximately 10-20 nM, according to the method of any one of items 10-12. (Item 14) The BFXa, after the test sample is added, has a concentration of approximately 14-18 nM, as described in item 13. (Item 15) The method according to any one of items 10 to 14, wherein the direct fXa inhibitor is selected from the group consisting of betrixaban, apixaban, rivaroxaban, edoxaban, otamixaban, retaxaban, and eribaxaban. (Item 16) The method according to item 15, wherein the direct fXa inhibitor is betrixaban. (Item 17) The chromogenic fXa substrate is Spectrozyme-Xa, as described in any one of items 10 to 16.

Claims

[Claim 1] The invention described in the specification.

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