Method for measuring blood coagulation activity

Abstract

To provide a measuring method of blood coagulation activity capable of facilitating measurement of samples of patients to whom lonoctocog alfa has been administered and suppressing human errors.SOLUTION: A measuring method of blood coagulation activity includes the steps of: acquiring a parameter relating to derivative of coagulation waveform in a blood sample collected from a patient to whom a polypeptide is administered, the polypeptide including (1) a polypeptide containing an array represented by a sequence number 1 or (2) a polypeptide having 95% or more sequence identity with the array represented by the sequence number 1, and having an activity as a blood coagulation factor VIII; and acquiring an activity value of the blood coagulation factor VIII in the blood sample based on the acquired parameter.SELECTED DRAWING: None

Description

[Technical field]

[0001] Disclosed herein is a method for measuring blood coagulation activity. [Background technology]

[0002] Lonoctocog alfa, described in Patent Document 1, is a factor VIII analogue, which is one of the human blood coagulation factors produced by genetic recombination, and is used in patients with factor VIII deficiency to suppress bleeding tendency.

[0003] The coagulation activity of patients administered with lonoctocog alfa is generally measured and monitored by a one-stage coagulation method such as activated partial thromboplastin time (APTT). However, when the activity of lonoctocog alfa is measured by the one-stage coagulation method after administration, the measurement result shows an apparently low value, as described in AFSTYLA (trademark) HIGHLIGHTS OF PRESCRIBING INFORMATION (Non-Patent Document 1). For this reason, Non-Patent Document 1 instructs that when the activity of lonoctocog alfa in plasma is measured by the one-stage coagulation method, the measured value of factor VIII obtained by the one-stage coagulation method should be multiplied by a conversion factor of 2 to be used as the measured value of factor VIII of the patient. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] U.S. Patent No. 7,041,635 [Non-patent literature]

[0005] [Non-Patent Document 1] AFSTYLA(TM) HIGHLIGHTS OF PRESCRIBING INFORMATION (https: / / labeling.cslbehring.com / PI / US / Afstyla / EN / Afstyla-Prescribing-Information.pdf) Summary of the Invention [Problem to be solved by the invention]

[0006] However, in order to follow the instructions described in Non-Patent Document 1, it is necessary to identify samples from patients who have been administered Lonoctcog alfa from among the many measured values ​​and multiply the identified samples by a conversion factor, which is a cumbersome process and may be subject to human error. An objective of the present invention is to provide a method for measuring blood coagulation activity, which can facilitate the measurement of samples from patients administered lonoctocog alfa and can reduce human error. [Means for solving the problem]

[0007] One embodiment of the present invention relates to a method for measuring blood coagulation activity, comprising the steps of: acquiring parameters relating to the differential of a clot waveform in a blood sample collected from a patient administered with (1) a polypeptide comprising a sequence represented by SEQ ID NO: 1; or (2) a polypeptide comprising a polypeptide having 95% or more identity to the sequence represented by SEQ ID NO: 1 and having activity as blood coagulation factor VIII; and acquiring an activity value of blood coagulation factor VIII in the blood sample based on the acquired parameters. According to this embodiment, the factor VIII activity of lonoctocog alfa can be measured more accurately. Effect of the Invention

[0008] It is possible to provide a method for measuring blood coagulation activity that can facilitate measurement of samples from patients administered lonoctocog alfa and reduce human error. [Brief description of the drawings]

[0009] [Figure 1] (A) An example of a clot waveform. (B) An example of a corrected clot waveform. [Diagram 2]The factor VIII activity of each preparation was calculated based on the clotting time. (A) shows the activity when each preparation was added to factor VIII-deficient plasma to give 1.00 IU / dL. (B) shows the activity when each preparation was added to factor VIII-deficient plasma to give 0.30 IU / dL. (C) shows the activity when each preparation was added to factor VIII-deficient plasma to give 0.05 IU / dL. [Diagram 3] (A) shows the parameter values ​​of Advate and Eifstira. (B) shows the first derivative waveforms of the APTT clot waveforms of Advate and Eifstira. (C) shows the second derivative waveforms of the APTT clot waveforms of Advate and Eifstira. [Figure 4] (A) shows the first derivative of the APTT clot waveform after correction for Advate and Eifstilla. (B) shows the second derivative of the APTT clot waveform after correction for Advate and Eifstilla. [Diagram 5] (A) shows the standard curve for min1. (B) shows the standard curve for min2. [Figure 6] (A) shows the calibration curve for Ad|min1|. (B) shows the calibration curve for Max2. [Figure 7] (A) shows the activity value (IU / dL) of A. stearothermia calculated based on the parameter values ​​related to the derivative of each APTT clot waveform. (B) shows the recovery rate (%) of the spiked A. stearothermia. [Figure 8] The activity of factor VIII in each preparation was calculated based on the maximum clotting rate min1. (A) shows the activity when each preparation was added to factor VIII-deficient plasma to give 1.00 IU / dL. (B) shows the activity when each preparation was added to factor VIII-deficient plasma to give 0.30 IU / dL. (C) shows the activity when each preparation was added to factor VIII-deficient plasma to give 0.05 IU / dL. [Figure 9]The activity of factor VIII of each preparation calculated based on the corrected maximum clotting rate Ad|min1| is shown. Figure 9 (A) shows the activity when each preparation was added to factor VIII-deficient plasma to give 1.00 IU / dL. (B) shows the activity when each preparation was added to factor VIII-deficient plasma to give 0.30 IU / dL. (C) shows the activity when each preparation was added to factor VIII-deficient plasma to give 0.05 IU / dL. [Figure 10] The activity of factor VIII in each preparation was calculated based on the maximum clotting acceleration min2. (A) shows the activity when each preparation was added to factor VIII-deficient plasma to give 1.00 IU / dL. (B) shows the activity when each preparation was added to factor VIII-deficient plasma to give 0.30 IU / dL. (C) shows the activity when each preparation was added to factor VIII-deficient plasma to give 0.05 IU / dL. [Figure 11] The activity of factor VIII in each preparation was calculated based on the maximum coagulation deceleration rate max2. (A) shows the activity when each preparation was added to factor VIII-deficient plasma to give 1.00 IU / dL. (B) shows the activity when each preparation was added to factor VIII-deficient plasma to give 0.30 IU / dL. (C) shows the activity when each preparation was added to factor VIII-deficient plasma to give 0.05 IU / dL. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] 1. Explanation of terms In the present specification, the subject for which the activity value of blood coagulation factor VIII is obtained is a polypeptide that contains a specific amino acid sequence and has activity as blood coagulation factor VIII. In the present specification, blood coagulation factor VIII is sometimes referred to simply as "factor VIII" and a polypeptide that has activity as blood coagulation factor VIII is sometimes referred to simply as "polypeptide."

[0011] Preferably, the polypeptide is a polypeptide comprising the sequence shown in SEQ ID NO: 1. The polypeptide comprising the sequence shown in SEQ ID NO: 1 may be modified by disulfide bonds, glycosylation, sulfation, etc.

[0012] For example, a disulfide bond may be formed between at least one cysteine ​​residue selected from the following in SEQ ID NO: 1: between cysteine ​​residues at positions 153 and 179, between cysteine ​​residues at positions 248 and 321, between cysteine ​​residues at positions 528 and 554, between cysteine ​​residues at positions 630 and 711, between cysteine ​​residues at positions 944 and 971, between cysteine ​​residues at positions 1111 and 1115, between cysteine ​​residues at positions 1131 and 1281, and between cysteine ​​residues at positions 1286 and 1438.

[0013] At least one asparagine residue selected from positions 71, 239, 757, 764, 922, and 1230 of SEQ ID NO: 1 can be bound to a glycosylated residue.

[0014] At least one selected from the serine residue at position 741, the serine residue at position 743, the serine residue at position 746, the threonine residue at position 759, the threonine residue at position 760, the threonine residue at position 765, the threonine residue at position 766, the serine residue at position 769, and the serine residue at position 781 in SEQ ID NO: 1 may also be bound to a glycan.

[0015] At least one tyrosine residue selected from positions 346, 718, 719, 723, 776, and 792 of SEQ ID NO:1 may be sulfated.

[0016] As used herein, the term "residue" of various amino acids refers to the structural units of amino acids that constitute a polypeptide, and refers to a group formed by removing a hydrogen atom from the main chain amino group and / or removing -OH from the main chain carboxyl group of an amino acid.

[0017] The polypeptide may also include a polypeptide having at least a certain percentage of identity with the sequence shown in SEQ ID NO: 1. The "certain percentage" is not limited as long as it is a percentage having the same or higher factor VIII activity as a polypeptide comprising the sequence shown in SEQ ID NO: 1. For example, the "certain percentage" refers to 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the sequence shown in SEQ ID NO: 1. Preferably, the "certain percentage" refers to 95%, 98%, or 99%.

[0018] Amino acid substitutions that maintain the same or higher level of activity as factor VIII as the sequence represented by SEQ ID NO: 1 can include, for example, substitutions between amino acids of the class to which each amino acid belongs. For example, when the class is non-polar (hydrophobic) amino acids, alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine can be included. When the class is polar neutral amino acids, glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine can be included. When the class is basic amino acids, arginine, lysine, and histidine can be included. When the class is acidic amino acids, aspartic acid and glutamic acid can be included. The method for measuring the activity of factor VIII is described below. The above polypeptides can be produced by recombinant gene technology.

[0019] The amino acids constituting the polypeptide may be artificial amino acids. Furthermore, the polypeptide may be modified in a manner other than those described above. Examples of the modification include polyethylene glycol modification, phosphorylation modification, acetylation modification, methylation modification, fluorescent modification, biotinylation modification, sugar modification, lipid modification, acylation modification, reductive amination modification, azide modification, and ketene modification.

[0020] As a polypeptide, the most preferred example is lonoctocog alfa (trade name: Eifstira (trademark)) described in Non-Patent Document 1.

[0021] The patient is not limited as long as it is a person who requires administration of factor VIII. For example, the patient may be a patient with factor VIII deficiency. For example, the patient may be a patient with factor VIII deficiency, such as hemophilia A, disseminated intravascular coagulation, liver dysfunction, etc.

[0022] The blood specimen is a blood sample collected from a patient, and is not limited as long as it is a sample for which the blood coagulation activity described below can be measured. Examples of blood samples include whole blood and plasma. It is preferable to collect blood samples using an anticoagulant other than a heparin preparation when collecting blood. It is more preferable to collect blood samples using a citrate salt, such as a sodium citrate solution, as the anticoagulant. The most preferable blood sample is plasma separated from a whole blood sample in which a 3.1 to 3.3% (weight / volume) trisodium citrate solution is used as the anticoagulant, and the anticoagulant and the patient's whole blood are mixed at a volume ratio of about 1:8.5 to 9.5.

[0023] 2. Method for measuring blood coagulation activity One embodiment of the present invention relates to a method for measuring blood coagulation activity, the method comprising the steps of: (1) a polypeptide comprising a sequence represented by SEQ ID NO: 1, or

[0024] (2) The method may include a step of acquiring parameters relating to the differential of the clot waveform in a blood sample collected from a patient to whom a polypeptide having 95% or more identity to the sequence represented by SEQ ID NO:1 and activity as blood coagulation factor VIII is administered, and a step of acquiring an activity value of blood coagulation factor VIII in the blood sample based on the acquired parameters.

[0025] The clot waveform will be described with reference to Fig. 1(A). The clot waveform shown in Fig. 1(A) is obtained by a method for measuring blood coagulation activity, generally called the one-stage clotting method. The one-stage clotting method is a method for measuring the clotting time by adding calcium ions necessary for blood clotting and a specified test reagent to a blood sample containing fibrinogen for which the clotting time is to be measured (hereinafter referred to as the "test sample"), irradiating the reaction solution with light, and monitoring the optical change over time of the reaction solution.

[0026] An example of a clot waveform is shown in FIG. 1(A). In FIG. 1(A), the vertical axis (Y-axis) indicates the transmitted light intensity, and the horizontal axis (X-axis) indicates the measurement time (seconds: sec) during which the transmitted light intensity was monitored. The clot waveform can be generated by plotting the change in the monitored transmitted light intensity over time on two axes: transmitted light intensity and measurement time. Point I in FIG. 1(A) is the time when calcium ions and a test reagent were added to the test sample, and is the time when the measurement was started (t I ) at the start of the measurement. Since the fibrinogen in the reaction solution has not yet changed to fibrin and fibrin precipitation has not yet occurred in the reaction solution, the transmitted light intensity shows a high value. As the coagulation reaction subsequently progresses and fibrin begins to precipitate, the transmitted light intensity begins to decrease. This is because the precipitated fibrin blocks the light. This point is point II in Figure 1(A), which is the coagulation start time. The measurement time at which coagulation begins is called (t II ) As the reaction progresses and fibrin precipitation progresses, the transmitted light intensity decreases. When most of the fibrinogen in the test sample is converted to fibrin, the reaction converges and the change in transmitted light intensity plateaus. This point is point III in Figure 1(A), which is the end of coagulation. The measurement time when coagulation is complete is called (t III) The clotting time (CT) is generally expressed as CT (sec) = [(t III )-(t II )] / 2 measurement time. Here, "-" indicates subtraction and " / " indicates division. In other words, parameters related to coagulation activity such as the coagulation start time, coagulation end time, and coagulation time can be obtained from the clot waveform.

[0027] However, depending on the patient, the initial amount of fibrinogen contained in the test sample may be small, so it is preferable to normalize the clot waveform as shown in Fig. 1(B) and determine the clotting time. Normalization can be achieved, for example, by assuming that the change in transmitted light intensity (dH), which is the difference between the transmitted light intensity at point II, which is the start of the clotting reaction, and the transmitted light intensity at point III, which is the end of clotting, is 100%, and converting the change in transmitted light intensity into a relative value. In this case, the clotting time (CT) can be set as the point at which the change in transmitted light intensity (dH) is, for example, 30%, 40%, 50%, or 60%. In a preferred embodiment, the clotting time is the time when the change in transmitted light intensity (dH) becomes 50%.

[0028] In this specification, the "clotting waveform" may include a clot waveform generated without normalizing the monitored transmitted light intensity, and a clot waveform generated based on corrected monitoring data obtained by performing the above-mentioned normalization on the monitored transmitted light intensity (hereinafter referred to as a "corrected clot waveform"). It is preferable to use a corrected clot waveform as the clot waveform. In this specification, the clot time obtained from the corrected clot waveform is referred to as the "corrected clot time."

[0029] Examples of comprehensive methods for measuring blood coagulation activity that can be measured by the one-stage coagulation method include activated partial thromboplastin time (APTT) and prothrombin time (PT). In addition, various coagulation factor activities that can be measured using comprehensive measurement methods can also be measured by the one-stage coagulation method. Coagulation factors whose activities can be measured using APTT include factor VIII, factor V, factor X, factor IX, factor XI, factor XII, prekallikrein, high molecular weight kininogen, prothrombin, fibrinogen, etc. Coagulation factors whose activities can be measured using PT include factor VII, factor V, factor X, prothrombin, fibrinogen, etc.

[0030] In this embodiment, the activity value of factor VIII is obtained. The activity value of factor VIII can be obtained using APTT. Here, "obtaining" may include calculating the activity value or receiving the activity value from another source.

[0031] In the case of measuring APTT, the predetermined test reagent is an APTT reagent that may contain an activator such as silica, ellagic acid, celite, etc.; and animal-derived, plant-derived, or artificially synthesized phospholipids; etc. The test reagent for measuring APTT may be a commercially available test reagent. For example, Thrombocheck APTT series manufactured by Sysmex Corporation, Coagpia APTT-N manufactured by Sekisui Medical Co., Ltd., Dataphi APTT manufactured by Siemens Healthcare Diagnostics Products GmbH, etc. may be exemplified. Alternatively, calcium ions can be provided by a 20 mM calcium chloride solution according to international standards.

[0032] The test sample, the test reagent for APTT measurement, and calcium ions are mixed in a predetermined diluent. Examples of the diluent include a solution that is isotonic with human plasma but does not have a pH adjustment function, such as physiological saline, and a buffer solution. Examples of the buffer solution include an Oren-Veronal buffer solution and an imidazole buffer solution. As the diluent, an Oren-Veronal buffer solution is preferable.

[0033] In the above, an example is shown in which the precipitation of fibrinogen is detected based on the change in the intensity of transmitted light. However, instead of the intensity of transmitted light, the amount of scattered light, absorbance, etc. can be used.

[0034] The APTT can also be measured using a blood coagulation measuring device. Examples of the blood coagulation measuring device include fully automatic blood coagulation testers CN-6000, CN-3000, CS-2400, CS-2500, CS-5100, CS-1600, and CS-600 series manufactured by Sysmex Corporation; and semi-automatic blood coagulation measuring devices CA-101 and CA-104. In this embodiment, since it is necessary to obtain parameters related to the differentiation of the clot waveform, it is preferable to use a fully automatic blood coagulation testing device equipped with software that performs differentiation processing of the clot waveform.

[0035] The activity value of factor VIII is obtained based on a calibration curve prepared from a plurality of standard plasma samples having known activity values ​​of factor VIII and different activity values. According to a conventional method, the activity value of factor VIII using APTT is obtained based on parameters related to coagulation activity obtained from a test sample and a calibration curve of the activity value of factor VIII prepared for parameters corresponding to the parameters related to coagulation activity. The calibration curve is prepared from parameters related to coagulation activity obtained by APTT measurement of standard plasma samples prepared by diluting standard human plasma for blood coagulation tests in a plurality of stages with physiological saline or factor VIII-deficient plasma. When the standard human plasma for blood coagulation tests is used to prepare a calibration curve, the standard human plasma for blood coagulation tests is diluted with physiological saline or with factor VIII-deficient plasma so that the activity value is, for example, at least one selected from theoretically 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, and 10%, assuming that the factor VIII activity of the standard human plasma for blood coagulation tests is 100%, and the diluted sample is used as a standard plasma sample with a known factor VIII activity value. The standard plasma sample may include standard human plasma for blood coagulation tests as a standard plasma sample with a factor VIII activity value of 100%. The standard plasma sample may also include factor VIII-deficient plasma as a standard plasma sample with a factor VIII activity value of 0%. In this way, a plurality of standard plasma samples with different factor VIII activity values ​​from each other can be prepared from the standard human plasma for blood coagulation tests.

[0036] The standard human plasma for blood coagulation tests may be pooled plasma collected from multiple individuals with normal blood coagulation function, or standard human plasma for blood coagulation tests available from Siemens KK etc. Factor VIII-deficient plasma can be purchased from, for example, George King Bio-Medical, Inc. (USA) etc.

[0037] Alternatively, a blood coagulation factor VIII preparation such as Cross Eight MC (Japan Blood Products Organization), Kovaltrii (trademark) (Bayer Yakuhin, Ltd.), Advate (Takeda Pharmaceutical Co., Ltd.), Adynovate (trademark) (Takeda Pharmaceutical Co., Ltd.), Novo Eight (trademark) (Novo Nordisk Pharma Co., Ltd.), or Eloctate (trademark) (Sanofi K.K.) may be added to the factor VIII-deficient plasma to prepare a standard plasma sample. Since the activity value of factor VIII in blood coagulation factor VIII preparations is known, a blood coagulation factor VIII preparation can be added to the factor VIII-deficient plasma so as to obtain a desired activity value, and multiple standard plasma samples with different activity values ​​can be prepared.

[0038] The parameter related to the coagulation activity used to obtain the activity value of factor VIII is generally the coagulation time. However, in this embodiment, a parameter related to the differentiation of the coagulation waveform is used to obtain the activity value of factor VIII. The parameter related to the differentiation of the coagulation waveform is obtained by performing differentiation processing on the coagulation waveform (hereinafter referred to as the "raw coagulation waveform") obtained by monitoring the optical change over time of the reaction solution in the APTT measurement. The differentiation processing is described in U.S. Patent Application Publication No. 2018-0306820 and is incorporated herein.

[0039] For example, the first derivative clot waveform, obtained by first differentiation of the raw clot waveform, is a parameter of the clot velocity. The apex of the peak of the first derivative clot waveform indicates the maximum clot velocity. In this specification, the maximum clot velocity is sometimes expressed as "min1". The value of the first derivative clot waveform of the clot velocity may also be expressed as an absolute value, in which case the maximum clot velocity can be expressed as "|min1|".

[0040] Furthermore, a second-order differential clot waveform is obtained by performing a second-order differential process on the raw clot waveform. The second-order differential clot waveform is a parameter of clot acceleration and clot deceleration. In the second-order differential clot waveform, the apex of a peak obtained in the same axial direction as the peak of the first-order differential clot waveform indicates the maximum clot acceleration. In this specification, the maximum clot acceleration is sometimes expressed as "min2". The maximum clot acceleration is sometimes expressed as an absolute value, and in this case, it can be expressed as "|min2|". In the second-order differential clot waveform, a peak that appears in the axial direction opposite to the axial direction indicating the acceleration indicates the maximum clot deceleration. In this specification, the maximum clot acceleration is sometimes expressed as "max2". The maximum clot deceleration is sometimes expressed as an absolute value, and in this case, it can be expressed as "|max2|".

[0041] The above-mentioned first derivative can also be performed on the corrected raw clot waveform. In this specification, the first derivative clot waveform generated from the corrected clot waveform is called the corrected first derivative clot waveform. The apex of the peak of the corrected first derivative clot waveform indicates the corrected maximum clot velocity. In this specification, the corrected maximum clot velocity may be expressed as "Ad|min1|".

[0042] Furthermore, a corrected second-order differential clot waveform is obtained by performing a second-order differential process on the corrected raw clot waveform. In the corrected second-order differential clot waveform, the apex of the peak obtained in the same axial direction as the peak of the corrected first-order differential clot waveform indicates the corrected maximum clot acceleration. In this specification, the maximum clot acceleration is sometimes expressed as "Ad|min2|". In the corrected second-order differential clot waveform, the peak that appears in the axial direction opposite the axial direction indicating acceleration indicates the corrected maximum coagulation deceleration. In this specification, the maximum coagulation acceleration is sometimes expressed as "Ad|max2|".

[0043] The parameters related to the differentiation of the clot waveform may be parameters that reflect the shapes of the first derivative clot waveform, the corrected first derivative clot waveform, the second derivative clot waveform, and the corrected second derivative clot waveform. For example, in this embodiment, a calibration curve is created using a parameter related to the differentiation of at least one clot waveform selected from the group consisting of the maximum clot velocity, the maximum clot acceleration, the maximum clot deceleration, the corrected maximum clot velocity, the corrected maximum clot acceleration, and the corrected maximum clot deceleration. In addition, the area under the curve and the center of gravity of the area under the curve of the first derivative clot waveform, the corrected first derivative clot waveform, the second derivative clot waveform, and the corrected second derivative clot waveform are also used as parameters related to the differentiation of the clot waveform. Then, the parameter values ​​related to the differentiation of the clot waveform corresponding to the calibration curve obtained from the test sample are applied to the created calibration curve to obtain the activity value of factor VIII in the test sample.

[0044] The creation of a calibration curve, the acquisition of parameters related to the differentiation of the clot waveform, and the acquisition of the activity value of factor VIII contained in the test sample can be carried out using software installed in the fully automated blood coagulation test apparatus. EXAMPLES

[0045] The present embodiment will be described in more detail below with reference to examples. However, the present invention is not limited to the embodiments.

[0046] 1. Materials and Methods (1) Preparation of test samples Each test sample was prepared by adding a commercially available factor VIII preparation to Congenital Factor VIII deficient plasma (George King Bio-Medical, Inc. (USA)) so that the final activity value was 0.05 IU / dL, 0.30 IU / dL, or 1.00 IU / dL, based on the activity value stated in the package insert of each drug.

[0047] The factor VIII preparations used are as follows: Cross Eight MC is a plasma fraction preparation, and the other preparations are recombinant preparations. Cross Eight MC (Japan Blood Products Organization) Kovaltrii (trademark) (Octocog Beta; Bayer Yakuhin, Ltd.) Advate (Rurioctocog alfa; Takeda Pharmaceutical Company Limited) Adynovate™ (Rurioctocog alfa pegol; Takeda Pharmaceutical Company Limited) NovoEight (trademark) (Turoctocog alfa; Novo Nordisk Pharma K.K.) Eloctate™ (eflaroctocog alfa; Sanofi Corporation) Aifstira (trademark) (Lonoctocog alfa; CSL Behring K.K.)

[0048] (2) Measurement reagents The following reagents were used for the measurement. Thrombocheck APTT-SLA (Sysmex Corporation) 20mM calcium chloride solution (Sysmex Corporation) Coagulation factor VIII deficient plasma (Siemens KK) Oren Veronal Buffer Solution (Siemens) Standard human plasma for blood coagulation tests (Siemens)

[0049] (3) Measurement equipment and measurement protocol The measurement was performed using a fully automated blood coagulation analyzer CS-2400 (Sysmex Corporation) with the default protocol. The following steps were performed using a computer program installed in the analyzer to measure the activated partial thromboplastin time (APTT) using a one-stage coagulation method, and the activity value of factor VIII in the test sample was obtained.

[0050] STEP 1: Dilute the aspirated test sample 20-fold with Oren-Veronal buffer and dispense 40 μL into a reaction cuvette. STEP 2: Add 40 μL of coagulation factor VIII-deficient plasma to the diluted test sample and incubate to prepare the first reaction solution. STEP 3: Add 40 μL of ThromboCheck APTT-SLA to the first reaction solution and incubate to prepare the second reaction solution. STEP 4: Add 40 μL of 20 mM calcium chloride solution to the second reaction solution to start the coagulation reaction, measure the transmitted light at a wavelength of 660 nm for a specified time, and monitor the change in transmitted light intensity over time. STEP 5: Detect the coagulation start and end points from the monitoring data and calculate the coagulation time (CT). STEP 6: The calculated clotting time (CT) is applied to the standard curve for factor VIII to calculate the coagulation factor VIII activity of the sample.

[0051] Here, the calibration curve for factor VIII was prepared using standard plasma samples prepared by diluting standard human plasma for blood coagulation tests stepwise with coagulation factor VIII-deficient plasma. In this case, the activity value of factor VIII in undiluted standard human plasma for blood coagulation tests was set to 100%. The standard plasma samples were also measured in the same manner as the test samples, and the calibration curve was prepared by taking the coagulation time as the factor VIII activity corresponding to the dilution ratio.

[0052] (4) Acquisition of parameters related to the derivative of the APTT clot waveform As parameters related to the differential of the APTT clot waveform, min1, min2, Ad|min1|, and max2 were calculated. Calculation of parameters related to the differential of the APTT clot waveform was performed for the test samples and the standard plasma samples.

[0053] The monitoring data obtained in the above (3) STEP 4 was used as APTT clot waveform data. The APTT clot waveform was obtained by plotting the monitoring data with the X-axis representing measurement time and the Y-axis representing transmitted light intensity.

[0054] In addition, the APTT clot waveform was differentiated according to the method described in U.S. Patent Application Publication No. 2018-0306820 to generate a first differential clot waveform and a second differential clot waveform of the APTT clot waveform. The peak value min1 of the velocity waveform was obtained from the first differential clot waveform. min1 indicates the maximum clot velocity. The peak value min2 of the coagulation acceleration and the absolute value max2 of the peak value of the coagulation deceleration were obtained from the second differential clot waveform. min2 indicates the maximum coagulation acceleration. max2 indicates the maximum coagulation deceleration. In addition, the absolute value |min1| of the peak value of the velocity waveform was obtained from the corrected first differential clot waveform generated based on the normalized APTT clot waveform data, and was set as the corrected absolute value Ad|min1|. A corrected first differential clot waveform and a corrected second differential clot waveform were generated from the normalized APTT clot waveform.

[0055] Furthermore, based on the parameters related to the differential of the APTT clot waveform obtained from the standard plasma sample, a calibration curve of the factor VIII activity based on each parameter was created. Based on this calibration curve, the factor VIII activity of each test sample was calculated for each parameter.

[0056] 2.Results (1) Comparison of factor VIII activity of each preparation Figure 2 shows the activity value (IU / dL) of factor VIII of each preparation calculated based on the clotting time (CT) obtained by the APTT one-stage method. Measurements were performed for each test sample (n=3). Figure 2(A) shows the activity value when each preparation was added to congenital factor VIII deficient plasma to give 1.00 IU / dL. Figure 2(B) shows the activity value when each preparation was added to congenital factor VIII deficient plasma to give 0.30 IU / dL. Figure 2(C) shows the activity value when each preparation was added to congenital factor VIII deficient plasma to give 0.05 IU / dL. Only Aifstira showed low activity in all activities.

[0057] (2) Comparison of parameters related to the derivative of the APTT clot waveform Figure 3(A) shows the parameter values ​​for Advate and Aifstira, which had the largest difference in clotting time (CT) in the results of (1) above. The unit of CT is seconds, and other data is calculated values. Figure 3(B) shows the first derivative waveforms of the APTT clot waveforms of both. Figure 3(C) shows the second derivative waveforms of the APTT clot waveforms of both. Furthermore, Figure 4(A) shows the first derivative waveforms of the APTT clot waveforms after correction for both. Figure 4(B) shows the second derivative waveforms of the APTT clot waveforms after correction for both. In the first derivative waveform, the Y axis represents the clotting rate (dT / dt). Here, T is the time when the maximum transmitted light intensity is measured, and t is the time when the minimum transmitted light intensity is measured. In the second derivative waveform, the Y axis represents the clotting rate (dT 2 / dt 2 )

[0058] As shown in Figure 3(A), CT showed that Eifstira had a longer CT and lower factor VIII activity. However, comparison of min1, Ad|min1|, and max2 showed that the difference in factor VIII activity between Advate and Eifstira was smaller than the difference in factor VIII activity between Advate and Eifstira when comparing CT.

[0059] (3) Calibration curves for each parameter of the APTT clot waveform derivative Next, the calibration curves created for each parameter related to the derivative of the APTT clot waveform are shown in Figures 5 and 6. Figure 5(A) shows the calibration curve for min1, and Figure 5(B) shows the calibration curve for min2. Figure 6(A) shows the calibration curve for Ad|min1|, and Figure 6(B) shows the calibration curve for Max2. Each axis of the calibration curve is expressed in logarithmic scale. The Y-axis shows the value of each parameter, and the X-axis shows the activity value of factor VIII. The calibration curves for all parameters were well linear, demonstrating that the activity of factor VIII can be evaluated.

[0060] (4) Calculation of Aifstilla activity using parameters related to the derivative of the APTT clot waveform Based on the activity value described in the package insert, Aifustira was added to Congenital Factor VIII deficient plasma so that the final activity value was 5 IU / dL, 30 IU / dL, and 100 IU / dL, and APTT coagulation data was obtained. Based on this data, parameter values ​​related to the differential of each APTT clot waveform were obtained, and applied to a calibration curve to calculate the activity value of factor VIII. The results are shown in Figure 7. Figure 7(A) shows the activity value of factor VIII, and Figure 7(B) shows the addition recovery rate (%). The activity value of factor VIII of Aifustira calculated using the parameters related to the differential of the APTT clot waveform was higher than the activity value calculated from CT and was close to the theoretical value. The recovery rate was also good.

[0061] (5) Factor VIII activity calculated using parameters related to the derivative of the APTT clot waveform For the test samples whose activity was shown in FIG. 2, the activity of factor VIII was calculated using parameters related to the differential of the APTT clot waveform.

[0062] Figure 8 shows the activity of factor VIII for each formulation calculated based on the maximum coagulation rate min1. Figure 8(A) shows the activity when each formulation was added to Congenital Factor VIII deficient plasma to give 1.00 IU / dL. Figure 8(B) shows the activity when each formulation was added to Congenital Factor VIII deficient plasma to give 0.30 IU / dL. Figure 8(C) shows the activity when each formulation was added to Congenital Factor VIII deficient plasma to give 0.05 IU / dL.

[0063] Figure 9 shows the activity of factor VIII for each formulation calculated based on the corrected maximum coagulation rate Ad|min1|. Figure 9(A) shows the activity when each formulation was added to Congenital Factor VIII deficient plasma to give 1.00 IU / dL. Figure 9(B) shows the activity when each formulation was added to Congenital Factor VIII deficient plasma to give 0.30 IU / dL. Figure 9(C) shows the activity when each formulation was added to Congenital Factor VIII deficient plasma to give 0.05 IU / dL.

[0064] Figure 10 shows the activity of factor VIII for each formulation calculated based on the maximum clotting acceleration min2. Figure 10(A) shows the activity when each formulation was added to Congenital Factor VIII deficient plasma to give 1.00 IU / dL. Figure 10(B) shows the activity when each formulation was added to Congenital Factor VIII deficient plasma to give 0.30 IU / dL. Figure 10(C) shows the activity when each formulation was added to Congenital Factor VIII deficient plasma to give 0.05 IU / dL.

[0065] Figure 11 shows the activity of factor VIII for each formulation calculated based on the maximum coagulation deceleration rate max2. Figure 11(A) shows the activity when each formulation was added to Congenital Factor VIII deficient plasma to give 1.00 IU / dL. Figure 11(B) shows the activity when each formulation was added to Congenital Factor VIII deficient plasma to give 0.30 IU / dL. Figure 11(C) shows the activity when each formulation was added to Congenital Factor VIII deficient plasma to give 0.05 IU / dL. Eifstira showed a higher factor VIII activity value than that calculated based on the clotting rate, eliminating the difference in activity value with other preparations.

[0066] This demonstrated that it is useful to use parameters related to the derivative of the clot waveform rather than the coagulation rate to assess the efficacy of administration of Eifstira (lonoctocog alfa).

[0067] In addition, it is no longer necessary to identify samples from patients who were administered Eifstira (lonoctocog alfa) for the obtained activity values, making measurement easier and reducing human error.

Claims

1. (1) A polypeptide comprising the sequence represented by SEQ ID NO: 1, or (2) A polypeptide having 95% or more identity to the sequence represented by SEQ ID NO:1 and having activity as blood coagulation factor VIII; obtaining a parameter related to a differential of a clot waveform in a blood sample collected from a patient to whom the patient has been administered, the parameter being obtained based on a clot waveform measured by a one-stage clotting method; obtaining an activity value of blood coagulation factor VIII in the blood sample based on the obtained parameters; Including, the measurement by the one-stage coagulation method includes diluting the blood sample, mixing the diluted blood sample with coagulation factor VIII-deficient plasma and an activated partial thromboplastin time (APTT) measurement reagent, incubating the mixture to prepare a reaction solution, mixing a calcium solution with the reaction solution to initiate a coagulation reaction, irradiating the reaction solution mixed with the calcium solution with light, monitoring the light from the reaction solution irradiated with light, and acquiring the coagulation waveform. A method for measuring blood coagulation activity.

2. 2. The method for measuring blood coagulation activity according to claim 1, wherein the measurement by the one-stage coagulation method is measurement of activated partial thromboplastin time.

3. 3. The method for measuring blood coagulation activity according to claim 1 or 2, wherein the parameter is obtained from at least one selected from a waveform of a coagulation rate obtained by first differentiating a coagulation waveform, and a waveform of a coagulation acceleration obtained by second differentiating a coagulation waveform.

4. The method for measuring blood coagulation activity according to claim 3, wherein the parameter is a value indicating at least one selected from maximum coagulation rate, maximum coagulation acceleration, maximum coagulation deceleration, corrected maximum coagulation rate, corrected maximum coagulation acceleration, and corrected maximum coagulation deceleration.

5. The method for measuring blood coagulation activity according to any one of claims 1 to 4, wherein the activity value of blood coagulation factor VIII in the blood sample collected from the patient is obtained based on a calibration curve prepared from a plurality of standard plasma samples having known activity values ​​of blood coagulation factor VIII different from one another.

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