阅读说明：本技术 血液凝固活性的测定方法 (Method for measuring blood coagulation activity ) 是由 筱原翔 熊野穣 新井信夫 窗岩清治 藤森祐多 于 2021-03-26 设计创作，主要内容包括：本发明提供能使施用Lonoctocog Alfa的患者的受试体的测定变得容易的同时抑制人为失误的血液凝固活性的测定方法。由血液凝固活性的测定方法解决了上述课题,所述方法包括：取得基于从施用(1)含SEQ ID NO:1所表示的序列的多肽、或者(2)含与SEQ ID NO:1所表示的序列具有95％以上的相同性的多肽、且具有作为血液第VIII凝血因子的活性的多肽的患者采集的血液受试体中的凝固波形的微分相关参数的工序,及取得的上述参数而取得上述血液受试体中的血液第VIII凝血因子的活性值的工序。(The present invention provides a method for measuring blood coagulation activity, which can facilitate measurement of a subject of a patient to whom Lonocicepg Alfa is administered and can suppress human error. The above object is solved by a method for measuring blood coagulation activity, comprising: a step of obtaining a parameter related to differentiation of a coagulation waveform in a blood specimen collected from a patient to whom (1) a polypeptide comprising a sequence represented by SEQ ID NO:1 or (2) a polypeptide comprising 95% or more identity to a sequence represented by SEQ ID NO:1 and having an activity as a blood coagulation factor VIII, and a step of obtaining an activity value of the blood coagulation factor VIII in the blood specimen based on the obtained parameter.)

1. A method for measuring blood clotting activity, comprising:

a step of obtaining a parameter related to a differential of a coagulation waveform in a blood specimen collected from a patient to whom the following polypeptide is administered:

(1) 1 or a polypeptide having a sequence represented by SEQ ID NO

(2) A polypeptide comprising a polypeptide having a homology of 95% or more with the sequence represented by SEQ ID NO. 1 and having an activity as blood coagulation factor VIII, and

and obtaining an activity value of blood factor VIII in the blood subject based on the obtained parameter.

2. The method for measuring blood coagulation activity according to claim 1, wherein the parameter is obtained based on a coagulation waveform measured by a coagulation section method.

3. The method for measuring blood coagulation activity according to claim 2, wherein the measurement by the coagulation section method is a measurement of activated partial thromboplastin time.

4. The method of measuring blood coagulation activity according to any one of claims 1 to 3, wherein the parameter is obtained from at least one selected from the group consisting of a waveform of a coagulation velocity obtained by first-order derivation of a coagulation waveform and a waveform of a coagulation acceleration obtained by second-order derivation of a coagulation waveform.

5. The method for measuring blood coagulation activity according to claim 4, wherein the parameter is a value representing at least one selected from the group consisting of a maximum coagulation velocity, a maximum coagulation acceleration, a maximum coagulation deceleration, a corrected maximum coagulation velocity, a corrected maximum coagulation acceleration, and a corrected maximum coagulation deceleration.

6. The method for measuring blood coagulation activity according to any one of claims 1 to 5, wherein the activity value of blood factor VIII in the blood specimen collected from the patient is obtained based on a calibration curve prepared from a plurality of standard plasma samples in which the activity value of blood factor VIII is known and the activity values are different from each other.

[ technical field ] A method for producing a semiconductor device

In the present specification, a method for measuring blood coagulation activity is disclosed.

[ background of the invention ]

Lonocog Alfa described in patent document 1 is a factor VIII analog, which is one of human blood coagulation factors produced by gene recombination, and is used for a factor VIII deficient patient for the purpose of suppressing bleeding tendency.

Coagulation activity in patients administered Lonocicepg Alfa is typically monitored by coagulation-segment assays that Activate Partial Thromboplastin Time (APTT) and the like. However, when the activity of lonocog Alfa after application was measured by the one-stage clotting method, the measurement result showed an apparent low value of HIGHLIGHTS OF PRESCRIBING INFORMATION (non-patent document 1) of AFSTYLA (trademark). Therefore, non-patent document 1 indicates that, when the activity of lonocoag Alfa in plasma is measured by the coagulation one-stage method, a value obtained by multiplying the measurement value of factor VIII obtained by the coagulation one-stage method by a conversion factor of 2 is used as the measurement value of factor VIII of a patient.

[ Prior art documents ]

[ patent document ]

[ patent document 1 ] specification of U.S. Pat. No. 7041635

[ non-patent literature ]

[ non-patent document 1 ] AFSTYLA (trademark) HIGHLIGHTS OF PRESCRIBING INFORMATION (https:// labeling. cslbehring. com/PI/US/Afstyla/EN/Afstyla-Presscription-information. pdf)

[ SUMMARY OF THE INVENTION ]

[ problem to be solved by the invention ]

However, according to the instructions described in non-patent document 1, there is a need to specify a subject of a patient to whom lonocog Alfa is administered from among a large number of measurement values, and multiply the specified subject by a conversion factor, which not only requires a complicated process but also may cause a human error.

The present invention aims to provide a method for measuring blood coagulation activity, which can facilitate measurement of a subject of a patient to whom Lonocicepg Alfa is administered and can suppress human error.

[ MEANS FOR SOLVING PROBLEMS ] to solve the problems

Certain embodiments of the present invention relate to methods of measuring blood clotting activity. The measurement method comprises the following steps: a step of obtaining a parameter related to differentiation of a coagulation waveform in a blood specimen collected from a patient to whom (1) a polypeptide comprising a sequence represented by SEQ ID NO:1 or (2) a polypeptide comprising 95% or more identity to a sequence represented by SEQ ID NO:1 and having a polypeptide having a blood coagulation factor VIII activity is administered, and a step of obtaining a blood coagulation factor VIII activity value in the blood specimen based on the obtained parameter.

According to this embodiment, the factor VIII activity of Lonocicepg Alfa can be measured more accurately.

[ Effect of the invention ]

A method for measuring blood coagulation activity, which can facilitate the measurement of a subject of a patient to whom Lonocicepg Alfa is administered and can suppress human error, can be provided.

[ brief description of the drawings ]

Fig. 1(a) shows an example of a coagulation waveform. (B) An example of the coagulation waveform after the correction is shown.

FIG. 2 shows the factor VIII activity of each preparation calculated based on the clotting time. (A) The activity values for factor VIII deficient plasma at 1.00IU/dL for each preparation are shown. (B) The activity values for factor VIII deficient plasma at 0.30IU/dL for each preparation are shown. (C) The activity values for factor VIII deficient plasma at 0.05IU/dL for each preparation are shown.

Fig. 3(a) shows the respective parameter values of advance and Afstyla. (B) The first derivative waveform of the APTT coagulation waveform of Advate and Afstyla is shown. (C) The second derivative waveform of the APTT coagulation waveform of Advate and Afstyla is shown.

Fig. 4(a) shows the first derivative waveform of the modified APTT coagulation waveforms of advance and Afstyla. (B) The second derivative waveform of the modified APTT coagulation waveform of Advate and Afstyla is displayed.

Fig. 5(a) shows a calibration curve for min 1. (B) A calibration curve for min2 is shown.

Fig. 6(a) shows a calibration curve of Ad | min1 |. (B) A calibration curve for Max2 is shown.

FIG. 7A shows the activity value (IU/dL) of Afstyla obtained based on the differential correlation parameter value of each APTT coagulation waveform. (B) The addition recovery (%) is shown.

Fig. 8 shows the activity value of factor VIII of each preparation calculated based on the maximum coagulation rate min 1. (A) The activity values for factor VIII deficient plasma at 1.00IU/dL for each preparation are shown. (B) The activity values for factor VIII deficient plasma at 0.30IU/dL for each preparation are shown. (C) The activity values for factor VIII deficient plasma at 0.05IU/dL for each preparation are shown.

FIG. 9 shows the activity values of factor VIII of each preparation calculated based on the corrected maximum coagulation velocity Ad | min1 |. Figure 9(a) shows the activity values for factor VIII deficient plasma at 1.00IU/dL with each formulation. (B) The activity values for factor VIII deficient plasma at 0.30IU/dL for each preparation are shown. (C) The activity values for factor VIII deficient plasma at 0.05IU/dL for each preparation are shown.

Fig. 10 shows the activity value of factor VIII of each preparation calculated based on the maximum coagulation acceleration min 2. (A) The activity values for factor VIII deficient plasma at 1.00IU/dL for each preparation are shown. (B) The activity values for factor VIII deficient plasma at 0.30IU/dL for each preparation are shown. (C) The activity values for factor VIII deficient plasma at 0.05IU/dL for each preparation are shown.

Fig. 11 shows the activity value of factor VIII of each preparation calculated based on the maximum coagulation deceleration max 2. (A) The activity values for factor VIII deficient plasma at 1.00IU/dL for each preparation are shown. (B) The activity values for factor VIII deficient plasma at 0.30IU/dL for each preparation are shown. (C) The activity values for factor VIII deficient plasma at 0.05IU/dL for each preparation are shown.

[ detailed description ] embodiments

[ 1 ] description of wording ]

In the present specification, the target for obtaining the activity value of blood factor VIII is a polypeptide having a predetermined amino acid sequence and having an activity as blood factor VIII. In the present specification, blood factor VIII is simply referred to as "factor VIII", and a polypeptide having an activity as blood factor VIII may be simply referred to as "polypeptide".

Preferably, the polypeptide is a polypeptide comprising the sequence represented by SEQ ID NO. 1. The polypeptide having the sequence represented by SEQ ID NO. 1 may also be modified by disulfide bond, sugar chain binding, sulfation, or the like.

For example, disulfide bonds may be formed between at least 1 cysteine residue selected from the group consisting of 153 th and 179 th cysteine residues, 248 th and 321 th cysteine residues, 528 th and 554 th cysteine residues, 630 th and 711 th cysteine residues, 944 th and 971 th cysteine residues, 1111 th and 1115 th cysteine residues, 1131 th and 1281 th cysteine residues, and 1286 th and 1436 th and 1438 th cysteine residues of SEQ ID NO. 1.

At least 1 asparagine residue selected from 71, 239, 757, 764, 922, and 1230 of SEQ ID NO. 1 can have a sugar chain bound thereto.

At least 1 of the amino acid residues of SEQ ID NO. 1 selected from the group consisting of 741 th serine residue, 743 th serine residue, 746 th serine residue, 759 th threonine residue, 760 th threonine residue, 765 th threonine residue, 766 th threonine residue, 769 th serine residue, and 781 th serine residue may be bonded with a sugar chain.

At least 1 tyrosine residue of SEQ ID NO. 1 selected from the group consisting of 346 th, 718 th, 719 th, 723 th, 776 th and 792 may be sulfated.

In the present specification, the "residue" of each amino acid refers to a constituent unit of an amino acid constituting a polypeptide, and is a group obtained by removing a hydrogen atom from an amino group of a main chain and/or removing an-OH group from a carboxyl group of the main chain.

The polypeptide may have a certain proportion of homology with the sequence represented by SEQ ID NO. 1. The specific ratio is not limited as long as it is a ratio of the activity as factor VIII to the same extent as or more than that of a polypeptide having a sequence represented by SEQ ID NO. 1. For example, a certain ratio is 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the sequence represented by SEQ ID NO. 1. Preferably, a certain ratio means 95%, 98%, or 99%.

Examples of the amino acid substitution that maintains the same degree of or more than the sequence represented by SEQ ID NO. 1 and that is the activity of factor VIII include, for example, substitution between amino acids belonging to the class to which each amino acid belongs. For example, where the like is a non-polar (hydrophobic) amino acid, alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine may be included. When the polar neutral amino acid is used, the amino acid may contain glycine, serine, threonine, cysteine, tyrosine, asparagine, or glutamine. When the group is a basic amino acid, arginine, lysine, and histidine may be contained. When the amino acid is an acidic amino acid, aspartic acid and glutamic acid may be contained. The method for measuring the activity of factor VIII is as follows. The above polypeptides may be produced by gene recombination techniques.

In addition, the amino acids constituting the polypeptide may be artificial amino acids. Furthermore, the polypeptide may be modified in addition to the above. Examples of the modification include pegylation modification, phosphorylation modification, acetylation modification, methylation modification, fluorescence modification, biotinylation modification, sugar modification, lipid modification, acylation modification, reductive amination modification, azide modification, and enone modification.

The most preferred polypeptide is lonocoag Alfa [ trade name: afstyla (trade Mark) ].

The patient is not limited as long as administration of factor VIII is required. For example, a patient with factor VIII deficiency may be mentioned. Examples of the factor VIII deficiency include hemophilia a, disseminated intravascular coagulation syndrome, and liver dysfunction.

The blood specimen is a blood sample collected from a patient, and is not limited as long as it can measure blood coagulation activity described below. Examples of the blood sample include whole blood and plasma. The blood sample is preferably collected by using an anticoagulant other than heparin preparation at the time of blood collection. As the anti-coagulating agent, collection using a citrate, for example, a sodium citrate solution is more preferable. Most preferably, the blood sample is plasma separated from a whole blood sample mixed so that the volume of the anticoagulation agent and the patient's whole blood is about 1:8.5 to 9.5, using a 3.1 to 3.3% (w/v) trisodium citrate solution as the anticoagulation agent.

[ 2 ] method for measuring blood coagulation Activity

Certain embodiments of the present invention relate to methods of measuring blood clotting activity.

The assay method may comprise: a step of obtaining a parameter related to differentiation of a coagulation waveform in a blood specimen collected from a patient to whom (1) the polypeptide having a sequence represented by SEQ ID NO:1 or (2) the polypeptide having 95% or more identity to the sequence represented by SEQ ID NO:1 and having a polypeptide having an activity as a blood coagulation factor VIII as described in the above 1, and a step of obtaining an activity value of the blood coagulation factor VIII in the blood specimen based on the obtained parameter.

Referring to fig. 1(a), a coagulation waveform will be described. The coagulation waveform shown in fig. 1(a) is a coagulation waveform obtained by a method of measuring blood coagulation activity, which is generally called a one-stage coagulation method. The coagulation-one-stage method is a method in which calcium ions necessary for blood coagulation and a predetermined test reagent are added to a blood sample (hereinafter referred to as a "test sample") containing fibrinogen to be measured for coagulation time, light is irradiated to a reaction solution, and the change in optical properties of the reaction solution with time is monitored to measure the coagulation time.

Fig. 1(a) shows an example of a coagulation waveform. In fig. 1 a, the vertical axis (Y axis) represents transmitted light intensity, and the horizontal axis (X axis) represents monitoring light intensityThe measurement time (sec) of the intensity of transmitted light was measured. The coagulation waveform can be generated by plotting the temporal change in the monitored transmitted light intensity with a biaxial plot of the transmitted light intensity and the measurement time. The first point in FIG. 1A is the time point when calcium ions and the test reagent are added to the test sample, and the time point when the measurement is started (t)_I). Since fibrinogen in the reaction solution did not change into fibrin at the start of the measurement, fibrin deposition did not occur in the reaction solution, and the transmitted light intensity showed a high value. Thereafter, when the coagulation reaction proceeds and fibrin begins to precipitate, the intensity of transmitted light begins to decrease. This is because the precipitated fibrin blocks light. This point is point II in fig. 1(a), and is the coagulation start time. The measurement time of the initiation of coagulation is represented by (t)_II) And (4) showing. As the reaction proceeds, fibrin deposition proceeds, and the transmitted light intensity decreases. When most of fibrinogen in the test sample becomes fibrin, the reaction converges, and the change in the transmitted light intensity becomes a steady state. This point is the III point in FIG. 1(A), and is the coagulation completion time. The measurement time of the completion of coagulation is represented by (t)_III) And (4) showing. The Coagulation Time (CT) may be generally represented by CT (sec) [ (t)_III)-(t_II)]The measurement time/2 is shown. Where "-" means minus, "/" means dividing. That is, parameters relating to coagulation activity such as a coagulation start time, a coagulation end time, and a coagulation time can be obtained from the coagulation waveform.

However, since the initial fibrinogen content in the test sample may be low depending on the patient, it is preferable to determine the coagulation time by normalizing the coagulation waveform as shown in fig. 1 (B). The normalization can be achieved, for example, by assuming that the change amount (dH) of the transmitted light intensity, which is the difference between the transmitted light intensity at the point II when the coagulation reaction starts and the transmitted light intensity at the point III when the coagulation ends, is 100%, and making the change in the transmitted light intensity a relative value. In this case, the Coagulation Time (CT) may be set to a point of time when the amount of change (dH) in the transmitted light intensity is, for example, 30%, 40%, 50%, or 60%. In a preferred embodiment, the coagulation time is a time when the change amount (dH) of the transmitted light intensity becomes 50%.

In the present specification, the "coagulation waveform" may include a coagulation waveform generated without being normalized with respect to the monitored transmitted light intensity and a coagulation waveform (hereinafter, referred to as "corrected coagulation waveform") generated based on corrected monitoring data obtained by performing the above-described normalization with respect to the monitored transmitted light intensity. As the coagulation waveform, a modified coagulation waveform is preferably used. In the present specification, the coagulation time obtained from the corrected coagulation waveform is referred to as "corrected coagulation time".

Examples of the method for measuring the blood coagulation activity inclusively, which can be measured by the coagulation section method, include Activated Partial Thromboplastin Time (APTT), Prothrombin Time (PT), and the like. In addition, various coagulation factor activities can also be measured by the coagulation one-stage method, which can be measured by the inclusionary measurement method. The blood coagulation factors capable of determining the activity by APTT include factor VIII, factor V, factor X, factor IX, factor XI, factor XII, prekallikrein, polymeric kininogen, prothrombin, fibrinogen and the like. The coagulation factors whose activity can be measured by PT include factor VII, factor V, factor X, prothrombin, fibrinogen and the like.

The present embodiment obtains the activity value of factor VIII. Factor VIII activity values can be obtained using APTT. In the "obtaining", the activity value may be calculated or may be received from another source.

In the measurement of APTT, the specified inspection reagent may contain an activator of silica, ellagic acid, diatomaceous earth, or the like; animal-derived, plant-derived, or synthetic phospholipids, and the like. As the test reagent for APTT measurement, a commercially available test reagent can be used. Examples thereof include ThromboCheckAPTT series manufactured by Sysmex corporation, COAGPAAPTT-N manufactured by Water Medical corporation, and Datafi/APTT of Siemens Healthcare Diagnostics Products GmbH. In addition, according to the international standard method, calcium ions can be supplied from a 20mM calcium chloride solution.

The test sample, the test reagent for APTT measurement, and calcium ions were mixed in a predetermined diluent. Examples of the diluent include a solution or a buffer that is isotonic with human plasma like physiological saline but has no pH adjusting function. Examples of the buffer include Owren's Veronal buffer and imidazole buffer. As the diluent, Owren's Veronal buffer is preferable.

In the above, an example is shown in which the detection of the deposition of fibrinogen is detected from a change in the intensity of transmitted light, but the intensity of transmitted light may be changed to the intensity of transmitted light, and the amount of scattered light, absorbance, or the like may be used.

The measurement of APTT can also be measured using a blood coagulation measuring device. Examples of the blood coagulation measuring device include fully automatic blood coagulation test devices 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 the present embodiment, since it is necessary to acquire parameters relating to the differentiation of the coagulation waveform, it is preferable to use a fully automatic blood coagulation inspection apparatus equipped with software for performing differentiation processing of the coagulation waveform.

The factor VIII activity value is known and is obtained based on a calibration curve prepared from a plurality of standard plasma samples having different activity values. According to the conventional method, the factor VIII activity value using APTT is obtained based on a calibration curve of the factor VIII activity value prepared for the parameter relating to clotting activity obtained from the test sample and the parameter corresponding to the parameter relating to clotting activity. The calibration curve is prepared from parameters relating to the clotting activity obtained by APTT measurement of standard plasma samples diluted in multiple stages, such as normal saline or factor VIII deficient plasma, for blood clotting tests. When the standard human plasma for blood coagulation test is used for preparing the calibration curve, a sample in which the activity value of the factor VIII is known is used as a standard plasma sample in which the activity value of the factor VIII is known, so that the activity value of the factor VIII of the standard human plasma for blood coagulation test is at least 1 selected from 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, and 10% in theory, assuming that the activity of the factor VIII is 100%. Among the standard plasma samples, the standard plasma sample having a factor VIII activity value of 100% contains standard human plasma for blood coagulation test. In addition, among the standard plasma samples, the factor VIII deficient plasma can be contained as a standard plasma sample having a factor VIII activity value of 0%. Thus, a plurality of standard plasma samples having different factor VIII activity values can be prepared from standard human plasma in a blood coagulation test.

The standard human plasma for blood coagulation test may be a mixed plasma of plasma collected from a plurality of persons having no abnormality in blood coagulation function, or may be a standard human plasma for blood coagulation test sold by Siemens co. Factor VIII deficient plasma is commercially available, for example, from George King Bio-Medical, Inc. (USA), and the like.

Further, a blood factor VIII preparation such as crosseiightmc (japan blood products institute), Kovaltry (trademark) (Bayer chemical corporation), Advate (martial arts chemical corporation), Adynovate (trademark) (martial arts chemical corporation), NovoEight (trademark) (Novo Nordisk Pharma corporation), and elotate (trademark) (Sanofi corporation) may be added to the factor VIII deficient plasma to prepare a standard plasma sample. Since the activity value of factor VIII is known, the factor VIII preparation can be added to factor VIII-deficient plasma so that the activity value becomes a desired value, and a plurality of standard plasma samples having different activity values can be prepared.

The parameter relating to clotting activity used in the determination of the factor VIII activity value is generally clotting time. However, in the present embodiment, a differential correlation parameter of the coagulation waveform is used to obtain the activity value of the factor VIII. The differential correlation parameter of the coagulation waveform is obtained by performing differential processing on the coagulation waveform obtained by monitoring the change with time of the optical property of the reaction liquid in the APTT measurement (hereinafter referred to as "original coagulation waveform"). The differential processing is described in U.S. patent application publication No. 2018-0306820, which is incorporated herein by reference.

For example, a first-order-derived coagulation waveform obtained by first-order derivation of an original coagulation waveform is a parameter of a coagulation speed. The peak of the first-order derived coagulation waveform indicates the maximum coagulation velocity. In this specification, the maximum solidification rate is sometimes represented as "min 1". In addition, the value of the coagulation waveform derived from the first order of the coagulation velocity may be expressed in absolute value, and in this case, the maximum coagulation velocity may be expressed in "| min1 |".

Then, the second-order derivative solidification waveform is obtained by performing second-order derivative processing on the original solidification waveform. The second order derivative coagulation waveform is a parameter of coagulation acceleration and coagulation deceleration. In the second-order derived coagulation waveform, the peak point obtained in the coaxial direction with the peak of the first-order derived coagulation waveform indicates the maximum coagulation acceleration. In this specification, the maximum solidification acceleration is sometimes represented as "min 2". The maximum coagulation acceleration is sometimes expressed in absolute value, and in this case, "| min2 |". In the second-order derived coagulation waveform, a peak in the axial direction on the opposite side with respect to the axial direction representing the acceleration represents the maximum coagulation deceleration. In this specification, the maximum solidification acceleration is sometimes indicated as "max 2". The maximum coagulation deceleration is sometimes expressed in absolute values, and in this case may be expressed as "| max2 |".

The first order derivation described above may also be performed to correct the original coagulation waveform. In this specification, a first-order derived coagulated waveform generated from a modified coagulated waveform is referred to as a modified first-order derived coagulated waveform. In addition, correcting the peak of the first-order derived coagulation waveform indicates correcting the maximum coagulation velocity. The corrected maximum coagulation speed is sometimes expressed in the present specification by "Ad | min1 |".

Further, a second-order derivation process is performed on the corrected original coagulation waveform to obtain a corrected second-order derivation coagulation waveform. In the modified second-order derived solidification waveform, a peak apex obtained in the coaxial direction with the peak of the modified first-order derived solidification waveform indicates a modified maximum solidification acceleration. In the present specification, the maximum coagulation acceleration is sometimes expressed as "Ad | min2 |". In the corrected second-order derived coagulation waveform, a peak in the axial direction on the opposite side with respect to the axial direction indicating the acceleration indicates the corrected maximum coagulation deceleration. In the present specification, the maximum coagulation acceleration is sometimes expressed as "Ad | max2 |".

The differential correlation parameter of the coagulation waveform may be any parameter that reflects the shape of the first-order derived coagulation waveform, the modified first-order derived coagulation waveform, the second-order derived coagulation waveform, and the modified second-order derived coagulation waveform. For example, in the present embodiment, the calibration curve is prepared using differential-related parameters of at least 1 coagulation waveform selected from the group consisting of the maximum coagulation speed, the maximum coagulation acceleration, the maximum coagulation deceleration, the corrected maximum coagulation speed, the corrected maximum coagulation acceleration, and the corrected maximum coagulation deceleration. Further, the first-order derived coagulated waveform, the corrected first-order derived coagulated waveform, the second-order derived coagulated waveform, and the corrected second-order derived coagulated waveform have an Area under the curve (Area under curve) and a center of gravity position of an Area under the curve, and the like, which are used as differential correlation parameters of the coagulated waveform. Further, the value of the parameter relating to the differential coagulation waveform corresponding to the calibration curve obtained from the test sample is applied to the prepared calibration curve, and the activity value of the factor VIII in the test sample is obtained.

The preparation of the calibration curve, the acquisition of the parameter related to the differentiation of the coagulation waveform, and the acquisition of the activity value of the factor VIII contained in the test sample can be performed by software installed in the fully automatic blood coagulation test apparatus.

[ examples ] A method for producing a compound

Next, the present embodiment will be described in more detail with reference to examples. However, the present invention is not limited to the embodiment.

[ 1 ] materials and methods ]

[ 1 ] preparation of test sample ]

Each test sample was prepared by adding a commercially available factor VIII preparation to an innate factor VIII deficient plasma (George King Bio-Medical, Inc. (USA)) to a final activity value of 0.05IU/dL, 0.30IU/dL, or 1.00IU/dL, based on the activity value described in the attached document for each drug.

The factor VIII preparation used is as follows. Crosseiightmc is a plasma fractionation preparation, and further the preparation is a recombinant preparation.

CrossEightMC (Japan blood preparation agency)

Kovaltry (trademark) (Octocog Beta; Bayer drug Co., Ltd.)

Advate (Ruriocotog Alfa; Wutian chemical industry Co., Ltd.)

Adynovate (trade mark) (Ruriotocog Alfa Pegol; Wutian chemical industry Co., Ltd.)

NovoEight (trademark) (Turococog Alfa; Novo Nordisk Pharma corporation)

Elockate (trade mark) (Efractocog Alfa; Sanofi Kabushiki Kaisha)

Afstyla (trade mark) (Lonococog Alfa; CSLBehring Co., Ltd.)

[ 2 ] measurement reagent

The following reagents were used for the measurement.

ThromboCheckAPTT-SLA (Sysmex corporation)

20mM calcium chloride solution (Sysmex Co., Ltd.)

Factor VIII deficient plasma (Siemens Co., Ltd.)

Owren's Veronal buffer (Siemens Co., Ltd.)

Standard human plasma for blood coagulation test (Siemens Co., Ltd.)

[ 3 ] measurement device and measurement procedure

For measurement, measurement was carried out by a default flow using a fully automatic blood coagulation measuring apparatus CS-2400(Sysmex Co., Ltd.). The Activated Partial Thromboplastin Time (APTT) was measured by performing the following one-step coagulation method using a computer program loaded on a measuring apparatus, and the factor VIII activity value of the test sample was obtained.

Step 1: diluting the aspirated test sample by 20 times with Owren's Veronal buffer solution, and dispensing 40. mu.L of the diluted sample into a reaction cuvette;

step 2: adding 40. mu.L of the factor VIII deficient plasma to the diluted test sample, incubating the mixture, and preparing a 1 st reaction solution;

and step 3: adding ThromboCheckAPTT-SLA 40. mu.L to the prepared 1 st reaction solution, incubating, and preparing a 2 nd reaction solution;

and 4, step 4: 40. mu.L of a 20mM calcium chloride solution was added to the 2 nd reaction solution to start the coagulation reaction, and the change in the intensity of transmitted light with time was monitored by measuring the transmitted light at a wavelength of 660nm for a prescribed time;

and 5: detecting a coagulation start point and a coagulation end point from the monitoring data, and calculating a Coagulation Time (CT);

step 6: the calculated Clotting Time (CT) is applied to a calibration curve for factor VIII to calculate factor VIII activity of the subject.

In this case, a calibration curve for factor VIII was prepared using a standard plasma sample obtained by diluting standard human plasma for a blood coagulation test with factor VIII-deficient plasma in stages. At this time, the factor VIII activity value of the undiluted human plasma for blood coagulation test was set to 100%. The standard plasma sample was also measured in the same manner as the test sample, and the factor VIII activity of the coagulation time and the dilution ratio was used as the coagulation time to prepare a calibration curve.

[ 4 ] acquisition of differential correlation parameters of APTT coagulation waveform

Min1, min2, Ad | min1| and max2 were calculated as parameters related to the differentiation of the APTT coagulation waveform. The differential correlation parameters of the APTT coagulation waveform were calculated for the test sample and the standard plasma sample.

And (4) taking the monitoring data obtained in the step (3) and the step 4 as APTT solidification waveform data. The APTT coagulation waveform was plotted with the X-axis as the measurement time and the Y-axis as the transmitted light intensity to obtain monitoring data.

Further, according to the method described in U.S. patent application No. 2018-0306820, the first-order derivative coagulation waveform and the second-order derivative coagulation waveform of the APTT coagulation waveform are generated by differentiating the APTT coagulation waveform. The peak min1 of the waveform of the velocity is further derived from the first order derivative coagulation waveform. min1 denotes maximum coagulation speed. The peak value min2 of the coagulation acceleration and the absolute value max2 of the peak value of the coagulation deceleration are obtained from the second order derivative of the coagulation waveform. min2 represents the maximum coagulation acceleration. max2 represents the maximum coagulation deceleration. Further, the absolute value | min1| of the peak value of the waveform of the velocity is obtained as a corrected absolute value Ad | min1| from the corrected first-order derived coagulated waveform generated based on the normalized APTT coagulated waveform data. A modified first-order derived coagulation waveform and a modified second-order derived coagulation waveform are generated from the normalized APTT coagulation waveform.

Further, a calibration curve of the factor VIII activity value based on each parameter was prepared based on the parameter relating to the differentiation of the APTT coagulation waveform obtained from the standard plasma sample. Based on this calibration curve, the factor VIII activity value of each test sample was calculated for each parameter.

[ 2. results ]

[ 1 ] comparison of factor VIII Activity of the preparations

FIG. 2 shows the factor VIII activity values (IU/dL) of each preparation calculated based on the Clotting Time (CT) determined by the APTT method. The measurement was performed on each test sample n ═ 3. FIG. 2(A) shows the activity values for the addition of each preparation to an innate factor VIII deficient plasma at 1.00 IU/dL. FIG. 2(B) shows the activity values at 0.30IU/dL for the addition of each preparation to the congenital factor VIII deficient plasma. FIG. 2(C) shows the activity values at 0.05IU/dL for each preparation added to the congenital factor VIII deficient plasma. In any activity, only Afstyla showed low values.

[ 2 ] data comparison between differential correlation parameters of APTT coagulation waveforms

Fig. 3(a) shows the respective parameter values of Advate and Afstyla having the largest difference in the set time (CT) among the results of (1) above. The units of CT are seconds, and the additional data represent calculated values. In addition, fig. 3(B) shows the first derivative waveform of the APTT coagulation waveform of both. Fig. 3(C) shows the second derivative waveform of the APTT coagulation waveform of both. Fig. 4(a) shows the first derivative waveform of the corrected APTT coagulation waveform of both. Fig. 4(B) shows the second derivative waveform of the modified APTT coagulation waveform of both. In the first derivative waveform, the Y-axis is represented by the coagulation rate (dT/dT). Where 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 is at the solidification velocity (dT)²/dt²) And (4) showing.

As shown in FIG. 3(A), when CT is observed, the square CT of Afstyla is long, indicating that the activity of factor VIII is low. However, when min1, Ad | min1|, and max2 are compared, the difference in the factor VIII activity values representing Advate and Afstyla is smaller than the difference in the factor VIII activity values of Advate and Afstyla when CT is compared.

[ 3 ] calibration curve of differential correlation parameter per APTT coagulation waveform

Next, calibration curves prepared for the differential-related parameters of each APTT coagulation waveform are shown in fig. 5 and 6. Fig. 5(a) shows a calibration curve for min1, and fig. 5(B) shows a calibration curve for min 2. Fig. 6(a) shows a calibration curve of Ad | min1|, and fig. 6(B) shows a calibration curve of Max 2. The axes of the calibration curve are shown in logarithmic scale. The Y-axis represents the value of each parameter and the X-axis represents the activity value of factor VIII. The linearity of the calibration curve for any parameter is good, indicating an evaluable factor VIII activity value.

[ 4 ] calculation of Activity value of Afstyla Using differential correlation parameter of APTT coagulation waveform ]

APTT coagulation data was obtained by adding Afstyla to an innate factor VIII deficient plasma to final activity values of 5IU/dL, 30IU/dL, and 100IU/dL, based on the activity values described in the accompanying documents. Based on this data, values of differential correlation parameters of the respective APTT coagulation waveforms are obtained, and applied to a calibration curve to calculate the activity value of factor VIII. The results are shown in FIG. 7. Fig. 7(a) shows the activity value of factor VIII, and fig. 7(B) shows the addition recovery rate (%). The activity value of factor VIII of Afstyla calculated using the differential-related parameter of the APTT coagulation waveform shows a value higher than the activity value calculated from CT, and shows a value close to the theoretical value. In addition, the recovery rate is also good.

[ 5 ] factor VIII Activity calculated Using differential correlation parameters of APTT coagulation waveforms

In fig. 2, for the test samples showing activity, the activity value of factor VIII was calculated using the differential correlation parameter of the APTT coagulation waveform.

Fig. 8 shows the activity value of factor VIII of each preparation calculated based on the maximum coagulation rate min 1. FIG. 8(A) shows the activity values for the addition of each preparation to an innate factor VIII deficient plasma at 1.00 IU/dL. FIG. 8(B) shows the activity values for the addition of each preparation to an innate factor VIII deficient plasma at 0.30 IU/dL. FIG. 8(C) shows the activity values for the addition of each preparation to an innate factor VIII deficient plasma at 0.05 IU/dL.

FIG. 9 shows the activity values of factor VIII of the respective preparations calculated based on the corrected maximum coagulation velocity Ad | min1 |. FIG. 9(A) shows the activity values for the addition of each preparation to an innate factor VIII deficient plasma at 1.00 IU/dL. Figure 9(B) shows the activity values for the addition of each preparation to 0.30IU/dL for the native factor VIII deficient plasma. FIG. 9(C) shows the activity values for the addition of each preparation to an innate factor VIII deficient plasma at 0.05 IU/dL.

Fig. 10 shows the activity value of factor VIII of each preparation calculated based on the maximum coagulation acceleration min 2. FIG. 10(A) shows the activity values for the addition of each preparation to an innate factor VIII deficient plasma at 1.00 IU/dL. FIG. 10(B) shows the activity values for the addition of each preparation to an innate factor VIII deficient plasma at 0.30 IU/dL. FIG. 10(C) shows the activity values for the addition of each preparation to an innate factor VIII deficient plasma at 0.05 IU/dL.

Fig. 11 shows the activity value of factor VIII of each formulation calculated based on the maximum coagulation deceleration max 2. FIG. 11(A) shows the activity values for the addition of each preparation to an innate factor VIII deficient plasma at 1.00 IU/dL. FIG. 11(B) shows the activity values for the addition of each preparation to an innate factor VIII deficient plasma at 0.30 IU/dL. FIG. 11(C) shows the activity values for the addition of each preparation to an innate factor VIII deficient plasma at 0.05 IU/dL. Afstyla shows a value higher than the activity value of factor VIII calculated based on the coagulation rate, and the difference from the activity value of other preparations is eliminated.

Therefore, it is useful to use the differential-related parameter of the coagulation waveform without using the coagulation speed in determining the application effect of afstyla (lonocog alfa).

In addition, it becomes unnecessary to specify the subject of the patient to whom the Afstyla (Lonocicepg Alfa) is administered with respect to the obtained activity value, making it possible to easily measure and suppress human error.

Sequence listing

<110> KEIO UNIVERSITY

TOKYO SAISEIKAI CENTRAL HOSPITAL

SYSMEX CORPORATION

<120> method for measuring blood coagulation activity

<130> 19-048JP

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1444

<212> PRT

<213> Artificial sequence

<220>

<223> recombinant factor VIII

<400> 1

Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser Trp Asp Tyr

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Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg Phe Pro Pro

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Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val Tyr Lys Lys

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Thr Leu Phe Val Glu Phe Thr Val His Leu Phe Asn Ile Ala Lys Pro

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Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln Ala Glu Val

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Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser His Pro Val

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Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser Glu Gly Ala

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Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp Asp Lys Val

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Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu Lys Glu Asn

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Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser Tyr Leu Ser

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His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile Gly Ala Leu

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Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr Gln Thr Leu

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His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly Lys Ser Trp

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His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp Ala Ala Ser

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Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr Val Asn Arg

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Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val Tyr Trp His

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Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile Phe Leu Glu

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Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser Leu Glu Ile

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Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met Asp Leu Gly

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Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His Asp Gly Met

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Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro Gln Leu Arg

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Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp Leu Thr Asp

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Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser Pro Ser Phe

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Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr Trp Val His

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Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro Leu Val Leu

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Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn Asn Gly Pro

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Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met Ala Tyr Thr

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Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu Ser Gly Ile

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Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu Leu Ile Ile

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Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro His Gly Ile

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Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys Gly Val Lys

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His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe Lys Tyr Lys

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Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp Pro Arg Cys

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Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg Asp Leu Ala

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Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu Ser Val Asp

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Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val Ile Leu Phe

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Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu Asn Ile Gln

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Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp Pro Glu Phe

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Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val Phe Asp Ser

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Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp Tyr Ile Leu

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Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe Ser Gly Tyr

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Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr Leu Phe Pro

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Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro Gly Leu Trp

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Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly Met Thr Ala

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Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp Tyr Tyr Glu

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Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys Asn Asn Ala

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Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Pro Ser Thr Arg

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Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro Glu Asn Thr Thr Leu Gln

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Ser Asp Gln Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu Met

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Lys Lys Glu Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro

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Arg Ser Phe Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu

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Arg Leu Trp Asp Tyr Gly Met Ser Ser Ser Pro His Val Leu Arg Asn

820 825 830

Arg Ala Gln Ser Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln

835 840 845

Glu Phe Thr Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu

850 855 860

Asn Glu His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu

865 870 875 880

Asp Asn Ile Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser

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Phe Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala

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Glu Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe

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Trp Lys Val Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys

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Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His

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Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn

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Pro Ala His Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe

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Thr Ile Phe Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu

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Arg Asn Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr

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Phe Lys Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met

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Asp Thr Leu Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg

1040 1045 1050

Trp Tyr Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile

1055 1060 1065

His Phe Ser Gly His Val Phe Thr Val Arg Lys Lys Glu Glu Tyr

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Lys Met Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val

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Glu Met Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys Leu

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Ile Gly Glu His Leu His Ala Gly Met Ser Thr Leu Phe Leu Val

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Tyr Ser Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His

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Ile Arg Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp

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Ala Pro Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala

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Trp Ser Thr Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu

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Ala Pro Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln

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Lys Phe Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser

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Leu Asp Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly

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Thr Leu Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys

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His Asn Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu

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His Pro Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu

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Met Gly Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu

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Ser Lys Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe

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Thr Asn Met Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg Leu His

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Leu Gln Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn Asn Pro

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Lys Glu Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val Thr

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Gly Val Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr

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Val Lys Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp

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Thr Leu Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn

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Gln Asp Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu

1400 1405 1410

Leu Thr Arg Tyr Leu Arg Ile His Pro Gln Ser Trp Val His Gln

1415 1420 1425

Ile Ala Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu

1430 1435 1440

Tyr