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WO2022003349A1 - Method for measuring the activity of a blood clotting factor - Google Patents

Method for measuring the activity of a blood clotting factor Download PDF

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Publication number
WO2022003349A1
WO2022003349A1 PCT/GB2021/051654 GB2021051654W WO2022003349A1 WO 2022003349 A1 WO2022003349 A1 WO 2022003349A1 GB 2021051654 W GB2021051654 W GB 2021051654W WO 2022003349 A1 WO2022003349 A1 WO 2022003349A1
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WO
WIPO (PCT)
Prior art keywords
blood clotting
clotting factor
sample
factor
activity
Prior art date
Application number
PCT/GB2021/051654
Other languages
French (fr)
Inventor
Jonathan Foley
Erald SHEHU
Original Assignee
Freeline Therapeutics Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Freeline Therapeutics Limited filed Critical Freeline Therapeutics Limited
Publication of WO2022003349A1 publication Critical patent/WO2022003349A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins

Definitions

  • composition or kit of the invention comprising FVIIIa and/or FIXa may comprise FVIII and an agent for activating FVIIIa, and/or FIXa or FIX and an agent for activating FIX.
  • the amount of FX that is converted to FXa is proportional to the intrinsic tenase activity in the sample, under conditions where FX is non-limiting. However, under conditions where FX is limiting, the amount of FX that is converted to FXa may be an under-estimate of the intrinsic tenase activity in the sample. As mentioned elsewhere herein, the present invention is based on the observation that it may be necessary to provide an amount of FX in a sample relative to the total expected intrinsic tenase activity in the sample that is non-limiting in order to accurately determine the activity of a blood clotting factor in a sample using a chromogenic assay.
  • the relative intrinsic tenase activity in a sample compared to a known standard when measured using the chromogenic assay may be compared with the relative intrinsic tenase activity in a sample compared to a known standard when measured by a one-stage clotting assay to determine whether a given amount of FX is non-limiting.
  • a length of time for contacting a sample with an agent for activating a blood clotting factor for which an amount of FX is non-limiting may be determined where the percentage decrease in measured activity of the blood clotting factor is linear as the length of the incubation step is decreased.
  • both the first length and the second length allow the amount of FX to be non-limiting.
  • a length of the incubation step for which an amount of FX is non-limiting may be determined where the percentage decrease in measured activity of the blood clotting factor is linear as the length of the incubation step is decreased.
  • the amount of FX is or the amount of FX used must be at least 100 times the expected amount of the blood clotting factor (the activity of which is to be measured, i.e. the blood clotting factor in the sample) in the composition.
  • amount of FX is or the amount of FX used must be at least 110 times, at least 120 times, at least 130 times, at least 140 times, at least 150 times, at least 160 times, at least 170 times, at least 180 times, at least 190 times, at least 200 times, at least 210 times, at least 220 times, at least 230 times, at least 240 times, at least 250 times, at least 260 times, at least 270 times, at least 280 times, at least 290 times, at least 300 times, at least 310 times, at least 320 times, at least 330 times, at least 340 times, at least 350 times, at least 360 times, at least 370 times, at least 380 times, at least 390 times, at least 400 times, at least 410 times, at least 420
  • amount of FX is or the amount of FX used must be between 100 and 5000 times, between 300 and 2500 times, between 500 and 1500 times, or between 750 and 1200 times the expected amount of the blood clotting factor in the composition.
  • the amount of FX is or the amount of FX used must be around 1000 times the expected amount of the blood clotting factor in the composition.
  • the amount of FX is or the amount of FX used may be 1000 times the expected amount of the blood clotting factor in the composition.
  • Contacting' in this context refers to the combination of the sample and the desired component, e.g. by admixing, and refers to the addition of the desired component to the sample, or the addition of the sample to the desired component unless otherwise specified.
  • contacting the “ sample ” with a second or subsequent component e.g. following a step of contacting the sample with a first component
  • contacting the “ sample ” with a second or subsequent component is used herein as shorthand for contacting an admixture of the sample and a first component with a second or subsequent component, and is not intended to be limiting on the order of contacting the sample with components for measuring tenase activity or as requiring the sample simultaneously to be contacted with said components unless otherwise specified.
  • the concentration of FX is or the concentration of FX used must be at least lOOnM.
  • the concentration of FX is or the concentration of FX used must be at least 110 nM, at least 120 nM, at least 130 nM, at least 140 nM, at least 150 nM, at least 160 nM, at least 170 nM, at least 180 nM, at least 190 nM, at least 200 nM, at least 210 nM, at least 220 nM, at least 230 nM, at least 240 nM, at least 250 nM, at least 260 nM, at least 270 nM, at least 280 nM, at least 290 nM, at least 300 nM, at least 310 nM, at least 320 nM, at least 330 nM, at least 340 nM, at least 350 nM, at least 360 nM, at least 370 nM, at least 380 nM,
  • the concentration of FX is or the concentration of FX used must be at least 50 nM/min, at least 100 nM/min, around 60 nM/min, around 120 nM/min, between 50 nM/min and 333 nM/min, or between 100 nM/min and 333 nM/min.
  • the concentration of FX is or the concentration of FX used must be at least 50 nM/min, at least 100 nM/min, around 60 nM/min, around 120 nM/min, between 50 nM/min and 333 nM/min, or between 100 nM/min and 333 nM/min.
  • the composition or kit comprises thrombin, prothrombin and/or bovine FVa.
  • a composition or kit for performing the same reaction as that of the Rox Factor IX (Rossix) kit may comprise thrombin, prothrombin and/or bovine FVa.
  • the composition or kit comprises thrombin but does not comprise prothrombin and/or bovine FVa.
  • a composition or kit for performing the same reaction as that of the BIOPHENTM (Hyphen Biomed) kit may comprise thrombin but not prothrombin and/or bovine FVa.
  • the amount of FX is: i. non-limiting relative to the total expected activity of intrinsic tenase; and/or ii. at least 100 times the expected amount of FIXa in the composition; and/or iii. at least 100 pmol per IU of FIXa; and/or
  • the present invention provides a method for measuring FIX activity in sample comprising FIX having increased specific activity relative to wild-type FIX, the method comprising: a. providing a composition comprising: i. Factor X (FX); ii. Factor VIII, an agent for activating FVIII, and an agent for activating FIX; iii. the sample; and iv. a chromogenic substrate of FX; and b. detecting a dye formed by cleavage of the chromogenic substrate, thereby to measuring FIX activity; wherein:
  • the method of the invention may further comprise a step of determining whether the patient has been partially or fully treated (e.g. determining that the patient has been partially or fully treated for the symptoms of haemophilia) by administration of the gene therapy vector.
  • Partially or fully treating haemophilia may refer to improving the clotting of a patient suffering from haemophilia, and reducing the risk of an uncontrolled bleeding event.
  • Partially or fully treating haemophilia may refer to reducing the number and/or frequency of uncontrolled bleeding events or internal bleeding e.g. in joints. A patient who is partially or fully treated may suffer fewer uncontrolled bleeding events per year.
  • the concentration of Ca 2+ may be between 100 mM and 25 mM, between 200 mM and 15 mM, between 300 pM and 10 mM, or between 400 pM and 5 mM.
  • the concentration of Ca 2+ may be around 10 mM.
  • the concentration of Ca 2+ may be 600 mM.
  • the user of the method will be able to look at the activity of the intrinsic tenase/the amount of the blood clotting factor in plasma samples taken from a small group of patients that have been administered the therapy.
  • the user of the method may then take that activity as the expected total activity of the intrinsic tenase and use that expected total activity to calculate the amount of FX which is non-limiting relative to the total expected activity of intrinsic tenase, or the user of the method may then take that amount of the blood clotting factor as the expected amount of the blood clotting factor to calculate how much FX is 100 times the expected amount of the blood clotting factor in the sample.
  • the blood clotting factor has increased specific activity relative to a corresponding reference wild-type blood clotting factor.
  • the blood clotting factor having increased specific activity relative to the corresponding reference wild-type blood clotting factor may have a specific activity which is at least 1.1 fold, at least 1.2 fold, at least 1.5 fold, at least 1.7 fold, at least 1.8 fold, at least 2 fold, at least 2.2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5, at least 10 fold, at least 11 fold, at least 12 fold, at least 13 fold, at least 14 fold or at least 15 fold higher than the specific activity of the corresponding reference wild-type blood clotting factor.
  • the agent for activating FVIII is a combination of FV and FII, and the incubation step to allow the formation of FXa is less than 5 minutes, less than 4 minutes, less than 3 minutes, between 0 minutes and 5 minutes, between 1 minute and 4 minutes, or between 1 minute and 3 minutes; or
  • the agent for activating FVIII is Flla
  • the incubation step to allow the formation of FXa is less than 3 minutes, less than 2.5 minutes, less than 2 minutes, between 0 minutes and 3 minutes, between 1 minute and 2.5 minutes, or between 1 minute and 2 minutes.
  • the change in the amount of dye formed may be measured up to 30 seconds, up to 40 seconds, up to 50 seconds, up to 60 seconds, up to 70 seconds, up to 80 seconds, up to 90 seconds, up to 100 seconds, up to 110 seconds, up to 120 seconds, up to 3 minutes, up to 4 minutes, up to 5 minutes, up to 6 minutes, up to 7 minutes, up to 8 minutes, up to 9 minutes or up to 10 minutes following contacting the sample with the chromogenic substrate.
  • the change in the amount of dye formed may be measured from 1 second to 10 minutes, from 5 seconds to 5 minutes, or from 10 seconds to 100 seconds following contacting the sample with the chromogenic substrate.
  • a calibration curve may be prepared.
  • a calibration curve may be prepared by carrying out the method of the invention for measuring activity of a blood clotting factor on a series of samples having a different (and known) amounts of procoagulant activity (e.g . using pooled human plasma or a commercial calibrator plasma to prepare a series of samples having known procoagulant activity).
  • procoagulant activity e.g . using pooled human plasma or a commercial calibrator plasma to prepare a series of samples having known procoagulant activity.
  • the amount of dye formed may be measured for each of the samples having a different known procoagulant activity, and thus the relationship between the amount of dye and the procoagulant activity in a sample may be established.
  • a calibration curve may be pre-determined, i.e. it may be prepared prior to performing the methods of the present invention.
  • kits of the invention may comprise FX, FVIIIa and/or FIXa, and a chromogenic substrate, and may comprise instructions for performing a method of the invention.
  • a kit may be for measuring the activity of a blood clotting factor in a sample.
  • Rl was aliquoted into two 3mL tubes. FX was spiked into each tube separately to increase the concentration by 360nM or 1200nM. This provided a concentration of 180nM or 460nM FX in the mixture following the addition of R2 (i.e. in the mixture containing Rl, the diluted plasma sample and R2). FX buffer was added to Rl instead of the additional FX as a control.
  • the modified versions of Rl were subsequently used in the kit as described below.
  • the chromogenic substrate comprises a chromogen or fluorogen joined by a peptide bond to the carboxyl carbon atom of the amino acid at the C-terminus of a FXa cleavage site, preferably wherein the FXa cleavage site comprises the amino acid sequence IDGR or IEGR or chemical derivatives thereof.

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Abstract

The present invention provides methods for measuring the activity of a blood clotting factor in a sample comprising providing a composition comprising Factor X, Factor VIIIa and/or Factor IXa, the sample and a chromogenic substrate of FXa, and detecting a dye formed by cleavage of the chromogenic substrate thereby measuring the activity of the blood clotting factor, wherein the amount of FX is non-limiting relative to the total expected activity of intrinsic tenase, at least 100 times the expected amount of the blood clotting factor in the composition and/or at least 100 pmol per IU of the blood clotting factor, and/or the concentration of FX is at least 100 nM. Also provided are a compositions provided in such a method and kits for use in such a method, as well as methods for determining whether an amount of FX used in methods for measuring the activity of a blood clotting factor is non-limiting.

Description

Method for measuring the activity of a blood clotting factor
Field of the invention
The present invention relates to a method for measuring the activity of a blood clotting factor in a sample. More particularly, the present invention provides methods and kits for measuring the activity of a blood clotting factor in a sample, which provide a sufficient amount of Factor X to allow blood clotting factor activity in a sample to be determined more accurately in a chromogenic assay, particularly where the blood clotting factor has increased specific activity relative to the corresponding reference wild-type blood clotting factor.
Background of the invention
The measurement of blood clotting factor activity in a sample is particularly important for predicting the risk to patients suffering from haemophilia of suffering an uncontrolled bleeding event, and ensuring that patients suffering from haemophilia receive the appropriate treatment to restore sufficient blood clotting factor activity. The number of annualised joint bleeds is negatively correlated with blood clotting factor activity; a patient with FIX activity levels of at least 5% FIX activity is categorised as having “mild” haemophilia and may suffer from only a low number of bleeds per year, whilst patients with less than around 1% FIX activity may be categorised as having “severe” haemophilia, which is generally associated with a far higher number of bleed events.
Blood clotting factor activity in a sample is generally measured by one of two assays: a one-stage assay (OSA), or a chromogenic substrate assay (CSA).
One-stage assays directly measure fibrin clot formation following activation of the clotting cascade by a one-stage assay activator. A sample is mixed with platelet poor plasma which is deficient in the blood clotting factor of interest, but which provides all other components required for clotting to occur, and phospholipid and a contact activation pathway activator such as Kaolin, ellagic acid or silica-based reagents are added. Calcium is then added, and the user measures the time taken for clotting to occur. Clot formation can be assessed directly by a magnetic steel ball method. Clot formation may be measured using a rotating cuvette assay in which a steel ball remains stationary in a magnetic field until the formation of fibrin strands around the ball produces movement and a change in the magnetic field can be detected. Alternatively, clot formation may be measured using a rotating steel ball assay in which a steel ball is rotated under the influence of a magnet until the formation of fibrin strands around the ball stops it rotating, which can be detected by a sensor. In either event, coagulation time may be recorded. Blood clotting factor activity may be determined by comparing the coagulation time with the coagulation time for a control sample, for example a control sample reconstituted using components purified from healthy plasma.
By contrast, chromogenic substrate assays measure the activity of blood clotting factors such as Factor VIII blood clotting factors (FVIII) and Factor IX blood clotting factors (FIX) indirectly by measuring the activity of Factor Xa (FXa) which is generated following formation of the intrinsic tenase. In an example chromogenic substrate assay, components for clotting, including blood clotting factors, phospholipids and calcium, are supplied as reagents, and thrombin (Factor Ila (Flla) is used to activate FVIII to form FVIIIa. The thrombin-activated Factor VIII polypeptide forms an enzymatic complex with Factor IXa polypeptide, phospholipids and calcium, and the resulting enzymatic complex can catalyse the conversion of Factor X polypeptide to Factor Xa polypeptide. The activated Factor Xa polypeptide can catalyse cleavage of a chromogenic substrate. For example, cleavage of SXa-11 produces the dye pNA. The level of pNA generated can be measured by determining colour development at 405 nm (e.g. measured by absorbance). The level of pNA generated is proportional to Factor Xa activity. Factor X polypeptide is provided in excess, thus the measured FXa activity is considered to be proportional to the activity of the blood clotting factor in the sample, the activity of which is being measured. Exemplary CSAs in the art include the BIOPHEN™ (Hyphen Biomed) and Rox Factor IX (Rossix) kits.
To-date, adoption of CSAs remains limited, and currently approximately less than 10% of laboratories use CSAs to measure FIX activity. However, the number of laboratories providing CSAs to measure blood clotting factor activity is increasing, and the use of CSAs is becoming more routine. CSAs ultimately may provide a more rapid and cost- effective means for measuring blood clotting factor activity in a sample.
It has been observed there may be some discrepancy between different assays for measuring blood clotting factor activity in a sample. Although the variation between individual OSAs is generally limited and different OSAs are generally likely to provide results which lead to similar clinical interventions, it has recently been identified that there may be substantial differences between the activity of FIX and FVIII measured by OSAs and by CSAs.
Summary of the invention
In the course of making the present invention, it was identified that measuring the activity of a mutant FIX blood clotting factor known to have increased specific activity relative to a wild-type FIX blood clotting factor by OSAs and CSAs provided divergent results. Specifically, the indicated activity of the FIX blood clotting factor having increased specific activity by CSAs was found to be lower than the indicated activity measured by OSAs. This may have significant implications when monitoring blood clotting factor activity in a patient where a variant blood clotting factor with increased specific activity compared to a reference wild-type blood clotting factor is used to treat haemophilia, for example, by direct administration of a recombinant blood clotting factor or in gene therapy.
Blood clotting factor activity in CSAs is determined by spectrophotometrically measuring the amount of a chromogenic substrate that is converted into a dye by proteolytic cleavage by FXa. The amount of FXa that is generated is assumed to be proportional to the enzymatic activity of the intrinsic tenase. However, without wishing to be bound by theory, it is believed that the observed discrepancy between CSAs and OSAs may have arisen at least in part from CSAs providing an amount of Factor X which may be limiting, and thus the activity level measured by a CSA might not reflect the true enzymatic activity of the FIX blood clotting factor. The present invention aims to rectify this deficiency in CSAs for measuring blood clotting factor activity in a sample by providing an amount of Factor X that is not limiting for the activity of the blood clotting factor in the sample (i.e. is not limiting relative to the total expected activity of intrinsic tenase provided by mixing the sample with FIXa or FVIIIa).
In a first aspect, the present invention provides a method for measuring the activity of a blood clotting factor in a sample, comprising: a. providing a composition comprising i. Factor FX (FX); ii. Factor Villa (FVIIIa) and/or Factor IXa (FIXa); iii. the sample; and iv. a chromogenic substrate of FXa, and b. detecting a dye formed by cleavage of the chromogenic substrate, thereby measuring the activity of the blood clotting factor, wherein:
1. the amount of FX i s : i. non-limiting relative to the total expected activity of intrinsic tenase; ii. at least 100 times the expected amount of the blood clotting factor in the composition; and/or iii. at least 100 pmol per IU of the blood clotting factor; and/or
2. the concentration of FX is at least 100 nM.
In a second aspect, the present invention provides a composition comprising: a. Factor X (FX); b. Factor Villa (FVIIIa) and/or Factor IXa (FIXa); c. a sample; and d. a chromogenic substrate of FXa; wherein:
1. the amount of FX i s : i. non-limiting relative to the total expected activity of intrinsic tenase; ii. at least 100 times the expected amount of the blood clotting factor in the composition; and/or iii. at least 100 pmol per IU of the blood clotting factor; and/or 2. the concentration of FX is at least 100 nM.
In a third aspect, the present invention provides a kit for measuring the activity of a blood clotting factor in a sample comprising: a. Factor X (FX); b. Factor Villa (F Villa) and/or Factor IXa (FIXa); and c. a chromogenic substrate of FXa; wherein:
1. the amount of FX i s : i. non-limiting relative to the total expected activity of intrinsic tenase; ii. at least 100 times the amount of expected FIXa; and/or iii. at least 100 pmol per IU of the blood clotting factor; and/or
2. the concentration of FX is at least 100 nM.
In a fourth aspect, the present invention provides a kit comprising the components required for performing the method of the invention.
In a fifth aspect, the present invention provides a kit comprising: i. Factor X (FX); ii. Factor Villa (F Villa) and/or Factor IXa (FIXa); iii. a chromogenic substrate of FXa; and iv. instructions for performing the method of the invention.
In a sixth aspect, the present invention provides a kit for measuring the activity of a blood clotting factor in a sample comprising: i. Factor X (FX); ii. Factor Villa (F Villa) and/or Factor IXa (FIXa); iii. a chromogenic substrate for FXa; and iv. instructions for performing a chromogenic assay which indicate that: 1.the amount of FX used must be: i. non-limiting relative to the expected total activity of intrinsic tenase; ii. at least 100 times the expected amount of the blood clotting factor; and/or iii. at least 100 pmol per IU of the blood clotting factor; and/or 2. the concentration of FX must be at least 100 nM.
In a seventh aspect, the present invention provides a kit comprising the components required for performing the method of the invention.
In an eighth aspect, the present invention provides a method for determining an amount of FX which is non-limiting relative to the total expected activity of intrinsic tenase in a method for measuring the activity of blood clotting factor in a sample, said method comprising performing a series of chromogenic assays in which the amount of FX is varied such that various concentrations of FX are used, and determining an amount of FX that is non-limiting, wherein each assay comprises: a. providing a composition comprising: i. Factor X (FX) ii. Factor Villa (F Villa) and/or Factor IXa (FIXa); iii. the sample; and iv. a chromogenic substrate of FXa, and b. detecting a dye formed by cleavage of the chromogenic substrate thereby measuring the activity of the blood clotting factor; wherein the amount of FX is non-limiting at any concentrations that provide a less than linear increase in the measured activity of the blood clotting factor when compared to the increase in the FX concentration .
In a ninth aspect, the present invention provides a method for determining a length of time for contacting a sample with an agent for activating a blood clotting factor in a method for measuring the activity of a blood clotting factor in a sample for which the amount of FX is non-limiting, said method comprising performing a series of chromogenic assays in which the length of time for which the sample is contacted with an agent for activating a blood clotting factor is varied such that various lengths of time may be used and determining a length of time for contacting the sample with an agent for activating a blood clotting factor for which the amount of FX is non-limiting, wherein each assay comprises: a. providing a composition comprising i. Factor X (FX); ii. Factor Villa (F Villa) and/or Factor IXa (FIXa); iii. the sample; and iv. a chromogenic substrate of FXa; and b. detecting a dye formed by cleavage of the chromogenic substrate thereby measuring the activity of the FIXa; wherein the amount of FX is non-limiting for a length of time for which the sample is contacted with an agent for activating a blood clotting factor if decreasing the length of time decreases the measured activity of the blood clotting factor proportionately.
In a tenth aspect, the present invention provides a method for determining a length of an incubation step to allow the formation of FXa in a method for measuring the activity of a blood clotting factor in a sample for which the amount of FX is non-limiting, said method comprising performing a series of chromogenic assays in which the length of the incubation step is varied such that various lengths of the incubation step may be used and determining a length of the incubation step for which the amount of FX is non-limiting, wherein each assay comprises: a. providing a composition comprising i. Factor X (FX); ii. Factor Villa (F Villa) and/or Factor IXa (FIXa); iii. the sample; and iv. a chromogenic substrate of FXa; and b. detecting a dye formed by cleavage of the chromogenic substrate thereby measuring the activity of the FIXa; wherein the amount of FX is non-limiting for a length of the incubation step if decreasing the length of the incubation step decreases the measured activity of the blood clotting factor proportionately. Description of the Figures
Figure 1 shows part of the coagulation cascade (intrinsic pathway) tested in one stage and chromogenic assays (OSA and CSA respectively). OSA begins by activation of FXII into FXIIa by means of a negatively charged surface in the presence of lipids and calcium, and culminates with fibrin clot formation. The % of FIX blood clotting factor activity of a particular FIX polypeptide is related to the amount of time taken for clot formation relative to the amount of time taken for clot formation for a pre-determined standard amount of FIX polypeptide generated using FIX polypeptide purified from healthy plasma or pooled healthy plasma. Chromogenic assays (CSAs) include a defined number of intrinsic coagulation cascade proteins in purified form, as shown inside the dashed-grey triangle. Formation of a reporter released upon processing of a chromogenic substrate by FXa is measured spectrophotometrically to measure FXa activity. FXa activity is proportional to the activity of the blood clotting factor being measured, e.g. the activity of a FIX blood clotting factor.
Figure 2 shows that the measurement of the activity of FIX-Padua (a FIX blood clotting factor comprising a leucine at position 338) varies within one-stage assays, and between one stage and chromogenic assays. A) The activity of the FIX-Padua variant was measured with four one-stage assays (SynthASil™, SynthaFax™, Actin FS and Pathromtin™), and two chromogenic assays (ROX (Rossix FIX) and Biophen (Hyphen FIX)). The activity of FIX (FIX:C, %) is plotted against all assays. B) FIX-Padua activity measured by one-stage assays correlates with that measured with chromogenic assays, but absolute values differ.
Figure 3 shows a comparison of measured FIX-Padua activity as between Synthasil (an OSA) and 12 other OSAs as well as two CSAs using field study samples. A) Plot showing the relationship between the SynthASil™ one stage assay and 12 OSAs as well as 2 CSAs. FIX-deficient plasma was spiked with various amounts of recombinant FIX-Padua protein to yield samples with 5, 20, 50, 100, and 150% FIX:C by SynthASil™ OSA, before measuring with the other assays. Linear regression enables comparison of values between assays. B) Overall differences in assay results relative to SynthASil™ together with their associated inter-lab variabilities (CVs, %). C) Comparison of the activity of plasma- derived wild-type FIX protein (Scientific and Standardisation Committee (SSC) FIX Lot 4 (a plasma standard that has been assayed and assigned a nominal 105% FIX activity)) by SynthASil™ assay, (referred to herein as “SSC Lot 4” and “SSC”) and recombinant FIX- Padua variant (100% nominal activity by SynthASil™) in 13 one stage and 2 chromogenic assays. Plotted values indicate the FIX polypeptide activity is non-limiting to the Synthasil™ results for wild-type FIX or FIX-Padua. Wild-type FIX polypeptide returned similar activity regardless of the assay used. FIX-Padua blood clotting factor returned 66- 120% activity for other OSAs relative to FIX-Padua blood clotting factor activity measured by SynthASil™, whereas FIX-Padua blood clotting factor returned only approximately 40% activity when measured by CSAs relative to FIX-Padua blood clotting factor activity measured by SynthASil™.
Figure 4 shows a comparison of recombinant wild-type (rFIX-Wt) and Padua (rFIX-Padua) FIX blood clotting factor activity with standard or modified chromogenic assays, using plasma-derived wild-type FIX blood clotting factor as a control (SSC Lot 4 and pdFIX- Wt). Samples spiked with recombinant FIX-Padua blood clotting factor or wild-type FIX blood clotting factor with 50, 100 or 150 % FIX activity (when measured by SynthASil™
) were tested with two chromogenic kits (Rossix and Hyphen) in parallel with reconstituted SSC Lot 4 (lyophilised plasma at 105% activity) or pdFIX-WT (plasma-derived wild-type WHO FIX standards (NIBSC 14/148) spiked in HaemB plasma to achieve 125% activity (when measured by SynthASil™)). The chromogenic kits were used either as prescribed by the manufacturer or by spiking in additional FX so that the concentration of the FX following the addition of second reagent was 180 nM (A) or 460 nM (C) for the Hyphen Biophen kit, or was 180nM (B) or 230 nM (D) for the Rossix ROX kit. The relative change in the measured FIX polypeptide activity (i.e. the FIX polypeptide activity measured when additional FX has been used (“excess”) divided by the FIX activity measured when using the standard amount of FX prescribed by the kit (“stock”)) is plotted for each sample as the average of at least 3 independent experiments (mean +/- 1 SD).
Figure 5 shows the effect of the length of the reaction on R338L-FIX activity in the ROX FIX chromogenic assay. Recombinant FIX-WT (WT) and recombinant R338L-FIX (R338L) spiked samples with 50, 100 and 150% FIX:C as measured by SynthASil one- stage assay were tested alongside SSC Lot 4 and pdFIX-WT (“WHO” standard NIBSC 14/148). Absorbance values from the ROX FIX kit with a standard length of time between adding “reagent B” and adding “FXa substrate” of 8 minutes (A) or a shortened length of time between adding “reagent B” and adding “FXa substrate” of 3 minutes (B). The standard curves for each length of time between adding “reagent B” and adding “FXa substrate” are shown (C). Interpolated FIX:C of each sample where length of time between adding “reagent B” and adding “FXa substrate” was 8 minutes (D) or 3 minutes (E). Mean + SD of at least 3 independent experiments. Means were compared using a paired two-tailed t-test (*p<0.05, ns = not significant).
Figure 6 is a sequence listing.
Description of the Sequence Listing
SEQ ID NO: 1 - Wild type FVIII SEQ ID NO: 2 - Wild type FIX (Malmo)
SEQ ID NO: 3 - IEGR SEQ ID NO:4 - IDGR
Detailed Description
General definitions
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention belongs.
In general, the term “comprising” is intended to mean including but not limited to. For example, a method comprising certain steps may form part of a larger method which comprises additional steps either before, during or after the steps recited therein. Similarly, a kit or a composition comprising certain reagents may contain further reagents in addition to those specified. In some embodiments of the invention, the word “comprising” is replaced with the phrase “consisting essentially o . The term “consisting essentially of” means that specific further components can be present, namely those not materially affecting the essential characteristics of the subject matter.
In some embodiments of the invention, the word “comprising” is replaced with the phrase “consisting of” . The term “consisting of’ is intended to be limiting. For example, a method consisting of certain steps should be interpreted to mean that the method contains those defined steps and does not comprise any additional steps. Similarly, a kit or composition consisting of certain reagents does not comprise any further reagents in addition to those specified.
In some embodiments of the invention, the word “have ” is replaced with the word “comprise ” or the phrase “consist of” .
The terms “protein ” and “polypeptide ” are intended to refer to a polymeric chain of amino acids of any length.
The terms “nucleic acid molecule “nucleic acid sequence “polynucleotide ” and “nucleotide sequence ” are intended to refer to a polymeric chain of nucleotides of any length.
The terms “substitution mutation ” and “amino acid substitution ” are used interchangeably herein, and are intended to mean the substitution of one amino acid in an amino acid sequence with a different amino acid. In the phrases “substitution of’ amino acid X, or “amino acid X that is (to be) substituted” amino acid X is the original or native amino acid that is present within an amino acid sequence and that is to be replaced. For example, substitution of methionine means that a native methionine amino acid is replaced by another amino acid. In the phrase “substitution with” amino acid Y, amino acid Y is the different amino acid which replaces the original or native amino acid in an amino acid sequence. For example, substitution with methionine refers to replacement of a native (non-methionine) amino acid with methionine. The standard shorthand nomenclature used to define a substitution mutation lists the original or native amino acid at a position within an amino acid sequence that is to be substituted, and the amino acid which replaces the original or native amino acid. For example, a Factor IX amino acid sequence comprising the substitution mutation R338L refers to a Factor IX amino acid sequence which comprises a substitution of the arginine residue at a position corresponding to position 338 with a leucine residue {i.e. the Factor IX amino acid sequence comprises a leucine residue at position 338).
The term “IU” refers to international units, an international biological standard for harmonising blood clotting factors in a sample. 1 IU/ml is defined as the activity of a blood clotting factor in normal pooled plasma. 1 IU/ml of FVIII is equivalent to approximately 1.2 nM of wild-type FVIII (approximately 200 ng/ml). 1 IU/ml of FIX is equivalent to approximately 90 nM of wild-type FIX (approximately 5 pg/ml).
The term “the expected amount of ” of a blood clotting factor refers to an amount of a blood clotting factor which is known or predicted to be present, i.e. in a composition or in a sample. For example, an estimate for the amount of blood clotting factor present in a sample may be the expected amount of the blood clotting factor. Similarly, “ the expected activity of intrinsic tenase ” refers to an activity of intrinsic tenase which is known or predicted to be present. . “ The expected amount ” or “ expected activity” does not need to precise, but can represent a range.
Reference herein to “a FVIII blood clotting factor or “a FIX blood clotting factor ”
(present) in a sample or composition includes the respective blood clotting factor which is capable of providing a respective blood clotting factor activity (i.e. when an agent for activating the blood clotting factor is provided) as well as the active blood clotting factor (e.g. FVIIIa or FIXa) per se. Put another way, the terms FVIII blood clotting factor and FIX blood clotting factor are not limited to only the active form of the blood clotting factor, but also includes the blood clotting factor if an agent for activating said blood clotting factor is or is to be provided. Where an agent for activating the blood clotting factor is not provided or is not to be provided (e.g. where a composition may comprise inactive FIX and no agent for activating FIX) and the blood clotting factor cannot or will not be activated within a composition, any blood clotting factor which might be present is not considered to be “ expected blood clotting factor” according to this definition.
The terms “convertase “Xa convertase “ Xase “ intrinsic tenase ” and “FVIIIa/FIXa convertase ” are used interchangeably herein to refer to the enzymatically active complex formed between FVIIIa and FIXa which is capable of cleaving FX to form FXa. FVIIIa and FIXa may be referred to as the components of the intrinsic tenase.
The term “non-limiting” refers to an amount of FX which is sufficiently high as to allow the user of the method to perform the method of the invention to their desired level of accuracy. For example, a “ non-limiting’ ’ amount of FX may be an amount of FX which ensures that the method of the invention gives a measurement for the activity of a blood clotting factor that less than 25%, less than 15%, or less than 10% lower than the measurement provided by a one-stage clotting assay performed on a sample comprising the total expected activity of intrinsic tenase. Optionally, a “ non-limiting ’ amount of FX ensures that the amount of FX is sufficient for the activity of intrinsic tenase in the composition is not limited by FX when measuring the activity of a blood clotting factor in a sample.
The term “incubation step to allow the formation of FXa ” refers to the step of a chromogenic assay method in which FVIIIa and FIXa are provided in the presence of FX to allow the formation of FXa. Optionally, this incubation step takes place prior to providing a chromogenic substrate of FXa.
Reference herein to the “amount” of FX relative to an amount of a blood clotting factor refers to the relative molar (mol) amounts of FX and the blood clotting factor. For example, if the expected amount of the blood clotting factor in the composition is 1 nanomole, then an amount of FX which is at least 100 times the expected amount of the blood clotting factor in the composition will be 100 nanomoles. Reference to the “concentration” of FX or FIXa refers to the molar (M or mol/1) concentrations of FX or FIXa and reference to the “concentration” of FX relative to the concentration of FIXa refers to the relative molar (M or mol/1) concentrations of FX and FIXa.
The terms “wild-type ” and “native ” are used interchangeably herein, and are intended to describe something which is commonly found in nature. A “wild-type Factor IX amino acid sequence ” is an amino acid Factor IX sequence having the amino acid sequence set forth in SEQ ID NO:2.
The singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “an amino acid” includes two or more instances or versions of such amino acids.
All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.
Measuring the activity of a blood clotting factor
The methods and kits of the invention use a sufficient amount of FX such that the amount of FX is not limiting on the amount of chromogenic substrate for FXa which may be converted into a detectable dye in a chromogenic assay. The present invention thus uses an amount of FX in a CSA which allows a more accurate measure of the activity of a blood clotting factor in a sample.
In the context of the present invention, measuring the activity of a blood clotting factor in a sample preferably refers to measuring the activity of a Factor VIII (FVIII) or a Factor IX (FIX) blood clotting factor. This is achieved indirectly via measurement of the enzymatic activity of FXa, by measuring the amount of a dye generated by FXa by proteolytic cleavage of a chromogenic substrate for FXa. The intrinsic tenase converts FX to its active form FXa; measuring FXa activity thus serves as a proxy measure for FVIIIa/FIXa activity and allows the activity of the blood clotting factor to be measured. The methods of the invention comprise providing a composition comprising FX; FVIIIa and/or FIXa; the sample; and a chromogenic substrate. Such compositions, and kits for forming such compositions (i.e. when combined with the sample) are also provided by the present invention.
Providing a composition comprising FVIIIa and/or FIXa may comprise providing a sample comprising FVIII or FIX. Where a sample comprises FVIII (e.g. where a method is for measuring the activity of a FVIII blood clotting factor in a sample), providing a composition comprising FVIIIa and/or FIXa may comprise providing (e.g. contacting the sample with) an agent for activating FVIII, and providing FIXa. Providing FIXa may comprise providing (e.g. contacting the sample with) FIXa or FIX and an agent for activating FIX. Where a sample comprises FIX (e.g. where a method is for measuring FIX activity in a sample), providing a composition comprising FVIIIa and/or FIXa may comprise providing (e.g. contacting the sample with) an agent for activating FIX, FVIII and an agent for activating FVIII.
Similarly, a composition or kit of the invention comprising FVIIIa and/or FIXa may comprise FVIII and an agent for activating FVIIIa, and/or FIXa or FIX and an agent for activating FIX.
Optionally, the amount of FX (i.e. the amount of FX in the composition provided in the methods of the invention, the composition of the invention, or by the kits of the invention) is or used must be non-limiting relative to the total expected activity of intrinsic tenase.
Optionally, the amount of FX may be non-limiting relative to the activity of the intrinsic tenase formed through the activation of the blood clotting factor(s) in the method of the invention.
Without wishing to be bound by theory, the amount of FX that is converted to FXa is proportional to the intrinsic tenase activity in the sample, under conditions where FX is non-limiting. However, under conditions where FX is limiting, the amount of FX that is converted to FXa may be an under-estimate of the intrinsic tenase activity in the sample. As mentioned elsewhere herein, the present invention is based on the observation that it may be necessary to provide an amount of FX in a sample relative to the total expected intrinsic tenase activity in the sample that is non-limiting in order to accurately determine the activity of a blood clotting factor in a sample using a chromogenic assay. As the amount of FX may optionally be non-limiting relative to the total expected activity of intrinsic tenase, both the amount of FX in the sample and the total expected intrinsic tenase activity may affect whether FX is non-limiting; whilst increasing the amount of FX may ensure that the amount of FX is non-limiting on intrinsic tenase activity, it may also be possible to ensure that the amount of FX is non-limiting on intrinsic tenase activity by reducing the intrinsic tenase activity in the sample in order to ensure that FX does not become limiting during the course of performing the method.
In one embodiment, an amount of FX that is non-limiting relative to the total expected activity of intrinsic tenase is provided. Optionally, a sample is supplemented with an amount of FX that is non-limiting relative to the total expected activity if intrinsic tenase. Optionally, the amount of FX in a sample may be non-limiting if the total expected intrinsic tenase activity is reduced. Optionally, the amount of FX in a sample may be non limiting if less intrinsic tenase is generated in the incubation step to allow the formation of FXa (i.e. if less of the blood clotting factor(s) in the sample are activated). For example, the amount of FX may be made non-limiting by reducing the time allowed for the activation of the blood clotting factor(s) in the incubation step, in order to ensure that less intrinsic tenase is formed. Thus, optionally, the amount of FX may be made non-limiting by reducing the length of time for which the sample is contacted with an agent for activating a blood clotting factor. Optionally, the length of time for which the sample is contacted with an agent for activating a blood clotting factor may be reduced. Optionally the amount of FX may be made non-limiting by reducing the length of the incubation step to allow the formation of FXa. Optionally, the length of the incubation step to allow the formation of FXa may be reduced such that the amount of FX is non-limiting. Optionally, both the length of time for which the sample is contacted with an agent for activating a blood clotting factor and the length of the incubation step to allow the formation of FXa may be reduced. As outlined in greater detail below, the period of time for which the sample is contacted with an agent for activating a blood clotting factor may optionally overlap with the incubation step to allow the formation of FXa. Optionally, the incubation step to allow the formation of FXa may comprise the period of time for which the sample is contacted with an agent for activating a blood clotting factor. Optionally, the incubation step to allow the formation of FXa and the period of time for which the sample is contacted with an agent for activating a blood clotting factor may coincide fully, i.e. both may start and finish at the same time and run concurrently (or in other words, the formation of FXa may occur simultaneously with the activation of a blood clotting factors by agent(s) for activating the blood clotting factor(s)). Thus, optionally, reducing the length of time for which the sample is contacted with an agent for activating a blood clotting factor may also reduce the length of the incubation step to allow the formation of FXa, and vice-versa.
Optionally, the amount of FX that is non-limiting on intrinsic tenase activity may be determined experimentally. Optionally, the amount of FX that is non-limiting on intrinsic tenase activity may be determined experimentally by investigating the concentration of FX and/or the intrinsic tenase activity generated in the incubation step.
Optionally, the amount of FX that is non-limiting on intrinsic tenase activity may be determined by performing a series of chromogenic assays in which the amount of FX is varied such that various concentrations of FX may be used and determining that the amount of FX is non-limiting at any concentrations that provide a less than linear increase in the measured activity of FIXa when compared to the increase in the FX concentration, wherein each assay comprises: a. providing a composition comprising i. Factor X (FX); ii. Factor Villa (F Villa); iii. FIXa at a level corresponding to the total expected activity of FIXa; and iv. a chromogenic substrate of FXa; and b. detecting a dye formed by cleavage of the chromogenic substrate thereby measuring the activity of the FIXa.
For example, the user could perform the assay using a first concentration of FX and a second concentration of FX that is 2 times greater than the first concentration. If the measured activity of FIXa using the second concentration is 2 or more times greater than the measured activity of FIXa using the first concentration, then the first concentration is limiting. However, if the measured activity of FIXa using the second concentration is less than 2 times greater than the measured activity of FIXa using the first concentration then both the first concentration and the second concentration are non-limiting.
Optionally, a non-limiting amount of FX may be determined where the percentage increase in measured activity of FIXa is 50% or less than the percentage increase in the FX concentration. Optionally, a non -limiting amount of FX may be determined where the percentage increase in measured activity of FIXa may be 40% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less or 1% or less than the percentage increase in the FX concentration. Optionally, the amount of FX is non-limiting at a concentration that provides (or would provide e.g. if activity levels at a given FX concentration are fitted to a curve) the same results for the measured activity of the blood clotting factor when the FX concentration is increased, optionally within an error range (e.g. a confidence interval or the standard deviation of the measured activity) of the method.
Alternatively, the amount of FX that is non-limiting may be determined by performing a series of chromogenic assays in which the amount of FX is varied such that various concentrations of FX may be used, in parallel with performing a one-stage clotting assay, and determining that the amount of FX is non-limiting at any concentrations for which the measured activity of intrinsic tenase by a chromogenic assay is comparable to the measured activity of intrinsic tenase by the one-stage assay, wherein each chromogenic assay comprises: a. providing a composition comprising i. Factor X (FX); ii. intrinsic tenase at a level corresponding to the total expected activity of intrinsic tenase; and iii. a chromogenic substrate of FXa; and b. detecting a dye formed by cleavage of the chromogenic substrate thereby measuring the activity of the intrinsic tenase.
Optionally, the measured activity of intrinsic tenase by a chromogenic assay is comparable to the measured activity of intrinsic tenase by the one-stage assay if the measured activity of intrinsic tenase by the chromogenic assay is the same as the measured activity of intrinsic assay within an error range (e.g. a confidence interval or the standard deviation of the measured activity) of the method.
Optionally, whether a given amount of FX is non-limiting may be determined by performing a chromogenic assay using that amount of FX and comparing the activity of the intrinsic tenase measured by the chromogenic assay with the activity of the intrinsic tenase measured by performing a one-stage clotting assay, where the chromogenic assay comprises: a. providing a composition comprising i. Factor X (FX) at the given amount; ii. intrinsic tenase at a level corresponding to the total expected activity of intrinsic tenase; and iii. a chromogenic substrate of FXa; and b. detecting a dye formed by cleavage of the chromogenic substrate thereby measuring the activity of the intrinsic tenase.
Optionally, a non-limiting amount of FX may be determined or a given amount of FX is non-limiting where the measured activity of intrinsic tenase by the chromogenic assay is at least 50% of the measured activity of intrinsic tenase by the one-stage assay. Optionally, a non-limiting amount of FX may be determined where the measured activity of intrinsic tenase by the chromogenic assay is at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 100% of the measured activity of intrinsic tenase by the one-stage assay. Optionally, a known standard may be used for comparing the activity of the intrinsic tenase measured by the chromogenic assay with the activity of the intrinsic tenase measured by performing a one-stage clotting assay for determining whether an amount of FX is limiting. Optionally, the standard may be a plasma-derived wild-type FIX protein standard. Optionally, the standard is a pooled plasma standard. Optionally, the standard is the Scientific and Standardisation Committee (SSC) FIX Lot 4 standard, a plasma standard that has been assayed and assigned a nominal 105% FIX activity. Optionally, the standard is the 5th WHO International Standard (IS) for Blood Coagulation FIX, Concentrate (NIBSC code: 14/148). Optionally, the relative intrinsic tenase activity in a sample compared to a known standard when measured using the chromogenic assay may be compared with the relative intrinsic tenase activity in a sample compared to a known standard when measured by a one-stage clotting assay to determine whether a given amount of FX is non-limiting.
More generally, the present invention provides a method for determining an amount of FX which is non-limiting relative to the total expected activity of intrinsic tenase in a method for measuring the activity of blood clotting factor in a sample, said method comprising performing a series of chromogenic assays in which the amount of FX is varied such that various concentrations of FX are used, and determining an amount of that FX is wherein each assay comprises: a. providing a composition comprising: i. Factor X (FX) ii. Factor Villa (F Villa) and/or Factor IXa (FIXa); iii. the sample; and iv. a chromogenic substrate of FXa, and b. detecting a dye formed by cleavage of the chromogenic substrate thereby measuring the activity of the blood clotting factor; wherein the amount of FX is non-limiting at any concentrations that provide a less than linear increase in the measured activity of the blood clotting factor when compared to the increase in the FX concentration. For example, the user could perform the assay using a first concentration of FX and a second concentration of FX that is 2 times greater than the first concentration. If the measured activity of the blood clotting factor using the second concentration is 2 or more times greater than the measured activity of the blood clotting factor using the first concentration, then the first concentration is limiting. However, if the measured activity of the blood clotting factor using the second concentration is less than 2 times greater than the measured activity of the blood clotting factor using the first concentration then both the first concentration and the second concentration are non-limiting.
Optionally, a non-limiting amount of FX may be determined where the percentage increase in measured activity of the blood clotting factor is 50% or less than the percentage increase in the FX concentration. Optionally, a non-limiting amount of FX may be determined where the percentage increase in measured activity of the blood clotting factor may be 40% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less or 1% or less than the percentage increase in the FX concentration. Optionally, the amount of FX is non-limiting at a concentration that provides (or would provide e.g. if activity levels at a given FX concentration are fitted to a curve) the same results for the measured activity of the blood clotting factor when the FX concentration is increased, optionally within an error range (e.g. a confidence interval or the standard deviation of the measured activity) of the method.
Optionally, if an amount of FX is found to be limiting by the method outlined above (i.e. for a particular length of the incubation step), the length of time for contacting a sample with an agent for activating a blood clotting factor and/or the length of the incubation step may be adjusted (e.g. reduced) and the method for determining whether an amount of FX is non-limiting may be repeated for the shorter time period. Optionally, this process may be repeated until a length of time for contacting a sample with an agent for activating a blood clotting factor and/or the incubation step is identified for which the amount of FX is non-limiting.
The method for determining whether an amount of FX is non-limiting on intrinsic tenase activity may be adapted to determine whether a particular length of time for contacting a sample with an agent for activating a blood clotting factor in a method for measuring the activity of a blood clotting factor in a sample for which that amount of FX is non-limiting. This may be done by performing a series of chromogenic assays in which the length of time for which the sample is contacted with an agent for activating a blood clotting factor is varied such that various lengths of time may be used and determining that the amount of FX is non-limiting for a length of time for which the sample is contacted with an agent for activating a blood clotting factor if decreasing the length of time for which the sample is contacted with an agent for activating a blood clotting factor decreases the measured activity of FIXa to a corresponding extent, wherein each assay comprises: a. providing a composition comprising i. Factor X (FX); ii. Factor Villa (FVIIIa) and/or Factor IXa (FIXa); iii. the sample; and iv. a chromogenic substrate of FXa; and b. detecting a dye formed by cleavage of the chromogenic substrate thereby measuring the activity of the FIXa.
For example, the user could perform the assay using a first length of the time period for contacting a sample with an agent for activating a blood clotting factor and a second length of the time period for contacting a sample with an agent for activating a blood clotting factor that is half the length of the first length. If the measured activity of FIXa using the second length is more than 50% the measured activity of FIXa using the first length, then the first length does not allow the amount of FX to be non-limiting. However, if the measured activity of FIXa using the second length is 50% or less the measured activity of FIXa using the first length, then both the first length and the second length allow the amount of FX to be non-limiting.
Optionally, a length of time for contacting a sample with an agent for activating a blood clotting factor for which an amount of FX is non-limiting may be determined where the percentage decrease in measured activity of FIXa is linear as the length of the incubation step is decreased. The present invention also provides a method for determining a length of time for contacting a sample with an agent for activating a blood clotting factor in a method for measuring the activity of a blood clotting factor in a sample for which the amount of FX is non-limiting, said method comprising performing a series of chromogenic assays in which the length of time for which the sample is contacted with an agent for activating a blood clotting factor is varied such that various lengths of time may be used and determining a length of time for contacting the sample with an agent for activating a blood clotting factor for which the amount of FX is non-limiting, wherein each assay comprises: a. providing a composition comprising i. Factor X (FX); ii. Factor Villa (F Villa) and/or Factor IXa (FIXa); iii. the sample; and iv. a chromogenic substrate of FXa; and b. detecting a dye formed by cleavage of the chromogenic substrate thereby measuring the activity of the FIXa; wherein the amount of FX is non-limiting for a length of time for which the sample is contacted with an agent for activating a blood clotting factor if decreasing the length of time decreases the measured activity of the blood clotting factor proportionately.
For example, the user could perform the assay using a first length of time for contacting a sample with an agent for activating a blood clotting factor and a second time for contacting a sample with an agent for activating a blood clotting factor that is half the length of the first length. If the measured activity of the blood clotting factor using the second length is more than 50% the measured activity of the blood clotting factor using the first length, then the first length does not allow the amount of FX to be non-limiting. However, if the measured activity of the blood clotting factor using the second length is 50% or less the measured activity of blood clotting factor using the first length, then both the first length and the second length allow the amount of FX to be non-limiting.
Optionally, a length of time for contacting a sample with an agent for activating a blood clotting factor for which an amount of FX is non-limiting may be determined where the percentage decrease in measured activity of the blood clotting factor is linear as the length of the incubation step is decreased.
The method outlined above for determining whether an amount of FX is non-limiting on intrinsic tenase activity may also be adapted to determine whether a particular length of the incubation step to allow the formation of FXa is suitable for the amount of FX to be used in a chromogenic assay, in order for that amount of FX to be non-limiting. This may be done by performing a series of chromogenic assays in which the length of the incubation step is varied such that various lengths of the incubation step may be used and determining that the amount of FX is non-limiting for a length of the incubation step if decreasing the length of the incubation step decreases the measured activity of FIXa to a corresponding extent, wherein each assay comprises: a. providing a composition comprising i. Factor X (FX); ii. Factor Villa (F Villa); iii. FIXa; and iv. a chromogenic substrate of FXa; and b. detecting a dye formed by cleavage of the chromogenic substrate thereby measuring the activity of the FIXa..
For example, the user could perform the assay using a first length of the incubation step and a second length of the incubation step that is half the length of the first length. If the measured activity of FIXa using the second length is more than 50% the measured activity of FIXa using the first length, then the first length does not allow the amount of FX to be non-limiting. However, if the measured activity of FIXa using the second length is 50% or less the measured activity of FIXa using the first length, then both the first length and the second length allow the amount of FX to be non-limiting.
Optionally, a length of the incubation step for which an amount of FX is non-limiting may be determined where the percentage decrease in measured activity of FIXa is linear as the length of the incubation step is decreased. It will therefore be seen that the amount of FX in the sample, and the length of the incubation step to allow the formation of FXa may optionally both be taken into account when optimising the chromogenic assay when carrying out the method of the invention. Optionally, the amount of FX in the sample may be non-limiting for a particular length of the incubation step.
The present invention also provides a for determining a length of an incubation step to allow the formation of FXa in a method for measuring the activity of a blood clotting factor in a sample for which the amount of FX is non-limiting, said method comprising performing a series of chromogenic assays in which the length of the incubation step is varied such that various lengths of the incubation step may be used and determining a length of the incubation step for which the amount of FX is non-limiting, wherein each assay comprises: a. providing a composition comprising i. Factor X (FX); ii. Factor Villa (FVIIIa) and/or Factor IXa (FIXa); iii. the sample; and iv. a chromogenic substrate of FXa; and b. detecting a dye formed by cleavage of the chromogenic substrate thereby measuring the activity of the FIXa; wherein the amount of FX is non-limiting for a length of the incubation step if decreasing the length of the incubation step decreases the measured activity of the blood clotting factor proportionately. For example, the user could perform the assay using a first length of the incubation step and a second length of the incubation step that is half the length of the first length. If the measured activity of the blood clotting factor using the second length is more than 50% the measured activity of the blood clotting factor using the first length, then the first length does not allow the amount of FX to be non-limiting. However, if the measured activity of the blood clotting factor using the second length is 50% or less the measured activity of blood clotting factor using the first length, then both the first length and the second length allow the amount of FX to be non-limiting. Optionally, a length of the incubation step for which an amount of FX is non-limiting may be determined where the percentage decrease in measured activity of the blood clotting factor is linear as the length of the incubation step is decreased.
Optionally, the amount of FX is, or the amount of FX used must be, in excess relative to the total expected activity of intrinsic tenase. Put another way, optionally, the amount of FX used is greater than the non-limiting amount of FX. Optionally, the amount of FX is or the amount of FX used must be at least 2-fold, at least 3-fold, at least 4-fold or at least 5- fold in excess relative to the total expected activity of intrinsic tenase. Optionally, the amount of FX is or the amount of FX used must be between 1-fold and 5-fold, between 2- fold and 5-fold, or between 3-fold and 5-fold excess relative to the total expected activity of intrinsic tenase. Optionally, the amount of FX is around 2-fold in excess relative to the total expected activity of intrinsic tenase.
Optionally, the amount of FX is or the amount of FX used must be at least 100 times the expected amount of the blood clotting factor (the activity of which is to be measured, i.e. the blood clotting factor in the sample) in the composition. Optionally, amount of FX is or the amount of FX used must be at least 110 times, at least 120 times, at least 130 times, at least 140 times, at least 150 times, at least 160 times, at least 170 times, at least 180 times, at least 190 times, at least 200 times, at least 210 times, at least 220 times, at least 230 times, at least 240 times, at least 250 times, at least 260 times, at least 270 times, at least 280 times, at least 290 times, at least 300 times, at least 310 times, at least 320 times, at least 330 times, at least 340 times, at least 350 times, at least 360 times, at least 370 times, at least 380 times, at least 390 times, at least 400 times, at least 410 times, at least 420 times, at least 430 times, at least 440 times, at least 450 times, at least 460 times, at least 470 times, at least 480 times, at least 490 times, at least 500 times, at least 510 times, at least 520 times, at least 530 times, at least 540 times, at least 550 times, at least 600 times, at least 650 times, at least 700 times, at least 750 times, at least 800 times, at least 850 times, at least 900 times, at least 950 times, at least 1000 times, at least 1100 times, at least 1200 times, at least 1300 times, at least 1400 times, at least 1500 times, at least 2000 times, at least 2500 times, at least 3000 times, at least 3500 times, at least 4000 times, at least 4500 times, at least 5000 times, at least 10,000 times, at least 20,000 times, at least 50,000 times, at least 100,000 times, at least 150,000 times, or at least 180,000 times the expected amount of the blood clotting factor in the composition. Optionally, amount of FX is or the amount of FX used must be between 100 and 5000 times, between 300 and 2500 times, between 500 and 1500 times, or between 750 and 1200 times the expected amount of the blood clotting factor in the composition. Optionally, the amount of FX is or the amount of FX used must be around 1000 times the expected amount of the blood clotting factor in the composition. Optionally, the amount of FX is or the amount of FX used may be 1000 times the expected amount of the blood clotting factor in the composition.
Optionally, the amount of FX is or the amount of FX used must be at least 100 pmol per IU of the blood clotting factor (the activity of which is to be measured, i.e. the blood clotting factor in the sample) in the composition. Optionally, amount of FX is or the amount of FX used must be at least 110 pmol, at least 120 pmol, at least 130 pmol, at least 140 pmol, at least 150 pmol, at least 160 pmol, at least 170 pmol, at least 180 pmol, at least 190 pmol, at least 200 pmol, at least 210 pmol, at least 220 pmol, at least 230 pmol, at least 240 pmol, at least 250 pmol, at least 260 pmol, at least 270 pmol, at least 280 pmol, at least 290 pmol, at least 300 pmol, at least 310 pmol, at least 320 pmol, at least 330 pmol, at least 340 pmol, at least 350 pmol, at least 360 pmol, at least 370 pmol, at least 380 pmol, at least 390 pmol, at least 400 pmol, at least 410 pmol, at least 420 pmol, at least 430 pmol, at least 440 pmol, at least 450 pmol, at least 460 pmol, at least 470 pmol, at least 480 pmol, at least 490 pmol, at least 500 pmol, at least 510 pmol, at least 520 pmol, at least 530 pmol, at least 540 pmol, at least 550 pmol, at least 600 pmol, at least 650 pmol, at least 700 pmol, at least 750 pmol, at least 800 pmol, at least 850 pmol, at least 900 pmol, at least 950 pmol, at least 1000 pmol, at least 1100 pmol, at least 1200 pmol, at least 1300 pmol, at least 1400 pmol, at least 1500 pmol, at least 2000 pmol, at least 2500 pmol, at least 3000 pmol, at least 3500 pmol, at least 4000 pmol, at least 4500 pmol, at least 5000 pmol, at least 10,000 pmol, at least 20,000 pmol, at least 50,000 pmol, at least 100,000 pmol, at least 150,000 pmol, or at least 180,000 pmol per IU of the blood clotting factor in the composition. Optionally, the amount of FX is or the amount of FX used must be between 100 and 5000 pmol, between 300 and 2500 pmol, between 500 and 1500 pmol, or between 750 and 1200 pmol per IU of the blood clotting factor in the composition. Optionally, the amount of FX is or the amount of FX used must be around 1000 pmol per IU of the blood clotting factor in the composition. Optionally, the amount of FX is or the amount of FX used must be 1000 pmol per IU of the blood clotting factor in the composition.
Optionally, the method or kit of the invention may allow the activity of a FVIII blood clotting factor to be measured, and the amount of FX may be an amount relative to the expected amount of the FVIII blood clotting factor in the composition. Thus, optionally, the amount of FX may be at least lOOx the expected amount of the FVIII blood clotting factor in the composition. Optionally, the amount of FX relative to the amount of the FVIII blood clotting factor may be any of the values or ranges indicated above.
Optionally, the method or kit of the invention may allow the activity of a FIX blood clotting factor to be measured, and the amount of FX may be an amount relative to the expected amount of the FIX blood clotting factor in the composition. Thus, optionally, the amount of FX may be at least lOOx the expected amount of the FIX blood clotting factor in the composition. Optionally, the amount of FX relative to the amount of the FIX blood clotting factor may be any of the values or ranges indicated above.
The sample may optionally be contacted with the other components required for the activity of the blood clotting factor to be measured to provide the composition.
“ Contacting' ’ in this context refers to the combination of the sample and the desired component, e.g. by admixing, and refers to the addition of the desired component to the sample, or the addition of the sample to the desired component unless otherwise specified. Furthermore, contacting the “ sample ” with a second or subsequent component (e.g. following a step of contacting the sample with a first component) is used herein as shorthand for contacting an admixture of the sample and a first component with a second or subsequent component, and is not intended to be limiting on the order of contacting the sample with components for measuring tenase activity or as requiring the sample simultaneously to be contacted with said components unless otherwise specified.
The methods of the present invention provide compositions comprising components which, in combination with the blood clotting factor in the sample (the activity of which is to be determined), allow a CSA to be performed. The methods may therefore comprise providing a sample comprising one or more blood clotting factors, and providing FX, FVIIIa and/or FIXa (i.e. the remaining components which, when combined with the one or more blood clotting factors in the sample allow the FVIIIa/FIXa convertase to form), and a chromogenic substrate. The methods may optionally comprise contacting the sample with FX, FVIIIa and/or FIXa and a chromogenic substrate.
The methods of the invention may comprise contacting the sample with FX and the kits of the invention may comprise FX. The amount of FX which is to be provided or which is present in the kit may depend on the concentration of other components present in the assay, and/or the reaction volume used to measure blood clotting activity. However, a skilled person may calculate, estimate or determine the amount of FX which is to be provided in order to achieve a desired amount of FX relative to the amount of the blood clotting factor.
Optionally, the method of the invention may comprise contacting the sample with Factor X. Optionally, following contacting the sample with Factor X, the concentration of FX in the sample is at least 100 times the expected concentration of the blood clotting factor. Optionally, the concentration of FX is at least 110 times, at least 120 times, at least 130 times, at least 140 times, at least 150 times, at least 160 times, at least 170 times, at least 180 times, at least 190 times, at least 200 times, at least 210 times, at least 220 times, at least 230 times, at least 240 times, at least 250 times, at least 260 times, at least 270 times, at least 280 times, at least 290 times, at least 300 times, at least 310 times, at least 320 times, at least 330 times, at least 340 times, at least 350 times, at least 360 times, at least 370 times, at least 380 times, at least 390 times, at least 400 times, at least 410 times, at least 420 times, at least 430 times, at least 440 times, at least 450 times, at least 460 times, at least 470 times, at least 480 times, at least 490 times, at least 500 times, at least 510 times, at least 520 times, at least 530 times, at least 540 times, at least 550 times, at least 600 times, at least 650 times, at least 700 times, at least 750 times, at least 800 times, at least 850 times, at least 900 times, at least 950 times, at least 1000 times, at least 1100 times, at least 1200 times, at least 1300 times, at least 1400 times or at least 1500, at least
2000 times, at least 2500 times, at least 3000 times, at least 3500 times, at least 4000 times, at least 4500 times, at least 5000 times, at least 10,000 times, at least 20,000 times, at least 50,000 times, at least 100,000 times, at least 150,000 times or at least 180,000 times the expected concentration of the blood clotting factor. Optionally, the concentration of FX is between 100 and 5000 times, between 300 and 2500 times, between 500 and 1500 times, or between 750 and 1200 times the expected concentration of the blood clotting factor. Optionally, the concentration of FX is around 1000 times the expected concentration of the blood clotting factor. Optionally, the concentration of FX is 1000 times expected concentration of the blood clotting factor.
Optionally, the concentration of FVIII in the composition is 0.00025 to 0.0625 IU/ml. Optionally, the concentration of FVIII is 0.0005 to 0.05 IU/ml, 0.00075 to 0.03725 IU/ml, 0.001 to 0.025 IU/ml or 0.00125 to 0.0125 IU/ml. Optionally, the concentration of FVIII in the composition is around 0.025 IU/ml. Optionally, the concentration of FVIII in the sample is 0.025 IU/ml.
Optionally, the concentration of FIX in the composition is 0.0001 to 0.025 IU/ml. Optionally, the concentration of FIX in the composition is 0.0002 to 0.02 IU/ml, 0.0003 to 0.015 IU/ml, 0.0004 to 0.01 IU/ml or 0.0005 to 0.005 IU/ml. Optionally, the concentration of FIX in the composition is around 0.0025 IU/ml. Optionally, the concentration of FIX in the sample is 0.0025 IU/ml.
Optionally the concentration of FX is or the concentration of FX used must be at least lOOnM. Optionally, the concentration of FX is or the concentration of FX used must be at least 110 nM, at least 120 nM, at least 130 nM, at least 140 nM, at least 150 nM, at least 160 nM, at least 170 nM, at least 180 nM, at least 190 nM, at least 200 nM, at least 210 nM, at least 220 nM, at least 230 nM, at least 240 nM, at least 250 nM, at least 260 nM, at least 270 nM, at least 280 nM, at least 290 nM, at least 300 nM, at least 310 nM, at least 320 nM, at least 330 nM, at least 340 nM, at least 350 nM, at least 360 nM, at least 370 nM, at least 380 nM, at least 390 nM, at least 400 nM, at least 410 nM, at least 420 nM, at least 430 nM, at least 440 nM, at least 450 nM, at least 500 nM, at least 550 nM, at least 600 nM, at least 650 nM, at least 700 nM, at least 750 nM, at least 800 nM, at least 850 nM, at least 900 nM, at least 950, or at least 1000 nM. Optionally the concentration of FX is or the concentration of FX used must be between 100 nM and 1000 nM, between 250 nM and 750 nM, between 350 nM and 600 nM, or between 450 nM and 500 nM. Optionally, the concentration of FX is or the concentration of FX used must be around 460 nM. Optionally, the concentration of FX is or the concentration of FX used must be 460 nM. Optionally, the concentration of FX is or the concentration of FX used must be at least 150 nM, at least 300 nM, around 180 nM, around 360nM , between 150 nM and 1000 nM, or between 300 nM and 1000 nM.
The concentration of FX in normal human plasma is around 180 nM. Optionally, the concentration of FX is or the concentration of FX used must be at least 50% of the concentration in normal human plasma. Optionally, the concentration of FX is or the concentration of FX used must be at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 550%, at least 600%, at least 650%, at least 700% or at least 750% the concentration in normal human plasma. Optionally the concentration of FX is or the concentration of FX used must be between 70% and 750%, between 80% and 500%, between 85% and 400%, or between 100% and 300% the concentration in normal human plasma. Optionally, the concentration of FX is or the concentration of FX used must be around 100% the concentration in normal human plasma. Optionally, the concentration of FX is or the concentration of FX used must be 100% the concentration in normal human plasma. Optionally, the concentration of FX is or the concentration of FX used must be around 300% the concentration in normal human plasma. Optionally, the concentration of FX is or the concentration of FX used must be 300% the concentration in normal human plasma.
Optionally, the amount of FX which is non-limiting on intrinsic tenase activity may be expressed in terms of an amount of FX per unit time, to reflect the fact that the length of various steps of the methods of the invention which result in the formation of FXa may be considered when calculating the amount of FX to use in the methods of the invention.
Optionally, the amount of FX may be an amount of FX per minute. Optionally, the amount of FX may be an amount of FX per minute for which the sample is contacted with an agent for activating blood clotting factor. Optionally, the amount of FX may be an amount of FX per minute of the incubation step to allow the formation of FXa.
Optionally, the amount of FX is or the amount of FX used must be at least 33 times the expected amount of the blood clotting factor per minute for which the sample is contacted with an agent for activating a blood clotting factor. Optionally, the amount of FX is or the amount of FX used must be at least 37 times, at least 40 times, at least 43 times, at least 47 times, at least 50 times, at least 53 times, at least 57 times, at least 60 times, at least 63 times, at least 67 times, at least 60 times, at least 63 times, at least 67 times, at least 70 times, at least 73 times, at least 77 times, at least 80 times, at least 83 times, at least 87 times, at least 90 times, at least 93 times, at least 97 times, at least 100 times, at least 103 times, at least 107 times, at least 110 times, at least 113 times, at least 117 times, at least 120 times, at least 123 times, at least 127 times, at least 130 times, at least 133 times, at least 137 times, at least 140 times, at least 143 times, at least 147 times, at least 150 times, at least 153 times, at least 157 times, at least 160 times, at least 163 times, at least 167 times, at least 170 times, at least 173 times, at least 177 times, at least 180 times, at least 183 times, at least 200 times, at least 217 times, at least 233 times, at least 250 times, at least 267 times, at least 283 times, at least 300 times, at least 317 times, at least 333 times, at least 367 times, at least 400 times, at least 433 times, at least 467 times, at least 500 times, at least 667 times, at least 833 times, at least 1000 times, at least 1167 times, at least 1333 times, at least 1500 times, at least 1667 times, at least 3333 times, at least 6667 times, at least 16,667 times, at least 33,333 times, at least 50,000 times, or at least 60,000 times the expected amount of the blood clotting factor in the composition per minute for which the sample is contacted with an agent for activating a blood clotting factor. Optionally, amount of FX is or the amount of FX used must be between 33 and 1667 times, between 100 and 833 times, between 167 and 500 times, or between 250 and 400 times the expected amount of the blood clotting factor in the composition per minute for which the sample is contacted with an agent for activating a blood clotting factor. Optionally, the amount of FX is or the amount of FX used must be around 333 times the expected amount of the blood clotting factor in the composition per minute for which the sample is contacted with an agent for activating a blood clotting factor. Optionally, the amount of FX is or the amount of FX used may be 333 times the expected amount of the blood clotting factor in the composition per minute for which the sample is contacted with an agent for activating a blood clotting factor.
Optionally, the amount of FX is or the amount of FX used must be at least 33 times the expected amount of the blood clotting factor per minute of the incubation step to allow the formation of FXa. Optionally, the amount of FX is or the amount of FX used must be at least 37 times, at least 40 times, at least 43 times, at least 47 times, at least 50 times, at least 53 times, at least 57 times, at least 60 times, at least 63 times, at least 67 times, at least 60 times, at least 63 times, at least 67 times, at least 70 times, at least 73 times, at least 77 times, at least 80 times, at least 83 times, at least 87 times, at least 90 times, at least 93 times, at least 97 times, at least 100 times, at least 103 times, at least 107 times, at least 110 times, at least 113 times, at least 117 times, at least 120 times, at least 123 times, at least 127 times, at least 130 times, at least 133 times, at least 137 times, at least 140 times, at least 143 times, at least 147 times, at least 150 times, at least 153 times, at least 157 times, at least 160 times, at least 163 times, at least 167 times, at least 170 times, at least 173 times, at least 177 times, at least 180 times, at least 183 times, at least 200 times, at least 217 times, at least 233 times, at least 250 times, at least 267 times, at least 283 times, at least 300 times, at least 317 times, at least 333 times, at least 367 times, at least 400 times, at least 433 times, at least 467 times, at least 500 times, at least 667 times, at least 833 times, at least 1000 times, at least 1167 times, at least 1333 times, at least 1500 times, at least 1667 times, at least 3333 times, at least 6667 times, at least 16,667 times, at least 33,333 times, at least 50,000 times, or at least 60,000 times the expected amount of the blood clotting factor in the composition per minute of the incubation step. Optionally, amount of FX is or the amount of FX used must be between 33 and 1667 times, between 100 and 833 times, between 167 and 500 times, or between 250 and 400 times the expected amount of the blood clotting factor in the composition per minute of the incubation step. Optionally, the amount of FX is or the amount of FX used must be around 333 times the expected amount of the blood clotting factor in the composition per minute of the incubation step. Optionally, the amount of FX is or the amount of FX used may be 333 times the expected amount of the blood clotting factor in the composition per minute of the incubation step. Optionally, the method or kit of the invention may allow the activity of a FVIII blood clotting factor to be measured, and the amount of FX may be an amount relative to the expected amount of the FVIII blood clotting factor in the composition. Thus, optionally, the amount of FX may be at least 33x the expected amount of the FVIII blood clotting factor in the composition per minute for which the sample is contacted with the agent for activating FVIII. Optionally, the amount of FX may be at least 33x the expected amount of the FVIII blood clotting factor in the composition per minute of the incubation step. Optionally, the amount of FX relative to the amount of the FVIII blood clotting factor per minute for which the sample is contacted with the agent for activating FVIII may be any of the values or ranges indicated above. Optionally, the amount of FX relative to the amount of the FVIII blood clotting factor per minute of the incubation step may be any of the values or ranges indicated above.
Optionally, the method or kit of the invention may allow the activity of a FIX blood clotting factor to be measured, and the amount of FX may be an amount relative to the expected amount of the FIX blood clotting factor in the composition. Thus, optionally, the amount of FX may be at least 33x the expected amount of the FIX blood clotting factor in the composition per minute for which the sample is contacted with the agent for activating FIX. Optionally, the amount of FX may be at least 33x the expected amount of the FIX blood clotting factor in the composition per minute of the incubation step. Optionally, the amount of FX relative to the amount of the FIX blood clotting factor per minute for which the sample is contacted with the agent for activating FIX may be any of the values or ranges indicated above. Optionally, the amount of FX relative to the amount of the FIX blood clotting factor per minute of the incubation step may be any of the values or ranges indicated above.
Optionally, the amount of FX is or the amount of FX used must be at least 33 pmol per minute for which the sample is contacted with an agent for activating a blood clotting factor (pmol/min) per IU of the blood clotting factor (the activity of which is to be measured, i.e. the blood clotting factor in the sample) in the composition. Optionally, amount of FX is or the amount of FX used must be at least 37 pmol/min, at least 40 pmol/min, at least 43 pmol/min, at least 47 pmol/min, at least 50 pmol/min, at least 53 pmol/min, at least 57 pmol/min, at least 60 pmol/min, at least 63 pmol/min, at least 67 pmol/min, at least 70 pmol/min, at least 73 pmol/min, at least 77 pmol/min, at least 80 pmol/min, at least 83 pmol/min, at least 87 pmol/min, at least 90 pmol/min, at least 93 pmol/min, at least 97 pmol/min, at least 100 pmol/min, at least 103 pmol/min, at least 107 pmol/min, at least 110 pmol/min, at least 113 pmol/min, at least 117 pmol/min, at least 120 pmol/min, at least 123 pmol/min, at least 127 pmol/min, at least 130 pmol/min, at least 133 pmol/min, at least 137 pmol/min, at least 140 pmol/min, at least 143 pmol/min, at least 147 pmol/min, at least 150 pmol/min, at least 153 pmol/min, at least 157 pmol/min, at least 160 pmol/min, at least 163 pmol/min, at least 167 pmol/min, at least 170 pmol/min, at least 173 pmol/min, at least 177 pmol/min, at least 180 pmol/min, at least 183 pmol/min, at least 200 pmol/min, at least 217 pmol/min, at least 233 pmol/min, at least 250 pmol/min, at least 267 pmol/min, at least 283 pmol/min, at least 300 pmol/min, at least 317 pmol/min, at least 333 pmol/min, at least 367 pmol/min, at least 400 pmol/min, at least 433 pmol/min, at least 467 pmol/min, at least 500 pmol/min, at least 667 pmol/min, at least 833 pmol/min, at least 1000 pmol/min, at least 1167 pmol/min, at least 1333 pmol/min, at least 1500 pmol/min, at least 1667 pmol/min, at least 3333 pmol/min, at least 6667 pmol/min, at least 16,667 pmol/min, at least 33,333 pmol/min, at least 50,000 pmol/min, or at least 60,000 pmol/min per IU of the blood clotting factor in the composition. Optionally, the amount of FX is or the amount of FX used must be between 33 and 1667 pmol/min, between 100 and 833 pmol/min, between 167 and 500 pmol/min, or between 250 and 400 pmol/min per IU of the blood clotting factor in the composition. Optionally, the amount of FX is or the amount of FX used must be around 333 pmol/min per IU of the blood clotting factor in the composition. Optionally, the amount of FX is or the amount of FX used must be 333 pmol/min per IU of the blood clotting factor in the composition.
Optionally, the amount of FX is or the amount of FX used must be at least 33 pmol per minute of the incubation step (pmol/min) per IU of the blood clotting factor (the activity of which is to be measured, i.e. the blood clotting factor in the sample) in the composition. Optionally, amount of FX is or the amount of FX used must be at least 37 pmol/min, at least 40 pmol/min, at least 43 pmol/min, at least 47 pmol/min, at least 50 pmol/min, at least 53 pmol/min, at least 57 pmol/min, at least 60 pmol/min, at least 63 pmol/min, at least 67 pmol/min, at least 70 pmol/min, at least 73 pmol/min, at least 77 pmol/min, at least 80 pmol/min, at least 83 pmol/min, at least 87 pmol/min, at least 90 pmol/min, at least 93 pmol/min, at least 97 pmol/min, at least 100 pmol/min, at least 103 pmol/min, at least 107 pmol/min, at least 110 pmol/min, at least 113 pmol/min, at least 117 pmol/min, at least 120 pmol/min, at least 123 pmol/min, at least 127 pmol/min, at least 130 pmol/min, at least 133 pmol/min, at least 137 pmol/min, at least 140 pmol/min, at least 143 pmol/min, at least 147 pmol/min, at least 150 pmol/min, at least 153 pmol/min, at least 157 pmol/min, at least 160 pmol/min, at least 163 pmol/min, at least 167 pmol/min, at least 170 pmol/min, at least 173 pmol/min, at least 177 pmol/min, at least 180 pmol/min, at least 183 pmol/min, at least 200 pmol/min, at least 217 pmol/min, at least 233 pmol/min, at least 250 pmol/min, at least 267 pmol/min, at least 283 pmol/min, at least 300 pmol/min, at least 317 pmol/min, at least 333 pmol/min, at least 367 pmol/min, at least 400 pmol/min, at least 433 pmol/min, at least 467 pmol/min, at least 500 pmol/min, at least 667 pmol/min, at least 833 pmol/min, at least
1000 pmol/min, at least 1167 pmol/min, at least 1333 pmol/min, at least 1500 pmol/min, at least 1667 pmol/min, at least 3333 pmol/min, at least 6667 pmol/min, at least 16,667 pmol/min, at least 33,333 pmol/min, at least 50,000 pmol/min, or at least 60,000 pmol/min per IU of the blood clotting factor in the composition. Optionally, the amount of FX is or the amount of FX used must be between 33 and 1667 pmol/min, between 100 and 833 pmol/min, between 167 and 500 pmol/min, or between 250 and 400 pmol/min per IU of the blood clotting factor in the composition. Optionally, the amount of FX is or the amount of FX used must be around 333 pmol/min per IU of the blood clotting factor in the composition. Optionally, the amount of FX is or the amount of FX used must be 333 pmol/min per IU of the blood clotting factor in the composition.
Optionally, following contacting the sample with Factor X, the concentration of FX in the sample is at least 33 times the expected concentration of the blood clotting factor per minute for which the sample is contacted with an agent for activating a blood clotting factor. Optionally, the concentration of FX is at least 37 times, at least 40 times, at least 43 times, at least 47 times, at least 50 times, at least 53 times, at least 57 times, at least 60 times, at least 63 times, at least 67 times, at least 60 times, at least 63 times, at least 67 times, at least 70 times, at least 73 times, at least 77 times, at least 80 times, at least 83 times, at least 87 times, at least 90 times, at least 93 times, at least 97 times, at least 100 times, at least 103 times, at least 107 times, at least 110 times, at least 113 times, at least 117 times, at least 120 times, at least 123 times, at least 127 times, at least 130 times, at least 133 times, at least 137 times, at least 140 times, at least 143 times, at least 147 times, at least 150 times, at least 153 times, at least 157 times, at least 160 times, at least 163 times, at least 167 times, at least 170 times, at least 173 times, at least 177 times, at least 180 times, at least 183 times, at least 200 times, at least 217 times, at least 233 times, at least 250 times, at least 267 times, at least 283 times, at least 300 times, at least 317 times, at least 333 times, at least 367 times, at least 400 times, at least 433 times, at least 467 times, at least 500 times, at least 667 times, at least 833 times, at least 1000 times, at least 1167 times, at least 1333 times, at least 1500 times, at least 1667 times, at least 3333 times, at least 6667 times, at least 16,667 times, at least 33,333 times, at least 50,000 times, or at least 60,000 times the expected concentration of the blood clotting factor in the composition per minute for which the sample is contacted with an agent for activating a blood clotting factor. Optionally, the concentration of FX is between 33 and 1667 times, between 100 and 833 times, between 167 and 500 times, or between 250 and 400 times the expected concentration of the blood clotting factor in the composition per minute for which the sample is contacted with an agent for activating a blood clotting factor. Optionally, the concentration of FX is around 333 times the expected concentration of the blood clotting factor in the composition per minute for which the sample is contacted with an agent for activating a blood clotting factor. Optionally, the concentration of FX is 333 times the expected concentration of the blood clotting factor in the composition per minute for which the sample is contacted with an agent for activating a blood clotting factor.
Optionally, following contacting the sample with Factor X, the concentration of FX in the sample is at least 33 times the expected concentration of the blood clotting factor per minute of the incubation step. Optionally, the concentration of FX is at least 37 times, at least 40 times, at least 43 times, at least 47 times, at least 50 times, at least 53 times, at least 57 times, at least 60 times, at least 63 times, at least 67 times, at least 60 times, at least 63 times, at least 67 times, at least 70 times, at least 73 times, at least 77 times, at least 80 times, at least 83 times, at least 87 times, at least 90 times, at least 93 times, at least 97 times, at least 100 times, at least 103 times, at least 107 times, at least 110 times, at least 113 times, at least 117 times, at least 120 times, at least 123 times, at least 127 times, at least 130 times, at least 133 times, at least 137 times, at least 140 times, at least 143 times, at least 147 times, at least 150 times, at least 153 times, at least 157 times, at least 160 times, at least 163 times, at least 167 times, at least 170 times, at least 173 times, at least 177 times, at least 180 times, at least 183 times, at least 200 times, at least 217 times, at least 233 times, at least 250 times, at least 267 times, at least 283 times, at least 300 times, at least 317 times, at least 333 times, at least 367 times, at least 400 times, at least 433 times, at least 467 times, at least 500 times, at least 667 times, at least 833 times, at least 1000 times, at least 1167 times, at least 1333 times, at least 1500 times, at least 1667 times, at least 3333 times, at least 6667 times, at least 16,667 times, at least 33,333 times, at least 50,000 times, or at least 60,000 times the expected concentration of the blood clotting factor in the composition per minute of the incubation step. Optionally, the concentration of FX is between 33 and 1667 times, between 100 and 833 times, between 167 and 500 times, or between 250 and 400 times the expected concentration of the blood clotting factor in the composition per minute of the incubation step. Optionally, the concentration of FX is around 333 times the expected concentration of the blood clotting factor in the composition per minute of the incubation step. Optionally, the concentration of FX is 333 times the expected concentration of the blood clotting factor in the composition per minute of the incubation step.
Optionally, the concentration of FX is or the concentration of FX used must be at least 33 nM per minute for which the sample is contacted with an agent for activation a blood clotting factor (nM/min). Optionally, the concentration of FX is or the concentration of FX used must be at least 37 nM/min, at least 40 nM/min, at least 43 nM/min, at least 47 nM/min, at least 50 nM/min, at least 53 nM/min, at least 57 nM/min, at least 60 nM/min, at least 63 nM/min, at least 67 nM/min, at least 60 nM/min, at least 63 nM/min, at least 67 nM/min, at least 70 nM/min, at least 73 nM/min, at least 77 nM/min, at least 80 nM/min, at least 83 nM/min, at least 87 nM/min, at least 90 nM/min, at least 93 nM/min, at least 97 nM/min, at least 100 nM/min, at least 103 nM/min, at least 107 nM/min, at least 110 nM/min, at least 113 nM/min, at least 117 nM/min, at least 120 nM/min, at least 123 nM/min, at least 127 nM/min, at least 130 nM/min, at least 133 nM/min, at least 137 nM/min, at least 140 nM/min, at least 143 nM/min, at least 147 nM/min, at least 150 nM/min, at least 153 nM/min, at least 157 nM/min, at least 160 nM/min, at least 163 nM/min, at least 167 nM/min, at least 170 nM/min, at least 173 nM/min, at least 177 nM/min, at least 180 nM/min, at least 183 nM/min, at least 200 nM/min, at least 217 nM/min, at least 233 nM/min, at least 250 nM/min, at least 267 nM/min, at least 283 nM/min, at least 300 nM/min, at least 317 nM/min or at least 333 nM/min. Optionally, the concentration of FX is or the concentration of FX used must be between 33 and 333 nM/min, between 83 nM/min and 250 nM/min, between 117 and 200 nM/min, or between 150 and 167 nM/min. Optionally, the concentration of FX is or the concentration of FX used must be around 167 nM/min. Optionally, the concentration of FX is or the concentration of FX used must be 333 nM/min. Optionally, the concentration of FX is or the concentration of FX used must be at least 50 nM/min, at least 100 nM/min, around 60 nM/min, around 120 nM/min, between 50 nM/min and 333 nM/min, or between 100 nM/min and 333 nM/min.
Optionally, the concentration of FX is or the concentration of FX used must be at least 33 nM per minute of the incubation step (nM/min). Optionally, the concentration of FX is or the concentration of FX used must be at least 37 nM/min, at least 40 nM/min, at least 43 nM/min, at least 47 nM/min, at least 50 nM/min, at least 53 nM/min, at least 57 nM/min, at least 60 nM/min, at least 63 nM/min, at least 67 nM/min, at least 60 nM/min, at least 63 nM/min, at least 67 nM/min, at least 70 nM/min, at least 73 nM/min, at least 77 nM/min, at least 80 nM/min, at least 83 nM/min, at least 87 nM/min, at least 90 nM/min, at least 93 nM/min, at least 97 nM/min, at least 100 nM/min, at least 103 nM/min, at least 107 nM/min, at least 110 nM/min, at least 113 nM/min, at least 117 nM/min, at least 120 nM/min, at least 123 nM/min, at least 127 nM/min, at least 130 nM/min, at least 133 nM/min, at least 137 nM/min, at least 140 nM/min, at least 143 nM/min, at least 147 nM/min, at least 150 nM/min, at least 153 nM/min, at least 157 nM/min, at least 160 nM/min, at least 163 nM/min, at least 167 nM/min, at least 170 nM/min, at least 173 nM/min, at least 177 nM/min, at least 180 nM/min, at least 183 nM/min, at least 200 nM/min, at least 217 nM/min, at least 233 nM/min, at least 250 nM/min, at least 267 nM/min, at least 283 nM/min, at least 300 nM/min, at least 317 nM/min or at least 333 nM/min. Optionally, the concentration of FX is or the concentration of FX used must be between 33 and 333 nM/min, between 83 nM/min and 250 nM/min, between 117 and 200 nM/min, or between 150 and 167 nM/min. Optionally, the concentration of FX is or the concentration of FX used must be around 167 nM/min. Optionally, the concentration of FX is or the concentration of FX used must be 333 nM/min. Optionally, the concentration of FX is or the concentration of FX used must be at least 50 nM/min, at least 100 nM/min, around 60 nM/min, around 120 nM/min, between 50 nM/min and 333 nM/min, or between 100 nM/min and 333 nM/min.
Optionally, the concentration of FX is or the concentration of FX used must be at least 17% of the concentration in normal human plasma per minute for which the sample is contacted with an agent for activation a blood clotting factor. Optionally, the concentration of FX is or the concentration of FX used must be at least 20%, at least 23%, at least 27%, at least 30%, at least 33%, at least 37%, at least 40%, at least 43%, at least 47%, at least 50%, at least 53%, at least 57%, at least 60%, at least 63%, at least 67%, at least 70%, at least 73%, at least 77%, at least 80%, at least 83%, at least 100%, at least 117%, at least 133%, at least 150%, at least 167%, at least 183%, at least 200%, at least 217%, at least 233% or at least 250% the concentration in normal human plasma per minute for which the sample is contacted with an agent for activation a blood clotting factor. Optionally the concentration of FX is or the concentration of FX used must be between 23% and 250%, between 27% and 267%, between 28% and 133%, or between 33% and 100% the concentration in normal human plasma per minute for which the sample is contacted with an agent for activation a blood clotting factor. Optionally, the concentration of FX is or the concentration of FX used must be around 33% the concentration in normal human plasma per minute for which the sample is contacted with an agent for activation a blood clotting factor. Optionally, the concentration of FX is or the concentration of FX used must be 33%/min the concentration in normal human plasma per minute for which the sample is contacted with an agent for activation a blood clotting factor. Optionally, the concentration of FX is or the concentration of FX used must be around 100%/min the concentration in normal human plasma per minute for which the sample is contacted with an agent for activation a blood clotting factor. Optionally, the concentration of FX is or the concentration of FX used must be 100%/min the concentration in normal human plasma per minute for which the sample is contacted with an agent for activation a blood clotting factor. Optionally, the concentration of FX is or the concentration of FX used must be at least 17% of the concentration in normal human plasma per minute of the incubation step. Optionally, the concentration of FX is or the concentration of FX used must be at least 20%, at least 23%, at least 27%, at least 30%, at least 33%, at least 37%, at least 40%, at least 43%, at least 47%, at least 50%, at least 53%, at least 57%, at least 60%, at least 63%, at least 67%, at least 70%, at least 73%, at least 77%, at least 80%, at least 83%, at least 100%, at least 117%, at least 133%, at least 150%, at least 167%, at least 183%, at least 200%, at least 217%, at least 233% or at least 250% the concentration in normal human plasma per minute of the incubation step. Optionally the concentration of FX is or the concentration of FX used must be between 23% and 250%, between 27% and 267%, between 28% and 133%, or between 33% and 100% the concentration in normal human plasma per minute of the incubation step. Optionally, the concentration of FX is or the concentration of FX used must be around 33% the concentration in normal human plasma per minute of the incubation step. Optionally, the concentration of FX is or the concentration of FX used must be 33%/min the concentration in normal human plasma per minute of the incubation step. Optionally, the concentration of FX is or the concentration of FX used must be around 100%/min the concentration in normal human plasma per minute of the incubation step. Optionally, the concentration of FX is or the concentration of FX used must be 100%/min the concentration in normal human plasma per minute of the incubation step.
Optionally, the composition or kit comprises thrombin, prothrombin and/or bovine FVa. A composition or kit for performing the same reaction as that of the Rox Factor IX (Rossix) kit may comprise thrombin, prothrombin and/or bovine FVa. Optionally, the composition or kit comprises thrombin but does not comprise prothrombin and/or bovine FVa. A composition or kit for performing the same reaction as that of the BIOPHEN™ (Hyphen Biomed) kit may comprise thrombin but not prothrombin and/or bovine FVa.
Optionally, the blood clotting factor may be a Factor VIII (FVIII) blood clotting factor. Optionally, the sample may comprise a FVIII blood clotting factor. Optionally, the method may comprise providing a composition comprising FIXa. Optionally, the kit or composition may comprise FIXa. Optionally, the method may comprise providing a composition comprising FIX and an agent for activating FIX. Optionally, the kit or composition may comprise FIX and an agent for activating FIX. Optionally, the method may comprise providing a composition comprising an agent for activating FVIII. Optionally, the kit or composition may comprise an agent for activating FVIII.
The present invention provides a method for measuring FVIII activity in a sample comprising FVIII, the method comprising: a. providing a composition comprising: i. Factor X (FX) ii. Factor IXa (FIXa) and an agent for activating FVIII; iii. the sample; and iv. a chromogenic substrate of FX; and b. detecting a dye formed by cleavage of the chromogenic substrate, thereby measuring FVIII activity, wherein:
1. the amount of FX is: i. non-limiting relative to the total expected activity of intrinsic tenase; and/or ii. at least 100 times the expected amount of the FVIII blood clotting factor in the composition; and/or iii. at least 100 pmol per IU of FVIII; and/or
2. the concentration of FX is at least 100 nM.
Optionally, the method may comprise contacting the sample with FIXa. Optionally, the method may comprise contacting the sample with FIX and an agent for activating FIXa. Optionally, the method may comprise contacting the sample with an agent for activating FVIII. Optionally, the method may comprise contacting the sample with FIXa and an agent for activating FVIII simultaneously. Optionally, FIXa and the agent for activating FVIII, or the agent for activating FIX and the agent for activating FVIII, may be combined and provided as a single reagent (an activator reagent) and the sample may be contacted with said reagent, thereby contacting the sample with an agent for activating FVIII, and FIXa. Optionally, the Factor VIII blood clotting factor may comprise part of the B domain, or the Factor VIII blood clotting factor may not comprise the B domain. Optionally, the Factor VIII polypeptide does not comprise a B domain. The Factor VIII blood clotting factor may therefore be a beta domain deleted (BDD) Factor VIII polypeptide.
Optionally, the blood clotting factor may be a FVIII blood clotting factor having increased specific activity and/or increased stability relative to wild-type FVIII.
Optionally, the blood clotting factor may be a Factor IX (FIX) blood clotting factor. Optionally, the sample may comprise a FIX blood clotting factor. Optionally, the method may comprise providing a composition comprising FVIIIa. Optionally, the method may comprise providing a composition comprising FVIII and an agent for activating FVIII. Optionally, the kit or composition may comprise FVIII and an agent for activating FVIII. Optionally, the method may comprise providing a composition comprising an agent for activating FIX. Optionally, the kit or composition may comprise an agent for activating FIX.
The present invention provides a method for measuring FIX activity in a sample comprising FIX, the method comprising: a. providing a composition comprising: i. Factor X (FX); ii. Factor VIII, an agent for activating FVIII, and an agent for activating FIX; iii. the sample; and iv. a chromogenic substrate of FX; and b. detecting a dye formed by cleavage of the chromogenic substrate, thereby to measure FIX activity; wherein:
1. the amount of FX is: i. non-limiting relative to the total expected activity of intrinsic tenase; and/or ii. at least 100 times the expected amount of FIXa in the composition; and/or iii. at least 100 pmol per IU of FIXa; and/or
2. the concentration of FX is at least 100 nM. Optionally, the method may comprise contacting the sample with FVIII. Optionally, the method may comprise contacting the sample with an agent for activating FVIII.
Optionally, the method may comprise contacting the sample with an agent for activating FIX. Optionally, the method may comprise contacting the sample with an agent for activating FVIII and an agent for activating FIX simultaneously. Optionally, the agent for activating FVIII and the agent for activating FIX may be combined and provided as a single reagent (an activator reagent) and the sample may be contacted with said reagent, thereby contacting the sample with an agent for activating FVIII and an agent for activating FIX.
Optionally, in a method for measuring FIX activity in a sample, the sample may be contacted with FX and FVIII simultaneously. Optionally, the FX may be combined with the FVIII prior to contacting the sample with said FX and said FVIII. As described in greater detail below, optionally a first reagent may comprise FX and FVIII.
Optionally, and as discussed in greater detail below, the blood clotting factor is a FIX blood clotting factor having increased specific activity relative to wild-type FIX. Optionally, the blood clotting factor is a FIX blood clotting factor comprising an amino acid substitution at a position corresponding to position 338 of the mature wild-type FIX polypeptide. It is within the abilities of the person skilled in the art to determine whether a given polypeptide has a mutation at a position corresponding to position 338. The person skilled in the art merely needs to align the sequence of the polypeptide sequence with that of the wild-type mature FIX polypeptide of SEQ ID NO: 2 and determine whether the residue of the former that aligns with the 338th residue of the latter is an arginine. If not, the polypeptide has a mutation at a position corresponding to position 338 in wild-type FIX. The alignment may be performed using any suitable algorithm, such as that of Needleman and Wunsch. Optionally, the blood clotting factor is a FIX blood clotting factor comprising leucine at a position corresponding to position 338 of (the “Padua” mutation) of the mature wild-type FIX polypeptide. The present invention provides a method for measuring FIX activity in sample comprising FIX having increased specific activity relative to wild-type FIX, the method comprising: a. providing a composition comprising: i. Factor X (FX); ii. Factor VIII, an agent for activating FVIII, and an agent for activating FIX; iii. the sample; and iv. a chromogenic substrate of FX; and b. detecting a dye formed by cleavage of the chromogenic substrate, thereby to measuring FIX activity; wherein:
1. the amount of FX i s : i. non-limiting relative to the total expected activity of the intrinsic tenase; and/or ii. at least 100 times the expected amount of FIXa in the composition; and/or iii. at least 100 pmol per IU of FIXa; and/or
2. the concentration of FX is at least 100 nM.
Optionally, for methods for measuring FIX activity in a sample comprising FIX having increased specific activity relative to wild-type FIX, the amount of FX is non-limiting relative to the activity of the intrinsic tenase formed through the activation of the FIX. Optionally, the length of time for which the sample is contacted with the agent(s) for activating the blood clotting factor(s) is reduced such that the amount of FX is non limiting. Optionally, the length of the incubation step to allow formation of FXa is reduced such that the amount of FX is non-limiting. Optionally, the amount of FX is at least 33 times the expected amount of FIXa in the composition per minute of the incubation step. Optionally, the amount of FX is at least 33 pmol per IU of FIXa per minute of the incubation step. Optionally, the concentration of FX is at least 33 nM per minute of the incubation step. Optionally the agent for activating FVIII is a protease. Optionally, the agent for activating FVIII is a component of the blood clotting pathway. Optionally, the agent for activating FVIII is Factor Ila (Flla) (thrombin). Optionally, the agent for activating FVIII is human Flla. Optionally, the agent for activating FVIII may be a combination of FV and FII.
Optionally the agent for activating FIXa is a protease. Optionally, the agent for activating FIX is a component of the blood clotting pathway. Optionally, the agent for activating FIX is Factor XIa (FXIa). Optionally, the agent for activating FIX is human FXIa.
Optionally, the amount of FX is non-limiting relative to the total expected activity of intrinsic tenase formed by the agent(s) for activating the blood clotting factor(s) activating the blood clotting factor(s). Optionally, the method of the invention comprises contacting the sample with agent(s) for activating the blood clotting factor(s) for at least 30 seconds, at least 35 seconds, at least 40 seconds, at least 45 seconds, at least 50 seconds, at least 55 seconds or at least 1 minute, or up to 2 minutes, up to 3 minutes, up to 4 minutes, up to 5 minutes, up to 6 minutes, up to 7 minutes, up to 8 minutes, up to 9 minutes or up to 10 minutes. Optionally, the sample may be contacted with the agent(s) for activating the blood clotting factor(s) for less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2.5 minutes, less than 2 minutes, between 0 minutes and 5 minutes, between 0 minutes and 4 minutes, between 1 minute 4 minutes, between 1 minute and 3 minutes, between 1 minute and 2.5 minutes or between 1 minute and 2.5 minutes. Optionally, the sample may be contacted with the agent(s) for activating the blood clotting factor(s) for 1 minute to 10 minutes, 2 minutes to 6 minutes or 3 minutes to 5 minutes. Optionally, the sample may be contacted with the agent(s) for activating the blood clotting factor(s) for around 3 minutes. Optionally, the sample may be contacted with the agent(s) for activating the blood clotting factor(s) for 3 minutes. Optionally the sample may be contacted with the agent(s) for activating the blood clotting factor(s) for around 5 minutes. Optionally, the sample may be contacted with the agent(s) for activating the blood clotting factor(s) for 5 minutes.
Optionally, the method of the invention comprises contacting the sample with an agent for activating FVIII, wherein:
A. the agent for activating FVIII is a combination of FV and FII, and the sample is contacted with the agent for activating FVIII for less than 5 minutes, less than 4 minutes, less than 3 minutes, between 0 minutes and 5 minutes, between 1 minute and 4 minutes, or between 1 minute and 3 minutes; or
B. the agent for activating FVIII is Flla, and the sample is contacted with the agent for activating FVIII for less than 3 minutes, less than 2.5 minutes, less than 2 minutes, between 0 minutes and 3 minutes, between 1 minute and 2.5 minutes, or between 1 minute and 2 minutes.
In one embodiment, the present invention provides a method for measuring the activity of a blood clotting factor in a sample comprising FIX, the method comprising: a. providing a composition comprising: i. Factor X (FX); ii. Factor VIII, an agent for activating FVIII, and an agent for activating FIX; iii. the sample; and iv. a chromogenic substrate of FX; and b. detecting a dye formed by cleavage of the chromogenic substrate thereby measuring the activity of the Factor IX blood clotting factor wherein:
A. the agent for activating FVIII is a combination of FV and FII, and the sample is contacted with the agent for activating FVIII for less than 5 minutes, less than 4 minutes, less than 3 minutes, between 0 minutes and 5 minutes, between 1 minute and 4 minutes, or between 1 minute and 3 minutes; or
B. the agent for activating FVIII is Flla, and the sample is contacted with the agent for activating FVIII for less than 3 minutes, less than 2.5 minutes, less than 2 minutes, between 0 minutes and 3 minutes, between 1 minute and 2.5 minutes, or between 1 minute and 2 minutes.
Optionally, the sample may be contacted with the agent(s) for activating the blood clotting factors in the absence of Factor X. Optionally, the sample may be contacted with the agent(s) for activating the blood clotting factor(s) and may subsequently be contacted with Factor X. Optionally, the sample may be contacted with an inhibitor of an agent for activating a blood clotting factor to halt activation of a blood clotting factor. Optionally the sample may be contacted with an inhibitor of an agent for activating FVIII to halt activation of FVIII. Optionally, the sample may be contacted with an inhibitor of an agent for activating FIX to halt the activation of FIX. Optionally, the sample may be contacted with an inhibitor for an agent for activating FVIII and an inhibitor for an agent for activating FIX to halt the activation of FVIII and FIX. Optionally, the method is a method for measuring the activity of a FVIII blood clotting factor, and the sample may be contacted with an inhibitor of an agent for activating FVIII to halt the activation of FVIII. Optionally, the method is a method for measuring the activity of a FIX blood clotting factor, and the sample may be contacted with an inhibitor of an agent for activating FIX to halt the activation of FIX. Optionally, the method is a method for measuring the activity of a FIX blood clotting factor, and the sample may be contacted with an inhibitor of an agent for activating FVIII to halt the activation of FVIII. Optionally, the method is a method for measuring the activity of a FVIII blood clotting factor, and the sample may be contacted with a Flla (thrombin) inhibitor to halt the activation of FVIII. Optionally, the method is a method for measuring the activity of a FIX blood clotting factor, and the sample may be contacted with an inhibitor of FXIa to halt the activation of FIX. Optionally, the method is a method for measuring the activity of a FIX blood clotting factor, and the sample may be contacted with a Flla (thrombin) inhibitor to halt the activation of FVIII.
Optionally, contacting the sample with an inhibitor of an agent for activating a blood clotting factor may be considered to provide the end of the time period for which the sample is contacted with an agent for activating a blood clotting factor.
Optionally, the sample may be contacted with the agent(s) for activating the blood clotting factor(s) in the presence of Factor X. Optionally, the incubation step to allow the formation of FXa may be initiated by contacting the sample with an agent for activating a blood clotting factor. Optionally, the incubation step may comprise at least part of the period of time for which the sample is contacted with an agent for activating a blood clotting factor. Optionally, the incubation step may comprise the period of time for which the sample is contacted with an agent for activating a blood clotting factor. Optionally, the incubation step to allow the formation of FXa may be initiated by contacting the sample with an agent for activating FIX. Optionally, the incubation step to allow the formation of FXa may be initiated by contacting the sample with an agent for activating FVIII. Optionally, the incubation step to allow the formation of FXa may be initiated by contacting the sample with an agent for activating FVIII and an agent for activating FIX.
Optionally, the incubation step to allow the formation of FXa may be initiated by contacting the sample with an agent for activating a blood clotting factor before contacting the sample with a chromogenic substrate of FXa.
Optionally, the sample may be contacted with the inhibitor of an agent for activating a blood clotting factor prior to being contacted with the chromogenic substrate. Optionally, the sample may be contacted with the inhibitor of an agent for activating a blood clotting factor simultaneously with the chromogenic substrate. Optionally, the inhibitor of an agent for activating a blood clotting factor and the chromogenic substrate may be provided as a single reagent.
The sample (which is a constituent component of the composition of the invention) may be or may be derived from any sample comprising a blood clotting factor. Optionally, the sample may be or may be derived from a patient sample. Optionally, the patient sample may be a patient sample which been taken from a patient. Optionally, the patient sample may be a patient sample taken from a patient who has previously been administered a gene therapy vector comprising a polynucleotide encoding a blood clotting factor. Optionally, the patient sample may be a patient sample taken from a patient who has previously been administered a recombinant blood clotting factor (i.e. a recombinant blood clotting factor polypeptide). Optionally, the patient sample may be taken from a patient who has been administered a gene therapy vector comprising a polynucleotide encoding a blood clotting factor that has increased specific activity relative to a corresponding reference wild-type blood clotting factor, or a recombinant blood clotting factor that has increased specific activity relative to a corresponding reference wild-type blood clotting factor. Optionally, the blood clotting factor may be a FVIII blood clotting factor. Optionally, the blood clotting factor may be a FIX blood clotting factor. Optionally, the patient sample is taken from a patient who has been administered a gene therapy vector comprising a polynucleotide encoding a FIX blood clotting factor that comprises a leucine at a position corresponding to position 338 of wild-type FIX, or a recombinant FIX blood clotting factor that comprises a leucine at a position corresponding to position 338 of wild-type FIX. Optionally, the patient is a haemophiliac. Optionally, the method may further comprise a step of obtaining the patient sample from the patient. Optionally, the patient sample may be a blood sample or a fraction of a blood sample, for example a plasma sample.
Optionally, the sample may be or may be derived from a citrated plasma sample.
Optionally, the method of the invention may further comprise a step of determining whether the patient has been partially or fully treated (e.g. determining that the patient has been partially or fully treated for the symptoms of haemophilia) by administration of the gene therapy vector. Partially or fully treating haemophilia may refer to improving the clotting of a patient suffering from haemophilia, and reducing the risk of an uncontrolled bleeding event. Partially or fully treating haemophilia may refer to reducing the number and/or frequency of uncontrolled bleeding events or internal bleeding e.g. in joints. A patient who is partially or fully treated may suffer fewer uncontrolled bleeding events per year. Partially or fully treating haemophilia may refer to increasing the amount or activity of the blood clotting factor present in the plasma of the patient. Partially treating haemophilia may refer to converting a patient with severe (<1% normal blood clotting factor activity) or moderately severe haemophilia (<2% normal blood clotting factor activity) to a patient with mild haemophilia (5-40% normal blood clotting factor activity). Fully treating haemophilia may refer to increasing the blood clotting factor activity of a patient with severe, moderately severe or mild haemophilia to within the normal range (50%-150% normal blood clotting factor activity). In this context, a patient with haemophilia may refer to a patient with haemophilia A (associated with reduced FVIII blood clotting factor activity) or haemophilia B (associated with reduced FIX blood clotting factor activity).
A patient who is partially or fully treated may require a lower dose of a blood clotting factor (e.g. Factor VIII or FIX) to be administered. Optionally, a patient who is partially or fully treated may not require the on-going administration of a blood clotting factor. Optionally, the method may further comprise a step of assessing whether a patient who has been partially or fully treated for haemophilia no longer needs ongoing treatment or needs a reduced level of ongoing treatment with Factor IX or Factor VIII, and adjusting the patient’s treatment regimen appropriately, e.g. to reduce the dose or dose frequency of the Factor IX or Factor VIII that is to be administered, or to halt the administration of the Factor IX or Factor VIII.
In other embodiments, the sample may be or may be derived from a therapeutic concentrate or a therapeutic preparation or medicament. In yet other embodiments, the sample may be or may be derived from conditioned cell medium (i.e. collected from cultured cells expressing a blood clotting factor), or may be enriched for the blood clotting factor (e.g. following one or more purification steps). In further embodiments, the sample may comprise a substantially pure blood clotting factor.
Optionally, the sample may be derived from an initial sample (for example, a larger sample). Optionally, the sample may be or may comprise at least a portion of an initial (e.g. larger) sample (i.e. an aliquot of an initial/larger sample may be taken to provide the “sample” which is to be used for measuring the activity of a blood clotting factor). Optionally, the sample may be or may comprise at least a diluted portion of the initial/larger sample. The sample may thus optionally be diluted compared to the initial/larger sample.
The degree to which the sample may be diluted relative to the initial/larger sample may depend on the nature of the initial/larger sample, the procoagulant activity which is to be measured, and the concentration of the blood clotting factor of interest in the sample. Optionally, the initial/larger sample may be diluted to dilute the concentration of FVIII to the concentration indicated below. Optionally, the initial/larger sample may be diluted to dilute the concentration of FIX to a concentration indicated below.
Optionally, diluting the initial/larger sample may comprise a ‘pre-dilution’ step, in which the concentration of the blood clotting factor is adjusted to within a desired range, before a further dilution step is carried out to adjust the concentration of the blood clotting factor to a suitable concentration to allow the activity of the blood clotting factor to be measured. Optionally, a pre-dilution step may be used to adjust the concentration of the blood clotting factor to a physiological range or level. For example, a pre-dilution step may dilute the concentration of F VIII to approximately 0.2-2 IU/ml prior to a further dilution step to dilute the concentration of F VIII to a suitable level for measuring FVIII activity.
Optionally a pre-dilution step may be used to dilute the concentration of FVIII to 1 IU/ml prior to a further dilution step to dilute the concentration of FVIII to a suitable level for measuring FVIII activity. Optionally, a pre-dilution step may be used to dilute the concentration of FIX to approximately 0.2-2 IU/ml prior to a further dilution step to dilute the concentration of FIX to a level suitable for measuring FIX activity. Optionally, a pre dilution step may be used to dilute the concentration of FIX to 1 IU/ml prior to a further dilution step to dilute the concentration of FIX to a level suitable for measuring FIX activity.
If two or more separate dilution steps are carried out, the fold dilution in each step may be multiplied to calculate the overall fold dilution; by way of representative example, a 1.5 fold pre-dilution step may be combined with a subsequent 100 fold dilution, thereby to provide a 150 fold overall dilution of the initial sample.
The initial sample may optionally be diluted 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500 fold or any range therein. For example, the sample may be diluted 50-500 fold, 100-250 fold, or 200-300 fold.
The initial sample may be pre-diluted and/or diluted using any suitable liquid which does not inhibit the activity of the intrinsic tenase. Optionally, the initial sample may be diluted with a solution comprising a buffer (a dilution buffer).
Optionally, the dilution buffer may be buffered to have a pH of 6.5-8.5. Optionally, the dilution buffer may be buffered to have a pH of 6.8-8.2. Optionally the dilution buffer may be buffered to have a pH of 7.0-8.0. Optionally, the dilution buffer may be buffered to have a pH of 7.2-7.8. Optionally the dilution buffer may be buffered to have a pH of around 7.4. Optionally, the dilution buffer may be buffered to have a pH of 7.4. Optionally, the dilution buffer may comprise a suitable concentration of a buffer reagent to maintain its desired pH. Optionally, the buffer reagent may be selected from the list consisting of MES, Bis-Tris, ADA, ACES, PIPES, Bis-Tris Propane, BES, MOPS, TES, HEPES, DIPSO, MOBS, TAPSO, Tris, HEPPSO, Phosphate, POPSO, TEA, EPPS, Tricine, Gly-Gly, HEPBS, TAPS or AMPD.
If a dilution step is carried out (i.e. if blood clotting factor activity is measured using a sample diluted with respect to an initial sample), the measured activity may be multiplied by the dilution factor to calculate the procoagulant activity of the blood clotting factor in the initial sample.
Optionally, the concentration of FVIII in the sample is 0.001 to 0.25 IU/ml for measuring FVIII activity. Optionally, the concentration of FVIII is 0.002 to 0.2 IU/ml, 0.003 to 0.15 IU/ml, 0.004 to 0.1 IU/ml or 0.005 to 0.05 IU/ml for measuring FVIII activity. Optionally, the concentration of FVIII in the sample is around 0.1 IU/ml for measuring FVIII activity. Optionally, the concentration of FVIII in the sample is 0.1 IU/ml for measuring FVIII activity.
Optionally, the concentration of FIX in the sample is 0.0004 to 0.1 IU/ml for measuring FIX activity. Optionally, the concentration of FIX in the sample is 0.0008 to 0.08 IU/ml, 0.0012 to 0.06 IU/ml, 0.0016 to 0.04 IU/ml or 0.002 to 0.02 IU/ml for measuring FIX activity. Optionally, the concentration of FIX in the sample is around 0.01 IU/ml for measuring FIX activity. Optionally, the concentration of FIX in the sample is 0.01 IU/ml for measuring FIX activity.
Optionally, the composition may comprise Ca2+. Optionally, the concentration of Ca2+ may be at least 100 mM. Optionally, the concentration of Ca2+ may be at least 110 pM, at least 120 pM, at least 130 pM, at least 140 pM, at least 150 pM, at least 160 pM, at least 170 pM, at least 180 pM, at least 190 pM, at least 200 pM, at least 250 pM, at least 300 pM, at least 350 pM, at least 400 pM, at least 450 pM, at least 500 pM, at least 600 pM, at least 700 pM, at least 800 pM, at least 900 pM, at least 1 mM, at least 1.5 mM, at least 2 mM,at least 2.5 mM, at least 5 mM, at least 10 mM, at least 15 mM, at least 20 mM or at least 25 mM. Optionally, the concentration of Ca2+ may be between 100 mM and 25 mM, between 200 mM and 15 mM, between 300 pM and 10 mM, or between 400 pM and 5 mM. Optionally, the concentration of Ca2+ may be around 10 mM. Optionally, the concentration of Ca2+ may be 600 mM.
Optionally, the composition may further comprise Cu2+. Optionally, the concentration of Cu2+ may be at least 0.5 pM. Optionally, the concentration of Cu2+ may be at least 0.6 pM, at least 0.7 pM, at least 0.8 pM, at least 0.9 pM, at least 1 pM, at least 1.1 pM, at least 1.2 pM, at least 1.3 pM, at least 1.4 pM, at least 1.5 pM, at least 1.6 pM, at least 1.7 pM, at least 1.8 pM, at least 1.9 pM or at least 2 pM. Optionally, the concentration of Cu2+ may be between 0.5 pM and 2 pM, between 0.6 and 1.8 pM, between 0.7 and 1.6 pM, between 0.8 and 1.4 pM, or between 0.9 and 1.2 pM. Optionally, the concentration of Cu2+ may be around 1 pM. Optionally, the concentration of Cu2+ may be 1 pM.
Optionally, the composition may further comprise phospholipids. Optionally, the phospholipids may be synthetic phospholipids.
Optionally, the FX may be provided in or the kit may comprise a reagent comprising FX (a first reagent). Put another way, the first reagent may comprise FX. Optionally, the first reagent may further comprise a fibrin polymerisation inhibitor. Optionally, the first reagent may further comprise FVIII. Optionally the first reagent may further comprise Ca2+. Optionally, the first reagent may further comprise Cu2+. In one embodiment, a first reagent for measuring FVIII activity in a sample may comprise FX, a fibrin polymerisation inhibitor, FVIII, Ca2+ and Cu2+. In one embodiment, a first reagent for measuring FIX activity may comprise FX and a fibrin polymerisation inhibitor.
Optionally, the FX may be provided in or the kit may comprise two or more separate reagents ( e.g . in a first reagent and in an activator reagent). The total amount of FX that is provided is the sum of the amounts of FX in the two or more separate reagents. As outlined above, optionally, the agent for activating FVIII may be provided in or the kit may comprise an activator reagent. Optionally, the agent for activating FIX may be provided in or the kit may comprise an activator reagent. Optionally, the activator reagent may further comprise Ca2+. Optionally, the activator reagent may further comprise phospholipids. Optionally, the phospholipids may be synthetic phospholipids. In one embodiment, an activator reagent for measuring FVIII activity may comprise FIXa, thrombin, Ca2+ and phospholipids. In one embodiment, an activator reagent for measuring FIX activity may comprise FXIa, thrombin, Ca2+ and phospholipids. Optionally, the activator reagent may further comprise FX.
Optionally, the methods of the invention may comprise or be for or the kits of the invention may be for measuring specific activity of a blood clotting factor. “Specific activity” refers to the activity (e.g. intrinsic enzyme activity) per unit (e.g. per pg, or per antigen level), as a % of the level in normal human plasma. For example, the specific activity of a blood clotting factor in a sample may be calculated by measuring the concentration of the blood clotting factor in the sample, for example by using a standard ELISA assay, and dividing the measured procoagulant activity of the blood clotting factor by the concentration of the blood clotting factor.
Optionally, the reference to an amount or concentration of a blood clotting factor (including reference to expected amount or concentration) may refer to the amount or concentration of the blood clotting factor which may be provided or formed. Optionally, the amount or concentration of a FVIII blood clotting factor or a FIX blood clotting factor may refer to FVIIIa or FIXa present e.g. in a sample or a composition. Optionally, the amount or concentration of a FVIII blood clotting factor or a FIX blood clotting factor may refer to inactive (i.e. unactivated or activatable) FVIII or FIX when an agent for activating FIX is also provided or is to be provided. Optionally, the amount or concentration of a FVIII blood clotting factor or a FIX blood clotting factor may refer to the total of FVIIIa and FVIII or FIXa and FIX when an agent for activating FVIII or FIX is provided or is to be provided. For example, in methods for determining FVIII activity in a sample, the amount of the FVIII blood clotting factor in the sample is the amount of FVIII in the sample, and in methods for determining FIX activity in a sample, the amount of the FIX blood clotting factor in the sample is the amount of FIX in the sample, as agents for activating FIX are provided.
Optionally, the expected amount of a blood clotting factor in the sample may be determined.
Optionally the method further comprises determining the expected amount of a blood clotting factor by measuring the activity of the blood clotting factor (e.g. a FVIII blood clotting factor or a FIX blood clotting factor) in a group of patients expected to have the same characteristics as a group of patients from which the patient sample will be taken. For example, this method may be used to determine whether a therapy (e.g. administration of a gene therapy vector comprising a polynucleotide encoding a blood clotting factor or a recombinant blood clotting factor) is effective. In such cases, the user of the method will be able to look at the activity of the intrinsic tenase/the amount of the blood clotting factor in plasma samples taken from a small group of patients that have been administered the therapy. The user of the method may then take that activity as the expected total activity of the intrinsic tenase and use that expected total activity to calculate the amount of FX which is non-limiting relative to the total expected activity of intrinsic tenase, or the user of the method may then take that amount of the blood clotting factor as the expected amount of the blood clotting factor to calculate how much FX is 100 times the expected amount of the blood clotting factor in the sample.
Optionally, the blood clotting factor has increased specific activity relative to a corresponding reference wild-type blood clotting factor. Optionally, the blood clotting factor having increased specific activity relative to the corresponding reference wild-type blood clotting factor may have a specific activity which is at least 1.1 fold, at least 1.2 fold, at least 1.5 fold, at least 1.7 fold, at least 1.8 fold, at least 2 fold, at least 2.2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5, at least 10 fold, at least 11 fold, at least 12 fold, at least 13 fold, at least 14 fold or at least 15 fold higher than the specific activity of the corresponding reference wild-type blood clotting factor. The blood clotting factor having increased specific activity relative to a corresponding reference wild-type blood clotting factor comprises an amino acid sequence (for example, a FVIII amino acid sequence or a FIX amino acid sequence) which comprises one or more mutations or modifications relative to the amino acid sequence of the corresponding wild- type blood clotting factor.
Optionally, the mutation may be a substitution mutation. A substitution mutation substitutes one amino acid in an amino acid sequence with a different amino acid; an amino acid that is present at a position within an amino acid sequence may be replaced with a different amino acid, such that the amino acid sequence of the blood clotting factor having increased specific activity differs from the amino acid of the wild-type blood clotting factor at that position.
Optionally, the mutation may be a deletion mutation. A deletion mutation removes one or more amino acids in an amino acid sequence from the amino acid sequence of a blood clotting factor; amino acids which would otherwise be separated in the amino acid sequence of the wild-type blood clotting factor by the one or more amino acids deleted from the amino acid sequence of the blood clotting factor are adjacent to one-another in the amino acid sequence of the blood clotting factor having increased specific activity.
Optionally, the mutation may be an insertion mutation. An insertion mutation introduces one or more amino acids into an amino acid sequence of a blood clotting factor; amino acids which would otherwise be adjacent to one-another in the amino acid sequence of the wild-type blood clotting factor are separated by the one or more amino acids introduced into the amino acid sequence of the blood clotting factor having increased specific activity.
Optionally, the blood clotting factor is a FVIII blood clotting factor having increased specific activity and/or stability relative to wild-type FVIII. The present invention therefore provides a method for measuring FVIII activity in a sample, wherein the sample comprises a FVIII blood clotting factor which has increased specific activity and/or stability relative to a reference wild-type FVIII polypeptide. The FVIII blood clotting factor may comprise a FVIII amino acid sequence comprising one or more mutations relative to the amino acid sequence of the wild-type FVIII sequence.
Optionally, the FVIII blood clotting factor having increased specific activity and/or stability comprises a FVIII amino acid sequence, wherein the FVIII amino acid sequence comprises one or more substitution or deletion mutations in the PACE/furin cleavage site corresponding to the amino acids at positions 1645-1648 of SEQ ID NO: 1, represented by the sequence RHQR. Optionally, one or more amino acids in the PACE/furin cleavage site are deleted. Optionally, the amino acid corresponding to the amino acid at position 1648 of SEQ ID NO: 1 is deleted. Optionally, the amino acid corresponding to the amino acid at position 1645 of SEQ ID NO: 1 is deleted. Optionally, the amino acids corresponding to the amino acids at positions 1645-1647 of SEQ ID NO:l are deleted. Optionally, all four amino acids of the PACE/furin site are deleted, i.e. the amino acids corresponding to the amino acids at positions 1645-1648 of SEQ ID NO: 1 are deleted. Optionally, the FVIII comprises a larger deletion which encompasses the PACE/furin cleavage site.
Optionally, the FVIII blood clotting factor having increased specific activity and/or stability comprises a FVIII amino acid sequence, wherein the FVIII amino acid sequence comprises one or more substitution mutations at an inter-domain interface. Optionally, the one or more substitution mutations is at the A1/A2 domain interface, the A1/A3 domain interface, the A2/A3 domain interface or the A1/C2 domain interface.
Optionally, the one or more substitution mutations comprises substitution of a pair of amino acids in the respective domains with cysteine residues. Optionally, the pair of amino acids which are substituted with cysteine residues may be in the A1 and A3 domains. Optionally, the first amino acid may correspond to M147, S149 or S289 of SEQ ID NO:l and the second amino acid may correspond to E1969, E1970 or N1977 of SEQ ID NO: 1. Optionally, the one or more substitution mutations comprises a pair of substitution mutations selected from the list consisting of (i) S289C and N1977C, (ii) M147C and E1970C, and (iii) S149C and E1969C. Optionally, the pair of amino acids which are substituted with cysteine residues may be in the A2 and A3 domains.
Optionally, the first amino acid corresponds to T667, T669, N684, L687, 1689, S695 or F697 of SEQ ID NO: 1 and the second amino acid corresponds to S1791, G1799, A1800, R1803, El 844, S1949, G1981, V1982, or Y1979 of SEQ ID NO: 1. Optionally, the one or more substitution mutations comprises a pair of substitution mutations selected from the list consisting of (i) T669C and V1982C, (ii) L687C and A1800C, (iii) I689C and G1799C, (iv) F697C and S1949C, (v) T667C and FI 981C, (vi) T669C and Y1979C, (vii) N684C and S1791C, (viii) L687C and R1803C, and (ix) S695C and E1844C. Optionally, the pair of amino acids which are substituted with cysteine residues may be in the A1 and C2 domains. Optionally, the first amino acid corresponds to A108, T118 or V137 of SEQ ID NO: 1 and the second amino acid corresponds to N2172, Q2329 or Y2332 of SEQ ID NO: 1. Optionally, the one or more substitution mutations comprises a pair of substitution mutations selected from the list consisting of (i) A108C and Q2329C, (ii) T118C and N2172C, and (iii) V137C and Y2332C.
Optionally, the one or more substitution mutations comprises substituting a charged amino acid with hydrophobic amino acid. Optionally, the FVIII amino acid sequence comprises a substitution of the glutamic acid residue corresponding to the amino acid at position 287 (E287) of SEQ ID NO: 1, the aspartic acid residue corresponding to the amino acid position 302 (D302) of SEQ ID NO: 1, the aspartic acid residue corresponding to the amino acid at position 519 (D519) of SEQ ID NO:l, the glutamic acid residue corresponding to the amino acid at position 665 (E665) of SEQ ID NO: 1, and/or the glutamic acid residue corresponding to the amino acid at position 1984 (El 984) of SEQ ID NO: 1. Optionally, the FVIII amino acid sequence comprises a substitution of the glutamic acid residue corresponding to the amino acid at position 287 of SEQ ID NO: 1 with alanine (E287A) or valine (E287V), the aspartic acid residue corresponding to the amino acid position 302 of SEQ ID NO: 1 with alanine (D302A) or valine (D302V), the aspartic acid residue corresponding to the amino acid at position 519 of SEQ ID NO:l with alanine (D519A) or valine (D519V), the glutamic acid residue corresponding to the amino acid at position 665 of SEQ ID NO: 1 with alanine (E665A) or valine (E665V), and/or the glutamic acid residue corresponding to the amino acid at position 1984 of SEQ ID NO: 1 with alanine (El 984 A) or valine (El 984V). Optionally, the one or more substitution mutations comprises substitution of an amino acid with a more hydrophobic amino acid. Optionally, the Factor VIII amino acid sequence comprises a substitution of the methionine residue corresponding to the amino acid at position 662 (M662) of SEQ ID NO: 1, and/or the histidine residue corresponding to the amino acid at position 693 (H693) of SEQ ID NO: 1. Optionally the Factor VIII amino acid comprises a substitution of the methionine residue corresponding to the amino acid at position 662 of SEQ ID NO: 1 with a tryptophan residue (M662W). Optionally, the Factor VIII amino acid comprises a substitution of the histidine residue corresponding to the amino acid at position 693 of SEQ ID NO: 1 with a tryptophan residue (H693W).
Optionally, the blood clotting factor is a FIX blood clotting factor having increased specific activity relative to wild-type FIX. The present invention therefore provides a method for measuring FIX activity in a sample, wherein the sample comprises a FIX blood clotting factor which has increased specific activity relative to a reference wild-type FIX blood clotting factor. The FIX blood clotting factor may comprise a FIX amino acid sequence comprising one or more mutations relative to the amino acid sequence of the wild-type FIX sequence.
Optionally, the FIX blood clotting factor having increased specific activity comprises a FIX amino acid sequence, wherein the FIX amino acid sequence comprises one or more substitution mutations. Optionally, the FIX amino acid sequence comprises a substitution of the arginine residue corresponding to the amino acid at position 338 (R338) of SEQ ID NO:2. Optionally, the FIX amino acid sequence comprises a substitution of the arginine residue corresponding to the amino acid at position 338 of SEQ ID NO:2 with an alanine (R338A), valine (R338V), leucine (R338L) or glutamine (R338Q) residue. Optionally, the FIX amino acid sequence comprises a substitution of the lysine residue corresponding to the amino acid at position 301 (K301) of SEQ ID NO:2. Optionally, the FIX amino acid sequence comprises a substitution of the lysine residue corresponding to the amino acid at position 301 of SEQ ID NO:2 with an arginine (K301R) or a leucine (K301L) residue. Optionally, the FIX amino acid sequence comprises a substitution of the arginine residue corresponding to the amino acid at position 338 (R338) of SEQ ID NO:2 and a substitution of the lysine residue corresponding to the amino acid at position 301 (K301) of SEQ ID NO:2. Optionally, the FIX amino acid sequence comprises a substitution of the arginine residue corresponding to the amino acid at position 338 of SEQ ID NO:2 with an alanine (R338A), valine (R338V), or leucine (R338L) or glutamine (R338Q) residue and a substitution of the lysine residue corresponding to the amino acid at position 301 of SEQ ID NO:2 with an arginine (K301R) or leucine (K301L) residue. Optionally, the FIX amino acid sequence comprises a substitution of the arginine residue corresponding to the amino acid at position 338 of SEQ ID NO:2 with a leucine (R338L) residue and a substitution of the lysine residue corresponding to the amino acid at position 301 of SEQ ID NO:2 with an arginine (K301R) residue (a K301R/R338L double mutant).
The methods of the present invention may also be used to identify a blood clotting factor having increased specific activity relative to a corresponding reference wild-type blood clotting factor. Without wishing to be bound by theory, the methods of the present invention may provide a more accurate reflection of the procoagulant activity of a blood clotting factor than CSAs of the art, and thus may allow the detection of differences in the activity of a blood clotting factor which might otherwise not be detected in a CSA. As outlined above, CSAs may potentially be carried out more rapidly and at lower cost than OSAs, and thus CSAs may be a more attractive format for screening variant blood clotting factors for the purposes of identifying a blood clotting factor which has increased specific activity relative to a corresponding reference wild-type blood clotting factor.
The present invention thus provides a method for identifying a blood clotting factor having increased specific activity relative to a corresponding reference wild-type blood clotting factor, said method comprising: a. providing a composition comprising i. Factor X; ii. Factor Villa (F Villa) and/or Factor IXa (FIXa); iii. a sample comprising the blood clotting factor suspected of having increased specific activity; and iv. a chromogenic substrate for FXa; b. detecting a dye formed by cleavage of the chromogenic substrate, thereby measuring the activity of the blood clotting factor suspected of having increased specific activity; and c. comparing the measured activity of the blood clotting factor suspected of having increased specific activity with the activity of a corresponding reference wild-type blood clotting factor; wherein:
1. the amount of FX i s : i. non-limiting relative to the total expected activity of intrinsic tenase; ii. at least 100 times the expected amount of the blood clotting factor in the composition; and/or iii. at least 100 pmol per IU of the blood clotting factor;
2. the concentration of FX is at least 100 nM.
Preferably, the activity of the corresponding reference wild-type blood clotting factor is measured under the same conditions as the activity of the blood clotting factor suspected of having increased specific activity (e.g. by a method of the invention). Preferably, the amount of the blood clotting factor suspected of having increased specific activity used in such a method is controlled relative to the amount of the corresponding wild-type blood clotting factor.
Optionally, the blood clotting factor having increased specific activity relative to the corresponding reference wild-type blood clotting factor may have a specific activity which is at least 1.1 fold, at least 1.2 fold, at least 1.5 fold, at least 1.7 fold, at least 1.8 fold, at least 2 fold, at least 2.2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, at least 9.5 fold, at least 10 fold, at least 11 fold, at least 12 fold, at least 13 fold, at least 14 fold, or at least 15 fold higher than the specific activity of the corresponding reference wild-type blood clotting factor. Optionally, the mutation may be a substitution mutation. Optionally, the mutation may be a deletion mutation. Optionally, the mutation may be an insertion mutation.
Optionally, the blood clotting factor may be a FVIII blood clotting factor. Optionally, the blood clotting factor may be a FIX blood clotting factor.
Optionally, the activity of the corresponding reference wild-type blood clotting factor may be measured by a method of the present invention. Optionally, the activity of the corresponding reference wild-type blood clotting factor may be a measured under the same conditions as the tenase activity of the blood clotting factor suspected of having increased specific activity.
Optionally, the methods of the invention may comprise an incubation step after providing FX and FVIIIa and/or FXa to allow the formation of FXa. may be at least 30 seconds, at least 35 seconds, at least 40 seconds, at least 45 seconds, at least 50 seconds, at least 55 seconds or at least 1 minute, or up to 2 minutes, up to 3 minutes, up to 4 minutes, up to 5 minutes, up to 6 minutes, up to 7 minutes, up to 8 minutes, up to 9 minutes or up to 10 minutes. Optionally, incubation step may be 1 minute to 10 minutes, 2 minutes to 6 minutes or 3 minutes to 5 minutes. Optionally, the incubation step may be around 3 minutes. Optionally, the incubation step may be 3 minutes. Optionally the incubation step may be around 5 minutes. Optionally, the incubation step may be 5 minutes.
Optionally, the methods of the invention may comprise contacting the sample with an agent for activating FVIII, wherein:
A. the agent for activating FVIII is a combination of FV and FII, and the incubation step to allow the formation of FXa is less than 5 minutes, less than 4 minutes, less than 3 minutes, between 0 minutes and 5 minutes, between 1 minute and 4 minutes, or between 1 minute and 3 minutes; or
B. the agent for activating FVIII is Flla, and the incubation step to allow the formation of FXa is less than 3 minutes, less than 2.5 minutes, less than 2 minutes, between 0 minutes and 3 minutes, between 1 minute and 2.5 minutes, or between 1 minute and 2 minutes. In one embodiment, the present invention provides a method for measuring the activity of a blood clotting factor in a sample comprising FIX, the method comprising: a. providing a composition comprising: i. Factor X (FX); ii. Factor VIII, an agent for activating FVIII, and an agent for activating FIX; iii. the sample; and iv. a chromogenic substrate of FX; and b. detecting a dye formed by cleavage of the chromogenic substrate thereby measuring the activity of the Factor IX blood clotting factor wherein:
A. the agent for activating FVIII is a combination of FV and FII, and the incubation step to allow the formation of FXa is less than 5 minutes, less than 4 minutes, less than 3 minutes, between 0 minutes and 5 minutes, between 1 minute and 4 minutes, or between 1 minute and 3 minutes; or
B. the agent for activating FVIII is Flla, and the incubation step to allow the formation of FXa is less than 3 minutes, less than 2.5 minutes, less than 2 minutes, between 0 minutes and 3 minutes, between 1 minute and 2.5 minutes, or between 1 minute and 2 minutes.
Optionally, the incubation step to allow the formation of FXa may take place prior to providing a chromogenic substrate of FXa. Optionally, the methods of the invention may comprise an incubation step after providing FX and FVIIIa and/or FIXa, but prior to providing a chromogenic substrate of FXa, to allow the formation of FXa. Optionally, the incubation step may be at least 30 seconds, at least 35 seconds, at least 40 seconds, at least 45 seconds, at least 50 seconds, at least 55 seconds or at least 1 minute, or up to 2 minutes, up to 3 minutes, up to 4 minutes, up to 5 minutes, up to 6 minutes, up to 7 minutes, up to 8 minutes, up to 9 minutes or up to 10 minutes. Optionally, the incubation step may be 1 minute to 10 minutes, 2 minutes to 6 minutes or 3 minutes to 5 minutes. Optionally, the incubation step may be around 3 minutes. Optionally, the incubation step may be 3 minutes. Optionally the incubation step may be around 5 minutes. Optionally, the incubation step may be 5 minutes. Optionally, providing the composition may comprise providing FX, FVIIIa and/or FIXa and the sample. This may allow the formation of FXa. Optionally, this may take place prior to contacting the sample with the chromogenic substrate of FXa. Optionally, the concentration of FX when forming FXa (and prior to contacting the sample with the chromogenic substrate for FXa) may be at least lOOnM. Optionally, the concentration of FX may be at least 110 nM, at least 120 nM, at least 130 nM, at least 140 nM, at least 150 nM, at least 160 nM, at least 170 nM, at least 180 nM, at least 190 nM, at least 200 nM, at least 210 nM, at least 220 nM, at least 230 nM, at least 240 nM, at least 250 nM, at least 260 nM, at least 270 nM, at least 280 nM, at least 290 nM, at least 300 nM, at least 310 nM, at least 320 nM, at least 330 nM, at least 340 nM, at least 350 nM, at least 360 nM, at least 370 nM, at least 380 nM, at least 390 nM, at least 400 nM, at least 410 nM, at least 420 nM, at least 430 nM, at least 440 nM, at least 450 nM, at least 500 nM, at least 550 nM, at least 600 nM, at least 650 nM, at least 700 nM, at least 750 nM, at least 800 nM, at least 850 nM, at least 900 nM, at least 950, or at least 1000 nM . Optionally the concentration of FX may be between 100 nM and 1000 nM, between 250 nM and 750 nM, between 350 nM and 600 nM, or between 450 nM and 500 nM. Optionally, the concentration of FX may be around 460 nM. Optionally, the concentration of FX may be 460 nM. Optionally, the concentration of FX may be at least 150 nM, at least 300 nM, around 180 nM, around 360nM , between 150 nM and 1000 nM, or between 300 nM and 1000 nM
Incubation may take place at a suitable temperature to allow the formation of FXa by the intrinsic tenase. Optionally, incubation may take place at room temperature (around 20- 25°C). Optionally, incubation may take place at a temperature higher than room temperature. Optionally, incubation may take place at around 30°C, Optionally, incubation may take place at around 35°C. Optionally, incubation may take place at around 37°C.
Reference to a “ chromogenic substrate ofFX ’ herein is intended to include fluorogenic substrates of FX, i.e. substrates which provide a fluorophore following cleavage by FXa that may be detected by measuring an increase in fluorescence at a particular wavelength of light. The chromogenic substrate of FXa may therefore comprise a chromogen or fluorogen which may be released by proteolytic cleavage by FXa, (i.e. to provide a dye or fluorophore), and may be detected. Optionally, the chromogenic substrate comprises a chromogen or fluorogen joined by a peptide bond to the carboxyl carbon atom of an amino acid. Optionally, the a chromogen or fluorogen is joined by a peptide bond to the carboxyl carbon of an arginine residue. Optionally, the chromogenic substrate comprises amino acids forming a FXa cleavage site. Optionally, the chromogenic substrate comprises a chromogen or fluorogen joined by a peptide bond to the carboxyl carbon atom of the amino acid at the C-terminus of a FXa cleavage site. Optionally, the FXa cleavage site comprises the amino acid sequence IDGR or IEGR (the consensus cleavage recognition site for FIXa) or chemical derivatives thereof. Optionally, the chromogenic substrate comprises a chromogen or fluorogen joined by a peptide bond to the carboxyl carbon atom of the amino acid sequence IDGR or IEGR.
Optionally, the chromogenic substrate comprises p-nitroanilide (p-NA). Optionally, following cleavage by FXa, p-NA is released from the substrate to provide a dye. Optionally, the chromogenic substrate is CS-11 (BIOPHEN). Optionally, the chromogenic substrate is S-2222, S-2732 or S-2765 (Chromogenix). Optionally, the chromogenic substrate is F3301 or B7020 (Sigma Aldrich). Optionally, the chromogenic substrate is Spectrozyme® FXa (BioMedica Diagnostics).
Optionally, the chromogenic substrate comprises a fluorogen. Optionally, following cleavage by FXa, the fluorogen is released from the substrate to provide a fluorophore. Optionally, the chromogenic substrate is Spectrofluor® FXa (BioMedica Diagnostics).
Optionally the concentration of the chromogenic substrate of FXa may be at least 25 mM. Optionally, the concentration of the chromogenic substrate of FXa may be at least 30 pM, at least 40 pM, at least 50 pM, at least 60 pM, at least 70 pM, at least 80 pM, at least 90 pM, at least 100 pM, at least 125 pM, at least 150 pM, at least 175 pM, at least 200 pM, at least 250 pM, at least 300 pM, at least 350 pM, at least 400 pM, at least 450 pM, at least 500 pM, at least 600 pM, at least 700 pM, at least 800 pM, at least 900 pM or at least 1 mM. Optionally, the concentration of the chromogenic substrate of FXa may be between 25 mM to 1 mM, or between 50 mM and 500 mM. Optionally, the concentration of the chromogenic substrate of FXa may be at least 2x the Km of the substrate for FXa.
The dye (e.g. the chromophore or fluorophore) formed by cleavage of the chromogenic substrate (i.e. by proteolytic cleavage of the chromogenic substrate by FXa) may be detected, thereby to measure the intrinsic tenase activity in the sample. Optionally, the dye or fluorophore may be detected photometrically. Optionally, the dye may be detected using a spectrophotometer, photometer or analyser for a chromogenic assay. Optionally, p-NA may be detected following cleavage of a chromogenic substrate by measuring optical density (OD) at the wavelength 405 nm (OD405). Optionally, the fluorophore generated following cleavage of Spectrofluor® FXa may be detected at an emission wavelength of between 425-455 nm, optionally 440 nm, and using an excitation wavelength of 315-355 nm, optionally 342 nm.
Optionally, and for example, where a sample comprises no or minimal blood clotting factor activity, the method may comprise detecting the lack of the presence of the dye. Put another way, the method may comprise carrying out a step for detecting the dye (for example, by measuring OD405) but not detecting the presence of the dye.
The composition comprising FX, FVIIIa and/or FIXa, the sample and a chromogenic substrate for FXa may be incubated for a period of time to allow the generation of the dye before detecting the dye. Optionally, the composition may be incubated for at least 10 seconds, at least 15 seconds, at least 20 seconds, at least 25 seconds, at least 30 seconds, at least 35 seconds, at least 40 seconds, at least 45 seconds, at least 50 seconds, at least 55 seconds or at least 1 minute, or up to 2 minutes, up to 3 minutes, up to 4 minutes, up to 5 minutes, up to 6 minutes, up to 7 minutes, up to 8 minutes, up to 9 minutes or up to 10 minutes. Optionally, the sample may be incubated for 1 minute to 10 minutes, 2 minutes to 6 minutes or 3 minutes to 5 minutes. Optionally, the sample may be incubated for around 3 minutes. Optionally, the sample may be incubated for 3 minutes. Optionally the sample may be incubated for around 5 minutes. Optionally, the sample may be incubated for 5 minutes. Incubation may take place at a suitable temperature to allow the formation of the dye by the cleavage of the chromogenic substrate by FXa. Optionally, incubation may take place at room temperature (around 20-25°C). Optionally, incubation may take place at around 30°C. Optionally, incubation may take place at around 35°C. Optionally, incubation may take place at around 37°C.
Optionally, following a period of incubation, the reaction may be quenched to stop the formation of the dye. Optionally, the composition may be contacted with a quenching agent to inactivate FXa and stop the formation of the dye. Optionally, the quenching agent may be a calcium chelator. Optionally, the quenching agent may be a monovalent salt of EDTA, EGTA or citric acid. Optionally, the sample may be quenched by reducing the pH, i.e. by contacting the sample with an acid. Optionally, the quenching agent may be an acid. Optionally, the quenching agent may be hydrochloric acid, sulphuric acid, phosphoric acid, citric acid or acetic acid. Optionally, the composition may be contacted with 1 :4 volume 20g/L (2%) citric acid. Optionally, the composition may be contacted with 1 :4 volume 20% acetic acid. Optionally, the dye may be detected following quenching the sample. Detecting the dye following incubation and quenching may be referred to as carrying out an “end-point” assay.
Optionally, a spectrophotometric blank comprising all of the reaction components may be used. The spectrophotometric blank may be used to zero the reading in a spectrophotometric measuring device. The blank may be quenched {i.e. may comprise the quenching agent) prior to contacting the blank with the chromogenic substrate.
Optionally, the method may be performed by detecting a change in the amount of dye formed through cleavage of the chromogenic substrate by FXa ( e.g . a change in OD405 (DOϋ405)). Optionally, the change in the amount of dye formed may be measured over a period of time. Optionally, the change in the amount of dye formed may be measured from 1 second, from 2 seconds, from 3 seconds, from 4 seconds, from 5 seconds, from 6 seconds, from 7 seconds, from 8 seconds, from 9 seconds or from 10 seconds following contacting the sample with the chromogenic substrate. Optionally, the change in the amount of dye formed may be measured up to 30 seconds, up to 40 seconds, up to 50 seconds, up to 60 seconds, up to 70 seconds, up to 80 seconds, up to 90 seconds, up to 100 seconds, up to 110 seconds, up to 120 seconds, up to 3 minutes, up to 4 minutes, up to 5 minutes, up to 6 minutes, up to 7 minutes, up to 8 minutes, up to 9 minutes or up to 10 minutes following contacting the sample with the chromogenic substrate. Optionally, the change in the amount of dye formed may be measured from 1 second to 10 minutes, from 5 seconds to 5 minutes, or from 10 seconds to 100 seconds following contacting the sample with the chromogenic substrate.
Optionally a calibration curve may be prepared. Optionally, a calibration curve may be prepared by carrying out the method of the invention for measuring activity of a blood clotting factor on a series of samples having a different (and known) amounts of procoagulant activity ( e.g . using pooled human plasma or a commercial calibrator plasma to prepare a series of samples having known procoagulant activity). Thus, the amount of dye formed may be measured for each of the samples having a different known procoagulant activity, and thus the relationship between the amount of dye and the procoagulant activity in a sample may be established. Optionally, a calibration curve may be pre-determined, i.e. it may be prepared prior to performing the methods of the present invention. Optionally, a calibration curve may be prepared separately before measuring the procoagulant activity in a given (i.e. every) sample. Optionally, a calibration curve may be prepared and used for measuring procoagulant activity in multiple samples, or put another way, the procoagulant activity in multiple samples may be measured using the same calibration curve. Optionally, a new or fresh calibration curve may be prepared periodically, e.g. daily, weekly or monthly, or may be prepared batch-wise, e.g. before a new batch of reagents is used, and said new calibration curve may be used to measure procoagulant activity until it is required that a new calibration curve is to be prepared.
Kits for measuring FVIIIa/FIXa convertase activity
The kits of the invention provide FX, FVIIIa and/or FIXa and a chromogenic substrate. Components of the kits of the invention are discussed above, and the following paragraphs include additional discussion. Optionally, a kit of the invention may provide an amount of FX that is non-limiting relative to the total expected activity of FIXa; and/or that is at least 100 times the amount of expected FIXa.
Optionally, a kit of the invention may comprise the components required for performing the method of the invention. Optionally, a kit of the invention may provide FX, FVIIIa and/or FIXa, and a chromogenic substrate required for performing the method of the invention. Optionally, a kit of the invention may provide FX, FVIIIa and/or FIXa and a chromogenic substrate required for providing a composition of the invention. Optionally, such a kit may comprise FX, FVIIIa and/or FIXa, and a chromogenic substrate, wherein the amount of FX is non-limiting relative to the total expected activity of intrinsic tenase; and/or is at least 100 times the expected amount of the blood clotting factor.
Optionally, a kit of the invention may comprise FX, FVIIIa and/or FIXa, and a chromogenic substrate, and may comprise instructions for performing a method of the invention. Optionally, such a kit may be for measuring the activity of a blood clotting factor in a sample.
Optionally, a kit of the invention may comprise FX, FVIIIa and/or FIXa, and a chromogenic substrate, and may comprise instructions which indicate that the amount of FX that must be used must be non-limiting relative to the expected total activity of intrinsic tenase; and/or at least 100 times the expected amount of the blood clotting factor; and/or that concentration of FX that must be used must be at least 100 nM
Optionally, the amount of FX provided in the kit may be an amount of FX such that when the kit is used or when the kit is in use (i.e. when the kit is used for measuring the activity of a blood clotting factor in a sample), the kit provides an amount of FX that is non limiting relative to the total expected activity of intrinsic tenase, and/or that is at least 100 times the expected amount of the blood clotting factor in the sample. The term “when in use” refers to use of the kit in a method for measuring blood clotting factor activity in a sample. The kit of the invention provides an amount of FX such that when a sample is contacted with FX provided in the kit, the amount and/or concentration of FX in the resulting admixture is at the desired level for performing a method of measuring the activity of a blood clotting factor in a sample, optionally a method of the invention.
The present invention also extends to the use of the kits of the invention in the methods of the invention.
Optionally, the kit is for measuring the activity of a blood clotting factor in two or more samples. Optionally, the kit is for measuring activity of a blood clotting factor in three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, 10 or more, 11 or more, 12 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, or 50 or more samples.
Optionally, the kit may comprise sufficient amounts of the components for measuring the activity of a blood clotting factor in two or more samples. Optionally, the kit may comprise sufficient amounts of the components for measuring the activity of a blood clotting factor in three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, 10 or more, 11 or more, 12 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, or 50 or more samples.
Optionally, the kit may provide two or more times the amounts of each of FX, FVIIIa and/or FIXa, and the chromogenic substrate than are required for measuring the activity of a blood clotting factor in a single sample. Optionally, the kit may provide three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, 10 or more, 11 or more, 12 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, or 50 or more times the amounts of each of the components for measuring the activity of a blood clotting factor in a single sample.
Optionally, each of the FX, the components for forming the intrinsic tenase, and the chromogenic substrate of the kit may be intended for dividing into two or more unit formulations, wherein each unit formulation comprises an amount of the respective component for measuring the activity of a blood clotting factor in a single sample (for example, by the methods of the invention outlined above). Optionally, each of the FX, the components for forming the intrinsic tenase, and the chromogenic substrate may be intended for dividing into three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, 10 or more, 11 or more, 12 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, or 50 or more unit formulations.
Optionally, the kit may provide: a. two or more unit formulations of FX; b. two or more unit formulations of FVIIIa and/or FIXa; and c. two or more unit formulations of a chromogenic substrate of FXa; wherein each unit formulation of FX comprises an amount of FX that is non-limiting relative to total expected activity of intrinsic tenase and/or an amount at least 100 times the expected amount of the blood clotting factor in the composition when in use.
Optionally, each unit formulation of FX may comprise at least 100 times the amount of FX relative to the expected amount of the blood clotting factor in the sample which is to be investigated to measure the blood clotting factor activity in the sample. Optionally, each unit formulation of FX may comprise at least 110 times, at least 120 times, at least 130 times, at least 140 times, at least 150 times, at least 160 times, at least 170 times, at least 180 times, at least 190 times, at least 200 times, at least 210 times, at least 220 times, at least 230 times, at least 240 times, at least 250 times, at least 260 times, at least 270 times, at least 280 times, at least 290 times, at least 300 times, at least 310 times, at least 320 times, at least 330 times, at least 340 times, at least 350 times, at least 360 times, at least 370 times, at least 380 times, at least 390 times, at least 400 times, at least 410 times, at least 420 times, at least 430 times, at least 440 times, at least 450 times, at least 460 times, at least 470 times, at least 480 times, at least 490 times, at least 500 times, at least 510 times, at least 520 times, at least 530 times, at least 540 times, at least 550 times, at least 600 times, at least 650 times, at least 700 times, at least 750 times, at least 800 times, at least 850 times, at least 900 times, at least 950 times, at least 1000 times, at least 1100 times, at least 1200 times, at least 1300 times, at least 1400 times, at least 1500 times, at least 2000 times, at least 2500 times, at least 3000 times, at least 3500 times, at least 4000 times, at least 4500 times, at least 5000 times, at least 10,000 times, at least 20,000 times, at least 50,000 times, at least 100,000 times, at least 150,000 times or at least 180,000 times the amount of FX relative to the expected amount of the blood clotting factor in the sample. Optionally, each unit formulation of FX may comprise between 100 and 5000 times, between 300 and 2500 times, between 500 and 1500 times, or between 750 and 1200 times the amount of FX relative to the expected amount of the blood clotting factor in the sample. Optionally, each unit formulation of FX may comprise around 1000 times the amount of FX relative to the expected amount of the blood clotting factor in the sample. Optionally, each unit formulation of FX may comprise 1000 times the amount of FX relative to the expected amount of the blood clotting factor in the sample.
Optionally, the kit may be for measuring FVIII activity in a sample. Optionally, the kit may provide FX, FIXa and an agent for activating FVIII. Optionally, the kit may comprise FX, FIXa and an agent for activating FVIII. Optionally, the kit may be a kit may be for measuring FIX activity in a sample. Optionally, the kit may provide FX, FVIIIa and an agent for activating FIX, Optionally, the kit may comprise FX, FVIII, an agent for activating FVIII and an agent for activating FIX.
Optionally, the kit may comprise a first reagent, an activator reagent and/or a third reagent as described herein. Optionally, the kit may comprise a first reagent, an activator reagent and a third reagent as described herein. Optionally, the first reagent, activator reagent and/or third reagent may be intended for dividing into two or more unit formulations (i.e. to provide unit formulations of the first reagent, activator reagent and/or third reagents).
Optionally, the first reagent, activator reagent and/or third reagent may be provided in lyophilised form, and may be reconstituted prior to use.
Optionally, the concentration of FX in the first reagent (and thus in each unit formulation of the first reagent) may be at least 400 times the expected amount of the blood clotting factor in the sample. Optionally, the concentration of FX in the first reagent is at least 440 times, at least 4800 times, at least 520 times, at least 560 times, at least 600 times, at least 640 times, at least 680 times, at least 720 times, at least 760 times, at least 800 times, at least 840 times, at least 880 times, at least 920 times, at least 860 times, at least 1000 times, at least 1040 times, at least 1080 times, at least 1120 times, at least 1160 times, at least 1200 times, at least 1240 times, at least 1280 times, at least 1320 times, at least 1360 times, at least 1400 times, at least 1440 times, at least 1480 times, at least 1520 times, at least 1560 times, at least 1600 times, at least 1680 times, at least 1720 times, at least 1760 times, at least 1800 times, at least 1840 times, at least 1880 times, at least 1920 times, at least 1960 times, at least 2000 times, at least 2200 times, at least 2400 times, at least 2600 times, at least 2800 times, at least 3000 times, at least 3500 times, at least 4000 times, at least 4500 times, at least 5000 times, at least 5500 times or at least 6000 times the expected concentration of the blood clotting factor in the sample. Optionally, the concentration of FX in the first reagent is between 400 and 6000 times, between 1200 and 5500 times, between 2000 and 5000 times, or between 3500 and 4500 times the expected concentration of the blood clotting factor in the sample. Optionally, the concentration of FX in the first reagent is around 4000 times the expected concentration of the blood clotting factor in the sample. Optionally, the concentration of FX in the first reagent is 4000 times expected concentration of the blood clotting factor in the sample. If provided as a lyophilised reagent, the concentration of FX in the first reagent relative to the expected concentration of the blood clotting factor in the sample may be any of the values indicated above when reconstituted. Optionally, the instructions may indicate the volume for reconstituting a lyophilised reagent to provide such a suitable concentration.
Optionally, the kit provides FX, FVIIIa and/or FIXa and the chromogenic substrate of FXa in suitable ratios such that each of the components is used in corresponding amounts, such that the kit is not substantially depleted of one or more of the components individually (i.e. at a faster rate than other components).
The present invention may be better understood by reference to the following non-limiting examples. Examples
Overview
The Biophen FIX kit (Quadratech 221806) and ROX Factor IX kit (900020) provide chromogenic methods for the quantification of Factor IX (FIX) activity and follow the same principle. In brief, an appropriate dilution of the plasma sample, as specified in the kit, is incubated with a first reagent (“reagent 1” (Rl) from the Biophen FIX kit, or “reagent A” (RA) from the ROX FIX kit) containing FX and FVIII (and FV for the ROX kit). Following this, a second reagent (“reagent 2” from the Biophen FIX kit or “reagent B” from the ROX FIX kit) is added. Reagent 2 (R2) (Biophen FIX kit) contains FXIa (FIX activator), Flla (FVIII activator) and Ca2+. Reagent B (RB) contains FXIa (FIX activator), FII (FVIII activator) and Ca2+. The addition of the second reagent results in the cleavage of FIX and FVIII into the catalytically active versions FIXa and F Villa, which together form the FIXa-FVIIIa complex (Tenase) that converts FX into FXa. A final reagent (“reagent 3” from the Biophen FIX kit, or “FXa substrate” from the ROX FIX kit) containing a FXa specific chromogenic substrate and either a FXIa inhibitor (for the Biophen FIX kit) or a thombin inhibitor (for the ROX FIX kit) is added. The length of time between adding the second reagent and adding the final reagent is 3 minutes for the Biophen FIX kit and 8 minutes for the ROX FIX kit. The FXIa inhibitor prevents further activation of FIX and, therefore, prevents the formation of further FIXa-FVIIIa complexes which convert FX to FXa. The thrombin inhibitor prevents further activation of FVIII and, therefore, prevents the formation of further FIXa-FVIIIa complexes which convert FX to FXa. The FXa specific chromogenic substrate allows quantification of the FXa generated . Conversion of the chromogenic substrate of FXa by FXa is stopped with 20% acetic acid (Biophen kit) or 2% citric acid (ROX kit) and the absorbance is measured at 405nM.
Both methods are performed at 37°C in an opaque 96-well flat bottom plate. The plasma sample is diluted at a minimum of 1 :80 for Rossix (ROX Factor IX kit) and 1 : 100 for Hyphen (Biophen FIX kit) in the dilution buffer provided by each kit.
Example 1 Increasing FX concentration in chromogenic assays
The concentration of FX was determined to be 180nM (100% normal plasma) in reagent 1 (Rl) provided in the Biophen FIX kit and 270nM (150% normal plasma) in reagent A (RA) of the ROX Factor IX kit, respectively by ELISA (Stago, Asserachrom FX: Ag, 00956).
In the Biophen FIX kit assay, Rl is incubated with equal volumes (typically 50pL) of the diluted plasma sample and reagent 2 (R2) to generate FXa (i.e., typically, 50pL of Rl,
50pL of diluted plasma sample and 50pL of R2). As a result, the concentration of FX in Rl is reduced to one-third of initial concentration, and thus the concentration of FX is approximately 33% (60 nM) the level in normal plasma in the mixture containing Rl, the diluted plasma sample and R2. In the ROX Factor IX kit assay, 25pL of RA is incubated with 25pL diluted plasma sample and 150pL Reagent B (RB), diluting the concentration of FX in RA to an eighth of the initial concentration, and thus the concentration of FX is approximately 19% (34 nM) the level in normal plasma in the mixture containing RA, the diluted plasma sample and RB. In the mixture containing the first reagent, the diluted plasma sample and the second reagent, the FX provided from the plasma sample is <1% the level in normal plasma. This is because the plasma sample is pre-diluted at a minimum of 1 : 80 and then further diluted 1 in 3 (Biophen FIX kit) or 1 in 8 (ROX Factor IX kit).
As shown in Figure 2, the measurement of the activity of FIX polypeptide with a R338L mutation (“FIX-Padua”; “Padua mutant”) varied between one stage and chromogenic assays.
To test our hypothesis that the FX concentration was contributing to the discrepancy between one-stage and chromogenic assays when quantifying the activity of FIX-Padua , we increased the concentration of FX to 180 or 230 nM for the ROX FIX kit, or to 180 or 460 nM for the Biophen FIX kit in the mixture following the addition of the second reagent (i.e. for the Biophen FIX kit, in the mixture containing Rl, the diluted plasma sample and R2, and, for ROX FIX fit, in the mixture containing RA, the diluted plasma sample and RB). The assay methods are set out below. The results are shown in Figure 4.
Hyphen Biomed FIX activity assay method
Reagent preparation
Rl, R2 and regent 3 (R3) were reconstituted with 6mL of distilled water. A calibrator (Biophen Calibrator plasma (Quadratech 222101)) was reconstituted with lmL of distilled water. All reagents were left for 30 minutes at room temperature following reconstitution and were then vortexed.
To account for the 1 in 3 dilution of Rl when Rl, the diluted plasma and R2 are mixed, the concentration of FX in Rl was increased by 360nM or 1200 nM. The additional FX combined with the 180nM FX present already in Rl provided in the kit resulted in a concentration of 180nM or 460nM in the mixture containing Rl, the diluted plasma sample and R2 prior to the addition of R3. To increase the FX concentration in Rl, human FX (Enzyme Research Laboratories CAT# HFX 1010) was reconstituted as per manufacturer's instructions to a concentration of 19 mM. The human FX was stored in 20mM Tris-HCL,
0.1M NaCl, ImM Benzamidine, pH 7.4 buffer. Rl was aliquoted into two 3mL tubes. FX was spiked into each tube separately to increase the concentration by 360nM or 1200nM. This provided a concentration of 180nM or 460nM FX in the mixture following the addition of R2 (i.e. in the mixture containing Rl, the diluted plasma sample and R2). FX buffer was added to Rl instead of the additional FX as a control. The modified versions of Rl were subsequently used in the kit as described below.
Standards and sample preparation
Plasma samples were analysed in the high range. The plasma samples were prepared with a 1 : 100 dilution in Reagent 4 (R4) buffer (5 pL in 495 pL of R4) and vortexed thoroughly.
The standard curve was performed in the high range as shown in the table below:
Figure imgf000079_0001
Assay procedure
The assay was run according to manufacturer's instructions as follows: A 96 well plate and modified R1 (i.e. “Rl+FX” which either had a FX concentration of 540nM or 1380nM, or the corresponding control “R1 -buffer” which contained FX buffer in place of the additional FX), R2 and R3 were separately incubated at 37°C for 10 minutes. 50pL of each standard, sample or control was applied to the plate in duplicate. Subsequently, 50pL of R1 -buffer was added to columns 1-4 and 50pL of Rl+FX to columns 5-8 before another incubation for 2 minutes at 37°C (a typical plate layout is shown below). 50pL of R2 was then added to each well and the plate was incubated for 3 minutes at 37°C. 50pL of R3 was then added to each well, and the plate was incubated for 2 minutes at 37°C. Lastly, 50pL of 20% acetic acid was added to each well to quench the reaction before reading at 405nm.
Typical plate layout for hyphen FIX assay:
Figure imgf000080_0001
*PC = Positive control as provided in the kit
Since the concentration of FX in the Rl+FX was 540nM or 1380nM, the concentration of FX following the addition of R2 was 180nM or 460nM FX.
Rossix ROX FIX activity assay method:
Method/Protocol Reagent preparation
Two vials of RA were reconstituted with 1.4mL water and one vial of RB with 8mL water. The vials were incubated for 5 mins at room temperature. The FIX diluent buffer was diluted 1:10 with water to achieve a working stock. To account for the 1 in 8 dilution of RA when RA, the diluted plasma and RB are mixed, the concentration of FX in RA was increased by 1200nm or 1600nM. The additional FX combined with the 270nM FX present already in the RA provided in the kit resulted in a concentration of 180nM or 230nM in the mixture following the addition of RB (i.e. in the mixture containing RA, the diluted plasma sample and RB). To increase the FX concentration in RA, human FX was reconstituted as per manufacturer's instructions to a concentration of 19mM. The human FX was stored in 20mM Tris-HCL, 0.1M NaCl, ImM Benzamidine, pH 7.4 buffer. Human FX was spiked into one vial of RA and FX buffer was added to another vial of RA as a control. For the RA containing additional FX, the concentration of FX was increased by 1200 nM or 1600nM. . This provided a concentration of 180nM or 230nM FX in the mixture following the addition of RB (i.e. in the mixture containing RA, the diluted plasma sample and RB). FX buffer was added to RA instead of the additional FX as a control. The modified versions of RA were subsequently used in the kit as described below.
Sample preparation Plasma samples and controls were analysed in the high range. The plasma samples were prepared with a 1 :80 dilution in FIX diluent buffer (5 pL in 395 pL of FIX diluent buffer) and vortexed thoroughly. The standard curve was performed in the high range as shown in the table below:
Figure imgf000081_0001
Assay procedure
The assay was run according to manufacturer's instructions as follows: A 96 well plate, RB the FXa substrate were incubated separately at 37°C for 10 minutes. 25pL of each standard, sample or control was added to the plate in duplicate. Subsequently, 25pL of RA- buffer (corresponding control which contained FX buffer in place of the additional FX) was added to columns 1-4 and 25 pL of RA+FX (which either had a FX concentration of 1470nM or 1870nM) was added to columns 5-8 before another incubation for 4 minutes at 37°C (a typical plate layout is shown below). 150pL of RB was added to each well and the plate was incubated for 8 minutes at 37°C. 50pL of FXa substrate (containing FXa specific chromogenic substrate and thombin inhibitor) was then added to each well and the plate was incubated for 2 minutes at 37°C. Lastly, 50pL of 2% citric acid was added to each well to quench the reaction before reading at 405nm.
Typical plate layout for Rossix FIX assay:
Figure imgf000082_0001
*PC = Positive control as provided in the kit
Since the concentration of FX in the RA+FX was 1470nM or 1870nM, the concentration of FX following the addition of RB was 180nM or 230nM FX. SynthASil One-stage FIX assay:
Materials
• FIX-deficient plasma (HemosIL - 0020011900)
• S SC lot 4 standard (105 IU/dL)
• STart cuvettes (vessel) (38876) · Ball vial (26441)
• HemosIL Synthasil (0020006810), containing: o Synthasil APTT reagent o 20mM Calcium Chloride
Reagent preparation · Synthasil APTT reagent and Calcium Chloride were incubated for 15 mins at room temperature
• Plasma samples were thawed at 37°C prior to assaying
Assay procedure Briefly, the STart Max instrument is used to carry out the FIX Synthasil one stage assay. Calibration is carried out in cuvettes containing a detection ball and 100 pL volumes of 1/10, 1/20 and 1/40 dilutions of the SSC standard (Lot 4) in FIX-deficient plasma. 100 pL of Synthasil activating reagent is added and equilibrated to 37°C. The clotting reaction is started by adding 100 pL of 20 mM calcium chloride buffer and measuring the time of clot formation. Once the StartMax instrument has been calibrated, samples with unknown FIX activity are measured by diluting in FIX-deficient plasma and following the same protocol as for the SSC calibration. At least three dilutions are required for both the calibration reference plasma and the test sample plasma. Additional dilutions may be carried out if the clotting factor levels fall out of the assay range, i.e. if the test clotting factor level is very high or very low. In the table below are typical results for the calibration of SSC standard (Lot 4).
Figure imgf000083_0001
Example 2
Reducing the length of the activation reaction in chromogenic assays
We hypothesised that the apparent lower R338L-FIX activity reported by the ROX FIX assay compared to the typical one-stage assay could be related to the length of time between adding “reagent B” and adding “FXa substrate” (8 minutes) compared to the typical reaction time (30-120s) used in the one-stage assays.. To determine the effect of reaction time on FIX:C values, we measured matched recombinant R338L-FIX and FIX- WT samples with nominal (SynthASil) FIX:C values of 50, 100 and 150% in parallel with plasma-derived FIX-WT samples as control in a shortened version of the ROX methodology where the length of time between adding “reagent B” and adding “FXa substrate” was 3 minutes. The assay was conducted accorded to the manufacturer’s instructions and in line with the ROX assay in Example 1, except that the length of time between adding “reagent B” and adding “FXa substrate” was varied. Interestingly, in contrast with the typical 8 minute length of time, the activity of recombinant R338L-FIX with the 3 minute length of time was greater than that of the matched recombinant FIX- WT sample (Figure 5 A-B), translating to an increased apparent activity (Figure 5 D-E).
Aspects of the invention
1. A method for measuring the activity of a blood clotting factor in a sample, comprising: a. providing a composition comprising i. Factor X (FX); ii. Factor Villa (F Villa) and/or Factor IXa (FIXa); iii. the sample; and iv. a chromogenic substrate of FXa; and b. detecting a dye formed by cleavage of the chromogenic substrate thereby measuring the activity of the blood clotting factor, wherein:
1. the amount of FX is: i. non-limiting relative to the total expected activity of intrinsic tenase; ii. at least 100 times the expected amount of the blood clotting factor in the composition; and/or iii. at least 100 pmol per IU of the blood clotting factor; and/or
2. the concentration of FX is at least 100 nM.
2. The method of aspect 1, wherein the method comprises contacting the sample with an agent for activating FVIII, and wherein:
A. the agent for activating FVIII comprises a combination of FV and FII, and the sample is contacted with the agent for activating FVIII for less than 5 minutes, less than 4 minutes, less than 3 minutes, between 0 minutes and 5 minutes, between 1 minute and 4 minutes, or between 1 minute and 3 minutes; or B. the agent for activating FVIII is Flla, and the sample is contacted with the agent for activating FVIII for less than 3 minutes, less than 2.5 minutes, less than 2 minutes, between 0 minutes and 3 minutes, between 1 minute and 2.5 minutes, or between 1 minute and 2 minutes.
3. The method of aspect 1 or 2, wherein the method comprises contacting the sample with an agent for activating FVIII, wherein: A. the agent for activating FVIII is a combination of FV and FII, and the method comprises an incubation step to allow the formation of FXa that is less than 5 minutes, less than 4 minutes, less than 3 minutes, between 0 minutes and 5 minutes, between 1 minute and 4 minutes, or between 1 minute and 3 minutes; or
B. the agent for activating FVIII is Flla, and the method comprises an incubation step to allow the formation of FXa that is less than 3 minutes, less than 2.5 minutes, less than 2 minutes, between 0 minutes and 3 minutes, between 1 minute and 2.5 minutes, or between 1 minute and 2 minutes.
4. The method of aspect 2 or 3, wherein contacting the sample with the agent for activating FVIII is carried out before step (a).
5. A method for measuring the activity of a Factor IX blood clotting factor in a sample, comprising: a. providing a composition comprising: i. Factor X (FX); ii. Factor VIII, an agent for activating FVIII, and an agent for activating FIX; iii. the sample; and iv. a chromogenic substrate of FX; and b. detecting a dye formed by cleavage of the chromogenic substrate, thereby to measure FIX activity; wherein the method comprises contacting the sample with an agent for activating FVIII, wherein:
A. the agent for activating FVIII comprises a combination of FV and FII, and the sample is contacted with the agent for activating FVIII for less than 5 minutes, less than 4 minutes, less than 3 minutes, between 0 minutes and 5 minutes, between 1 minute and 4 minutes, or between 1 minute and 3 minutes; or
B. the agent for activating FVIII is Flla, and the sample is contacted with the agent for activating FVIII for less than 3 minutes, less than 2.5 minutes, less than 2 minutes, between 0 minutes and 3 minutes, between 1 minute and 2.5 minutes, or between 1 minute and 2 minutes. 6. A method for measuring the activity of a Factor IX blood clotting factor in a sample, comprising: a. providing a composition comprising: i. Factor X (FX); ii. Factor VIII, an agent for activating FVIII, and an agent for activating FIX; iii. the sample; and iv. a chromogenic substrate of FX; and b. detecting a dye formed by cleavage of the chromogenic substrate, thereby to measure FIX activity; wherein the method comprises contacting the sample an agent for activating FVIII, wherein:
A. the agent for activating FVIII is a combination of FV and FII, and the method comprises an incubation step to allow the formation of FXa that is less than 5 minutes, less than 4 minutes, less than 3 minutes, between 0 minutes and 5 minutes, between 1 minute and 4 minutes, or between 1 minute and 3 minutes; or
B. the agent for activating FVIII is Flla, and the method comprises an incubation step to allow the formation of FXa that is less than 3 minutes, less than 2.5 minutes, less than 2 minutes, between 0 minutes and 3 minutes, between 1 minute and 2.5 minutes, or between 1 minute and 2 minutes.
7. The method of aspect 5 or 6, wherein contacting the sample with the agent for activating FVIII is carried out before step (a).
8. A method for determining an amount of FX which is non-limiting relative to the total expected activity of intrinsic tenase in a method for measuring the activity of blood clotting factor in a sample, said method comprising performing a series of chromogenic assays in which the amount of FX is varied such that various concentrations of FX are used, and determining an amount of FX that is non-limiting, wherein each assay comprises: a. providing a composition comprising: i. Factor X (FX) ii. Factor Villa (FVIIIa) and/or Factor IXa (FIXa); iii. the sample; and iv. a chromogenic substrate of FXa, and b. detecting a dye formed by cleavage of the chromogenic substrate thereby measuring the activity of the blood clotting factor; wherein the amount of FX is non-limiting at any concentrations that provide a less than linear increase in the measured activity of the blood clotting factor when compared to the increase in the FX concentration.
9. A method for determining a length of time for contacting a sample with an agent for activating a blood clotting factor in a method for measuring the activity of a blood clotting factor in a sample for which the amount of FX is non-limiting, said method comprising performing a series of chromogenic assays in which the length of time for which the sample is contacted with an agent for activating a blood clotting factor is varied such that various lengths of time may be used and determining a length of time for contacting the sample with an agent for activating a blood clotting factor for which the amount of FX is non-limiting, wherein each assay comprises: a. providing a composition comprising i. Factor X (FX); ii. Factor Villa (F Villa) and/or Factor IXa (FIXa); iii. the sample; and iv. a chromogenic substrate of FXa; and b. detecting a dye formed by cleavage of the chromogenic substrate thereby measuring the activity of the FIXa; wherein the amount of FX is non-limiting for a length of time for which the sample is contacted with an agent for activating a blood clotting factor if decreasing the length of time decreases the measured activity of the blood clotting factor proportionately.
10. A method for determining a length of an incubation step to allow the formation of FXa in a method for measuring the activity of a blood clotting factor in a sample for which the amount of FX is non-limiting, said method comprising performing a series of chromogenic assays in which the length of the incubation step is varied such that various lengths of the incubation step may be used and determining a length of the incubation step for which the amount of FX is non-limiting, wherein each assay comprises: a. providing a composition comprising i. Factor X (FX); ii. Factor Villa (F Villa) and/or Factor IXa (FIXa); iii. the sample; and iv. a chromogenic substrate of FXa; and b. detecting a dye formed by cleavage of the chromogenic substrate thereby measuring the activity of the FIXa; wherein the amount of FX is non-limiting for a length of the incubation step if decreasing the length of the incubation step decreases the measured activity of the blood clotting factor proportionately.
11. The method of aspect 1, wherein the amount of FX that is non-limiting is determined by the method of aspect 8.
12. The method of aspect 1, wherein the method comprises a step of contacting the sample with an agent for activating a blood clotting factor, wherein the length of the step of contacting the sample with the agent for activating the blood clotting factor is determined by the method of aspect 9.
13. The method of aspect 1, wherein the method comprises an incubation step to allow the formation of FXa, wherein the length of the incubation step is determined by the method of aspect 10.
14. A composition comprising: i. Factor X (FX); ii. Factor Villa (F Villa) and/or Factor IXa (FIXa); iii. a sample; and iv. a chromogenic substrate of FXa, wherein:
1. the amount of FX is: i. non-limiting relative to the total expected activity of intrinsic tenase; ii. at least 100 times the expected amount of the blood clotting factor in the composition; and/or iii. at least 100 pmol per IU of the blood clotting factor; and/or 2. the concentration of FX is at least 100 nM.
15. A kit for measuring the activity of a blood clotting factor in a sample comprising: i. Factor X (FX); ii. Factor Villa (F Villa) and/or Factor IXa (FIXa);and iii. a chromogenic substrate of FXa, wherein:
1. the amount of FX is: i. non-limiting relative to the total expected activity of intrinsic tenase; ii. is at least 100 times the expected amount of the blood clotting factor; and/or iii. at least 100 pmol per IU of the blood clotting factor; and/or
2. the concentration of FX is at least 100 nM.
16. A kit comprising the components required for performing the method of any one of aspects 1 to 13.
17. The kit of aspect 16, wherein the kit comprises: i. Factor X (FX); ii. Factor Villa (F Villa) and/or Factor IXa (FIXa);and iii. a chromogenic substrate of FXa, wherein:
1. the amount of FX is: i. non-limiting relative to the total expected activity of intrinsic tenase; ii. is at least 100 times the expected amount of the blood clotting factor in a sample; and/or iii. at least 100 pmol per IU of the blood clotting factor; and/or
2. the concentration of FX is at least 100 nM. 18. A kit comprising: i. Factor X (FX); ii. Factor Villa (FVIIIa) and/or Factor IXa (FIXa); iii. a chromogenic substrate of FXa; and iv. instructions for performing the method of any one of aspects 1-13.
19. The kit of aspect 18, wherein:
1. the amount of FX is: i. non-limiting relative to the total expected activity of intrinsic tenase; ii. at least 100 times the expected amount of the blood clotting factor in a sample; and/or iii. at least 100 pmol per IU of the blood clotting factor; and/or
2. the concentration of FX is at least 100 nM.
20. The kit of any one of aspects 16 to 19, wherein the kit is for measuring the activity of a blood clotting factor in a sample.
21. A kit for measuring the activity of a blood clotting factor in a sample comprising: i. Factor X (FX); ii. Factor Villa (FVIIIa) and/or Factor IXa (FIXa); iii. a chromogenic substrate of FXa; and iv. instructions for performing a chromogenic assay which indicate that:
1. the amount of FX used must be: i. non-limiting relative to the total expected activity of intrinsic tenase; ii. at least 100 times the expected amount of the blood clotting factor in the sample; and/or iii. at least 100 pmol per IU of the blood clotting factor; and/or
2. the concentration of FX must be at least 100 nM. 22. The method, composition or kit of any one of the preceding aspects, wherein the amount of FX is or the amount of FX used must be non-limiting relative to the total expected activity of intrinsic tenase.
23. The method, composition or kit of aspect 22, wherein the amount of FX is or the amount of FX used must be in excess relative to the total expected activity of intrinsic tenase.
24. The method, composition or kit of aspect 23, wherein the amount of FX is or the amount of FX used must be an at least 2-fold, around 2-fold or between 1-fold and 5-fold excess relative to the total expected activity of intrinsic tenase.
25. The method, composition or kit of any one of the preceding aspects, wherein the amount of FX is or the amount of FX used must be at least 100 times the expected amount of the blood clotting factor in the composition.
26. The method, composition or kit of aspect 25, wherein the amount of FX is or the amount of FX used must be at least 250 times the expected amount of the blood clotting factor in the composition.
27. The method, composition or kit of aspect 25, wherein the amount of FX is or the amount of FX used must be between 100 times and 5000 times, between 300 times and 2500 times, between 500 times and 1500 times, or around 1000 times the expected amount of the blood clotting factor in the composition.
28. The method, composition or kit of any one of the preceding aspects, wherein the amount of FX is or the amount of FX used must be at least 33 times the expected amount of the blood clotting factor per minute for which the sample is contacted with an agent for activating a blood clotting factor.
29. The method, composition or kit of aspect 28, wherein the amount of FX is or the amount of FX used must be at least 84 times the expected amount of the blood clotting factor per minute for which the sample is contacted with an agent for activating a blood clotting factor.
30. The method, composition or kit of aspect 28, wherein the amount of FX is or the amount of FX used must be between 33 times and 1667 times, between 100 times and 833 times, between 167 times and 500 times, or around 333 times the expected amount of the blood clotting factor in the composition per minute for which the sample is contacted with an agent for activating a blood clotting factor.
31. The method, composition or kit of any one of the preceding aspects, wherein the amount of FX is or the amount of FX used must be at least 33 times the expected amount of the blood clotting factor per minute of the incubation step to allow the formation of FXa.
32. The method, composition or kit of aspect 31, wherein the amount of FX is or the amount of FX used must be at least 84 times the expected amount of the blood clotting factor per minute of the incubation step to allow the formation of FXa.
33. The method, composition or kit of aspect 31, wherein the amount of FX is or the amount of FX used must be between 33 times and 1667 times, between 100 times and 833 times, between 167 times and 500 times, or around 333 times the expected amount of the blood clotting factor in the composition per minute of the incubation step to allow the formation of FXa.
34. The method, composition or kit of any one of the preceding aspects, wherein the concentration of FX is or the concentration of FX used must be at least 100 nM.
35. The method, composition or kit of aspect 34, wherein the concentration of FX is or the concentration of FX used must be at least 180 nM, at least 230 nM, around 180 nM, around 230 nM, between 180 nM and 230 nM, or between 180 nM and 460 nM. 36. The method, composition or kit of aspect 34 or 35, wherein the concentration of FX is or the concentration of FX used must be at least 180 nM, around 180 nM or between 180 nM and 230 nM and the composition or the kit comprises thrombin, prothrombin and/or bovine FVa.
37. The method, composition or kit of any one of aspects 34-36, wherein the concentration of FX is or the concentration of FX used must be at least 180 nM, around 180 nM or between 180 nM and 460 nM and the composition or the kit does not comprise prothrombin and/or bovine FVa, optionally wherein the composition or kit comprises thrombin.
38. The method, composition or kit of any one of the preceding aspects, wherein the concentration of FX is or the concentration of FX used must be at least 33 nM per minute for which the sample is contacted with an agent for activating a blood clotting factor.
39. The method, composition or kit of aspect 38, wherein the concentration of FX is or the concentration of FX used must be at least 60 nM, at least 77 nM, around 60 nM, around 77 nM, between 60 nM and 77 nM, or between 60 nM and 153 nM per minute for which the sample is contacted with an agent for activating a blood clotting factor.
40. The method, composition or kit of aspect 38, wherein the concentration of FX is or the concentration of FX used must be at least 60 nM, around 60 nM or between 60 nM and 77 nM per minute for which the sample is contacted with an agent for activating a blood clotting factor and the composition or the kit comprises thrombin, prothrombin and/or bovine FVa.
41. The method, composition or kit of aspect 38, wherein the concentration of FX is or the concentration of FX used must be at least 60 nM, around 60 nM, or between 60 nM and 153 nM per minute for which the sample is contacted with an agent for activating a blood clotting factor and the composition or the kit does not comprise prothrombin and/or bovine FVa, optionally wherein the composition or kit comprises thrombin. 42. The method, composition or kit of any one of the preceding aspects, wherein the concentration of FX is or the concentration of FX used must be at least 33 nM per minute of the incubation step to allow the formation of FXa.
43. The method, composition or kit of aspect 42, wherein the concentration of FX is or the concentration of FX used must be at least 60 nM, at least 77 nM, around 60 nM, around 77 nM, between 60 nM and 77 nM, or between 60 nM and 153 nM per minute of the incubation step to allow the formation of FXa.
44. The method, composition or kit of aspect 42, wherein the concentration of FX is or the concentration of FX used must be at least 60 nM, around 60 nM or between 60 nM and 77 nM per minute of the incubation step to allow the formation of FXa and the composition or the kit comprises thrombin, prothrombin and/or bovine FVa.
45. The method, composition or kit of aspect 42, wherein the concentration of FX is or the concentration of FX used must be at least 60 nM, around 60 nM, or between 60 nM and 153 nM per minute of the incubation step to allow the formation of FXa and the composition or the kit does not comprise prothrombin and/or bovine FVa, optionally wherein the composition or kit comprises thrombin
46. The method, composition or kit of any one of aspects 1 to 13, 15 or 20 to 45, wherein the blood clotting factor is a Factor IX (FIX) or a Factor VIII (FVIII) blood clotting factor.
47. The method, composition or kit of any one of aspects 1 to 13, 15, or 20 to 46, wherein the blood clotting factor is a FIX blood clotting factor and the composition or the kit comprises FVIIIa.
48. The method, composition or kit of any one of aspects 1 to 13, or 20 to 47, wherein the blood clotting factor is a FIX blood clotting factor having increased specific activity relative to wild-type FIX. 49. The method, composition or kit of any one of aspects 1, to 13 or 20 to 48, wherein the blood clotting factor is a FIX blood clotting factor comprising a leucine at a position corresponding to position 338 of wild-type FIX.
50. The method, composition or kit of any one of aspects 1 to 13 15 or 20 to 46, wherein the blood clotting factor is a Factor VIII blood clotting factor and the composition or the kit comprises FIXa.
51. The method, composition or kit of any one of aspects 1 to 13, 20 to 46 or 50, wherein the blood clotting factor is a FVIII blood clotting factor having increased stability relative to wild-type FVIII.
52. The method, composition or kit of any one of aspects 1 to 13 or46 to 51, wherein the sample is derived from a patient sample taken from a patient.
53. The method, composition or kit of aspect 52, wherein the patient is a patient who has previously been administered (i) a gene therapy vector comprising a polynucleotide encoding a blood clotting factor, or (ii) a recombinant blood clotting factor.
54. The method, composition or kit of aspect 53, wherein the patient has previously been administered (i) a gene therapy vector comprising a polynucleotide encoding a blood clotting factor that has increased specific activity relative to corresponding reference wild- type blood clotting factor, or (ii) a recombinant blood clotting factor that has increased specific activity relative to a corresponding reference wild-type blood clotting factor.
55. The method, composition or kit of aspect 53 or aspect 54, wherein the blood clotting factor is a FVIII blood clotting factor or a FIX blood clotting factor.
56. The method, composition or kit of any one of aspects 53 to 55, wherein the patient has previously been administered (i) a gene therapy vector comprising a polynucleotide encoding a FIX blood clotting factor that comprises a leucine at a position corresponding to position 338 of wild-type FIX, or (ii) a recombinant FIX blood clotting factor that comprises a leucine at a position corresponding to position 338 of wild-type FIX.
57. The method of any one of aspects 53 to 56, wherein the patient had haemophilia prior to administration of the gene therapy vector or the recombinant blood clotting factor, and further comprising a step of determining that the patient has been partially or fully treated for haemophilia by administration of the gene therapy vector or the recombinant blood clotting factor.
58. The method of aspect 57, further comprising assessing that a patient who has 51?with Factor IX or Factor VIII or (ii) needs a reduced level of ongoing treatment with Factor IX or Factor VIII, and adjusting the patient’s treatment regimen appropriately.
59. The method, composition or kit of any one of aspects 1 to 13 or 20 to 58, wherein the sample comprises at least a portion of a larger sample or the patient sample.
60. The method, composition or kit of any one of aspects 1 to 13 or 20 to 59, wherein the sample is diluted compared to a larger sample or the patient sample.
61. The method, composition or kit of any one of aspects 1 to 13 or 20 to 60, wherein the sample or the patient sample is a blood sample, a plasma sample.
62. The method, composition or kit of aspect 61, wherein the sample or the patient sample is a citrated plasma sample.
63. The method, composition or kit of any one of aspects 1 to 13,20 to 51 or 59 to 60, wherein the sample is a therapeutic concentrate, therapeutic preparation or medicament, such as a medicament comprising a blood clotting factor.
64. The method, composition or kit of any one of aspects 1 to 13,20 to 51 or 59 to 60, wherein the sample or the larger sample is conditioned cell medium. 65. The method of any one of the preceding aspects, further comprising a step of obtaining the sample, the patient sample, or the larger sample.
66. The method of any one of aspects 52 to 65, further comprising a step of diluting the patient sample or the larger sample.
67. The method, composition or kit of any one of the preceding aspects, wherein the composition or the kit comprises an agent for activating FIX, FVIII and an agent for activating FVIII.
68. The method, composition or kit of any one of the preceding aspects, wherein the composition or kit comprises an agent for activating FVIII, and FIXa.
69. The method or kit or any one of the preceding aspects, wherein:
(i) the FX is provided in a first reagent;
(ii) the FVIIIa and an agent for activating FIX are provided in an activator reagent; and (ii) the chromogenic substrate is provided in a third reagent.
70. The kit of any one of aspects 15 to 56, 59 to 64 or 67 to 69, wherein the kit comprises:
(i) a first reagent comprising the FX;
(ii) an activator reagent comprising the FVIIIa and an agent for activating FIX; and
(iii) a third reagent comprising the chromogenic substrate.
71. The method, composition or kit of any one of aspects 67 to 70, wherein the agent for activating FVIII is thrombin.
72. The method, composition or kit of any one of aspects 67 to 71, wherein the agent for activating FIX is Factor XI (FXI).
73. The method of any one of any one of aspects 1, 22 to 69 or 71 to 72, wherein the method further comprises a step of determining the amount of FX which is non-limiting by performing the following method using various concentrations of FX and determining that the amount of FX is non-limiting at any concentrations that provide the same results for the measured activity of FIXa: a. providing a composition comprising i. Factor X (FX); ii. Factor Villa (F Villa); iii. FIXa at a level corresponding to the total expected activity of FIXa; and iv. a chromogenic substrate of FXa; and b. detecting a dye formed by cleavage of the chromogenic substrate thereby measuring the activity of the FIXa.
74. The method of any one of aspects 52 to 56, 59 to 62 or 65 to 66, wherein the method further comprises determining the expected amount of FIXa by measuring the activity of FIXa in a group of patients expected to have the same characteristics as a group of patients from which the patient sample will be taken.
75. The method, composition or kit of any one of the preceding aspects, wherein the chromogenic substrate comprises a chromogen or fluorogen joined by a peptide bond to the carboxyl carbon atom of the amino acid at the C-terminus of a FXa cleavage site, preferably wherein the FXa cleavage site comprises the amino acid sequence IDGR or IEGR or chemical derivatives thereof.
76. The method, composition or kit of aspect 75, wherein the chromogen is pNA, preferably wherein the chromogenic substrate is CS-11, S-2222, S-2732, S-2765, F3301, B7020 or Spectrozyme® FXa.
77. The method, composition or kit of aspect 75 or 76, wherein the chromogen is pNA and detecting pNA comprises measuring absorbance at 405 nm.
78. The method, composition or kit of aspect 75, wherein the chromogenic substrate comprises a fluorogen, preferably wherein the chromogenic substrate is Spectrofluor® FXa or SN-7. 79. A kit comprising the components required for performing the method of any one of aspects 1 to 13, 22 to 69 or 71 to 78.
80. The kit of any one of aspects 15 to 56, 59 to 64, 67 to 72, 76 or 79, wherein the kit comprises all the components required for performing the method of any one of aspects 1 to 13, 22 to 69 or 71 to 78.
81. The kit of any one of aspects 15 to 56, 59 to 64, 67 to 72, 76, or 79 to 80, wherein the kit comprises instructions for performing the method of any one of aspects 1 to 13, 22 to 69 or 71 to 78.
82. The kit of aspect 81, wherein the kit is for measuring the activity of a blood clotting factor in a sample.

Claims

Claims
1. A method for measuring the activity of a blood clotting factor in a sample, comprising: a. providing a composition comprising i. Factor X (FX); ii. Factor Villa (FVIIIa) and/or Factor IXa (FIXa); iii. the sample; and iv. a chromogenic substrate of FXa; and b. detecting a dye formed by cleavage of the chromogenic substrate thereby measuring the activity of the blood clotting factor, wherein:
1. the amount of FX i s : i. non-limiting relative to the total expected activity of intrinsic tenase; ii. at least 100 times the expected amount of the blood clotting factor in the composition; and/or iii. at least 100 pmol per IU of the blood clotting factor; and/or
2. the concentration of FX is at least 100 nM.
2. The method of claim 1, wherein the method comprises contacting the sample with an agent for activating FVIII, and wherein:
A. the agent for activating FVIII comprises a combination of FV and FII, and the sample is contacted with the agent for activating FVIII for less than 5 minutes, less than 4 minutes, less than 3 minutes, between 0 minutes and 5 minutes, between 1 minute and 4 minutes, or between 1 minute and 3 minutes; or B. the agent for activating FVIII is Flla, and the sample is contacted with the agent for activating FVIII for less than 3 minutes, less than 2.5 minutes, less than 2 minutes, between 0 minutes and 3 minutes, between 1 minute and 2.5 minutes, or between 1 minute and 2 minutes.
3. The method of claim 1 or 2, wherein the method comprises contacting the sample with an agent for activating FVIII, wherein: A. the agent for activating FVIII is a combination of FV and FII, and the method comprises an incubation step to allow the formation of FXa that is less than 5 minutes, less than 4 minutes, less than 3 minutes, between 0 minutes and 5 minutes, between 1 minute and 4 minutes, or between 1 minute and 3 minutes; or
B. the agent for activating FVIII is Flla, and the method comprises an incubation step to allow the formation of FXa that is less than 3 minutes, less than 2.5 minutes, less than 2 minutes, between 0 minutes and 3 minutes, between 1 minute and 2.5 minutes, or between 1 minute and 2 minutes.
4. The method of claim 2 or 3, wherein contacting the sample with the agent for activating FVIII is carried out before step (a).
5. A method for measuring the activity of a Factor IX blood clotting factor in a sample, comprising: a. providing a composition comprising: i. Factor X (FX); ii. Factor VIII, an agent for activating FVIII, and an agent for activating FIX; iii. the sample; and iv. a chromogenic substrate of FX; and b. detecting a dye formed by cleavage of the chromogenic substrate, thereby to measure FIX activity; wherein the method comprises contacting the sample with an agent for activating FVIII, wherein:
A. the agent for activating FVIII comprises a combination of FV and FII, and the sample is contacted with the agent for activating FVIII for less than 5 minutes, less than 4 minutes, less than 3 minutes, between 0 minutes and 5 minutes, between 1 minute and 4 minutes, or between 1 minute and 3 minutes; or
B. the agent for activating FVIII is Flla, and the sample is contacted with the agent for activating FVIII for less than 3 minutes, less than 2.5 minutes, less than 2 minutes, between 0 minutes and 3 minutes, between 1 minute and 2.5 minutes, or between 1 minute and 2 minutes.
6. A method for measuring the activity of a Factor IX blood clotting factor in a sample, comprising: a. providing a composition comprising: i. Factor X (FX); ii. Factor VIII, an agent for activating FVIII, and an agent for activating FIX; iii. the sample; and iv. a chromogenic substrate of FX; and b. detecting a dye formed by cleavage of the chromogenic substrate, thereby to measure FIX activity; wherein the method comprises contacting the sample with an agent for activating FVIII, wherein:
A. the agent for activating FVIII is a combination of FV and FII, and the method comprises an incubation step to allow the formation of FXa that is less than 5 minutes, less than 4 minutes, less than 3 minutes, between 0 minutes and 5 minutes, between 1 minute and 4 minutes, or between 1 minute and 3 minutes; or
B. the agent for activating FVIII is Flla, and the method comprises an incubation step to allow the formation of FXa that is less than 3 minutes, less than 2.5 minutes, less than 2 minutes, between 0 minutes and 3 minutes, between 1 minute and 2.5 minutes, or between 1 minute and 2 minutes.
7. The method of claim 5 or 6, wherein contacting the sample with the agent for activating FVIII is carried out before step (a).
8. A method for determining an amount of FX which is non-limiting relative to the total expected activity of intrinsic tenase in a method for measuring the activity of blood clotting factor in a sample, said method comprising performing a series of chromogenic assays in which the amount of FX is varied such that various concentrations of FX are used, and determining an amount of that FX is wherein each assay comprises: a. providing a composition comprising: i. Factor X (FX) ii. Factor Villa (F Villa) and/or Factor IXa (FIXa); iii. the sample; and iv. a chromogenic substrate of FXa, and b. detecting a dye formed by cleavage of the chromogenic substrate thereby measuring the activity of the blood clotting factor; wherein the amount of FX is non-limiting at any concentrations that provide a less than linear increase in the measured activity of the blood clotting factor when compared to the increase in the FX concentration.
9. A method for determining a length of time for contacting a sample with an agent for activating a blood clotting factor in a method for measuring the activity of a blood clotting factor in a sample for which the amount of FX is non-limiting, said method comprising performing a series of chromogenic assays in which the length of time for which the sample is contacted with an agent for activating a blood clotting factor is varied such that various lengths of time may be used and determining a length of time for contacting the sample with an agent for activating a blood clotting factor for which the amount of FX is non-limiting, wherein each assay comprises: a. providing a composition comprising i. Factor X (FX); ii. Factor Villa (FVIIIa) and/or Factor IXa (FIXa); iii. the sample; and iv. a chromogenic substrate of FXa; and b. detecting a dye formed by cleavage of the chromogenic substrate thereby measuring the activity of the FIXa; wherein the amount of FX is non-limiting for a length of time for which the sample is contacted with an agent for activating a blood clotting factor if decreasing the length of time decreases the measured activity of the blood clotting factor proportionately.
10. A method for determining a length of an incubation step to allow the formation of FXa in a method for measuring the activity of a blood clotting factor in a sample for which the amount of FX is non-limiting, said method comprising performing a series of chromogenic assays in which the length of the incubation step is varied such that various lengths of the incubation step may be used and determining a length of the incubation step for which the amount of FX is non-limiting, wherein each assay comprises: a. providing a composition comprising i. Factor X (FX); ii. Factor Villa (FVIIIa) and/or Factor IXa (FIXa); iii. the sample; and iv. a chromogenic substrate of FXa; and b. detecting a dye formed by cleavage of the chromogenic substrate thereby measuring the activity of the FIXa; wherein the amount of FX is non-limiting for a length of the incubation step if decreasing the length of the incubation step decreases the measured activity of the blood clotting factor proportionately.
11. The method of claim 1, wherein the amount of FX that is non-limiting is determined by the method of claim 8.
12. The method of claim 1, wherein the method comprises a step of contacting the sample with an agent for activating a blood clotting factor, wherein the length of the step of contacting the sample with the agent for activating the blood clotting factor is determined by the method of claim 9.
13. The method of claim 1, wherein the method comprises an incubation step to allow the formation of FXa, wherein the length of the incubation step is determined by the method of claim 10.
14. A composition comprising: a. Factor X (FX); b. Factor Villa (FVIIIa) and/or Factor IXa (FIXa); c.a sample; and d. a chromogenic substrate of FXa, wherein:
1. the amount of FX i s : i. non-limiting relative to the total expected activity of intrinsic tenase; ii. at least 100 times the expected amount of the blood clotting factor in the composition; and/or iii. at least 100 pmol per IU of the blood clotting factor; and/or
2. the concentration of FX is at least 100 nM.
15. A kit:
(a) for measuring the activity of a blood clotting factor in a sample comprising: a. Factor X (FX); b.Factor Villa (FVIIIa) and/or Factor IXa (FIXa);and c.a chromogenic substrate of FXa, wherein:
1. the amount of FX i s : i. non-limiting relative to the total expected activity of intrinsic tenase; ii. is at least 100 times the expected amount of the blood clotting factor; and/or iii. at least 100 pmol per IU of the blood clotting factor; and/or
2. the concentration of FX is at least 100 nM; and/or
(b) comprising the components required for performing the method of any one of claims 1 to 13; and/or
(c) comprising: i. Factor X (FX); ii. Factor Villa (FVIIIa) and/or Factor IXa (FIXa); iii. a chromogenic substrate of FXa; and iv. instructions for performing the method of any one of claims 1 to 13; and/or
(d) for measuring the activity of a blood clotting factor in a sample comprising: i. Factor X (FX); ii. Factor Villa (FVIIIa) and/or Factor IXa (FIXa); iii. a chromogenic substrate of FXa; and iv. instructions for performing a chromogenic assay which indicate that:
1. the amount of FX used must be: i. non-limiting relative to the total expected activity of intrinsic tenase; ii. at least 100 times the expected amount of the blood clotting factor in the sample; and/or iii. at least 100 pmol per IU of the blood clotting factor; and/or
2. the concentration of FX must be at least 100 nM.
16. The kit of claim 15(b), wherein the kit comprises: i. Factor X (FX); ii. Factor Villa (FVIIIa) and/or Factor IXa (FIXa); and iii. a chromogenic substrate of FXa, wherein:
1. the amount of FX i s : i. non-limiting relative to the total expected activity of intrinsic tenase; ii. is at least 100 times the expected amount of the blood clotting factor in a sample; and/or iii. at least 100 pmol per IU of the blood clotting factor; and/or
2. the concentration of FX is at least 100 nM.
17. The kit of claim 15(c), wherein:
1. the amount of FX i s : i. non-limiting relative to the total expected activity of intrinsic tenase; ii. at least 100 times the expected amount of the blood clotting factor in a sample; and/or iii. at least 100 pmol per IU of the blood clotting factor; and/or
2. the concentration of FX is at least 100 nM.
18. The kit of any one of claims 15 to 17, wherein the kit is for measuring the activity of a blood clotting factor in a sample.
19. The method, composition or kit of any one of the preceding claims, wherein the amount of FX is or the amount of FX used must be: (i) non-limiting relative to the total expected activity of intrinsic tenase; and/or (i) in excess relative to the total expected activity of intrinsic tenase; and/or
(iii) an at least 2-fold, around 2-fold or between 1-fold and 5-fold excess relative to the total expected activity of intrinsic tenase; and/or
(iv) at least 100 times the expected amount of the blood clotting factor in the composition; and/or
(v) at least 250 times the expected amount of the blood clotting factor in the composition; and/or
(vi) between 100 times and 5000 times, between 300 times and 2500 times, between 500 times and 1500 times, or around 1000 times the expected amount of the blood clotting factor in the composition.
20. The method, composition or kit of any one of the preceding claims, wherein the amount of FX is or the amount of FX used must be:
(i) at least 33 times the expected amount of the blood clotting factor per minute for which the sample is contacted with an agent for activating a blood clotting factor; and/or
(ii) at least 84 times the expected amount of the blood clotting factor per minute for which the sample is contacted with an agent for activating a blood clotting factor; and/or
(iii) between 33 times and 1667 times, between 100 times and 833 times, between 167 times and 500 times, or around 333 times the expected amount of the blood clotting factor in the composition per minute between 33 times and 1667 times, between 100 times and 833 times, between 167 times and 500 times, or around 333 times the expected amount of the blood clotting factor in the composition per minute for which the sample is contacted with an agent for activating a blood clotting factor; and/or
(iv) at least 33 times the expected amount of the blood clotting factor per minute of the incubation step to allow the formation of FXa; and/or
(v) at least 84 times the expected amount of the blood clotting factor per minute of the incubation step to allow the formation of FXa; and/or (vi) between 33 times and 1667 times, between 100 times and 833 times, between 167 times and 500 times, or around 333 times the expected amount of the blood clotting factor in the composition per minute of the incubation step to allow the formation of FXa.
21. The method, composition or kit of any one of the preceding claims, wherein the concentration of FX is or the concentration of FX used must be:
(i) at least 100 nM; and/or
(ii) at least 180 nM, at least 230 nM, around 180 nM, around 230 nM, between 180 nM and 230 nM, or between 180 nM and 460 nM; and/or
(iii) at least 180 nM, around 180 nM or between 180 nM and 230 nM and the composition or the kit comprises thrombin, prothrombin and/or bovine FVa and/or
(iv) at least 180 nM, around 180 nM or between 180 nM and 460 nM and the composition or the kit does not comprise prothrombin and/or bovine FVa, optionally wherein the composition or kit comprises thrombin.
22. The method, composition or kit of any one of the preceding claims, wherein the concentration of FX is or the concentration of FX used must be:
(i) at least 33 nM per minute for which the sample is contacted with an agent for activating a blood clotting factor; and/or
(ii) at least 60 nM, at least 77 nM, around 60 nM, around 77 nM, between 60 nM and 77 nM, or between 60 nM and 153 nM per minute for which the sample is contacted with an agent for activating a blood clotting factor; and/or
(iii) at least 60 nM, around 60 nM or between 60 nM and 77 nM per minute for which the sample is contacted with an agent for activating a blood clotting factor and the composition or the kit comprises thrombin, prothrombin and/or bovine FVa; and/or
(iv) at least 60 nM, around 60 nM, or between 60 nM and 153 nM per minute for which the sample is contacted with an agent for activating a blood clotting factor and the composition or the kit does not comprise prothrombin and/or bovine FVa, optionally wherein the composition or kit comprises thrombin; and/or (v) at least 33 nM per minute of the incubation step to allow the formation of FXa; and/or
(vi) at least 60 nM, at least 77 nM, around 60 nM, around 77 nM, between 60 nM and 77 nM, or between 60 nM and 153 nM per minute of the incubation step to allow the formation of FXa; and/or
(vii) at least 60 nM, around 60 nM or between 60 nM and 77 nM per minute of the incubation step to allow the formation of FXa and the composition or the kit comprises thrombin, prothrombin and/or bovine FVa; and/or
(viii) at least 60 nM, around 60 nM, or between 60 nM and 153 nM per minute of the incubation step to allow the formation of FXa and the composition or the kit does not comprise prothrombin and/or bovine FVa, optionally wherein the composition or kit comprises thrombin.
23. The method, composition or kit of any one of claims 1 to 13, 15 or 18 to to 22, wherein:
(i) the blood clotting factor is a Factor IX (FIX) or a Factor VIII (FVIII) blood clotting factor; or
(ii) the blood clotting factor is a FIX blood clotting factor and the composition or the kit comprises F Villa; or
(iii) the blood clotting factor is a FIX blood clotting factor having increased specific activity relative to wild-type FIX; or
(iv) the blood clotting factor is a FIX blood clotting factor comprising a leucine at a position corresponding to position 338 of wild-type FIX; or
(v) the blood clotting factor is a Factor VIII blood clotting factor and the composition or the kit comprises FIXa; or
(vi) the blood clotting factor is a FVIII blood clotting factor having increased stability relative to wild-type FVIII.
24. The method, composition or kit of any one of claims 1 to 13, 15 or 18 to 23, wherein the sample is derived from a patient sample taken from a patient.
25. The method, composition or kit of claim 24, wherein the patient: (i) is a patient who has previously been administered (i) a gene therapy vector comprising a polynucleotide encoding a blood clotting factor, or (ii) a recombinant blood clotting factor; and/or
(ii) has previously been administered (i) a gene therapy vector comprising a polynucleotide encoding a blood clotting factor that has increased specific activity relative to corresponding reference wild-type blood clotting factor, or (ii) a recombinant blood clotting factor that has increased specific activity relative to a corresponding reference wild-type blood clotting factor; and/or
(iii) is a patient who has previously been administered (i) a gene therapy vector comprising a polynucleotide encoding a blood clotting factor, or (ii) a recombinant blood clotting factor and the blood clotting factor is a FVIII blood clotting factor or a FIX blood clotting factor; and/or
(iv) has previously been administered (i) a gene therapy vector comprising a polynucleotide encoding a blood clotting factor that has increased specific activity relative to corresponding reference wild-type blood clotting factor, or (ii) a recombinant blood clotting factor that has increased specific activity relative to a corresponding reference wild-type blood clotting factor and the blood clotting factor is a FVIII blood clotting factor or a FIX blood clotting factor; and/or
(v) has previously been administered (i) a gene therapy vector comprising a polynucleotide encoding a FIX blood clotting factor that comprises a leucine at a position corresponding to position 338 of wild-type FIX, or (ii) a recombinant FIX blood clotting factor that comprises a leucine at a position corresponding to position 338 of wild-type FIX; and/or
(vi) had haemophilia prior to administration of the gene therapy vector or the recombinant blood clotting factor, and further comprising a step of determining that the patient has been partially or fully treated for haemophilia by administration of the gene therapy vector or the recombinant blood clotting factor, optionally further comprising assessing that a patient who has been partially or fully treated for haemophilia (i) no longer needs ongoing treatment with Factor IX or Factor VIII or (ii) needs a reduced level of ongoing treatment with Factor IX or Factor VIII, and adjusting the patient’s treatment regimen appropriately.
26. The method, composition or kit of any one of claims 1 to 13 or 18 to 25, wherein:
(i) the sample comprises at least a portion of a larger sample or the patient sample; and/or
(ii) the sample is diluted compared to a larger sample or the patient sample; and/or
(iii) wherein the sample or the patient sample is:
(a) a blood sample or a plasma sample; or
(b) a citrated plasma sample; or
(c) a therapeutic concentrate, therapeutic preparation or medicament, such as a medicament comprising a blood clotting factor; or
(d) conditioned cell medium.
27. The method of any one of the preceding claims, further comprising a step of obtaining the sample, the patient sample, or the larger sample.
28. The method of any one of claims 24 to 27, further comprising a step of diluting the patient sample or the larger sample.
29. The method, composition or kit of any one of the preceding claims, wherein:
(a) the composition or the kit comprises an agent for activating FIX, FVIII and an agent for activating FVIII; and/or
(b) the composition or kit comprises an agent for activating FVIII, and FIXa; and/or
(c) (i) the FX is provided in a first reagent;
(ii) the FVIIIa and an agent for activating FIX are provided in an activator reagent; and
(iii) the chromogenic substrate is provided in a third reagent; and/or
(d) the kit comprises:
(i) a first reagent comprising the FX;
(ii) an activator reagent comprising the FVIIIa and an agent for activating FIX; and
(iii) a third reagent comprising the chromogenic substrate, optionally wherein the agent for activating FVIII is thrombin and/or the agent for activating FIX is Factor XI (FXI).
30. The method of any one of any one of claims 1 to 13 or 19 to 29, wherein the method further comprises a step of determining the amount of FX which is non-limiting by performing the following method using various concentrations of FX and determining that the amount of FX is non-limiting at any concentrations that provide the same results for the measured activity of FIXa: a. providing a composition comprising i. Factor X (FX); ii. Factor Villa (F Villa); iii. FIXa at a level corresponding to the total expected activity of FIXa; and iv. a chromogenic substrate of FXa; and b. detecting a dye formed by cleavage of the chromogenic substrate thereby measuring the activity of the FIXa.
31. The method of any one of claims 24 to 30, wherein the method further comprises determining the expected amount of FIXa by measuring the activity of FIXa in a group of patients expected to have the same characteristics as a group of patients from which the patient sample will be taken.
32. The method, composition or kit of any one of the preceding claims, wherein the chromogenic substrate comprises a chromogen or fluorogen joined by a peptide bond to the carboxyl carbon atom of the amino acid at the C-terminus of a FXa cleavage site, preferably wherein the FXa cleavage site comprises the amino acid sequence IDGR or IEGR or chemical derivatives thereof, optionally wherein the chromogen:
(i) is pNA, preferably wherein the chromogenic substrate is CS-11, S-2222, S- 2732, S-2765, F3301, B7020 or Spectrozyme® FXa; and/or
(ii) is pNA and detecting pNA comprises measuring absorbance at 405 nm; or the fluorogen is Spectrofluor® FXa or SN-7.
33. A kit comprising the components required for performing the method of any one of claims 1-13 or 197 to 32.
34. The kit of any one of claims 15 to 33, wherein the kit comprises: (i) all the components required for performing the method of any one of claims 1 to
13 or 19 to 32; and/or
(ii) instructions for performing the method of any one of claims 1 to 13 orl9 to 32, optionally wherein the kit is for measuring the activity of a blood clotting factor in a sample.
PCT/GB2021/051654 2020-06-29 2021-06-29 Method for measuring the activity of a blood clotting factor WO2022003349A1 (en)

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Citations (3)

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