EP2368124A1 - Procédé de détermination d'activité de facteur de coagulation sanguine pégylé dans un essai de temps de thromboplastine partielle activée à base de silice - Google Patents

Procédé de détermination d'activité de facteur de coagulation sanguine pégylé dans un essai de temps de thromboplastine partielle activée à base de silice

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Publication number
EP2368124A1
EP2368124A1 EP09828376A EP09828376A EP2368124A1 EP 2368124 A1 EP2368124 A1 EP 2368124A1 EP 09828376 A EP09828376 A EP 09828376A EP 09828376 A EP09828376 A EP 09828376A EP 2368124 A1 EP2368124 A1 EP 2368124A1
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European Patent Office
Prior art keywords
peg
blood coagulation
conjugated
coagulation factor
factor
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EP09828376A
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German (de)
English (en)
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EP2368124A4 (fr
Inventor
Liang Tang
Heiner Apeler
Philip Ramsey
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Bayer Healthcare LLC
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Bayer Healthcare LLC
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Publication of EP2368124A1 publication Critical patent/EP2368124A1/fr
Publication of EP2368124A4 publication Critical patent/EP2368124A4/fr
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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
    • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors

Definitions

  • [001] Provided is a method for measuring coagulation activity of polyethylene glycol- conjugated ("PEGylated”) blood coagulation factors.
  • the method comprises the addition of PEG to a silica-based activated partial thromboplastin time (APTT) assay.
  • APTT silica-based activated partial thromboplastin time
  • Blood coagulation is a complex chemical and physical reaction that occurs when blood comes into contact with an activating agent.
  • the whole blood coagulation process can be generally viewed as three activities: platelet adhesion, platelet aggregation, and formation of a fibrin clot.
  • platelets flow through the blood vessels in an inactivated state because the blood vessel lining, the endothelium, prevents activation of platelets.
  • the endothelium loses its integrity and platelets are activated by contact with tissue underlying the damaged site. Activation of the platelets causes them to become “sticky” and adhere together. Additional platelets then adhere to the activated platelets and also become activated. This process continues until a platelet "plug" is formed. This platelet plug then serves as a matrix upon which blood clotting or coagulation proceeds.
  • thrombin is then produced that causes fibrinogen to convert to fibrin, which forms the major portion of the clot mass.
  • additional platelets are activated and trapped in the forming clot, contributing to clot formation.
  • polymerization and cross-linking of fibrin results in the permanent clot.
  • Clotting may be initiated by either the intrinsic pathway, which involves the activation of Factor XII, or by the extrinsic pathway, which involves the release of tissue factor.
  • Several different in vitro assays can be used to assess coagulation activity, including a chromogenic assay, a thrombin generation assay, a whole blood coagulation assay, and an Activated Partial Thromboplastin Time (APTT) assay.
  • the APTT assay measures activity of blood coagulation factors of the intrinsic pathway. These factors include Factors I (fibrinogen), II (prothrombin), V, VIII, IX, X, XI, and XII.
  • Factors I fibrinogen
  • II prothrombin
  • V Activated Partial Thromboplastin Time
  • proenzyme Factor XII is converted to its enzyme XIIa which in turn converts the zymogen Factor XI to the enzyme Factor XIa, which then activates Factor IX in the presence of calcium.
  • the enzyme Factor IXa in the presence of Factor Villa and phospholipids activates Factor X.
  • This reaction is greatly increased by the prior exposure of Factor VIII to thrombin or Factor Xa. Therefore, APTT assays require several components in addition to blood plasma, including a source of phospholipids, calcium, and an intrinsic pathway activator, such as micronized silica, ellagic acid, or kaolin.
  • Micronized silica beads are used as an activator of the intrinsic pathway, initiating the conversion of FXII to FXIIa.
  • the APTT assay is commonly used in the clinic for screening patients' plasma for coagulation factor deficiencies and for monitoring treatment with anticoagulants such as heparin. This assay is also used for quality control practices during the manufacture of coagulation factors for therapeutic use.
  • covalent modification of therapeutic proteins can potentially interfere with the APTT assay, limiting its use in clinical applications and manufacturing.
  • one covalent modification that is used to increase the in vivo half-life of a protein is PEGylation.
  • PEGylation is the covalent attachment of long- chained polyethylene glycol (PEG) molecules to a protein or other molecule.
  • PEGylation has been used to reduce antibody development, protect the protein from protease digestion and keep the material out of the kidney filtrate (Harris et al., 2001, Clinical Pharmacokinetics 40, pp. 539-51). In addition, PEGylation may also increase the overall stability and solubility of the protein.
  • conjugation of polyethylene glycol (PEG) to blood coagulation factors, such as Factor VIII (FVIII) extends half-life in animals, increasing their efficacy as therapeutic agents.
  • FVIII Factor VIII
  • PEGylated FVIII shows normal biological activity in other coagulation assays
  • initial tests with PEGylated Factor VIII in APTT assays with micronized silica indicated that the APTT assay does not accurately measure the coagulation activity of PEGylated blood coagulation factors.
  • Activity in the micronized silica-based APTT assay is severely compromised. Since the APTT assay is in widespread use, there was a need to adapt this assay for the measurement of PEGylated blood coagulation factors to support manufacturing and clinical applications.
  • a method for determining coagulation activity of a sample containing or suspected of containing a PEG-conjugated blood coagulation factor comprising: a) providing a thromboplastin reagent mixture comprising a thromboplastin reagent having silica as the activator and free polyethylene glycol and b) adding a sample that contains or is suspected of containing PEG-conjugated blood coagulation factor to said thromboplastin reagent mixture to form a resulting reaction mixture and c) determining coagulation time of said resulting reaction mixture, thereby determining the coagulation activity of said sample.
  • the present invention also provides a method for determining activated partial thromboplastin time of a sample containing or suspected of containing a PEG-conjugated blood coagulation factor comprising : a) adding free polyethylene glycol to an activated partial thromboplastin time assay reaction mixture containing a sample containing or suspected of containing PEG-conjugated blood coagulation factor and a thromboplastin reagent comprising silica particles to form a resulting reaction mixture, and b) determining coagulation time of said resulting reaction mixture, thereby determining the activated partial thromboplastin time of said sample.
  • the present invention further provides a method of restoring the activity of a PEG- conjugated blood coagulation factor in an activated partial thromboplastin time assay that uses silica as the coagulation activator, comprising adding free polyethylene glycol to an activated partial thromboplastin time assay reaction mixture comprising: a) a sample containing or suspected of containing said PEG-conjugated blood coagulation factor and b) a thromboplastin reagent comprising silica particles.
  • the present invention provides a method for determining the coagulation activity in a patient treated with a PEG-conjugated coagulation factor comprising: a) providing an activated partial thromboplastin time assay reaction mixture containing a blood or plasma sample from the patient, a thromboplastin reagent comprising silica particles as the activator, and free polyethylene glycol, and b) determining coagulation time of said reaction mixture, thereby determining the coagulation activity in the patient.
  • the PEG-conjugated blood coagulation factor is a PEG- conjugated Factor I, ⁇ , V, VIII, IX, X, XI, or XII.
  • the PEG-conjugated blood coagulation factor is Factor VIII or B domain deleted Factor VIII.
  • the PEG-conjugated blood coagulation factor is a recombinant PEG-conjugated blood coagulation factor.
  • Figure 1 shows the coagulation time (APTT Time) of B domain deleted FVIII (BDD) and a BDD mutein (L491C/K1804C) which is PEGylated at positions 491 and 1804 (PEG2+14).
  • FV ⁇ i activity was tested at two concentrations, 0.025 and 0.0125 IU/ml. Free PEG was added directly to the APTT reagent (PEG + APTT) or to the FVIII sample (+ PEG).
  • FIG. 2 shows a Western blot of BDD and PEGylated BDD (PEG2+ 14) that was incubated with micronized silica in a modified APTT assay.
  • the micronized silica was spun down, washed with PBS, and analyzed by Western blotting using a rabbit anti-FVIII polyclonal antibody.
  • Lanes 1 and 3 represent assays with no free PEG while lanes 2 and 4 represent assays with free PEG added to a final concentration of 500 ⁇ M.
  • Figure 3 shows FVHI coagulation (COAG) activity of PEGylated BDD (PEG2+14) in a micronized silica-based APTT assay with four different sizes of free PEG added (2 kD, 35 kD,
  • Free PEG was added at a final concentration 0.2 and 2 million-fold greater than the final concentration of FVIII.
  • Figure 4 shows the coagulation (COAG) activity of BDD and two PEGylated BDD muteins (PEG-14 and PEG-2+14) in a micronized silica based APTT assay (BioMerieux).
  • PEG-14 is a BDD mutein (K1804C) which is PEGylated at position 1804.
  • Figure 5 shows the coagulation (COAG) activity of BDD and two PEGylated BDD muteins (PEG-14 and PEG-2+14) in an ellagic acid based APTT assay containing the clot endpoint reagent AMAX AlexinTM.
  • Figure 6 shows a comparison of coagulation activity of PEGylated BDD-PEG 14 in a chromogenic assay and in two different APTT assays.
  • One of the APTT assays contains micronized silica (BioMerieux) and the other contains ellagic acid provided by the clot endpoint reagent AMAX AlexinTM.
  • PEG and "polyethylene glycol” as used herein are interchangeable and include any water-soluble poly(ethylene oxide).
  • PEGs for use in accordance with embodiments of the invention comprise the following structure “-(OCH 2 CH 2 ) n — " where (n) is 2 to 4000.
  • PEG also includes "-CH 2 CH 2 -O(CH 2 CH 2 O) n -CH 2 CH 2 -” and “— (OCH 2 CH 2 ) n O— ,” depending upon whether or not the terminal oxygens have been displaced.
  • PEG includes structures having various terminal or “end capping” groups, such as without limitation a hydroxyl or a C 1-2O alkoxy group.
  • PEG also means a polymer that contains a majority, that is to say, greater than 50%, of -OCH 2 CH 2 — repeating subunits.
  • free polyethylene glycol or “free PEG” refers to a PEG molecule which has not been conjugated to another molecule. With respect to specific forms, the PEG can take any number of a variety of molecular weights, as well as structures or geometries such as branched, linear, forked, and multifunctional.
  • the free PEG may be the same PEG used for conjugation of the PEGylated coagulation factor.
  • the molecular weight of PEG added to the APTT reaction mixture is about 2 to 10OkD. In some embodiments, the molecular weight of PEG added to the APTT reaction mixture is about 2 to 35 kD, in some embodiments the molecular weight of PEG is about 35 to 64 kD, and in some embodiments the molecular weight of PEG is about 64 tolOO kD.
  • PPG polypropylene glycol
  • PBG polybutylene glycol
  • Free PEG can be added to the APTT reaction mixture in a range of different concentrations.
  • concentration of PEGylated blood coagulation factor in a sample is known, free PEG can be added to the reaction mixture in a specific fold excess of the PEGylated coagulation factor.
  • concentration of free PEG is inversely correlated to the molecular weight of PEG. For a small molecular weight PEG, a higher concentration of free PEG can be used. Whereas, for a large molecular weight PEG, a lower concentration of free PEG can be used.
  • the final concentration of free PEG in the reaction mixture is about 50 to 100, about 100 to 200, about 200 to 500, about 1000 to 5000, about 5000 to 10,000, about 10,000 to 200,000, or about 200,000 to 10,000,000 times greater than the final concentration of PEGylated coagulation factor. In some embodiments, the final concentration of free PEG in the reaction mixture is about 200,000 to 2,000,000 times greater than the final concentration of PEGylated coagulation factor. In some cases, the concentration of PEGylated coagulation factor in a sample may not be known, for example, when the sample is taken from a subject administered with PEGylated coagulation factor. Therefore, free PEG can also be added at concentrations that are not dependent on coagulation factor concentration.
  • the final concentration of free PEG in the reaction mixtures is around 1-200 nM, 200-1000 nM, 1-10 ⁇ M, 10-1000 ⁇ M, or higher.
  • the concentration could be high enough to reach the saturation point of the reaction mixture with the solubility of free PEG as the limit.
  • the final concentration of free PEG in the reaction mixture is around 10-200 ⁇ M.
  • a PEG-conjugated protein is a protein which is covalently attached to one or more polyethylene glycol (PEG) molecules.
  • PEG polyethylene glycol
  • PEG-conjugated and “PEGylated” are used herein interchangeably.
  • This invention relates to PEG-conjugated blood coagulation factors. Methods for PEG conjugation of blood coagulation factors are described, for example in US Pat. No. 5,766,897, US Pat. No. 6,753,165, WO90/12874, and US Application No. 20060115876.
  • Proteins can be PEG-conjugated in several different ways, such as by random modification of primary amines (N-terminus and lysines) or by site-directed strategies such as incorporation of unnatural amino acids followed by the addition of a PEG derivative that will react specifically with the unnatural amino acid. Another approach to site-specific PEG-conjugation of proteins is by targeting N-terminal backbone amine with PEG- aldehydes. Proteins can also be PEG- conjugated by inserting or substituting a cysteine for another amino acid, then adding a PEG moiety that has a sulfhydryl reactive group.
  • Examples of PEG-conjugated proteins include but are not limited to BDD-PEG- 14 and BDD-PEG- 14+2.
  • BDD-PEG-14 is a BDD mutein with a mutation at amino acid position 491 (L491C).
  • BDD-PEG- 14+2 is a BDD mutein with mutations at amino acid positions 491 (L491C) and 1804 (K1804C).
  • the amino acid position numbers of the BDD muteins refer to positions in the full-length FVIII protein.
  • Blood coagulation factors include proteins involved in blood coagulation and clotting and biologically active fragments thereof.
  • the assay can be used to determine the coagulation activity of PEGylated blood coagulation factors of the intrinsic pathway, including but not limited to Factors I, II, V, VIII, IX, X, XI, and XII.
  • Factor VII is the only factor not affected by the partial thromboplastin time and therefore its activity is not detectable in the APTT assay.
  • the invention is used to determine the coagulation activity of FVIII.
  • the coagulation activity of B domain deleted (BDD) FVIII is determined.
  • the protein is a recombinant protein.
  • Blood clotting Factor VIII (FVIII) is a glycoprotein synthesized and released into the bloodstream by the liver. In the circulating blood, it is bound to von Willebrand factor (vWF, also known as Factor Vlll-related antigen) to form a stable complex. Upon activation by thrombin, it dissociates from the complex to interact with other clotting factors in the coagulation cascade, which eventually leads to the formation of a thrombus.
  • vWF von Willebrand factor
  • Factor Vlll-related antigen Upon activation by thrombin, it dissociates from the complex to interact with other clotting factors in the coagulation cascade, which eventually leads to the formation of a thrombus.
  • the term "Factor V ⁇ i" as used herein includes active variants of naturally occurring factor VIII.
  • BDD B domain deleted FVIII
  • BDD B domain deleted FVIII
  • BDD is characterized by having the amino acid sequence which contains a deletion of all but 14 amino acids of the B domain of FVIII such that the first 4 amino acids of the B domain are linked to the last 10 residues of the B domain.
  • the activated partial thromboplastin time (APTT) assay tests coagulation factor activity of a sample by determining the increase in turbidity or viscosity of a sample caused by the conversion of fibrinogen to fibrin during clot formation.
  • APTT assays employ an intrinsic pathway activator, such as micronized silica, ellagic acid, or kaolin, and a phospholipid component of a thromboplastin reagent (without tissue factor) for evaluating coagulation associated with the intrinsic pathway.
  • Blood coagulation is initiated by adding calcium. The calcium acts to reverse the anticoagulant effect of oxalate or citrate that is typically added to the sample when it is drawn from the patient.
  • Coagulation time can be determined by visual inspection or by automated coagulation instruments. Typically most instruments detect the formation of a clot by monitoring either optical turbidity or electrical conductivity. Optical turbidity may be sensed by the decrease in light transmission due to the formation of a clot, while increasing electrical conductivity may be correlated to the formation of clots.
  • the coagulation activity of a sample corresponds to its coagulation time, with shorter coagulation times reflecting greater coagulation activity.
  • the methods of the present invention can be used to determine the coagulation activity of a PEGylated blood coagulation factor before it has been administered to a patient, for example, as part of a quality control step during manufacturing.
  • the invention could be used to test coagulation activity in a sample taken from a patient administered with a PEGylated blood coagulation factor.
  • Thromboplastin reagents are comprised of a coagulation activator such as, for example, silica, ellagic acid, or kaolin and a phospholipid source such as, for example, rabbit brain phospholipids.
  • the thromboplastin reagent also comprises one or more of a buffer, an antimicrobial agent, and calcium (to reverse the anticoagulation effect of oxalate or citrate that often are added to a plasma or blood sample).
  • the thromboplastin reagents are mixed with the sample for determination of blood coagulation activity in the APTT assay.
  • the present invention relates to APTT assays conducted with thromboplastin reagents containing silica.
  • silica-based thromboplastin reagents examples include CEPHALINEXTM (Bio/Data Corporation) and Automated APTT reagent (Biomerieux).
  • CEPHALINEXTM is a lyophilized preparation of rabbit brain phospholipids (cephalin) and particulate microsilica activator.
  • Automated APTT reagent contains rabbit brain phospholipids and micronized silica in a suitable buffer.
  • the term plasma generally refers to a solution comprising proteins having procoagulant activity when combined with an APTT reagent. Proteins in the plasma include blood clotting factors, thrombin, and fibrinogen. The plasma also contains other plasma proteins, sugars, and/or salts.
  • the plasma can be whole plasma that is obtained from humans or other animals. The plasma may also be deficient in one or more blood clotting factors, for example FV ⁇ i-deficient plasma from a hemophilia A patient.
  • the plasma can also be a plasma derivative that has procoagulant activity and is derived from one or more whole plasmas.
  • the plasma derivative can be, for example, a plasma fraction or a plasma that has been purified or otherwise treated to remove some protein, sugar, salt or other components thereof.
  • the plasma can alternatively be a plasma substitute formed from components obtained from separate sources, including natural or man-made components. Exemplary man-made components include plasma proteins that are substantially isolated and/or purified from natural sources and plasma proteins that are prepared using recombinant technology.
  • One plasma that may be used is a primate plasma, and in some embodiments is a human plasma.
  • blood generally refers to whole blood, citrated blood, platelet concentrate, or control mixtures of plasma and blood cells.
  • PEGylated Factor VIII has normal or close to normal biological activity in a chromogenic (FXa-substrate) assay. However, their activity is severely compromised in a micronized silica-based APTT assay using Automated APTT reagent (Biomerieux) in an Automatic Coagulation Analyzer (Electra 1800C or ACL Advance, Instrumentation Laboratory Co). Because a PEGylated FVIII such as BDD-PEG14 shows normal FVIII activity in other in vitro assays (e.g.
  • the reagents used were Tris-buffered saline with 1% human serum albumin, FVIII-deficient plasma from a hemophilia A patient, 25 mM CaCl 2 , and one of two APTT reagents, the Platelet Factor 3 reagent Automated APTT (Biomerieux) which contains micronized silica as the activator or the clot endpoint reagent AMAX ALEXINTM (Trinity Biotech) which contains ellagic acid as the activator.
  • a B-domain-deleted FVIII and two PEGylated BDD molecules, BDD-PEG 14 and BDD-PEG2+14 are used in the investigation.
  • the FVIII samples were tested at two different final concentrations of 0.0250 IU/ml and 0.0125 IU/ml.
  • 64 kD free PEG (Sigma- Aldrich) was used at a final concentration of 43 ⁇ M. Free PEG was either added to the FVIII samples directly or to the APTT reagent before addition of FVIII into the reaction mix.
  • the assay was conducted according to the manufacturer's instructions for the Automatic Coagulation Analyzer (Electra 1800C, Instrumentation Laboratory Co). The clotting time of each sample was measured and collected automatically by the instrument.
  • Example 2 Addition of free PEG blocks binding of PEGylated Factor VIII to micronized silica
  • micronized silica-based APTT reagent was mixed with 100 kD linear free PEG at a final PEG concentration of 500 ⁇ M.
  • FVIII was then added to the samples at a final concentration of 12 IU/ml as determined by chromo genie assay.
  • the APTT assay was modified by omitting the addition of CaCl 2 to avoid quick coagulation which would inhibit separation of micronized silica from the assay mixture. After a 3 min. incubation at RT, the micronized silica were spun down, washed with PBS, and analyzed by Western blotting using a rabbit anti-FVIII polyclonal antibody.
  • Example 3 The effect of free PEG in restoring activity of PEGylated FVIII in a micronized silica based APTT assay is dependent on PEG size and concentration
  • BDD-PEG 2+14 was used at a final concentration of 0.077 nM (0.1 IU/mL).
  • One of four different sized PEGs (2, 35, 64 and 100 kD) was added at one of 7 different fold concentrations in large excess of BDD-PEG 2+14 (50, 100, 200, 500, 1,000, 5,000 and 10,000 fold greater). These 7 different fold concentrations correspond to final PEG concentrations of 3.8, 7.7, 15.4, 38.5, 77.0, 385, and 770 nM, respectively.
  • the four different sized PEGs were added at fold concentrations of 200,000 and 2,000,000 in excess of BDD-PEG 2+14 (as shown in Figure 3).
  • the reaction was carried out as described in Example 1. Clotting times of each sample were determined using the micronized silica product (Automated APTT, Biomerieux) as the APTT reagent. Non-PEGylated BDD was used as the control.
  • Figure 3 shows that the effect of free PEG in restoring activity of PEGylated FVIII in a micronized silica-based APTT assay was dependent on the size and concentration of PEG, with larger PEG molecules and higher concentrations showing the greatest effect. Since PEG is a polymer made up of repeated identical moieties, the effect of free PEG in restoring activity of PEGylated FVIII is probably a function of PEG moiety (CH 2 CH 2 O-) concentration. The high ratio of free PEG to PEGylated FVIII at the million fold range was needed in order to rescue the activity of BDD-PEG2+14, indicating that the interaction of free PEG with micronized silica may have had a relatively low binding affinity. It is also possible that there was a great deal of excess surface area on the micronized silica beads that could interact with the PEG relative to the amount of PEGylated FVIII.
  • Example 4 Reduced activity of PEGylated FVIII in the APTT assay is specific to a micronized silica-based assay
  • an APTT assay reagent that does not use micronized silica as the contact activator was investigated. Automated APTT reagent containing micronized silica (Biomerieux) and the clot endpoint reagent AMAX ALEXINTM without micronized silica (Trinity Biotech) were used in the investigation. Various dilutions of BDD, BDD- PEG 14, and BDD-PEG2+14 were used in the study to measure their coagulation activities by these two assay systems. The APTT assays for all FVIII samples were conducted on the MLA 1800 instrument. The assays were conducted as described in Example 1.

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Abstract

La présente invention concerne un procédé de mesure de l’activité de coagulation d’un facteur de coagulation sanguine pégylé par un essai de temps de thromboplastine partielle activée (APTT) à base de silice. Il a été observé qu’un facteur de coagulation sanguine pégylé présente une activité réduite dans un essai APTT à base de silice, et il a été démontré que l’ajout de PEG au mélange de réaction APTT restaure son activité. La présente invention concerne des procédés pour déterminer l’activité de coagulation d’un facteur de coagulation sanguine pégylé dans un essai APTT.
EP09828376A 2008-11-24 2009-11-24 Procédé de détermination d'activité de facteur de coagulation sanguine pégylé dans un essai de temps de thromboplastine partielle activée à base de silice Withdrawn EP2368124A4 (fr)

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PCT/US2009/065732 WO2010060081A1 (fr) 2008-11-24 2009-11-24 Procédé de détermination d’activité de facteur de coagulation sanguine pégylé dans un essai de temps de thromboplastine partielle activée à base de silice

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