WO2016137431A1 - Procédés robustes permettant de déterminer des concentrations d'héparine - Google Patents

Procédés robustes permettant de déterminer des concentrations d'héparine Download PDF

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Publication number
WO2016137431A1
WO2016137431A1 PCT/US2015/017076 US2015017076W WO2016137431A1 WO 2016137431 A1 WO2016137431 A1 WO 2016137431A1 US 2015017076 W US2015017076 W US 2015017076W WO 2016137431 A1 WO2016137431 A1 WO 2016137431A1
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solution
heparin
concentration
working
factor
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PCT/US2015/017076
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English (en)
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Ralph Yamamoto
Yumi Kim
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Nantpharma, Llc
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Priority to PCT/US2015/017076 priority Critical patent/WO2016137431A1/fr
Publication of WO2016137431A1 publication Critical patent/WO2016137431A1/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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/38—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence, e.g. gluco- or galactomannans, e.g. Konjac gum, Locust bean gum, Guar gum
    • G01N2400/40—Glycosaminoglycans, i.e. GAG or mucopolysaccharides, e.g. chondroitin sulfate, dermatan sulfate, hyaluronic acid, heparin, heparan sulfate, and related sulfated polysaccharides

Definitions

  • Heparin was first used as a natural anticoagulant in 1916 and continues to be a clinically important drug.
  • many uses are the treatment and prevention of thromboembolic disorders, such as acute venous thrombosis and pulmonary embolism, the prevention of blood clotting within extracorporeal circuits, such as those used during cardiopulmonary bypass surgery and renal dialysis, the prevention of blood clotting in long term intravenous lines, and the prevention of clotting of blood drawn for the determination of blood cell counts.
  • heparin is the source of a new generation of anticoagulants, the "low molecular weight heparins," (“LMWHs").
  • LMWHs are salts of sulfated
  • glycosaminoglycans having a molecular weight of less than 8,000 Daltons.
  • the LMWHs have become widely used and have been found to have fewer serious side effects than heparin.
  • Fibrin clots proteinaceous plugs
  • Factor II proteinaceous plugs
  • thrombin thrombin
  • Factor Xa activated Factor X
  • Thrombin then acts on fibrinogen to form a primary blood clot.
  • the same processes which lead to beneficial blood clots can also be pathological when they occur in blood vessels to form thrombi which disrupt the normal flow of blood.
  • the coagulation system also acts to prevent excessive clotting.
  • Anti-thrombin is component of the coagulation system that controls the clotting process.
  • the controls on clotting can fail and excessive or "pathologic blood clotting" often causes significant morbidity and even death. Consequentially, treatments have been developed that maintain or restore normal hemostatsis.
  • Heparin is one the most important of these treatments. Heparin is composed of long unbranched acidic GAG chains with repeating disaccharide units. These structures allow heparin to bind other molecules. Most importantly, heparin binds to and activates
  • antithrombin or "AT.”
  • AT antithrombin
  • the heparin-AT complex inactivates Factor Xa and Factor Ila and, indirectly, thrombin's activation of fibrinogen.
  • heparin Most of the heparin used clinically is extracted from slaughterhouse remnants, e.g., from porcine intestine.
  • a typical heparin extraction process employs five general steps. First, the particular tissue is collected from an animal at a slaughterhouse. Second, heparin is extracted from this tissue via a combination of elevated temperatures, high pressures, hydrolysis at an alkaline pH and proteolytic enzymes to produce a "crude heparin extract.” Third, raw heparin is bound to and recovered from an anion-exchange resin, wherein heparin is selectively adsorbed and eluted from the resin, and then filtered, precipitated, and vacuum- dried.
  • heparin is further extracted either via dissolution in purified water, followed by filtration at low pH, and oxidation to alkaline pH, or via cation exchange chromatography.
  • purified heparin is recovered either by precipitating and vacuum drying or by various filtration and freeze-drying steps.
  • heparin extracted by the above protocol is fractionated by oxidative deamination, enzymatic elimination, or chemical elimination methods.
  • the yield of purified heparin from current methods is typically 10-25 mg/g wet tissue, or 30,000-50,000 U/animal.
  • the typical yield using conventional protocols is well below the potential yield.
  • the examination of three standard protocols (those of Charles and Scott, Max, and Volpi) for preparing heparin have reported yields as low as 0.3-0.5%.
  • the current invention satisfies the long felt need for more robust methods of determining heparin concentration including, but not limited to, determining the
  • concentration of heparin in complex liquids such as those characteristic of the earlier stages of heparin extraction including crude extracts from animal tissue.
  • the current invention can determine the "known and unknown" concentrations of heparin and that this method can be run as a manual or automated method.
  • the invention provides methods for determining the heparin concentration of a liquid, including a complex liquid, such as a crude heparin extract from an animal source.
  • the invention takes advantage of prior art assays that exploited heparin's binding and activation of AT (see, e.g., USP Heparin Sodium Monograph, USP 37, 3222-27).
  • the current invention can be used to determine the heparin concentration at all points during extraction.
  • These methods comprise preparing aliquots of known heparin concentrations for the generation of a standard curve, preparing a positive control solution, and preparing test solutions from samples of unknown heparin concentration (SOUHC). These preparations are used in an assay comprising binding any heparin present to Antithrombin (AT), which then inhibits Factor Xa or Thrombin (Factor Ila). Any excess uninhibited Factor Xa or Thrombin activity cleaves the chromogenic substrate resulting in a substance that has an optical density at 405nm.
  • AT Antithrombin
  • Factor Xa or Thrombin Factor Xa or Thrombin
  • the current invention has many advantages over the prior art including, but not limited to, being able to use liquid samples, providing a concentration rather than a potency, and being resistant to the effects of impurities in a test sample (such as the crude extracts obtained at the early stages of extraction).
  • FIG. 1 is a schematic representation of the intrinsic and extrinsic coagulation cascades.
  • UH refers to "unfractionated heparin”
  • LMWH refers to "low molecular weight heparin.”
  • FIG. 2 depicts a typical standard curve for use in accordance with the invention.
  • FIG. 3 depicts a typical standard curve (boxes) from a Factor Xa version of the USP Potency assay and plotted unknowns (triangles).
  • FIG. 4 depicts a typical standard curve (boxes) from a Factor Ila version of the USP Potency assay and plotted unknowns (triangles).
  • FIG. 5 depicts a comparison between the standard curve (boxes) and a curve made up of the determined heparin concentrations produced by the prior art USP method using liquid samples at the crude extract stage (triangles).
  • the current invention takes advantage of ATs heparin-stimulated inhibition of Thrombin or Factor Xa in reactions wherein the excess Thrombin or Factor Xa cleaves a chromogenic substrate to produce a color generating compound.
  • the reaction results in an inverse relationship between the amount of heparin present and the optical density at OD405.
  • the invention carefully controls the amount of interfering substances are present in the chromogenic reaction by diluting the sample studied so that such any substances are no longer present in amounts that can interfere with the reaction.
  • heparin concentration means the anti-factor Xa or anti- factor Ila activity (units per unit volume) generally expressed as units/milliliter.
  • the term "the determined heparin concentration” means a heparin concentration obtained by the use of a method in accordance with the current invention.
  • complex liquid means liquid derived from an animal tissue, e.g., from intestinal mucosa or lung, containing, contaminants such as
  • glycosaminoglycans nucleic acids, lipids, proteins, small organic molecules, and inorganic molecules, metals and inorganic ions which may distort the results of prior art heparin assays.
  • standard curve means the dose-response curve of the logOD 4 o 5 versus concentration constructed from samples with a predefined range of concentrations prepared from USP Heparin Sodium Assays Reference Standards.
  • a working stock standard solution means a solution containing known USP heparin units (units per unit volume) prepared by the dilution of a stock standard solution with buffer.
  • a stock standard solution means a solution containing known USP heparin units (units per unit volume) prepared from reconstitution of a lyophilized USP Heparin Sodium for Assays Reference Standard with water for injection (“WFI") in accordance with the invention.
  • stock positive control solution means a solution containing known USP heparin units (units per unit volume) prepared by dilution of a stock standard solution with buffer in accordance with the invention.
  • first working positive control solution means a solution containing known USP heparin units (units per unit volume) prepared from stock control solution with buffer.
  • second working positive control solution means a solution containing known USP heparin units (units per unit volume) prepared from the stock positive control solution or the first working positive control with buffer such that the first working positive control solution is twice the concentration of the second working positive control solution
  • first test solution means predetermined dilution of a complex liquid of unknown heparin concentration.
  • second test solution means predetermined dilution of a complex liquid of unknown heparin concentration which is more dilute than the "first test solution.”
  • working solution Factor Xa solution or "working solution Thrombin solution” means solutions of specified concentration of reagents prepared for use in either the anti-factor Xa or the anti-factor Ila assays, respectively.
  • working chromogenic substrate solution means solution of specified concentration of chromogenic reagent.
  • S-2765 is used for the anti-Factor Xa assay and S-2238 (aka., "Ila Substrate”) is used for the anti-Factor Ila assay.
  • OD at 405 nm and "OD405" mean absorbance or milli- absorbance of a sample quantitatively measured by a detection system at a wavelength of 405 nm.
  • sample Response means a measurement of the amount of light at a wavelength of 405 nm that can pass through a defined volume of a liquid.
  • the Antithrombin Solution for use in Factor Xa embodiments, reconstitute a vial of Antithrombin (10 IU) with 2 mL of water for injection ("WFI") to obtain 5 IU/mL solution. Then combine 1 mL of 5 IU/mL Antithrombin solution with 4 mL of pH 8.4 Buffer to obtain a 1 IU/mL solution. Incubate at 37°C for at least 15 minutes before using
  • S-2765 Chromogenic Substrate Solution reconstitute a vial of S-2765 Chromogenic Substrate (25 mg) with 1 1.66 mL of water to obtain 3 mM solution. Then combine 5 mL of 3 mM S-2765 Chromogenic Substrate solution with 10 mL of WFI to obtain 1 mM solution and mix well.
  • the invention provides methods for determining the heparin concentration of a liquid sample containing heparin.
  • Suitable heparin containing samples for use in accordance with the invention include solutions and suspensions.
  • any suitable heparin-containing sample can be used in accordance with the invention, including, but not limited to, complex liquids from any source, e.g., a crude heparin extract, whole blood, serum or plasma; a partially purified heparin extract or substantially pure heparin liquid.
  • embodiments of the current invention allow for the use of liquid heparin samples resulting from the reconstitution of heparin in a solid form, e.g., reconstitution of heparin powder with WFI.
  • the invention provides for the preparation of aliquots of known heparin concentration for the generation of a standard curve by a method comprising preparing 1 ,000 to 5,000 U/mL stock standard solution, preferably a 500 to 2,500 U/mL stock standard solution, more preferably a 2,000 U/mL stock standard solution. Said stock standard solution is then diluted with a suitable pH 8.0 to 8.6 buffer, preferably a suitable pH 8.2 to 8.5 buffer, more preferably with a preferred pH 8.4 buffer, to prepare a intermediate standard solution.
  • This intermediate standard solution has a heparin concentration of 1 to 20 U/mL, preferably a heparin concentration of 12 to 17 U/mL, more preferably a heparin concentration of 15 U/mL.
  • Said intermediate standard solution is then diluted with a suitable pH 8.0 to 8.6 buffer, preferably a suitable pH 8.2 to 8.5 buffer, more preferably a preferred pH 8.4 buffer resulting in at least five, preferably 7, more preferable 10 aliquots of differing heparin concentrations of working standard solutions for the generation of a standard curve.
  • each aliquot has a heparin concentration in the range of 0.01 U/mL to 0.5 U/ml, preferably 0.02 U/mL to 0.4 U/mL, more preferably within the range of 0.05 U/mL to 0.25 U/mL.
  • each aliquot has a heparin concentration within the range of 0.001 U/mL to 0.05 U/mL, preferably 0.002 U/mL to 0.04 U/mL, more preferably within the range of 0.005 U/mL to 0.025 U/mL.
  • Positive control solutions are prepared by a method comprising preparing a 0.1 U/mL to 100 U/mL stock positive control solution, preferably preparing a 1 U/mL to 50 U/mL stock positive control solution, more preferably 20 U/mL positive stock control solution for FXa assay and 2 U/mL for Factor Ila assay, and diluting the stock control solution with a suitable pH 8.0 to 8.6 buffer, preferably a suitable pH 8.2 to 8.5 buffer, more preferably a preferred pH 8.4 buffer, to prepare a 0.03 to 0.7 U/mL, preferably a 0.05 to 0.5 U/mL, more preferably a 0.2 U/ml for FXa assay and 0.003 to 0.06 U/mL, preferably 0.005 to 0.05 U/mL, more preferably a 0.02 U/mL for Factor Ila assay, first working positive control solution, and a second positive working control solution, which has a different heparin concentration from the first positive working control solution
  • This second positive working control solution preferably has a heparin concentration of 0.05 to 0.5 U/mL, preferably 0.07 to 0.4 U/mL, more preferably 0.1 U/mL for FXa assay and 0.005 to 0.05 U/mL, preferably 0.007 to 0.04 U/mL, more preferably 0.01 U/mL for Factor Ila assay.
  • the invention further calls for preparing a test solution by a method comprising obtaining a sample of unknown heparin concentration (SOUHC) and preparing first test solution by diluting the SOUHC 1 :2 to 1 :250,000with a suitable pH 8.2 to 8.6 buffer, preferably pH a suitable pH 8.3 to 8.5 buffer, more preferably a preferred pH 8.4 buffer.
  • a second test solution is prepared by diluting the first test solution with a suitable pH 8.2 to 8.6 buffer, preferably a preferred pH 8.4 buffer, where the first test solution is twice as concentrated as the second test solution.
  • the anti-Factor Ila assay comprises adding 50 ⁇ , of the pH 8.4 buffer, the working control solution, working standard solutions and test solutions, respectively, to reaction vessels (vessels in which the following actions can take place which can also be used in the determination of the OD ⁇ sunknown heparin samples), adding 50 ⁇ , antithrombin working solution to each reaction vessel, mixing and incubating for 2 minutes at 37 °C, adding 50 ⁇ _, working Thrombin solution to each reaction vessel, mixing and incubating for 2 minutes at 37 °C, adding 50 iL Ila
  • Chromogenic Substrate Solution (S-2238) to each reaction vessel, mixing and incubating for 2 minutes at 37 °C (not required when an automated spectrophotometer is used), and reading the optical density OD at 405nm using any suitable device, e.g. a spectrophotometer.
  • the invention provides, for both the Factor Xa and Factor Ila embodiments, for methods of determining the heparin concentration of the positive control and SOUHCs by a method comprising generating a standard curve, and applying equation (I) as set forth above (see “Summary of the Invention.")
  • % Difference ⁇ 1: 3 ⁇ 4 x200 (II), Where: X] is the heparin concentration from the first point within the standard curve, and X 2 is the heparin concentration from the second point within the standard curve. If the % Difference is greater than 10%, the heparin concentration determination should be repeated.
  • a further optional assessment of the quality of the results produced in accordance with the invention is the use of correlation coefficient of the standard curve of the log mAbs/min versus the working standard solution concentration from 0.0 to 0.25 U/mL. If the R I or R (as determined by conventional statistical methods) is less than 0.95, the heparin concentration determination should be repeated.
  • Control Concentration is the average concentration from the dilutions of control, where 2000 U/mL is the nominal concentration of the stock standard. If percent adjustment is greater than ⁇ 10% from the nominal (2000 U/mL), the heparin concentration determination should be repeated. If the % Adjustment is within ⁇ 10% then it can be used to adjust the determined concentration as follows:
  • Tris(hydroxymethyl)aminomethane (ACS or equivalent), Sodium Chloride (SigmaUltra, >99.5% or equivalent), EDTA Sodium (99+% or equivalent), Polyethylene Glycol 6,000 (Ultra or equivalent), Hydrochloric Acid, 1.0 N, Antithrombin (Chromogenix or equivalent), Factor Xa (Chromogenix or equivalent), Chromogenic Substrate, S-2765 (Chromogenix or equivalent), Water for Injection (“WFI”), and USP Heparin Sodium for Assays Reference Standard.
  • EXAMPLES 1-5 demonstrate that the current invention, in an embodiment that employs Factor Xa, is suitable for determining heparin concentrations.
  • EXAMPLES 6-1 1 demonstrate that the current invention, in a Factor Ila embodiment is suitable for determining heparin concentrations.
  • EXAMPLE 12 demonstrates that the USP potency assay cannot be used with impure samples.
  • an embodiment of the invention that employs Factor Xa has the accuracy needed from a method for determining heparin concentration in a manufacturing or clinical setting.
  • a pH 8.4 Buffer was made by dissolving 6.057 g of tris(hydroxymethyl)-aminomethane, 2.7918 g of EDTA disodium, 10.227 g of sodium chloride, and 1 g of polyethylene glycol 6,000 in about 1000 mL of purified water. The pH was adjusted to 8.4 with 1.0 N hydrochloric acid.
  • An antithrombin solution was made by reconstituting a vial of antithrombin with 2 mL of WFI to obtain 5 IU/mL solution.
  • a working antithrombin solution was made by diluting the reconstituted antithrombin solution with a pH 8.4 buffer (e.g. 1000 ih of 5 IU/mL solution plus 4000 of pH 8.4 Buffer).
  • a Factor Xa solution was made by reconstituting a vial of Factor Xa with 10 mL of WFI to obtain 7.1 nkat/mL solution.
  • a working Factor Xa solution was made by diluting the reconstituted Factor Xa solution with a pH 8.4 Buffer (e.g., 5000 ih of Factor Xa 7.1 nkat/mL solution plus 5000 iL of pH 8.4 Buffer) resulting in a solution which has an OD405 of 0.65 and 1.25 when the "blank" is assayed.
  • a pH 8.4 Buffer e.g., 5000 ih of Factor Xa 7.1 nkat/mL solution plus 5000 iL of pH 8.4 Buffer
  • a Chromogenic Substrate (S-2765) Solution was prepared by reconstituting a vial of Chromogenic Substrate (S-2765) with WFI to obtain 3 mM solution ⁇ e.g. 1 1.66 mL of WFI per 25 mg of substrate). Then 5 mL of 3 mM Chromogenic Substrate solution was mixed with 10 mL of WFI and incubated at 37°C for at least 15 minutes to prepare 1 mM solution.
  • the heparin concentration assay was carried out as follows. First, aliquots of known heparin concentration for the generation of a standard curve were prepared as follows: a 2,000 U/mL stock standard solution was made from USP Heparin Sodium for Assays Reference Standard followed by diluting this stock standard solution with a pH 8.4 buffer to prepare a 15 U/mL intermediate standard solution. The intermediate standard solution was then used to prepare five working standard solutions for the generation of the standard curve. The working stock standard solutions were made according to TABLE 1. TABLE 1
  • a positive control solution was made by a method comprising: preparing a 20 U/mL positive stock control solution, diluting the positive stock control solution to prepare a 0.2 U/mL first positive working control solution, and diluting the first positive working control solution with the pH 8.4 buffer to prepare a 0.1 U/mL second positive working control solution;
  • test solutions were prepared for each SOUHC by a method comprising: obtaining liquid samples of known heparin concentration (to act as SOUHCs), a first test solution was prepared by diluting each SOUHC 1 : 80,000 with a pH 8.4 buffer, preparing a second test solutions for each SOUHC by diluting the first test solution to final 1 : 160,000 with the pH 8.4 buffer.
  • the heparin concentration was determined by a process comprising the following ordered steps: Mixing 24 of pH 8.4 buffer to a plurality of suitable assay vessels with 6iL of pH 8.4 buffer (as a blank) or working standard solutions (for generating an OD450 versus heparin concentration curve), working control solutions or working test solutions to each assay vessel, adding 30 ⁇ ⁇ antithrombin working solution to each assay vessel, mixing and incubating for 2 minutes at 37 °C; adding 60 L Factor Xa working solution to each assay vessel, mixing and incubating for 2 minutes at 37 °C, adding 60 working S-2765 chromogenic substrate solution to each assay vessel, mixing and incubating for 2 minutes at 37 °C, and stopping the reaction to read the optical density (OD) at 405nm .
  • Rigorous quality control standards allow the current invention to detect heparin at low concentrations.
  • the OD405 was used to determine the heparin concentration of the controls and SOUHCs by: generating a standard curve of OD 05 versus heparin concentration from the aliquots of the first step above, and determining the heparin concentrations with Equation I.
  • sample response is a log of the response, provided that it is within the standard curve's end points
  • y int is the y-intercept of the standard curve
  • m is the slope of the standard curve from
  • DF is the dilution factor corresponding to the sample dilution level.
  • Average % Recoveries ranged from 99 - 108% (TABLE 2), which met the average % recovery goal of 70% - 130%).
  • the "repeatability" was also determined by preparing six replicate sample preparations from a composite amount and analyzing the 1 : 80,000 and 1 : 160,000 dilutions for each replicate.
  • the heparin concentrations were determined using the anti-Factor Xa protocol used in EXAMPLE 1.
  • IP intermediate precision
  • the quantitation limit (QL) of this embodiment of the invention was evaluated by preparing and analyzing 0.05 U/mL solution six times.
  • the 0.05 U/mL solutions were prepared using 15 U/mL intermediate standard solution.
  • Table 9 summarizes the system suitability results from the precision, accuracy, QL and linearity studies for the current invention.
  • EXAMPLES 1-4 demonstrate that the current invention is suitable for determining heparin concentrations.
  • WFI Water for injection
  • USP Water for injection
  • USP Heparin Sodium for Assays Reference Standard Purified Water.
  • any suitable water maybe used in accordance with the invention, as stated herein, WFI is preferred for those applications where it is expressly set forth.
  • the pH 8.4 Buffer Dissolve 6.10 g of Tris(hydroxymethyl)armnomethane, 2.80 g of Edetate Disodium, 10.20 g of Sodium Chloride in about 700 mL of purified water. Adjust the pH to 8.4 with 1.0N Hydrochloric Acid and dilute to volume with purified water (or equivalent) to 1000-mL. Mix well and filter.
  • the 20% (v/v) Acetic Acid Solution Transfer 20 mL of Glacial Acetic Acid into a 100-mL volumetric flask. Dilute to volume with water and mix.
  • the Antithrombin Solution (0.3125 IU/mL): Reconstitute a vial of Antithrombin with 8 mL of pH 8.4 Buffer. Incubate at 37°C for at least 15 minutes.
  • Thrombin Solution (5 IU/mL): Reconstitute a vial of Thrombin with 8 mL of pH 8.4 Buffer. Incubate at 37°C for at least 15 minutes.
  • the Factor Ila Substrate Solution (0.78 mg/niL): Reconstitute a vial of Ila Substrate with 8 mL of WFI. Incubate at 37°C for at least 15 minutes.
  • the Test Solution is made by obtaining a 1 : 1000 dilution of sample with pH 8.4 Buffer (e.g. 5 ⁇ L of sample is added to 4995 ⁇ , of pH 8.4 Buffer). Perform 1 :2 serial dilutions using the 1 : 1000 diluted sample with pH 8.4 Buffer in duplicate.
  • the Stock Control Solution Perform 1 : 1000 dilution of Stock Standard Solution (2000 Units/mL) with pH 8.4 Buffer (e.g. 5 ⁇ . of Stock Standard Solution is added to 4995 ⁇ . of pH 8.4 Buffer).
  • pH 8.4 Buffer e.g. 5 ⁇ . of Stock Standard Solution is added to 4995 ⁇ . of pH 8.4 Buffer.
  • the Working Control Solution is made by making 1 :2 serial dilutions of Stock Control Solution with pH 8.4 Buffer in duplicate (e.g. 125 xL of Stock Control Solution is added to 125 ⁇ , of pH 8.4 Buffer).
  • the correlation coefficient (r) was determined for each set of Working Standard Solution.
  • the reaction times can be varied slightly from 2 minutes as long as the standards and samples are assayed together using a suitable reaction vessel (e.g. 96-well plate).
  • the USP heparin Sodium anti-Factor Xa Activity assay unlike the current invention, assumes that the lnOD ⁇ (or "specific activity") of test article will behave similar to the USP Heparin Sodium for Assays Reference Standard in the same concentration range. Samples with similar specific activities generate similar curves when In-y is plotted against heparin concentration. When this comparison is done for either the anti-Factor Xa (FIG. 3) and anti- Factor Ila (FIG. 4) versions of the USP assays the slopes are highly similar.

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Abstract

L'invention concerne des procédés permettant de déterminer la concentration d'héparine d'un liquide, comprenant notamment mais non exclusivement, un liquide ayant des impuretés qui affectent la précision des procédés de l'état de la technique. L'invention consiste à lier toute l'héparine présente à l'antithrombine (AT) exogène, à améliorer l'inhibition du facteur Xa ou de la thrombine par l'antithrombine. Un quelconque excès du facteur Xa ou de la thrombine clive un substrat chromogène pour créer un composé chromatique présentant une densité optique à 405 nm. En outre, l'invention concerne des mesures de contrôle de qualité rigoureuses qui assurent des déterminations de concentration précises.
PCT/US2015/017076 2015-02-23 2015-02-23 Procédés robustes permettant de déterminer des concentrations d'héparine WO2016137431A1 (fr)

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CN112816615A (zh) * 2021-01-07 2021-05-18 沈阳力创环保科技有限公司 氯离子检测仪及氯离子检测方法
CN114279989A (zh) * 2021-12-30 2022-04-05 江苏大同盟制药有限公司 一种低分子量肝素钙注射液抗Xa因子效价测定方法及其系统

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CN112816615A (zh) * 2021-01-07 2021-05-18 沈阳力创环保科技有限公司 氯离子检测仪及氯离子检测方法
CN112816615B (zh) * 2021-01-07 2023-06-20 沈阳力创环保科技有限公司 氯离子检测仪及氯离子检测方法
CN114279989A (zh) * 2021-12-30 2022-04-05 江苏大同盟制药有限公司 一种低分子量肝素钙注射液抗Xa因子效价测定方法及其系统

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