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10152025303540CA 02265270 l999-03- ll1998/B001 - Ma 1161Dr. Pfe/ZiDade Behring Marburg GmbHprothrombin time based onrecombinant tissue factorReady-to-use reagentThe present invention relates to a readyâtoâuseprothrombin time reagent which is stable longâterm,based on recombinant tissue factor, and its use inclotting tests.The time (PT) is the mostscreening test in the field of clotting diagnosis. Inprothrombin customarythis test, patient plasma is mixed with a reagent whichcontains at least tissue factor, phospholipids andcalcium. The tissue factor can be isolated from tissueor prepared by recombinant means (Hoppenstaedt, D.A. etal. (1995) Lab. Med. 26(3), 198-203); thisactivates the extrinsic reaction pathway of clotting.proteinThe clotting time measured depends on the concentrationof the factors II, V, VII and X. Theevaluation of the PT is seconds ofclottingcarried out inclotting time or in International Standardized Ratios(ISR). The ISR is PRISI, theprothrombin ratio PR is the quotient of the clottingcalculated as wheretime of the sample and a mean normal clotting time, andthe International Sensitivity Index ISI is a constantwhich depends on the reagent and on the measuringapparatus used. The sensitivity of a reagent is all thethe smaller the ISI An ISIbetween 0.9 and 1.2 results in an optimum sensitivitygreater, is numerically.for disorders of the extrinsic system. Even smaller ISIvalues lead to extremely long clotting times in thepathological field, which for manyâ measuring systemsare no longer readily measurable.The PT is employed:0 as a screening test for the extrinsic clotting system101520253035CA 02265270 l999-03- ll_ 2 -0 for checking oral anticoagulation0 for the diagnosis of liver disorders.Customarily, most PT reagents which are available aresupplied in freezeâdried form and reconstituted beforeuse using a reconstitution medium (as a rule distilledwater or a saline solution). The reason for this is thelack of stability of the reagents in the liquid state.describe arabbitabout the(US 3,522,148)based onAdam & Eberhard liquidthromboplastinthromboplastin. Nosensitivity of the reagent.reagent brainstatements are madeButler et al. (US 5,385,853) describe the preparationof a PT reagent from rabbit brain thromboplastin.According to the invention, polyethylene glycol (PEG)is added. and. calciun1 gluconate is used as a calciumsource and. gentamycin as an antimicrobial agent. TheISI value achieved, however, is very high at, onaverage, 2.0; this corresponds to an inadequately lowsensitivity of the reagent.Two liquid thromboplastin reagents are available on themarket, one from Pacific Hemostasis (USA), the otherfrom Diamed (Basle, Switzerland). Both reagents arebased on rabbit brain thromboplastin, exhibit a lowsensitivity (ISI > 1.6) and have a life of 1 month and1 year respectively.Three different recombinant PT reagents are on themarket. All are highly sensitive (ISI about 1.0) andare supplied in freeze-dried form.already beenThe following processes have thusdescribed:0 conventional freezeâdried thromboplastin reagentsl01520253035CA 02265270 l999-03- ll_ 3 _0 conventional ready-toâuse rabbit brain thromboplastinreagents with low sensitivity0 recombinant freezeâdried thromboplastin reagents withhigh sensitivity.Using the processes known to date, it has not beenpossible until now to stabilize recombinantthromboplastin such that it can be employed in a liquidreagent.The object on which the present invention is basedtherefore consists in making available a liquidformulation of a recombinant thromboplastin reagentwhich is stable longâterm. Such a reagent is extremelythecombines the advantages of easy handleabilityadvantageous for clotting laboratory, since it(ready-toâuse state) with the advantages of high sensitivityand good reproducibility.Surprisingly, it has been found that a combination ofan antioxidant in a high concentration. with a serumalbumin in a likewise high concentration is successful,whereas the individual stabilizers are not sufficientlyeffective. The combination of ascorbic acid with humanserum albumin, in each case in millimolarconcentration, is particularly advantageous. A lowerconcentration. of one of the two substances led to alower stability in each case.crucial for theA stability of at4°C issuch a reagent.The aboututilizability ofstability atleast 12 months at 4°C is advantageous, a stability ofat least 18 months is particularly advantageous and astability of at least 24 months is very particularlyadvantageous.Stability is defined here as the time constancy of themeasured result for the determination of the PT of ae.g. of a normal plasma. The measureddefined plasma,101520253035CA 02265270 l999-03- ll_4_result counts as constant if it decreases less than10%, preferably less than 5%.about theit proves to be nmre favorable inIn order to obtain rapid informationstabilityâ at 4°C,first subject the stresspractice to reagents toloading at 37°C.The stability of a biological material can be estimatedfrom these loading experiments. The equation:(1) At = A0 eâktapplies where At = activity at the time t, A0 = activityat theconstant. Thestart, t = time and k = degradation rateconstant k is dependent on thetemperature. The Arrhenius equation:(2) log k = const 1 + [const 2/T]approximately applies where T = absolute temperature.In practice, the stability, for example, at 4°C isestimated by a loading experiment, for example at 37°C.For example, a preparation whose activity has fallen byless than 5% counts as stable. For the correspondingperiod, after transformation of equation (1) in eachcase:(3) ln (A1-_/A0) = ln 0.95 = -k t = ââk37ot37o = âk4ot4oor:(4) C40/t37° = k37°/k4°The life at aa specific temperature is thus inverselyproportional to the constant k at this temperature.l015202530CA 02265270 l999-03- ll_ 5 _For the relative size of the rate constants at varioustemperatures, the rule of thumb has proven that anincrease in the temperature by 10° leads to a doublingof the rate constants. Comparative investigations onthe stability oftemperatures are still not available.liquid thromboplastins at variousIn a comparativecase (liquid clotting factor IX), observations of thedegradation rate constants at various temperatures were(Kirkwood, T.B.L. (1995)made byâ Kirkwood Biometrics33, 736-742). He found the following constants:Temperature k[104 month'1]-20°C 0.3+ 4°C 1.0+20°C 7.4+37°C 30.2In this example, the ratio of the constants kyw/k4 isthus about 30.preparation at 4°C is thirty times greater than thestability at 37°C. This that thedetermined at 37°C in days corresponds to the stabilityThis means that the stability" of themeans stabilityat 4°C in months.Loading data at 37°C are available for thromboplastintime reagents prepared according to the invention (seeIf loading took place at 37°C,the activity could. be detected over a period. of 30theExample 3). no fall indays. According to the preceding considerations,stability of the preparation at 4°C can therefore beestimated as at least 30 months.However, not only the stability, but in principle alsothe sensitivity, is crucial for a. PT reagent. It isundesirable that an optionally high stability has to bebought at the expense of a loss in sensitivity. In thecase of the process according to the invention, it waspossible to set an ISI value of about 1.0, which isCA 02265270 l999-03- ll-5-seen by experts as an expression of optimumsensitivity.The following examples are intended to illustrate theinvention.101520253035CA 02265270 l999-03- 11Example 1Preparation of a prothrombin time reagent1 part of 20% Triton X 100 is mixed with 12 parts of10% phospholipid suspension (Phospholipon 25 P,Nattermann, Germany) and 6 parts of purifiedrecombinant human tissue factor from E. coli (about2 mg/ml, Behring Diagnostics, Germany). Afterincubation at room temperature for two hours forthe batch is diluted with a 500âfoldThe buffer consists of 50 mM HEPESrelipidation,excess of buffer.pH 7.0, 100 mM glycine and 13 mM calcium chloride, andfurthermore contains the following stabilizers:2 mM ascorbic acid1% mannitol1% human serum albumin (Behring Diagnostics, Germany)Example 2ISR reference curve of a prothrombin time reagentA reagent according to Example 1 was prepared. Theclotting times for the AK calibration plasmas (Immuno,Austria) were determined on a Behring coagulation timer(Behring Diagnostics). These are lyophilized Marcumarplasmas having declared ISR values.MNPT and ISI can be estimated from the data (seeFig. 1)Example 3Stability of a prothrombin time reagentReagent 1 was prepared according to Example 1.Differing from Example 1, reagent 2 only contains 0.1%CA 02265270 l999-03- ll-3-human serum albumin. The ISI was determined accordingto Example 2.Reagents were loaded at 37°C. Samples were taken in thecourse of time and the prothrombin time of lyophilizednormal plasma and lyophilized pathological plasma(standard human plasma and Pathoplasma II, BehringDiagnostics) were determined cn1 a Behring coagulationtimer (see Fig. 2).
