Ready-to-use prothrombin time reagent based on recombinant tissue factor

The present invention relates to a ready-to-use long-term stable prothrombin time reagent based on recombinant tissue factor and its use in coagulation tests.

The prothrombin time (PT) is the most common screening test in the field of coagulation diagnostics. Patient plasma is mixed with a reagent containing at least tissue factor, phospholipids and calcium. The tissue factor can be isolated from tissue or produced recombinantly (Hoppenstaedt, DA et al. (1995) Lab. Med. 26 (3), 198-203); this protein activates the extrinsic pathway of coagulation. The measured clotting time depends on the concentration of coagulation factors II, V, VII and X. The PT is evaluated in seconds clotting time or in International Standardized Ratios (INR). The INR is calculated as PR^ISI, where the prothrombin ratio PR is the quotient of the clotting time of the sample and an average normal clotting time and the International Sensitivity Index ISI is a constant which depends on the reagent and the measuring instrument used. The sensitivity of a reagent is greater the numerically smaller the ISI is. An ISI between 0.9 and 1.2 provides optimal sensitivity for extrinsic system perturbations. Even lower ISI values ​​lead to extremely long clotting times in the pathological area, which are no longer readily measurable for many measuring systems.

The PT is used:<ul id="ul0001" list-style="bullet" compact="compact"><li>as a screnning test for the extrinsic coagulation system</li><li>for the control of oral anticoagulation</li><li>for the diagnosis of liver diseases.</li></ul>

Typically, most PT reagents that are available are offered in lyophilized form and reconstituted with a reconstitution medium (usually distilled water or saline solution) before use. The reason for this is the lack of stability of the reagents in the liquid state.

Adam &amp; Eberhard (US 3,522,148) describe a rabbit thromboplastin-based liquid thromboplastin reagent. No statements are made about the sensitivity of the reagent.

Butler et al. (US 5,385,853) describe the preparation of a PT reagent from rabbit brain thromboplastin. According to the invention, the addition of polyethylene glycol (PEG) and the use of calcium gluconate as calcium source and gentamycin as antimicrobial agent. However, the ISI value achieved is very high at 2.0; this corresponds to an insufficiently low sensitivity of the reagent.

Two liquid thromboplastin reagents are available on the market, one from Pacific Hemostasis (USA), the other from Diamed (Basel, Switzerland). Both reagents are based on rabbit brain thromboplastin, have low sensitivity (ISI&gt; 1.6) and run for 1 month or 1 year.

Three different recombinant PT reagents are on the market. All are highly sensitive (ISI approx. 1.0) and are offered in freeze-dried form.

Thus, the following methods have already been described:<ul id="ul0002" list-style="bullet" compact="compact"><li>conventional freeze-dried thromboplastin reagents</li><li>conventional ready-to-use low-sensitivity rabbit brain thromboplastin reagents</li><li>recombinant freeze-dried thromboplastin reagents with high sensitivity.</li></ul>

With the previously known methods, it has hitherto not been possible to stabilize recombinant thromboplastin so that it can be used in a liquid reagent.

The object underlying the present invention was therefore to provide a long-term stable liquid formulation of a recombinant thromboplastin reagent. Such a reagent is extremely advantageous for the coagulation laboratory because it combines the advantages of ease of handling (convenience of use) with the advantages of high sensitivity and good reproducibility.

Surprisingly, it has been found that a combination of an antioxidant in high concentration with a serum albumin in equally high concentration is successful, while the individual stabilizers are not sufficiently effective. Particularly advantageous is the combination of ascorbic acid with human serum albumin, each in millimolar concentration. Lower concentration of one of the two substances resulted in a lower stability.

Decisive for the usability of such a reagent is the stability at about 4 ° C. A stability of at least 12 months at 4 ° C., particularly advantageously a stability of at least 18 months, very particularly advantageously a stability of at least 24 months, is advantageous.

As stability, the temporal constancy of the measurement result for the determination of the PT of a defined plasma, eg a normal plasma, is defined here. The measurement result is considered constant if it decreases less than 10, preferably less than 5%.

In order to gain a quick information about the stability at 4 ° C, it proves in practice to be more favorable to first subject the reagents to a stress load at 37 ° C.

The stability of a biological material can be estimated from these stress experiments. The equation applies:<maths id="math0001" num="(1)"><math display="block"><mrow><msub><mrow><mtext> A</mtext></mrow><mrow><mtext>t</mtext></mrow></msub><msub><mrow><mtext> = A</mtext></mrow><mrow><mtext>O</mtext></mrow></msub><msup><mrow><mtext> e </mtext></mrow><mrow><mtext>kt</mtext></mrow></msup></mrow></math><img file="EP0942284A2_D0001.tif" /></maths> with A_t = Activity at time t, A_O = Activity at the beginning, t = time and k = degradation rate constant. The constant k depends on the temperature. As an approximation, the Arrhenius equation applies:<maths id="math0002" num="(2)"><math display="block"><mrow><mtext> log k = const 1 + [const 2 / T]</mtext></mrow></math><img file="EP0942284A2_D0002.tif" /></maths> with T = absolute temperature.

In practice, the stability is estimated at about 4 ° C by a loading experiment, say at 37 ° C. For example, a preparation whose activity decreased by less than 5% is regarded as stable. For the corresponding period after conversion of equation (1), in each case:<maths id="math0003" num="(3)"><math display="block"><mrow><msub><mrow><mtext> ln (A</mtext></mrow><mrow><mtext>t</mtext></mrow></msub><msub><mrow><mtext> / A</mtext></mrow><mrow><mtext>O</mtext></mrow></msub><msub><mrow><mtext> ) = ln 0.95 = -kt = -k </mtext></mrow><mrow><mtext>37 °</mtext></mrow></msub><msub><mrow><mtext> t</mtext></mrow><mrow><mtext>37 °</mtext></mrow></msub><msub><mrow><mtext> = -k</mtext></mrow><mrow><mtext>4 °</mtext></mrow></msub><msub><mrow><mtext> t</mtext></mrow><mrow><mtext>4 °</mtext></mrow></msub></mrow></math><img file="EP0942284A2_D0003.tif" /></maths> or.:<maths id="math0004" num="(4)"><math display="block"><mrow><msub><mrow><mtext> t </mtext></mrow><mrow><mtext>4 °</mtext></mrow></msub><msub><mrow><mtext> / t </mtext></mrow><mrow><mtext>37 °</mtext></mrow></msub><msub><mrow><mtext> = k </mtext></mrow><mrow><mtext>37 °</mtext></mrow></msub><msub><mrow><mtext> / k</mtext></mrow><mrow><mtext>4 °</mtext></mrow></msub></mrow></math><img file="EP0942284A2_D0004.tif" /></maths>

The running time at a certain temperature is thus inversely proportional to the constant k at this temperature.

For the relative size of the rate constants at different temperatures, the rule of thumb has been proven that increasing the temperature by 10 ° leads to a doubling of the rate constants. Comparative studies on the stability of liquid thromboplastins at different temperatures are not yet available. In a comparable case (Liquid Coagulation Factor IX), observations of degradation rate constants at various temperatures were made by Kirkwood (Kirkwood, TBL (1995) Biometrics 33, 736-742). He found the following constants: <tables id="tabl0001" num="0001"><table frame="all"><tgroup cols="2" colsep="1" rowsep="1"><colspec colnum="1" colname="col1" colwidth="78.75mm" /><colspec colnum="2" colname="col2" colwidth="78.75mm" /><thead valign="top"><row><entry namest="col1" nameend="col1" align="center"><b>temperature</b></entry><entry namest="col2" nameend="col2" align="center"><b>k</b> [10^-3 month^-1]</entry></row></thead><tbody valign="top"><row><entry namest="col1" nameend="col1" align="right">- 20 ° C</entry><entry namest="col2" nameend="col2" align="center">0.3</entry></row><row><entry namest="col1" nameend="col1" align="right">+ 4 ° C</entry><entry namest="col2" nameend="col2" align="center">1.0</entry></row><row><entry namest="col1" nameend="col1" align="right">+ 20 ° C</entry><entry namest="col2" nameend="col2" align="center">7.4</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="right">+ 37 ° C</entry><entry namest="col2" nameend="col2" align="center">30.2</entry></row></tbody></tgroup></table></tables>

In this example, the ratio of the constants k _{37 °} / k₄ This means that the stability of the preparation at 4 ° C is thirty times greater than the stability at 37 ° C. That is, the stability determined at 37 ° C in days corresponds to stability at 4 ° C in months.

For thromboplastin time reagents prepared according to the invention, exposure data are available at 37 ° C. (see Example 3). When loaded at 37 ° C, no drop in activity could be detected over a period of 30 days. According to the preceding considerations, therefore, the stability of the preparation at 4 ° C can be set at least 30 months.

However, not only the stability but also the sensitivity is decisive for a PT reagent. It is undesirable that any high stability would have to be paid for with a loss of sensitivity. In the case of the method according to the invention, an ISI value of about 1.0, which is regarded by experts as an expression of optimum sensitivity, could be set.

The following examples are intended to illustrate the invention.

<b>example 1</b>

Preparation of a prothrombin time reagent

1 Part 20% Triton-X 100 is diluted with 12 parts of 10% phospholipid suspension (Phospholipon 25P, Nattermann, Germany) and 6 parts of purified recombinant human tissue factor from E. coli (ca. 2 mg / mL, Behring Diagnostics, Germany). After incubation for two hours at room temperature for relipidation, the batch is diluted with a 500-fold excess of buffer. The buffer consists of 50 mM HEPES pH 7.0, 100 mM glycine, 13 mM calcium chloride and also contains the following stabilizers: <tables id="tabl0002" num="0002"><table frame="all"><tgroup cols="3" colsep="1" rowsep="0"><colspec colnum="1" colname="col1" colwidth="52.50mm" /><colspec colnum="2" colname="col2" colwidth="52.50mm" /><colspec colnum="3" colname="col3" colwidth="52.50mm" /><tbody valign="top"><row><entry namest="col1" nameend="col1" align="right">2</entry><entry namest="col2" nameend="col2" align="left">mM</entry><entry namest="col3" nameend="col3" align="left">ascorbic acid</entry></row><row><entry namest="col1" nameend="col1" align="right">1</entry><entry namest="col2" nameend="col2" align="left">%</entry><entry namest="col3" nameend="col3" align="left">mannitol</entry></row><row rowsep="1"><entry namest="col1" nameend="col1" align="right">1</entry><entry namest="col2" nameend="col2" align="left">%</entry><entry namest="col3" nameend="col3" align="left">Human serum albumin (Behring Diagnostics, Germany)</entry></row></tbody></tgroup></table></tables>

<b>Example 2</b>

INR reference curve of a prothrombin time reagent

A reagent according to Example 1 was prepared. The coagulation times for the AK calibration plasmas (Immuno, Austria) were determined on the Behring Coagulation Timer (Behring Diagnostics). These are lyophilized Marcumarplasmen with declared INR value.

From the data MNPT and ISI can be estimated (see Fig. 1)

<b>Example 3</b>

Stability of a prothrombin time reagent

Reagent 1 was prepared according to Example 1. Deviating from Example 1, reagent 2 contains only 0.1% human serum albumin. The ISI was determined according to Example 2.

Reagents were loaded at 37 ° C. Over time, samples were taken and the prothrombin time of lyophilized normal plasma and lyophilized pathological plasma (standard human plasma and Pathoplasma II, Behring Diagnostics) determined on the Behring Coagulation Timer (see Fig. 2).