Calibrator for prothrombin time (PT) assays

Abstract

This invention pertains to a PT Assay Calibrator and a method of preparing a PT Assay Calibrator including a coagulation factor such as recombinant FVII or recombinant FVIIa that will allow preparation of PT calibration curves with values about 100% and which will give results analogous to those obtained using fresh normal plasma.

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a method of preparing a commercial plasma preparation that will allow preparation of PT calibration curves with values about 100% and which will give results analogous to those obtained using fresh normal pooled plasma.

2. Description of the Related Art

The Prothrombin Time (PT) is used as a screening test for blood coagulation factor deficiencies and for monitoring oral anti-coagulant therapy using, e.g., coumadin. Thromboplastin reagents activate the "extrinsic" pathway of coagulation and are the basis for the PT test. Thromboplastin contains lipidated tissue factor (TF), which is the activator of the extrinsic pathway. This activation centers on Factor VII (FVII) and activated Factor VII (Factor VIIa), the TF-FVII Complex activates Factor X, which with Factor V activates Factor II to produce thrombin, which creates the fibrin clot.

There are several ways of expressing the results of the PT test. One system, the INR system, is recommended by the World Health Organization. However, many countries have not adopted this system for expressing PT results. Moreover, the INR system has only been validated for patients on oral anticoagulant control, but should not be used in expressing results from patients with other disease states, such as liver disease. Another system, commonly used in the United States, expresses the time in seconds for the blood to begin to coagulate. Still another system expresses the results in terms of a percentage PT ("% PT") which is read from a standard calibration (or dilution) curve prepared by diluting fresh normal pool plasma ("FNP") in 0.9% saline. (Other diluents work, but by convention, only saline is used.) The curve allows for the conversion of results from time in seconds to percent of normal activity (% PT). Unfortunately, in order for this system to be used, most laboratories have to prepare their own pool plasma and keep it frozen, usually in liquid nitrogen or frozen at -80.degree. C. Moreover, due to the inherent variation found in different plasma pools, there is no standardization between the plasma pools of different laboratories. Moreover, it has been shown that if a pool of plasma is prepared, the mean % PT value obtained from the pool is different than the mean % PT value obtained from the individual samples that were used to make the pool. It has also been shown that the collection of bulk collections of blood, as would be required to commercially prepare a lyophilized standard, causes a reduction in the measured % PT when compared with blood collected by venipuncture. See Important Differences Encountered in the Normal Plasma Pools used for the Control of Oral Anticoagulation. M. Burgess-Wilson, R. Burri and B. Woodhams, Thromb. Haemost. 69 Abstract 2081 (1993). Moreover, FNP cannot be sold until lyophilized. Lyophilization results in a plasma which, when reconstituted, has a % PT value lower than that found in a normal hospital pool of plasma. This reconstituted FNP is then used to prepare the standard curve. The dilutions usually used for the standard calibration curve are undiluted, 1:1, 1:2 and 1:4. Where reconstituted FNP is used, the undiluted sample is assigned a value of 100% PT. A PT assay is performed and the results (in seconds) are plotted on hyperbolic or reciprocal graph paper against the dilution (in %). See FIG. 1. Patient samples are tested undiluted and then read from this standard curve. However, using reconstituted FNP as a calibrator means that values for normal samples are above the top calibration point of the standard curve made using the reconstituted FNP. (By definition, 50% of all normal values would be above the top point of the standard curve.)

The % PT curve is not a straight line. Although a polynomial plot gives the most realistic curve through the data, many laboratories and users do not have the computer software required for such a procedure. Therefore, a linear curve through the points is commonly used. To make the results more accurate around the 100% region of the curve, the line is forced through the 100% point. One type of assay machine, the Medical Laboratory Automation ("MLA") Electra automated coagulometers, does not calculate % PT outside of certain ranges (above about 125% PT and below about 12% PT).

The recommended method of calculating % PT varies between the instrument manufacturers. There is no universally used standard procedure. Some instrument manufacturers, such as MLA, recommend forcing a straight line through 100%. Others recommend polynomial or non-forced straight lines. This introduces variability into the procedure, especially if the calibration plasma has a value of % PT much lower than 100%. See FIG. 2. In the examples that follow, the method of calculating the % PT was to use a forced linear curve through the 100% point using the SigmaPlot transformation.

SUMMARY OF THE PRESENT INVENTION

This invention relates to a method for preparing a commercial plasma preparation that will allow calibration curves to be prepared that will have % PT values of about 100% and will give results analogous to those obtained with FNP. In summary, the invention involves the addition of recombinant human FVII or FVIIa (or any other source of FVII, provided it is of high enough purity and behaves in a similar fashion to human FVII) to normal human citrated plasma to give the required PT %. For an article discussing the purification of recombinant human Factor VII, please see Kemball--Cook, Mcvey, Garner, Martin, O'Brien and Tuddenham, Stable High Level Expression of Recombinant Human Factor VII In Mammalian Cell Culture, Thromb. Haemostatis 69 (6) 1993, Abstract 253. The resulting plasma is lyophilized and calibrated. It is expected that the addition of other recombinant factors such as rFVIII, rFV or rFXI could be made to a plasma that would also act as a calibrator for other coagulation assays, e.g. FVIII, FV, FXI, derived fibrinogen, FIX, FII, FX, Protein-C, Protein-S, and APTT (clotting and chromogenic) assays. For example, rFV is obtained from available sources and can be added to the plasma such that a level of about 100% rFV is achieved.

The composition for calibration of a PT assay of the present invention (hereinafter `Calibrator` can be used with thromboplastin reagents such as THROMBOPLASTIN IS lyopholized acetone dehydrated rabbit brain thromboplastin, calcium ions, buffers and stabilizers, hereinafter THROMBOPLASTIN IS, THROMBOPLASTIN C reagent, lyophilized acetone-dehydrated rabbit rain thromboplasint, calcium ions, stabilizers and preservatives, hereinafter THROMBOPLASTIN C reagent, and THROMBOPLASTIN C+ reagent, lyophilized acetone-dehydrated rabbit brain thromboplastin, calcium, buffer, antimicrobial and stabilizers, hereinafter THROMBOPLASTIN C+ reagent. Particularly, the Calibrator of the present invention is designed for use with a recombinant tissue factor PT reagent such as Baxter Diagnostics Inc.'s Dade INNOVIN.TM. Reagent lyophilized recombinant human tissue factor and phospholipids (thromboplastin), calcium ions, buffers and stabilizers, hereinafter "INNOVIN reagent" and Ortho Diagnostics Systems Ortho.RTM. RECOMBOPLASTIN .TM. which are used as reagents in the PT determinations and PT-based assays. Recombinant tissue factor reagents, and in particular, INNOVIN.TM. reagent, was found to have increased sensitivity, when compared to other reagents used in PT determination and PT-based assays, to various factor deficiencies and oral anticoagulant-treated patient samples. The increased sensitivity of such reagents is such that they differentiate much more between FNP collected by syringe or by blood bag than traditional thromboplastins (prepared from animal or human tissue extracts). A calibration plasma should be collected in a fashion similar to clinical samples, i.e., syringe drawn. However, until the Calibrator of the present invention, commercial preparations of a calibration plasma with a PT of 100% were difficult, if not impossible, to prepare. Lyophilized normal plasma has a % PT of 85% or less when measured with INNOVIN.TM. reagent. The use of a plasma sample with such a low % PT value makes calculations of the % PT value of normal samples difficult and introduces a large amount of variation according to the method used to calculate the % PT, as explained further below. As shown in FIG. 2, the boxed area shows the two curves which can be drawn (polynomial and extrapolated). The enlarged boxed area shown in FIG. 3 demonstrates that the two curves will give very different results as they diverge. The divergence increases above the top calibration point. If the top calibration point is at 85%, then the calibration of normal results (130-70% PT) will be more strongly influenced by the choice of curve as the 85-100% PT part of the curve will have to be extrapolated. The use of the calibrator of the present invention will keep the % PT close to 100% and avoid using the diverging areas of the curves. The resulting calibrated plasma preparation can be used on the MLA Electra, KC, and ACL range of instruments.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a calibration curve of clotting time (in seconds) vs. 1/PT % for dilutions of FNP in saline.

FIG. 2 depicts the problem posed by calculating % PT using PT dilution curves when the clotting time of the test plasma is shorter than that of the calibration plasma.

FIG. 3 depicts an enlarged portion of FIG. 2.

FIG. 4 depicts the effect of the addition of rFVIIa in different concentrations on PT Clotting Time in seconds from the data in Table 1a.

FIG. 5 depicts the PT calibration curves of FNP alone and FNP with the addition of rFVIIa (1/10.sup.3 dilution), from the data in Table 1b.

DETAILED DESCRIPTION OF THE INVENTION

Recombinant FVIIa did raise the % PT of the plasma pool. Recombinant FVII also raised the % PT. The amount of recombinant material needed to be added to a large pool of plasma to produce a % PT of about 100% was determined.

The FVII levels achieved (as measured using the one stage clotting assay) did not usually parallel the rise in PT %. Two lots of rFVII showed quite different relationships between PT % and FVII level rise. The difference was thought to be due to "contamination" of the rFVII with the more active rFVIIa. As described later herein, the rFVIIa material did not have this problem. Without limiting the scope of the invention, it is believed that rFVIIa is preferable as a calibrator because the "contamination" factor is not present.

Either rFVII or rFVIIa was added to a pool of HEPES buffered citrated plasma. While HEPES buffer was chosen for these examples because it lyophilizes well, it is believed that most buffers which work in the physiological pH range could be used, except for phosphate type buffers. Examples of buffers which should work include Good's Buffers: PIPES, ACES, BES, MOPS, TES, and TRICINE. The resulting plasma plus recombinant material was tested for PT % prior to lyophilization. Two lots of the plasma plus rFVII had a PT % of about 100% prior to lyophilization. After lyophilization, the PT % was about 85%. The PT % calibration curve from such reconstituted plasma was used to calculate PT % results. Values were very similar to those obtained using a calibration curve from COAG CAL ("CCN") plasma, a lyophilized, intrated normal plasma containing all clotting factors.

Three lots of the calibrator plasma were produced by adding rFVIIa to a pool of HEPES buffered citrated plasma. The accelerated stability studies showed that after 35 days at 37.degree. C. (equivalent to 2 years at 4.degree. C.), the results were similar to those of CCN plasma and suggests they will have a similar stability. In two lots, the PT % was adjusted to approximately 100% before lyophilization. Lyophilization appeared to reduce the PT % to between 90-95%. The pre-lyophilization target for the third lot was changed to between 105% and 108%, inclusive. Post-lyophilization, the third lot had a PT % of about 100%. The reconstituted third lot was stable for 8 hours at 4.degree. C. and room temperature. The PT % calibration curves from such lot were stable for 30 minutes.

As more fully explained in the examples, one or more of the following reagents were used in the examples that follow. (These examples are intended for purposes of illustration of the invention, not for limitation of the invention. For instance, the addition of HEPES is referred to as "dropwise" in an example. The invention obviously is not limited to use of the HEPES buffer or its dropwise addition.)

______________________________________Recombinant material:Material Lot No. Concentration Source______________________________________rFVIIa 29491 1 mg/ml (Novo) DudingenrFVIIa 8293 1.2 mg/ml (Novo) HarrowrFVII 28193 30-40 U/ml HarrowrFVII 9393 24 U/ml HarrowrFVII 10393 15 U/ml HarrowrFVIIa 21593 2500 U/ml Harrow______________________________________

Harrow refers to the Haemostasis Research Group, Clinical Research Centre, Watford Road, Harrow, Middlesex, England.

Other reagents:

TIS THROMBOPLASTIN IS lots TPS--46 and 59 (Baxter's dried rabbit brain with calcium PT assay reagent) INNOVIN.TM. INNOVIN.TM. reagent lots TFS--12, 13, 14 and 24 Saline NaCl (0.9%) lots H1-75 Owrens Buffer Owrens Buffer lots 550.029, 550.030 and 550.032 Factor VII Immuno Absorbed Plasma ("IAP") Factor VII IAP lots IAP7-25A and 26A FVII(a) -Tris Buffer Tris Buffer pH 7.4 lots H1-85 Buffer used to dilute rFVII (Although TRIS buffer is used in these examples, it is believed that any buffer of the same pH can be used.) 0.05M Tris (hydroxymethyl)-aminomethane 0.15M NaCl

Several lots of CCN plasma, a lyophilized normal plasma containing all clotting factors, were tested for PT % using THROMBOPLASTIN IS and INNOVIN.TM. reagents. They were also tested for the FVII % level. The results are tabulated below. The five lots of CCN plasma were combined to make FNP 870.003.

______________________________________ PT %CCN lot No. PT % TIS Innovin .TM. FVII % level______________________________________540.042 92 -- 98540.049 91 85 105540.050 100 85 --540.053 97 85 97540.054 -- -- --FNP 870.003 100 100 100______________________________________ Machines and software:

MLA Electra 1000C: No 572--Software Version 3 Rev. E

MLA Electra 900C: No 1753--Software Version 4 Rev. 1

MLA Electra 1000C: Software Version 5.0: Munchen

Methods:

Prothrombin Time (PT)

The PT testing assays were performed as per the Box Inserts for THROMBOPLASTIN IS and INNOVIN.TM. reagents, and the MLA Electra 900C or 1000C operating manuals.

Factor VII assay

Factor VII assays were performed as per the Box Insert of the FVII IAP and the MLA Electra 900C or 1000C operating manuals. The dilutions of the plasma or concentrate were selected so that the clotting times obtained were within the range obtained using the calibration curve dilutions. In general, the 1 in 10 dilution was assigned as 100% Factor VII.

EXAMPLE I

Recombinant FVIIa lot 29491 was diluted in Owrens Buffer 1/10.sup.2, 1/10.sup.3, 1/10.sup.4, 1/10.sup.5, 1/10.sup.6, and 1/10.sup.7. Five 500 ul aliquots of FNP 870.003 were prepared. To each of the aliquots of FNP 870.003 were added one 20 ul aliquot of one rFVIIa dilution. The PT % of the resulting plasmas were tested using the MLA Electra 900C.

When measured using THROMBOPLASTIN IS and INNOVIN.TM. reagents, it was possible to reduce the PT clotting time of FNP, thus increasing the % PT. (See Table la). Using the 1/10.sup.4 dilution of the rFVIIa, the Factor VII % level in the FNP was raised by 13-20%. The calibration curves (Clotting Time, in seconds, vs. 1/PT %) of FNP and FNP plus rFVIIa were nearly parallel, indicating that the modified plasma (FNP plus rFVIIa) can be used as a calibrator. See Table 1b and FIG. 5.

TABLE 1______________________________________Effect of different concentrations rFVIIa in FNP onPTTable 1a: rFVIIa dilutions in FNP PT Clotting Time (seconds)Dilution added In FNP THROMBOPLASTIN IS INNOVIN .TM.______________________________________None 14.4 11.41/100 9.8 8.51/1000 10.1 9.71/10,000 13.6 11.11/100,000 14.6 11.51/1,000,000 14.7 11.6Buffer 14.8 11.5______________________________________ TABLE 1b:______________________________________Data for rFVIIa 10 3 dilution in FNP, calibrationcurve, compared with data for FNP curve PT Clotting Time (seconds) FNP FNP + rFVIIa 10 3Dilution TIS INNOVIN .TM. TIS INNOVIN .TM.______________________________________Neat 14.5 11.5 12.6 10.51 in 2 20.2 15.0 17.4 13.71 in 4 32.0 25.5 27.9 22.4.1 in 8 56.1 44.3 51.7 41.2______________________________________

EXAMPLE II

Recombinant FVIIa Lot 8293 was diluted in CCN plasma lot 049 by adding 50 ul of concentrated rFVIIa to 5 ml of CCN plasma, resulting in a 1 in 100 dilution. Then a range of 1 in 10 dilutions were produced by adding 500 ul of the resulting plasma to 4.5 ml of the CCN plasma. Three further dilutions were made, resulting in 1/10.sup.3, 1/10.sup.4 and 1/10.sup.5 dilutions. The CCN plasma lots and the four dilutions were tested using TIS and INNOVIN.TM. reagents. The results are set forth below:

TABLE 2______________________________________Addition of rFVIIa to CoagCal N plasmaTHROMBOPLASTIN IS INNOVINSample Neat 1 in 2 1 in 4 1 in 8 Neat 1 in 2 1 in 4 1 in 8______________________________________CCN 042 15.1 22.5 40.2 73.4 11.9 16.3 27.3 53.1CCN 049 15.0 22.6 37.6 75.6 12.0 16.0 27.5 51.11/100,000 12.2 17.3 29.7 57.6 10.3 13.5 21.8 40.61/10,000 10.7 15.4 25.6 50 9.7 12.2 19.1 35.61/1,000 9.8 13.6 22.0 41.5 8.9 11.0 16.3 28.510 2 9.5 13.2 21.9 42.6 -- 10.9 16.3 --______________________________________ CCN plasma, like FNP, experienced a reduction in PT, thus increasing the % PT by the addition of rFVIIa.

EXAMPLE III

Testing was done on rFVII material. Reagents included FNP 870.003 and CCN plasma lot 042. Testing was performed on the MLA Electra 1000C.

Different volumes of the three lots of rFVII were added to CCN plasma lot 042. Because the rFVII preparation had lower FVII activity than the rFVIIa preparation, instead of diluting the FVII preparation and adding the dilution to the plasma as in Example I, a different method was used as described below. This was done by reducing the amount of distilled water added to reconstitute the CCN plasma by the volume of rFVII added. For example, when 100 ul rFVII was added, the vial of CCN plasma was reconstituted with only 900 ul of distilled water.

The % PT and FVII % of the reconstituted CCN plasma lot 042 samples were calculated using the FNP calibration curve assigned as 100% PT activity. The mean % PT for all calibration curve dilutions was used. All lots of rFVII raised the % PT and the Factor VII % levels. The effect on the % PT was not proportional to the rise in FVII activity. It was thought that the lots of "rFVII" may have activated rFVII present in variable amounts which led to a variable effect on the % PT which was not related to the assigned Factor VII level. Because of the variability in rFVII, the use of rFVIIa would be preferable as it would be a more consistent reagent.

TABLE 3__________________________________________________________________________Addition of rFVII to CoagCal N plasma PT % Factor VII %Sample THROMBOPLASTIN IS INNOVIN TISM. INNOVIN .TM.__________________________________________________________________________FNP 100 100 -- --CCN 90 95 98 98rFVII 75 ul 92 98 112 1109393 100 ul 97 102 118 116 100 ul 96 100 130 124rFVII 20 ul 108 108 116 10428192 30 ul 108 115 200 202 50 ul 121 120 284 304rFVII 150 ul 94 93 138 11610393 200 ul 97 94 134 118__________________________________________________________________________ Factor VII calculated using CCN plasma as calibrator PT % calculated assuming FNP=100%

EXAMPLE IV

The method of measuring the Factor VII in the concentrate was investigated and the relationship of the Factor VII levels and PT % in CCN plasma with different amounts of rFVIIa added was examined. Reagents included rFVIIa lot 21593, CCN plasma lots 042 and 049 and IAP7-26A.

Testing was performed on the MLA Electra 1000C. The Factor VII level of the rFVIIa preparation was measured in two ways, by adding the preparation to CCN plasma and assaying dilutions of 1/100 to 1/1000 in Owrens buffer.

A primary dilution of rFVIIa in CCN plasma was made (CCN plasma 5ml plus 20 ul rFVIIa), also referred to as "plasma+rFVIIa". Then the following dilutions were made from the plasma+rFVIIa and CCN plasma. See Table 4. The 10 ul dilution of Table 4, marked with the "*", is the same 10 ul dilution used in Table 5.

TABLE 4__________________________________________________________________________Dilutions of rFVIIa in CoagCal N plasma__________________________________________________________________________Amount 10 ul* 5 ul 4 ul 3 ul 2 ul 1 ul 0 ulofrFVIIain 5 mlCCNPrimary 1 ml 0.5 ml 0.4 ml 0.3 ml 0.2 ml 0.1 ml 0 mldilutionCCN 1 ml 1.5 ml 1.6 ml 1.7 ml 1.8 ml 1.9 ml 2.0 ml__________________________________________________________________________ TABLE 5______________________________________Further dilutions of rFVIIa______________________________________Amount 2 ul 1 ul 0.5 ml 0.25 ul 0.125 ul 0 ulof rFVIIain 5 mlCCN10 ul 1 ml 0 ml 0 ml 0 ml 0 ml 0 mldilution*CCN 4 ml 1 ml 1 ml 1 ml 1 ml 2 mlMix + 0 ml 1 ml 1 ml 1 ml 1 ml 0 mlTransferPreviousDilution______________________________________

The results are found in Tables 8 and 9. The following formula was used to calculate the FVII % concentration in U/ml.

______________________________________(FVII %/100 .times. 5)-5 .times. (1000/ul of rFVIIa added) =FVII of the concentrate (U/ml)______________________________________FVII %/100 100% FVII = 1 U/ml.times.5 5 ml of plasma-5 5 U/ml of FVII in this 5 ml of normal plasma1000/ulrFVIIa added Volume of rFVIIa compared to 1000 ul added______________________________________

Results were calculated for plasma+rFVIIa using the formula set forth above.

TABLE 6______________________________________Calculation of FVII levels - rFVIIa added toplasmaAmount rFVIIa rFVIIaadded FVII % FVII (U/ml)______________________________________5 ul 253 15324 ul 246 18303 ul 224 20702 ul 197 24371 ul 167 3345mean 218 2243______________________________________

Table 6 shows that the concentrate FVII level was 2243 U/ml when rFVII was added to plasma.

TABLE 7______________________________________Calculation of FVII levels - dilutions ofrFVIIa in buffer FVII %Dilution FVII % effective FVII (U/ml)______________________________________1/1000 441 441001/2000 292 584001/4000 190 76000mean 59500 5951/10000 93 930001/20000 51 1020001/40000 29 116000mean 103667 1037______________________________________

Table 7 shows that the concentrate FVII level was between about 600 and 1000 U/ml when rFVII diluted in buffer was tested.

When measuring rFVIIa in plasma, the result obtained (2243 U/ml) was similar to the quoted concentrated from Harrow (2500 U/ml). Estimates using diluted concentrate were lower (600-1000 U/ml) and we concluded that this method is not useful.

A progressive rise occurs in % PT and FVII levels with increasing the addition volume of rFVIIa to plasma (Tables 8 and 9). As seen from the data in Table 9, there was a relationship between the rise in Factor VII level and the rise in PT %, r=0.9661.

TABLE 8______________________________________Effect of rFVIIa on Prothrombin TimeLot rFVIIa 21593______________________________________Amount INNOVIN .TM.rFVIIa PT % Calibrator curve dilutions TestAdded Neat 1 in 2 1 in 4 1 in 8 Mode______________________________________10 ul 10.1 14.8 24.2 50.4 10.55 ul 10.4 15.3 25.5 49.7 10.54 ul 10.6 15.7 26.7 53.8 10.93 ul 10.8 16.5 28.3 55.9 10.82 ul 11.1 16.4 28.2 58.5 11.31 ul 11.5 17.4 30.8 60.6 11.4zero 12.4 19.2 34.2 67.2 12.52 ul 10.8 15.8 27.4 54.6 10.91 ul 11.0 16.7 29.2 61.5 11.00.5 ul 11.3 17.2 30.8 64.0 11.40.25 ul 11.6 17.7 31.6 64.6 11.70.125 ul 11.7 18.1 32.8 66.0 11.7zero 12.0 18.9 34.4 62.6 12.0______________________________________Amount THROMPBOPLASTIN ISrFVIIa PT % Calibrator curve dilutions TestAdded Neat 1 in 2 1 in 4 1 in 8 Mode______________________________________10 ul 11.4 17.7 30.5 62.1 --5 ul 12.0 19.0 32.0 66.9 --4 ul 12.3 19.0 33.6 66.5 --3 ul 12.4 19.3 33.7 71.4 --2 ul 12.8 20.4 34.4 73.0 --1 ul 13.3 21.2 36.3 76.3 --zero 15.2 24.7 41.9 85.9 --______________________________________ TABLE 9______________________________________Investigation of increasing PT % and FVII %level______________________________________INNOVIN .TM.Amount FVII %rFVIIa Dilution Rise in Rise inAdded 1 in 10 PT % PT % FVII %______________________________________10 ul -- 131 33 --5 ul 253 131 33 1424 ul 246 123 25 1353 ul 224 125 17 1132 ul 197 115 17 861 ul 167 114 16 56zero 111 98 0 02 ul -- 118 18 --1 ul -- 116 16 --0.5 ul -- 109 9 --0.25 ul -- 104 4 --0.125 ul -- 104 4 --zero -- 100 0 --______________________________________Thromboplastin ISAmount FVII %rFVIIa Dilution Rise In Rise InAdded 1 in 10 PT % PT % FVII %______________________________________10 ul -- 161 61 --5 ul -- 146 46 --4 ul -- 140 40 --3 ul -- 138 38 --2 ul -- 131 31 --1 ul -- 123 23 --zero -- 99.5 0 --______________________________________

EXAMPLE V

Stable lyophilized plasma which has had Factor rFVII added to be used as a calibrator in the PT % test was prepared as follows.

Reagents used were rFVII lots 28193 and 9393, plasma as described in Table 11a, and HEPES buffer H1-83.

The volume of rFVII needed was calculated as follows. It was expected that after lyophilization the PT % would be about 85-90%; thus a rise in PT % of 10-15% was required. Preliminary work with lot 28193 suggested that 20-30 ul rFVII per 5 ml of plasma created the desired rise in PT %; 25 ul rFVII per 5 ml of plasma was used. Lot 9393 had a lower Factor VII level and about 400 ul rFVII per 5 ml plasma was needed to raise the PT %.

Ten units of approximately 200 ml each of plasma were selected from each of the plasma bags described in Table 11a. All plasma had been collected into the anticoagulant CPD-A. The plasma was carefully thawed in a large waterbath at 37.degree. C. Bag contents were mixed until all ice had disappeared and the plasma was free from undissolved precipitate. Once thawed, the bags were kept in crushed ice. The entire contents of each bag were pooled and stirred thoroughly while kept cool by crushed ice. Four pools were prepared for lyophilization as described in Table 10.

TABLE 10______________________________________Preparation of different plasma pools Volume of Volume of VolumePool plasma HEPES recombinant FVII______________________________________Pool P 100 ml None NonePool P1 100 ml 3 ml NonePool P1 & 28193 100 ml 3 ml 500 ul of lot 28193Pool P1 & 9393 100 ml 3 ml 8 ml of lot 9393______________________________________

HEPES was added dropwise to the stirred plasma. The recombinant FVII was added last and the final mixture stirred thoroughly. The resulting plasmas were pipetted into separate 1.1 ml vials and stored at 4.degree.-8.degree. C. for about 1 hour before lyophilization. After lyophilization, the vials were kept at 4.degree.-8.degree. C. Several vials from each lot were not lyophilized but stored at -70.degree. C. storage. Prior to lyophilization, the different pools of plasma, CCN plasma lot 042 and CCN plasma lot 049, both freshly reconstituted, were tested using the MLA Electra 1000C. Both PT % and FVII % level assays were performed. Plasma samples were tested in the calibration curve mode and in the test mode. The 10 plasmas used to make up Pool P were all normal (see Table 11a). Testing of fresh pools suggest a PT % of approximately 100% in both pools (P1 & 28193, P1 & 9393). The rise in FVII was 140% and 190%, respectively. The conclusion is that the amount of rFVII needed to prepare a control with approximately 100% PT can be predicted. Prior to lyophilization, the addition of HEPES buffer reduced the PT % by about 5%.

After lyophilization, the different pools of plasma, CCN plasma lot 042 and CCN plasma lot 049 were tested using the same instrument and procedure as in their testing before lyophilization. After lyophilization, the pool without HEPES showed a loss of 11% PT whereas the pool with HEPES showed no difference. The two pools with rFVII showed a slight (2%) loss in % PT. No changes were seen in FVII % levels after lyophilization even in the pool without HEPES. (See Tables 12a-12b.)

TABLE 11a______________________________________PT Clotting Time of plasmas making upplasma Pool PBAG Clotting TimeNo. 1020 hrs 1344 hrs______________________________________1 11.6 11.22 11.5 11.13 12.8 12.44 11.8 11.45 12.7 12.36 11.8 11.37 12.3 11.68 12.3 11.79 12.1 11.610 11.2 11.0Mean 12.01 11.56______________________________________ TABLE 11b______________________________________PT Clotting Times before lyophilizationINNOVIN .TM. PT Reagent Calibration Curve Test PT %Calibrator Neat 1 in 2 1 in 4 1 in 8 Mode **______________________________________CCN 049 12.2 18.0 32.7 69.0 12.14 84.2CCN 042 12.2 18.3 31.9 68.3 12.16 84.0Pool P 11.6 17.6 32 66.9 11.56 91.3Pool P1 12.1 18.6 32.8 66.2 11.99 86.0Pool P1 & 28 11.1 15.7 28.4 58.1 10.95 100.1Pool P1 & 93 11.0 16.1 27.0 58.6 11.06 98.4______________________________________ TABLE 11c______________________________________Factor VII assay before lyophilizationINNOVIN .TM. PT ReagentCalibration Curve TestCali- 1 in 1 in 1 in 1 in 1 in Mode FVII % FVII %brator 10 20 40 80 160 1 in 10 1/10** 1/20**______________________________________CCN 23.1 21.4 41.2 53.6 69.7 23.2 110 104042CCN 23.4 31.5 42.1 54.1 70.4 23.1 111 103049Pool P 24.3 32.9 43.9 57.7 74.3 23.5 107 93Pool P1 24.5 33.9 43.6 56.5 72.4 23.6 105 86Pool 20.4 27.8 35.8 47.4 63.3 20.8 144 141P1 & 28Pool 18.1 23.6 NA 39.8 54.1 18.3 198 210P1 & 93______________________________________ **Calculated with CCN plasma 049 calibration Curve: PT % = 85%, FVII % = 105% "Test Mode" means that a sample can be tested as a calibrator whether the sample is diluted or just as neat plasma. TABLE 12a______________________________________PT Clotting time after lyophilizationINNOVIN .TM. PT ReagentINNOVIN .TM. Calibration Curve Test PT %Calibration Neat 1 in 2 1 in 4 1 in 8 Mode **______________________________________CCN 049 12 17.5 30.8 63.1 12.15 84.1CCN 042 12.2 17.8 30.9 65.7 12.05 85.3Pool P 12.5 18.9 33.1 66.9 12.55 79.9Pool P1 11.8 18.1 31 65.4 11.85 87.6Pool P1 & 28 11 16.3 27.4 57.9 11.05 98.5Pool P1 & 93 11.1 15.9 26.5 54.6 11.2 96.3______________________________________ TABLE 12b______________________________________Factor VII assay after lyophilizationCalibration Curve Test Mode 1 in 1 in 1 in 1 in 1 in 1 in 1 in FVII %Calibrator 10 20 40 80 160 10 10 **______________________________________CCN 049 23.1 30.2 40.9 54.2 71.7 23.5 23 112CCN 049 23.5 31.1 41.1 53.6 71.3 -- -- --CCN 042 23.6 31.7 42.9 56.1 72.6 23.6 23.6 105Pool P -- -- -- -- -- 23.2 24.1 100Pool P1 -- -- -- -- -- 23.9 23 112Pool P1 & -- -- -- -- -- 20.5 20.3 15328193Pool P1 & -- -- -- -- -- 18.2 18.1 2039393______________________________________ **Calculated with CCN plasma 049 calibration Curve: PT % = 85%, FVII % = 105%

EXAMPLE VI

Stable, lyophilized plasma to which rFVIIa has been added to be used as a calibrator in the PT % test was prepared as follows.

Reagents used were rFVIIa Lot 21593, Pool 2 (CCN plasma lot 053 just before lyophilization), and TRIS Buffer Lot H1-85. Four hundred milliliters of a plasma pool ready to use (containing HEPES) were used to prepare CCN plasma lot 053.

TABLE 13______________________________________Preparation of different plasma pools______________________________________Pool Name rFVIIa added Plasma pool 053 �FVII! added______________________________________Pool P2 None 100 ml NonePool P2/20 20 ul 100 ml 1 ul/5 mlPool P2/10 5 ul 50 ml 0.5 ul/5 ml______________________________________ Tris Buffer Plasma �Tris B.!Pool Name added pool 053 added______________________________________Pool P2/B 20 ul 100 ml 1 ul/5 ml______________________________________

Vials were filled with 1.1 ml pooled plasma and stored at -70.degree. C. for five days and then lyophilized. A certain number of vials were kept at -70.degree. C. and not lyophilized.

Prior to lyophilization, the four pools were tested on the MLA Electra 1000C. After lyophilization, the four pools were tested against the corresponding four frozen pools using the same instrument and procedure as in the testing prior to lyophilization.

The results found in Tables 14 and 15 were calculated using a previous CCN plasma lot 049 calibration curve (Table lib: 12.2, 18.0, 32.7 and 69.0 seconds assigned as 85% PT). The fresh results for two lots (Pool P2/20 and Pool P2/10) are 102% and 98% respectively. After lyophilization, there appears to be a 5-8% drop in the PT %, which did not occur with rFVII. The frozen samples did not show this drop. It appears that in plasmas where rFVIIa is used to increase the PT %, there is a 5-8% loss of PT % during lyophilization. This needs to be compensated for during the manufacturing process.

TABLE 14______________________________________Pool P2 fresh before the lyophilization Calibration Curve Test PT %INNOVIN .TM. Neat 1 in 2 1 in 4 1 in 8 Mode **______________________________________Pool P2 12.3 17.7 30.5 60.1 12.1 84.7Pool P2/B 12.1 17.6 30.1 59 12 85.5Pool P2/10 11.1 15.6 26 51.1 -- 97.8Pool P2/20 10.8 15.2 24.5 50.2 10.8 102.5______________________________________ **Calculated with CCN plasma lot 049 Calibration Curve: PT % = 85%, FVII = 105% TABLE 15______________________________________Pool P2 after the lyophilization______________________________________Lyophilized calibrator test with INNOVIN .TM. PT Reagent Calibration Curve Test PT %Calibrator Neat 1 in 2 1 in 4 1 in 8 Mode **______________________________________Pool P2 12.2 18.6 32.3 66.6 12.3 82.5Pool P2/B 12 18.6 32.1 65.1 12.3 82.5Pool P2/10 11.4 16.7 29.1 60.3 11.65 89.5Pool P2/20 11.1 16.6 28.6 57.7 11.3 94.8______________________________________Frozen calibrator test with INNOVIN .TM. PT Reagent Calibration Curve Test PT %Calibrator Neat 1 in 2 1 in 4 1 in 8 Mode **______________________________________Pool P2 11.7 17.4 30.2 59.3 11.65 901Pool P2/B 11.7 17.2 30.1 59.4 11.85 87.6Pool P2/10 11 15.5 26.3 52 11.1 97.8Pool P2/20 10.7 15.3 25.5 51 10.5 107.7______________________________________Lyophilized calibrator test with THROMBOPLASTIN IS PT Reagent Calibration Curve Test PT %Calibrator Neat 1 in 2 1 in 4 1 in 8 Mode **______________________________________Pool P2 15.2 20.1 34.3 66.2 15.7 --PooI P2/B 14.9 22.7 37.7 73.3 15 --PooI P2/10 14.1 21.1 34.3 67.8 14.1 --PooI P2/20 13.6 20.1 34.3 66.2 13.7 --______________________________________ TABLE 16______________________________________Frozen Pool P2Frozen calibrator test with THROMBOPLASTIN IS PT Reagent Calibration Curve Test PT %Calibrator Neat 1 in 2 1 in 4 1 in 8 Mode **______________________________________Pool P2 14.7 22.6 37.5 74.3 14.95 --Pool P2/B 14.9 22.5 37.3 72.6 15 --Pool P2/10 13.7 20.3 33.8 67.6 13.7 --Pool P2/20 13.1 19.5 32.9 64.6 13.1 --______________________________________ **Calculated with CCN plasma lot 049 calibration curve: PT % = 85%, FVII = 105%

EXAMPLE VII

The accelerated stability of Pool P2 (as prepared in Example VI) with rFVIIa added was tested and compared with two lots of CCN plasma. Reagents used were Pools P2, P2/13, P2/10, P2/20, CNN plasma lots 050 and 053. Several vials of the plasmas were stored at 37.degree. C. and tested after 10, 14, 26 and 35 days on the MLA Electra 1000C according to the Box Insert and the MLA Electra 1000C Handbook. Vials of the same plasmas stored at 4.degree. C. were tested for the same time periods. All plasma tested showed a progressive drop in the PT % on incubation at 37.degree. C. The plasma containing rFVIIa did not drop differently than those not containing rFVIIa. Adding rFVIIa does not change the stability of the plasma incubated at 37.degree. C. measured using the PT % assay. See Table 17. Subsequent analysis of further lots with Arrhenius stability testing has given a predicted shelf life of greater than 2 years.

TABLE 17__________________________________________________________________________Accelerated stability Pool P2 Prothrombin Time in % 10 days 14 days 26 days 35 daysCalibrator 4.degree. C. 37.degree. C. 4.degree. C. 37.degree. C. 4.degree. C. 37.degree. C. 4.degree. C. 37.degree. C.__________________________________________________________________________CCN 050 83.6 79.4 84.7 76.5 83.6 76.5 83.6 79.7CCN 053 83.6 78.4 83.6 76.5 82.5 75.7 83.6 73.0Pool P2 83.6 78.4 83.6 76.5 83.6 73.8 83.6 71.3Pool P2/B 84.7 78.4 84.7 76.5 83.6 73.8 84.7 73.0Pool P2/10 92.1 85.8 90.8 83.6 92.1 80.4 92.1 79.4Pool P2/20 94.8 89.5 96.3 85.8 94.8 83.6 96.3 81.4__________________________________________________________________________ PT % is calculated with CCN lot 049 Calibration Curve: PT %=85%.

EXAMPLE VIII

A previously prepared pool of citrated plasma, from 10 donors (See Table 6a), stored at -20.degree. C., was thawed in a 37.degree. C. waterbath and then stored at 4.degree. C. When the temperature of the thawed plasma reached 4.degree. C., then a HEPES solution was added slowly dropwise.

The HEPES solution was prepared by adding 40 mg of HEPES powder to 100 ml distilled water. The pH was adjusted to approximately 7.3 to 7.5 using 5M NaOH. (About 5 ml of 5M NaOH was needed.) This resulted in a 40% HEPES solution (lot H1-83).

For each liter of plasma in the pool, 30 ml of the 40% HEPES solution were added. The pooled plasma and the HEPES solution were mixed for 10 minutes, with care not to create foam.

All testing was performed using an MLA Electra 1000C. The PT % of the pool plus HEPES buffer (the "Buffered Pool") was determined. Recombinant Factor VIIa was then added to the Buffered Pool in a step-wise manner, as described below, until the PT % of the Buffered Pool plus rFVIIa was between 105% and 108%. It was adjusted 5-8% above 100% to allow for PT % loss of 5-8% during lyophilization. The rFVIIa had previously had its activity determined by adding dilutions to plasma (as described in Example IV), and this activity was used in the following formula to determine the amount (in ml) of rFVII to add per ml of Buffered Pool. ##EQU1## The total amount of rFVIIa that should be added to the Buffered Pool to achieve a PT % of 105-108% is about 0.6 Units per ml of plasma. If the PT % is as follows, then the amount of rFVIIa that is required is as follows:

______________________________________ <90% add 0.6 U/ml <100% add 0.3 U/ml <105% add 0.15 U/ml.______________________________________

Once the target PT % activity of the plasma pool with rFVIIa was achieved, the mixture was again thoroughly stirred for at least two minutes. Two aliquots of the mixture were tested and the mean of all 8 results was calculated. If the mean result was between 105-108% (inclusive), the material was accepted for lyophilization. If need be, further buffered plasma that has not had rFVIIa added to it can be added to the Buffered Pool to reduce the PT % to achieve the required value. See Tables 18-20. Data from a pilot production size run is shown in Tables 18, 19, and 20.

TABLE 18______________________________________Pre-Lyophilization Testing Calibration plasma curve Dilution of Plasma Neat 1/2 1/4 1/8______________________________________Sample 1 11.8 16.8 27.9 56.7 11.8 16.6 27.7 54.5Sample 2 11.5 16.7 29.8 55.7 11.3 16.2 28.3 56.5Sample 3 11.4 16.3 27.2 54.6 11.3 16.1 27.7 53.9Mean* 11.5 16.5 28.1 55.3PT % 88 44 22 11 COD 0.939______________________________________ TABLE 19______________________________________Reagents used______________________________________Calibration PlasmaLot No. R&D Pool P3PT % with INNOVIN 88rFVIIa conc. Lot No. 21593rFVIIa conc. (U/ml) 2500Innovin .TM. Lot No. TFS-12Saline Lot No. H1-86Machine type 1000CMachine No. 187Programme version 3.E______________________________________ Testing of Plasma Pool Measured volume (ml) 1200 Reserved plasma volumes (ml) 200 Numbers of donor units 10 TABLE 20______________________________________Results obtained______________________________________ Raw Data Clotting Time CalculatedMaterial Tested (Secs) PT % Mean______________________________________Initial Pool R&D Pool P3 12.5 12.3 75.6 77.8 77.1Calibration Plasma 12.2 12.5 78.9 75.6 12.3 12.4 77.8 76.7Plasma Pool plus 0.3 U/ml 11.2 10.9 92.5 97.5 95.0plasma 11.0 11.1 95.8 94.1rFVIIa Volume ofrFVIIa added = 0.12 ml______________________________________Further addition ofrFVIIa U/ml PlasmaVolume Plasma (ml)0.06 0.15 0 95.8 99.3 11.0 10.8 98.9 99.3 101.2 10.8 10.70.06 0.15 0 101.2 105.1 10.7 10.5 104.7 105.1 107.2 10.5 10.40.03 0.075 0 105.1 105.1 10.5 10.5 104.7 101.2 107.2 10.7 10.40.03 0.075 0 107.2 107.2 10.4 10.4 104.8 107.2 109.4 10.4 10.30 0 0 103.2 109.4 10.6 10.3 107.3 107.3 109.4 10.4 10.3______________________________________Final PT % 107.3______________________________________

Testing of the lyophilized product was performed using the MLA Electra 1000C. A lyophilized plasma that had been calibrated against FNP was used as a calibrator ("the Calibrator"). The PT % of the lyophilization product was calculated (using only the results from the undiluted plasma). This was assigned as the PT % of the product. A calibration curve was then obtained using the lyophilized product. As an in-process control check of the lyophilized product, a range of test results (see Tables 21-24) were calculated using the lyophilized product and the Calibrator, and the percentage difference was calculated. The lyophilized product was deemed to be acceptable if there were no differences greater than 15% (See Table 25).

TABLE 21______________________________________Post-Lyophilization Testing: Calibration plasma curve Dilution of Plasma Neat 1/2 1/4 1/8______________________________________Sample 1 11.5 16.3 27.9 53.5 11.5 16.2 26.4 53.7Sample 2 11.3 16.1 27.8 53.0 11.2 16.0 26.7 54.6Sample 3 11.2 16.1 27.5 52.8 11.2 16.1 26.6 53.3Mean 11.5 16.1 27.2 53.5PT % 86 44 22 11 COD 0.931______________________________________ TABLE 22______________________________________Reagents Used______________________________________Calibration PlasmaLot No. R&D Pool P3PT % with INNOVIN .TM. 88Innovin .TM. Lot No. TFS-12Saline Lot No. H1-86Machine Type 1000CMachine No. 187Programme version 3.E______________________________________ TABLE 23______________________________________INNOVIN PT Calibrator: Dilution of Plasma Neat 1/2 1/4 1/8______________________________________Sample 1 10.4 15.1 26.0 54.1 10.6 15.1 26.5 51.2Sample 2 10.6 15.5 26.8 51.1 10.7 15.3 26.1 50.8Sample 3 10.5 15.8 27.1 51.1 10.7 15.5 26.8 52.1Mean 10.6 15.4 26.5 51.2PT % 104 52 26 13 COD 0.947______________________________________ TABLE 24______________________________________Final Results of Pool P4______________________________________IPTC Lot R&D Pool P4PT % with INNOVIN .TM. 104rFVIIa conc Lot No. 21593Units added/ml plasma 0.75______________________________________ TABLE 25______________________________________Comparison of calculation with IPTC and CoagCal N: PT % PT %Test Calculated CalculatedResults Using Using % Difference(secs.) IPTC CoagCal N CCPT/CCN______________________________________ 9 142.5 151.0 5.6 9.5 127.4 131.7 3.210 115.2 116.8 1.411 96.7 95.3 1.512 83.3 80.4 3.613 73.1 69.6 5.014 65.1 61.33 6.1916 53.6 49.6 8.120 39.5 35.8 10.325 29.7 26.6 11.730 23.8 21.1 12.840 17.1 15.0 14.050 13.2 11.6 13.860 10.9 9.5 14.770 9.2 8.0 15.0______________________________________Final PT % 104______________________________________

EXAMPLE IX

The stability of the first Pilot production (identified here as Lot P4), when reconstituted, was tested using Innovin.TM. reagent lot TFS-12 and Saline H1-86. Dilution stability testing was performed on the MLA Electra 900C using programme version 4.1.

The testing was performed immediately after the dilutions had been prepared (t=0) and exactly 30 minutes after preparation (t=30). All reconstituted stability testing was performed using the E1000C using programme version 3E. Six vials were reconstituted. Three were left at room temperature for 8 hours and three at 4.degree. C. for 8 hours. After 8 hours, three more vials were freshly reconstituted and all nine vials had calibration curves produced. Innovin.TM. reagent was freshly reconstituted and tested immediately, after 4 and 8 hours stored on the E1000C (8.degree. C.) using freshly reconstituted reagents at each time point.

The material was deemed not to have failed stability testing if the clotting time in seconds was not more than 10% different from the clotting time obtained from lyophilized material stored at 4.degree. C. that had been freshly tested after reconstitution. The plasma dilutions were stable for 30 minutes. See Table 26. There was no significant variation between time 0 and time 30. The stability of INNOVIN.TM. reagent on the Electra 1000C is also good for 8 hours at 8.degree. C. (Table 27). The results do not give a variation from time t=0 until time t=8 hours. The PT calibrator reconstituted stability was also measured for 8 hours. There is little change between time t=0 and time t=8 hours at either 4.degree. C. (2%) or room temperature (4%). See Table 28.

The testing confirms the stability of the dilutions, the stability of INNOVIN.TM. TFS-12 and the reconstituted stability of Pilot lot P4.

TABLE 26______________________________________Stability of the Dilutions of PT Calibrator:Incubation Time0 Minutes 30 MinutesDilution of plasma Dilution of PlasmaNeat 1 in 2 1 in 4 1 in 8 Neat 1 in 2 1 in 4 1 in 8______________________________________Sample 1 11.1 13.5 20.0 36.0 11.1 13.1 20.9 37.0 10.9 13.0 19.2 36.6 10.8 13.0 19.3 35.9Sample 2 11.4 13.0 19.4 37.9 11.0 12.7 19.7 37.4 10.7 12.6 19.6 36.0 10.5 12.7 19.8 34.8Sample 3 10.7 12.5 21.0 37.5 10.9 12.8 20.0 35.0 10.4 12.5 19.1 38.4 10.4 12.9 18.9 24.5Mean 10.87 12.85 19.72 37.07 10.78 12.87 19.77 35.77______________________________________ TABLE 27__________________________________________________________________________Stability of INNOVIN .TM. Reagenton the Electra 1000CIncubation Time0 Hours 4 Hours 8 hoursDilution of plasma Dilution of plasma Dilution of plasmaSample Neat 1 in 2 1 in 4 1 in 8 Neat 1 in 2 1 in 4 1 in 8 Neat 1 in 2 1 in 4 1 in 8__________________________________________________________________________1 10.9 15.3 24.9 49.0 10.7 15.0 25.3 49.9 10.8 16.0 27.3 53.9 11.0 14.9 24.4 48.9 10.7 15.1 25.2 49.4 10.6 15.9 26.8 52.62 11.0 16.2 27.5 54.8 10.9 16.0 27.3 54.5 10.8 16.5 27.8 53.2 10.7 16.2 26.9 55.2 11.0 15.9 27.8 55.5 10.7 16.0 26.7 53.43 10.8 16.0 28.1 52.7 10.9 16.2 27.6 55.5 10.7 16.6 27.3 54.0 10.8 16.2 27.9 53.0 10.8 16.1 27.4 61.7 10.9 16.0 26.9 55.5Mean 10.87 15.80 26.62 52.27 10.83 15.72 26.77 54.42 10.75 16.17 27.13 53.77__________________________________________________________________________ TABLE 28__________________________________________________________________________PT Calibrator: reconstituted stability of 8 hoursIncubation Time0 hours 4 hours 8 hoursDilution of plasma Dilution of plasma Dilution of plasmaSample Neat 1 in 2 1 in 4 1 in 8 Neat 1 in 2 1 in 4 1 in 8 Neat 1 in 2 1 in 4 1 in 8__________________________________________________________________________1 10.90 15.90 26.80 54.80 11.30 17.30 28.80 56.10 10.90 15.90 27.20 56.90 10.70 16.00 27.30 56.00 11.20 16.50 28.60 55.90 10.80 15.90 27.50 52.302 10.90 15.90 28.10 54.20 11.30 16.10 28.70 NCD 11.10 16.10 27.80 54.40 10.70 15.80 27.80 56.10 10.90 17.30 27.80 56.80 10.90 16.50 26.60 55.403 10.70 16.60 26.60 NCD 11.10 16.60 28.60 54.50 11.00 16.10 27.20 55.60 10.50 16.00 27.50 53.50 10.90 16.50 27.40 55.10 10.80 15.70 28.80 52.90Mean 10.73 16.03 27.35 54.92 11.12 16.72 28.32 55.68 10.92 16.03 27.52 54.58__________________________________________________________________________ RT = Room Temperature = 24.degree. C. NCD = No Clot Detected

EXAMPLE X

Further stability testing was performed on Lot P4. The failure criterion was defined as a change of 10% in the Clotting Time (in seconds) as compared with the mean baseline value.

The accelerated stability calculation with the Arrhenius method was calculated with the SigmaPlot program as follows:

1. For each temperature plot decimal log of concentration (in this case--Clotting Time in seconds) (Y axis) against the time (in this case--days) (X axis) (Table 29).

2. For each temperature (graph) calculate the regression equation Y=m X+b.

3. Define a percent change at which the product is no longer acceptable, (in this case--+10% of Clotting time; mean baseline+10%=10.69+10%=11.76 seconds), convert the value of the zero time analyses to decimal log concentration (in this case--Log of Clotting Time(s)=log of 11.76-1.070).

4. Using the regression equations for each temperature, substitute the decimal log and calculate the day failure.

5. Plot decimal log days from section 4, against 1/absolute temperature (Table 30).

6. Calculate the regression equation Y=m X+b, for the graph in section 5.

7. Using the regression equation from section 6, calculate the expected shelf life at 4.degree. C.

Table 31 shows baseline date which demonstrates the reproducibility between different vials of Lot P4. Tables 32 and 33 show the results of testing of controls during the stability testing. Table 34 shows that the stability of Lot P4 failed after 45 days at room temperature (25.degree. C.). Table 35 shows the stability of Lot P4 at 30.degree. C.; Table 36 shows the stability of Lot P4 at 37.degree. C.; and Table 37 shows the stability of Lot P4 at 50.degree. C.

TABLE 29______________________________________Calculation of failure day for each temperature______________________________________ Y axis Decimal Clotting Log of X axis time CT FailureTemperature Days (secs.) (secs.) Statistics Day______________________________________at 25.degree. C. 0 10.69 1.029(Room temp.) 5 11.10 1.045 11 11.28 1.052 15 11.38 1.056 20 11.20 1.049 32 11.62 1.065 37 11.52 1.061 r = 0.933 45 11.73 1.069 I = 1.038 56 12.00 1.079 s = 44.4 0.00072 daysat 30.degree. C. 0 10.69 1.029 3 11.13 1.046 4 11.47 1.060 7 11.65 1.066 8 11.63 1.066 9 11.73 1.069 10 11.73 1.069 14 12.05 1.081 16 12.15 1.085 r = 0.96956 18 12.33 1.091 I = 1.039 20 12.60 1.100 s = 10.2 0.00304 daysat 37.degree. C. 0 10.69 1.029 1 11.30 1.053 2 11.45 1.059 3 11.62 1.065 4 11.68 1.067 r = 0.96541 5 12.08 1.082 I = 1.037 6 12.32 1.091 s = 0.009 3.67 daysat 50.degree. C. 0 10.69 1.029 0.083 11.12 1.046 (2 hrs) 0.167 11.32 1.054 r = (4 hrs) 0.97223 0.25 11.45 1.059 I = 1.033 (6 hrs) 0.333 11.67 1.067 s = 0.346 (8 hrs) 0.1068 day______________________________________Formula Y = 1.070Y = mX + b m = slope (s)X = Y - b/m b = Intercept (I)______________________________________ TABLE 30______________________________________Calculation of shelf life stability at 4.degree. C.______________________________________ Y axisX-axis logTemperature 1/temperature failure day failure day Statistics______________________________________25.degree. C. 0.04 44.4 1.6530.degree. C. 0.033 10.2 1.0086 r = 0.95337.degree. C. 0.027 3.67 0.56 I = 0.704650.degree. C. 0.02 0.346 -0.46 s = 18.875 (8.1 hours)______________________________________Formula X = 0.25 (1.4.degree. C.)Y = mX + b m = Slope (s) = 18.875 b = Intercept (I) = -0.7046______________________________________Y = 18.875 .times. 0.25 + (-0.7046) = 4.014= > inv. log of 4.014 = 10327 days stable>= > 28.3 years28 years - 33% = 18 years______________________________________

In conclusion, Lot P4 is stable 18 years at 4.degree. C.

TABLE 31______________________________________Stability testing - Baseline DataAssay: Prothrombin TimeReagents: INNOVIN PT Calibrator lot PILOT LOT 1 (P4) INNOVIN reagent lot TFS - 12 Saline 0.9% Lot H1 - 86Machine: MLA E1000C Software Version 5.00E P46 Clotting Time (Secs.) Neat 1 in 2 1 in 4 1 in 8______________________________________ Refer- enceVials Tested1 FIG. 14 10.4 10.6 15.1 15.1 26.0 26.5 54.1 51.2 NB CO82 P842 10.6 10.7 15.5 15.3 26.8 26.1 51.1 50.83 10.5 10.7 15.8 15.5 27.1 26.8 51.1 52.14 Table 10.6 10.4 15.4 15.4 26.8 26.2 53.0 51.6 32 NB Co82 P 845 10.6 10.5 15.5 15.4 26.1 26.1 52.3 52.66 10.6 10.5 15.4 15.4 26.3 25.8 53.0 51.87 Table 10.9 11.0 15.3 14.9 24.9 24.9 49.0 48.9 45 NB Co95 p28 11.0 10.7 16.2 16.2 27.5 26.9 54.8 55.29 10.8 10.8 16.0 16.2 28.1 27.9 52.7 53.010 Table 10.9 10.7 15.9 16.0 26.8 27.3 54.8 56.0 46 NB CO95 p311 10.9 10.7 15.9 15.8 28.1 27.8 54.2 56.1______________________________________Mean 10.69 15.60 26.67 52.70SD 0.178 0.382 0.902 1.996CV 1.666 2.447 3.384 3.787Mean + 10% 11.76 17.16 29.34 57.97Mean + 10% 9.62 14.04 24.00 47.43______________________________________ TABLE 32______________________________________Stability testing - ControlsCoag Cal NLot No: 540.053Innovin .TM. PT % 85Assay: Prothrombin TimeReagents: INNOVIN .TM. lot TFS-12 Saline 0.9% lot H1-86 H1-87Machine: MLA E1000C Software version 5.00 E P 46______________________________________ Clotting Time (Seconds)CoagCalN Neat 1 in 2 1 in 4 1 in 8______________________________________Sample 1 12.1 17.5 31.2 61.7 12.1 17.5 29.5 56.1Sample 2 12.2 17.6 29.9 57.7 12.0 17.9 30.0 58.6Sample 3 12.1 17.6 30.0 58.4 12.0 17.7 30.1 57.6Mean 12.08 17.63 30.12 58.35PT % 85 42.5 21.25 10.625______________________________________ Clotting TimeDate Control (seconds) Mean PT %______________________________________30.11.93 CTN 12.9 12.8 12.85 75.6 CTP 21.0 20.8 20.90 36.0 6.12.93 CTN 12.7 12.5 12.60 78.3 CTP 20.7 21.5 21.10 35.517.12.93 CTN 12.9 12.7 12.80 76.1 CTP 20.9 20.3 20.60 36.720.12.93 CTN 13.1 12.9 13.00 74.1 CTP 21.3 21.2 21.25 35.221.12.93 CTN 12.6 12.7 12.65 77.7 CTP 20.5 20.4 20.40 37.222.12.93 CTN 12.4 12.4 12.40 80.6 CTP 20.3 20.4 20.35 37.323.12.93 CTN 12.6 12.4 12.35 81.2 CTP 21.0 20.6 20.60 36.727.12.93 CTN 12.6 12.5 12.50 79.4 CTP 21.1 20.8 20.80 36.228.12.93 CTN 12.6 12.5 12.50 79.4 CTP 20.8 20.5 20.50 37.029.12.93 CTN 12.5 12.5 12.50 79.4 CTP 20.9 20.9 20.90 36.0______________________________________Controls Lot No. Assigned Value______________________________________CoagTrol N CTN 537.001 73-99%CoagTrol P CTP 541.034 29-39%______________________________________ TABLE 33______________________________________Stability testing - ControlsCoagCal NLot No: 540.053Innovin .TM. PT % 85Assay: Prothrombin TimeReagents: INNOVIN .TM. lot TFS-12 Saline 0.9% lot H1-86 H1-87Machine: MLA E100C Software version 5.00 E P 46______________________________________ Clotting Time (Seconds)CoagCalN Neat 1 in 2 1 in 4 1 in 8______________________________________Sample 1 12.1 17.5 31.2 61.7 12.1 17.5 29.5 56.1Sample 2 12.2 17.6 29.9 57.7 12.0 17.9 30.0 58.6Sample 3 12.1 17.6 30.0 58.4 12.0 17.7 30.1 57.6Mean 12.08 17.63 30.12 58.35PT % 85 42.5 21.25 10.625______________________________________ ClottingDate Control Time(s) Mean PT %______________________________________30.12 CTN 12.5 12.3 12.40 80.6 CTP 20.4 21.1 20.75 36.43.01.94 CTN 12.6 12.6 12.60 78.3 CTP 21.3 22.3 21.80 34.05.01.94 CTN 12.6 12.5 12.55 78.8 CTP 20.6 20.1 20.35 37.36.01.94 CTN 12.7 12.5 12.60 78.3 CTP 20.7 21.1 20.90 36.006.01.94 CTN 12.5 12.5 12.50 79.4MLA 900 CTP 20.0 20.1 20.05 38.07.01.94 CTN 12.7 12.5 12.60 78.3 CTP 21.0 20.9 20.95 36.010.01.94 CTN 12.6 12.6 12.60 78.3 CTP 20.9 20.8 20.85 36.0______________________________________Controls Lot No. Assigned Value______________________________________CoagTrol N CTN 537.001 73-99%CoagTrol P CTP 541.034 29-39%______________________________________ TABLE 34______________________________________Accelerated StabilityAssay: Prothrombin TimeReagents: INNOVIN .TM. PT Calibrator lot PILOT LOT 1 INNOVIN .TM. lot TFS-12 Saline 0.9% lot H1-86 H1-87 MLA E1000C Software version 5.00E P46TEMPERATURE INCUBATED: 20.degree. C.______________________________________Mean baseline clotting time (secs) 10.69 PT % 100______________________________________Clotting Time of neat plasma (seconds) % ChangeNumber from PTof Day Vial 1 Vial 2 Vial 3 Mean Mean %______________________________________ 5 11.2 11.3 11.3 11.10 3.84 92.8 10.9 10.9 11.011 11.4 11.5 11.3 11.28 5.52 90.3 11.3 11.1 11.115 11.5 11.6 11.5 11.38 6.45 88.9 11.3 11.2 11.220 11.3 11.2 -- 11.20 4.77 91.4 11.2 11.1 --32 11.5 11.9 11.7 11.62 8.70 85.8 11.5 11.5 11.637 11.7 11.7 11.6 11.52 7.76 87.1 11.4 11.4 11.345 11.9 11.8 11.9 11.73 9.73 84.5 11.7 11.5 11.656 12.1 -- -- 12.00 12.25 81.3 11.9 -- --______________________________________Failed stability after (days) 45______________________________________ TABLE 35______________________________________Accelerated StabilityAssay: Prothrombin TimeReagents: INNOVIN .TM. PT Calibrator lot PILOT LOT 1 INNOVIN .TM. lot TFS-12 Saline 0.9% lot H1-86 H1-87 MLA E1000C Software version 5.00E P46TEMPERATURE INCUBATED: 30.degree. C.______________________________________Mean baseline clotting time (secs) 10.69 PT % 100______________________________________Clotting Time of neat plasma (seconds) % ChangeNumber from PTof Day Vial 1 Vial 2 Vial 3 Mean Mean %______________________________________ 3 11.3 11.3 11.3 11.13 4.12 92.4 10.9 11.0 11.0 4 11.7 11.6 11.5 11.47 7.30 87.7 11.5 11.2 11.3 7 11.8 11.9 11.7 11.65 8.98 85.4 11.5 11.5 11.5 8 11.7 11.7 11.7 11.63 8.79 85.7 11.7 11.5 11.5 9 11.9 11.9 11.8 11.73 9.73 84.5 11.8 11.6 11.410 11.9 11.9 11.8 11.73 9.73 84.5 11.6 11.8 11.414 12.1 12.2 12.1 12.05 12.72 80.8 12.0 12.0 11.915 12.6 12.8 -- 12.65 18.33 74.6 12.5 12.7 --16 12.2 12.2 12.4 12.15 13.66 79.7 12.0 12.1 12.018 12.5 12.4 12.3 12.33 15.34 77.8 12.3 12.3 12.220 12.6 12.8 -- 12.60 17.87 75.1 12.5 12.5 --______________________________________Failed stability after (days) 10______________________________________ TABLE 36______________________________________Accelerated StabilityAssay: Prothrombin TimeReagents: INNOVIN .TM. PT Calibrator lot PILOT LOT 1 INNOVIN .TM. lot TFS-12 Saline 0.9% lot H1-86 H1-87 MLA E1000C Software version 5.00E P46TEMPERATURE INCUBATED: 37.degree. C.______________________________________Mean baseline clotting time (secs) 10.69 PT % 100______________________________________Clotting Time of neat plasma (seconds) % ChangeNumber from PTof Day Vial 1 Vial 2 Vial 3 Mean Mean %______________________________________1 11.4 11.5 11.3 11.30 5.70 90.0 11.2 11.2 11.22 11.6 11.6 11.6 11.45 7.11 88.0 11.3 11.3 11.33 11.6 11.8 11.7 11.62 8.70 85.8 11.6 11.5 11.54 11.8 11.9 11.9 11.68 9.26 85.1 11.7 11.7 11.65 12.1 12.3 12.2 12.08 13.00 80.4 12.0 12.0 11.96 12.6 12.3 12.5 12.32 15.25 77.9 12.2 12.0 12.3______________________________________Failed stability after (days) 4______________________________________ TABLE 37______________________________________Accelerated StabilityAssay: Prothrombin TimeReagents: INNOVIN .TM. PT Calibrator lot PILOT LOT 1 INNOVIN .TM. lot TFS-12 Saline 0.9% lot H1-86 H1-87 MLA E1000C Software version 5.00E P46TEMPERATURE INCUBATED: 50.degree. C.______________________________________Mean baseline clotting time (secs) 10.69 PT % 100______________________________________Clotting Time of neat plasma (seconds) % ChangeNumber from PTof Day Vial 1 Vial 2 Vial 3 Mean Mean %______________________________________2 11.3 11.2 11.3 11.12 4.02 92.6 11.1 11.0 10.94 11.5 11.6 11.4 11.32 5.89 89.7 11.2 11.1 11.16 11.6 11.6 11.7 11.45 7.11 88.0 11.2 11.2 11.48 11.7 11.7 11.8 11.67 9.17 85.2 11.6 11.5 11.7______________________________________Failed stability after (hours) 8______________________________________

Claims

1. A composition for calibration for a prothrombin time assay comprising:

a) normal pool plasma selected from the group consisting of citrated plasma and citrate based anticoagulated plasma;

b) a quantity of a coagulation factor selected from the group consisting of rFVII, rFVIIa, sufficiently purified FVII, and sufficiently purified FVIIa, which when added to said plasma, is sufficient to increase the % PT value of the plasma to about 100% after lyophilization of said normal pool plasma and added coagulation factor.

2. The composition of claim 1 for use with a PT reagent.

3. The composition of claim 1 for use with a recombinant tissue factor reagent.

4. The composition of claim 3 wherein the recombinant tissue factor PT reagent is selected from the group consisting of INNOVIN.TM. PT reagent and Ortho.RTM. RECOMBOPLASTIN.TM. PT reagent.

5. A method of preparing a composition for calibration for use in the prothrombin time assay comprising the steps of:

a) collecting a normal pool of plasma selected from the group consisting of citrated plasma and citrate based anticoagulant plasma;

b) adding a quantity of a coagulation factor selected from the group consisting of rFVII, rFVIIa, sufficiently purified FVII, and sufficiently purified FVIIa to said plasma, which is sufficient to increase the % PT of said plasma and added coagulation factor to about 100% PT after lyophilization.

6. The method of claim 5 wherein the composition is used with INNOVIN.TM. PT reagent.

7. The method of claim 5 wherein the calibrator is used with a recombinant tissue factor PT reagent.

8. The method of claim 7 wherein the recombinant PT reagent is selected from the group consisting of INNOVIN.TM. PT reagent and Ortho.RTM. RECOMBOPLASTIN.TM. reagent.

9. A composition for calibration for a coagulation factor assay comprising:

a) normal pool plasma selected from the group consisting of citrated plasma and citrate based anticoagulant plasma;

b) a quantity of coagulation factor selected from the group consisting of rFVII, rFVIIa, sufficiently purified FVII, and sufficiently purified FVIIa which when added to said plasma, is sufficient to increase the percentage of said coagulation factor of the plasma to about 100% after lyophilization of said normal pool plasma and added coagulation factor.

10. A method of preparing a composition for calibration for use in a coagulation factor assay comprising the steps of:

a) collecting a normal pool plasma selected from the group consisting of citrated plasma and citrate based anticoagulant plasma;

b) adding a quantity of coagulation factor selected from the group consisting of rFVII, rFVIIa, sufficiently purified FVII, and sufficiently purified FVIIa which when added to said plasma, is sufficient to increase the percentage of said coagulation factor of the plasma to about 100% after lyophilization of said normal pool plasma and added coagulation factor.