expert comm1'l-l'~ on biological standardization …

DISTR. : LIMITED

DISTR. : LJMITEE

B8/96.1846 Rev~ 1

WORLD HEALTH ORGANIZATION

ORGANISATION MONDIALE DE LA SANTE

ENGL- I:SH "~NL Y

EXPERT COMM1'l-l'~ ON BIOLOGICAL STANDARDIZATIONGeneva, 7 to n October 1996

INTERNATIONAL COLLABORATIVE STUDY FOR THE CALIBRATION OF APROPOSED REFERENCE PREPARATION FOR THROMBOPLASTIN,. -:~HUMAN RECOMBINANT, PLAIN ." .

Armando Tripodi

Angelo Bianchi Bonomi Haemophilia and Thrombosis Centre,Institute of Internal Medicine, University and IRCCS

Maggiore Hospital, Milano, Italy.

SUMMARY

A Preparation of human recombinant thromboplastin, provisionally coded X/95, is proposed as acandidate to the 3rd International Reference Preparation (IRP) for thromboplastin (human, plain),meant to replace the 2nd IRP coded BCT/253 (human, plain) whose stocks are now exhausted.The preparation was calibrated in an international collaborative study organized and carried outunder the auspices of the Scientific and Standardization Committee (SSC) of the InternationalSociety on Thrombosis and Haemostasis (lSTH). The study involved 20 laboratories from 13countries and the calibration was performed against the three existing IRPs (i.e., BCT/253,RBT /90 and OBT /79). An additional candidate recombinant thromboplastin coded as Y /95 wasalso included in the study. On the basis of predetermined criteria which included (i) the within-laboratory precision of calibration; (ii) the between-laboratory precision of the calibration and(iii) the confonnity to the calibration model, the SSC of the ISTH deliberated unanimously that

X/95 was to be preferred as the candidate IRP.Nineteen out 20 participants eventually submitted data for statistical analysis. Within-

laboratory precision of the calibration of X/95, expressed as the Coefficient of Variation (CV)for the estimation of the slope was below 3% in the vast majority of the cases (88%); between3% and 4% in 10% of cases and above 4% in one case. The mean International Sensitivity Index(lSI) value of the candidate X/95 calibrated against the three existing IRPs was 0.940 and the

./.. .

Ce document n'est pas destine a etre distribue au grand public et tausles droits V afferents sont reserves par l'Organisation mondiale de laSante (OMS). line peut etre commente, resume, cite, reproduit outraduit, partiellement ou en totalite, sans une autorisation prealableecrite de I'OMS. Aucune partie ne doit etre chargee dans un svsteme derecherche documentaire ou diffusee sous quelque forme ou par quelquemaven que ce soit .electronique, mecanique, ou autre. sans une

,---:-- __'_'~L'~ ,~_:.~ -'~ ""aa"

This document is not issued to the general public, and all rightsare reserved by the World Health Organization (WHO). Thedocument may not be reviewed, abstracted, quoted, reproduced ortranslated, in part or in whole, without the prior written permissionof WHO. No part of this document may be stored in a retrievalsystem or transmitted in any form or by any means -electronic,mechanical Dr Dther -without the prior written permission of'N~n I

BS/96.1846 Rev. 1

interlaboratory agreement was good (between-laboratory CV of the ISI was 4.7%). Accelerateddegradation studies performed in three different laboratories indicate adequate long term stability(prothrombin time ratio did not change significantly after exposure of X/95 at temperature ashigh as 440 C for 5 months). The homogeneity of the preparation X/95 and the precision of theprothrombin time measurement were satisfactory (inter-vial variation, expressed as the CV of theprothrombin time measurement was 1.32% and 1.12% for normal and coumarin plasma).

Overall, the preparation coded X/95 proved to be a suitable candidate to serve as the.replacement to the current 2nd IRP coded BCT /253.

INTRODUCTION

According to the recommendation issued by the World Health Organization (WHO),-working thromboplastins used in the prothrombin time (PT) test for the laboratory control of oralanticoagulant treatment must be calibrated against International Reference Preparations ~) todetermine the International Sensitivity Index (lSI) necessary to convert PT results intoInternational Normalized Ratio (INR) (1). The observation that the calibration of a given"thromboplastin is in general more precise when it is performed against an IRP of similarcomposition and from the same species, supports the recommendation made by the WHO ExpertCommittee on Biological Standardization (ECBS) that like vs. like calibration should beperformed and is one of the reasons to maintain IRPs from different species (1). Another reasonto maintain more than one IRP is that it permits to assess periodically the stability of the IRPs(2). The fIrst IRP named 67/40, was a human brain extract to which it was added adsorbedbovine plasma (combined reagent). In 1984, 67/40 was replaced by BCT/253, a human brainextract (plain reagent). Presently, there are two additionallRPs from different species availablefrom WHO: OBT/79 (combined, bovine brain) and RBT/90 (plain, rabbit brain). Each of thethree IRPs is characterized by a value of the ISI, being the slope of relationship with the primaryIRP 67/40. The stocks of the IRP for thromboplastin human, plain, BCT/253 are now practicallyexhausted. The present report deals with the results of an international collaborative studyorganized under the auspices of the Scientific and Standardization Committee (SSC) of theInternational Society on Thrombosis and Haemostasis (lSTH) for the calibration of thereplacement candidate.

DESIGN OF THE COLLABORATIVE STUDY

The candidates (X/95 and Y/95) and the current IRPs RBT/90, BCT/253, OBT/79 weretested in each laboratory by the same expert operator with the manual (tilt tube) technique. Testplasmas were freshly prepared from healthy subjects and patients stabilized on long termanticoagulant therapy. Participants were instructed to select patient plasmas with PTcorresponding to an interval of INR from 1.5 to 4.5. Also included in the series of measurementswere three lyophilized plasmas. To account for the effect of inter-day variation, PTmeasurements were performed in each laboratory on 10 different days (not necessarily consecu-tive). Participants were instructed to include on each day plasmas from 2 healthy individuals and6 anticoagulated patients. Healthy individuals and patients had to be different on each workingday. To minimize the effect of plasma instability on the relationship between the

BS/96 .1846 Rev~-:'l

"""'

MATERIAL PRO VIDFJ!

1.

2.

3.4.

5.6.

Study protocol with detailed instructions on how to collect and store fresh plasmas, toreconstitute lyophilized plasmas and thromboplastins and to do actual testing.Lyophilized plasmas (coded A, B and C); IRPs BCT/253, RBT/90 and OBT/79; candidatereplacement thromboplastins. .Vacuum tubes for blood collection containing 0.105 M Sodium citrate. ~-Appropriate diluents to reconstitute X/95 (Calcium Chloride solution attached to- thecandidate), BCT/253 (phenolized water attached to the IRP) and OBT/79 (Calc~chloride 3.2 mM). cSterile redistilled water to reconstitute RBT/90, Y/95 and lyophilized plasmas.Sterile 25 mM Calcium Chloride to recalcify plasma/thromboplastin mixtures for RBT /90and BCT/253.

STATISTICAL METHODS

The statistical methods were those employed for the calibration of previous IRPs (2, 3,4). In particular, orthogonal regression was used to estimate the slope of the relationship betweenthe log-transformed PTs obtained with the candidates and the IRPs. Values exceeding the interval1.5-4.5 INR as measured with the IRPs were excluded and outliers were rejected if theirorthogonal distance was more than 3 times the Standard Deviation (SD) from the orthogonalregression line (calculated with all points included).

The ISI value was calculated as the product of the slope of the orthogonal regression lineand the ISI value of the IRP. ISI values within each route of calibration were calculated as themean of the separate regression lines calculated for the different laboratories. The fInal ISI valueassigned to the candidates was the average value of all ISIs obtained with the three differentroutes of calibration after exclusion of outliers. These were detected within each route ofcalibration by the algorit.l}m described previously (5) and used for the calibration of RBT/90 (2).

INR values for fresh patients' samples were calculated by dividing PT from patients bythe geometric mean PT of normals and raising this ratio to the ISI value of the reagent (1, 6).

RESULTS

Nineteen out of 20 laboratories eventually provided results. The results reported in thissection are thosF obtained with the candidate X/95. Results of the calibration of Y /95 and theircomparison vs. 1X/95 are reported in Appendix IV and V.

I

BS/96.1846 Rev. 1

The geometric means of the PTs from fresh normal plasmas are shown in Table 1. The ."

values obtained with the IRPs are in close agreement with those obtained at the time when theywere calibrated against their predecessors (2, 3, 4). Slopes and their coefficients of variation(CV) for calibration of X/95 vs. different IRPs are shown in Table 2. The CV values whichestimate the within-laboratory precision of the calibration were within acceptable limits ~-3 %)(7) in the vast majority of laboratories. Laboratory 13 scored an usually high CV value (7.9%)in the calibration vs. OBT/79. That laboratory also scored high CV values for the lyophilized- ~

plasmas included in the study (not shown), suggesting that the operator might have had problems:'in dealing with OBT/79. It was, therefore, decided to exclude the ISI value obtained in -, '

laboratory 13 with OBT/79 from the fmal calculation. The ISI values obtained with calibration ofX/95 against the three IRPs are shown in Table 3. There were two outliers: one in thecalibration vs. BCT/253 (laboratory 14) and one in the calibration vs. OBT/79 (laboratory)).The between-laboratory CV values ranged from 5.0 % to 6.3 % (including the outliers) or from3.8% to 5.0% (excluding the outliers). The mean ISI value obtained vs. BCT/253 (i.e., 0.926after exclusion of the outliers) was 1.7% apart from that obtained in the calibration vs. RBT/90(i.e., 0.942) and 3.0% apart from that obtained vs. OBT/79 (i.e., 0.954). The fmal mean ISIvalue, i.e., the average value of the 54 calibrations generated in this study and retained fo~:calculation after exclusion of the outliers, was 0.940 with a Standard Error of 0.006. T~ple 4shows the geometric mean values of the INRs calculated for fresh patients' plasmas by u~ing themean ISI of all laboratories. They were in close agreement and ranged from 2.67 with BCT/253to 2.72 for OBT/79.

DISCUSSION

The study included all three existing IRPs. Following a previous deliberation of the SSCof the ISTH it was decided that the ISI of the candidate replacement be the average value ofthree calibrations against the three existing IRPs (8). This was deemed necessary in order tominimize small differences that have been observed between the three routes of calibration (2).This strategy that had already been implemented for the calibration of RBT /90 proved to befu1itful because the ISI values for X/95 calculated in the present study vs. BCT/253 and RBT/90are now very close each other (i.e., 1.7% difference), whereas the ISI values calculated vs.BCT/253 and OBT/79 are 3% apart. This difference, which is however negligible, might bereduced when OBT/79 will be replaced following the proposed calibration procedure. It is,therefore, reasonable to assume that the proposed scheme of calibration of new IRPs willconsiderably reduce the bias between the three routes of calibration. On the other hand, theproposed scheme does not substantially change the original scheme established by WHO. Therecommendation of like-vs.-like calibration will still remain in place for commercial reagents,thus reducing the variability of the ISI value, particularly when small number of laboratoriesperform the calibration. The rule of like-vs.-like calibration will be broken only for calibration ofthe IRPs. In this case the risk of increasing the interlaboratory standard deviation of the ISI maybe kept at bay by increasing the number of laboratories (at least twenty) and by well plannedstudy designs. As demonstrated in the present as well as in previous calibration studies (2, 4,9), the between-laboratory CV value of the ISI can be less than 5% even with "unlike" calibra-

tions .

BS/96.1846 Rev~:.l

"'"'

-,.,Potential candidates to replace BCT /253 were human brain, human placenta and humanrecombinant relipidated tissue factor. To avoid any risk of viral transmitted diseases the SSC ofthe ISTH after consultation with WHO decided to restrict the choice among available humanrecombinant preparations that previous studies have shown to be equivalent to the extractivepreparations (7, 10). Additional requirements were that the material should have to be providedin glass sealed ampoules and a suitable number of them (at least 10,000) donated to WHO afterthe evaluation was completed and the fmal choice of the most suitable candidate was made. Twocandidates that met the above requirements, were eventually submitted for evaluation. They werecoded as X/95 and Y /95 by people neither involved in the calibration exercise nor in thestatistical analysis and calibrated in an international collaborative study. Twenty laboratoriesfrom Europe, North America, South America and Australia were invited to participa~ in thestudy. The vast majority of them had already participated in the collaborative exercise tocalibrate RBT/90 (2) and had experience with the manual (tilt tube) technique for PT testing. Allexcept one laboratory eventually provided results.

The criteria used to judge the calibration of the two candidates were, the within,. andbetween-laboratory precision of the calibration and the conformity to the model of calibration.They were derived from the procedure recommended by WHO (7) and the literature thatappeared on this topic over the last 15 years (2-4, 7). The within-laboratory precision ofcalibration was better for X/95 than for Y /95 because the percentage of laboratories whichscored a CV value below the recommended 3% (7) were 88% vs. 65%. Also the between-laboratory precision was better for X/95 than for Y /95 with CV values of 4.7 % vs. 7.9 % .Finally, X/95 was more adequate to fulfill the requirements of the calibration model. Theserequire that in a given calibration the overall regression line describes patient and normal datapoints adequately. According to Tomenson (11) this can be achieved by testing the assumptionthat the mean log PT of the normals lies on the orthogonal regression line drawn through patientdata points. We tested this assumption in all calibration plots generated with the two candidatesand found that X/95 deviated from the assumption in 9% of the case, whereas Y/95 in 82%.

On the basis of these results the SSC of the ISTH during its Annual Meeting (Barcelona,Spain 22-24 June, 1996) deliberated unanimously that X/95 was the preferred candidate IRP tobe submitted to the ECBS of the WHO (12).

The material is presently stored at -20 °C under the care of the manufacturer (OrthoDiagnostic Systems, Raritan N.J.) who accepted to offer free of charge at least 10,000 ampoules(plus an equivalent number of ampoules containing the diluent for reconstitution) to WHO. Assoon as the material will be established as the 3rd IRP for thromboplastin, human, plain,ampoules will be labeled by the manufacturer according to the instructions of WHO (suggestedcode rTF/95) and transferred to the custodian laboratory (Central Laboratory, Netherlands RedCross Blood Transfusion Service, Plesmanlaan 125, Amsterdam, The Netherlands).

BS/96.1846 Rev. 1

REFERENCE~ ~.,;:;

1

2.

3. Hermans J, Ivan den Besselaar AMHP, Loeliger EA, van der Velde EA. A collaborativecalibration $tudy of reference materials for thromboplastins. Thromb Haemost 1983; 50:712-7.

4. Thomson 1M, Tomenson lA, Poller L. The calibration of the second primaryinternational reference preparation for thromboplastin (thromboplastin, human, plain;coded BCT/253). Thromb Haemost 1984; 52: 336-42. -

5. Reijnierse GLA, van den Besselaar AMHP, Hermans J. een nieuwverwerkingsprogramma van ingezonden uitslagen in bet kader van extemekwailiteisbewaking. Ervaringen in 1988. Tijdschr NVKC 1989; 14: 131-6.

6. Kirkwood ~L. Calibration of reference thromboplastins and standardization of theprothrombin I time ratio. Thromb Haemost 1983; 49: 238-44.

7 World Health Organization. Recommended methodology for using WHO InternationalReference Preparations for thromboplastins. WHO, 1211 Geneva 27, Switzerland.

8.

Tripodi A, Poller L, van den Besselaar AMHP, Mannucci PM. A proposed scheme forcalibration of international reference preparations of thromboplastin for the prothrombintime. Thromb Haemost 1995; 74: 1368-9.

9. Tripodi A, Chantarangkul V, Braga M, Poller L, ten Cate JW, van den Besselaar AMHP,Mannucci PM. Results of a multicenter study assessing the status of standardization of arecombinant thromboplastin for the control of oral anticoagulant therapy. ThrombHaemost 1994; 72: 261-7.

10. Bader R, MaIinucci PM, Tripodi A, Hirsh J, Keller F, Solleder EM, Hawkins P, PengM, Pelzer H, Teijidor LM, Ramirez IF. Kolde HJ. Multicenter evaluation of a new PTreagent based on recombinant human tissue factor and synthetic phospholipids. ThrombHaemostas 1994; 71: 292-9.

11 Tomenson JA. A statistician's independent evaluation. In Thromboplastin calibration.AMHP van den Besselaar, HR Gralnick, SM Lewis (ed.s). Martinus Nijhoff Publishers,Boston 1984: pp 87-108.

12, Scientific and Standardization Committee (SSC) of the International Society onThrombosis and Haemostasis (ISm). Report of Subcommittee on Control ofAnticoagulation. 22-24 June 1996, Barcelona (Spain). !

LAB BCT/253 RBTj90 OBT/79 Xj95

1234567891111111111

14.37916.14714.47114.14614.39714.64814.32114.41314.39714.01514.01913.55813.84714.01714.51815.02113.85413.99112.579

18.89119.96418.09518.74617.40718.53517.59618.38118.44618.02617.54616.62916.87218.82718.90118.74417.72716.68016.619

35.17541.11136.08037.78540.17335.44942.15935.22737.69036.63235.25534.23233.82134.39437.92938.12434.98732.96334.602

12.27916.02113.83013.03414.03712.56614.44112.56313.43712.92213.14312.19811.59412.43713.54313.38313.93512.46211.840

OverallMean 14.249 18.033 36.515 13.140

I ! BS/96.1846 Rev. 1

i ..

0123456789

BS/96.1846 Rev. t

calibra::::Table 2. Slopes and coefficients of variation (CV) fortion of X/95 vs. IRPs.

'".:;"

vs.BCT/253Slope CV

vs.RBT/90Slope CV

vs.OBTj79Slope CVLAB

12345678910111213141516171819

0.8900.8400.8330.8170.8810.8760.9030.7710.8320.8630.8630.8140.9001.0070.8340.8040.8510.8410.949

2.2.1,2.1.1.2,2.2.1.2.

2.2.1.

1.2.1.2.1.

0.8530.9720.9910.9360.9480.9600.9210.8840.9370.9301.0530.8960.9390.9770.9460.9220.9640.8700.990

2.4.I.3.2.2.3.2.3.2.3.2.2.2.2.3.

2.2.2.

0.7881.0250.9770.9320.9400.9720.8900.9520.9050.9710.9390.9230.9340.9690.9450.8790.9750.9260.924

2.2.1.2.1.1.1.2.2.1.2.1.7.1.1.3.2.3.

1.

.3

.8.2.2

.9

.8

.3

.7

.6

.5

,5

.2.1

,9

8.8

,5

,5

,5

.8

,0.8.3

.4

,1.3

.8

.4

,2.5

.7

.7

,03

.5

,2

85

.4

.8

.3

.4

.9

.5

.8~2

.2

.6

.6

.7

.9

.7

.7

,0.2

.0

.7

BS/96.1846 Rev

1

LAB VS.BCT/253 vs.RBT/90 VS.OBT/79

~3i4~

~89101112131415~6Ii

0.9660.9120.9040.8860.9560.9510.9800.8370.9020.9360.9360.8840.9761.093 *0.9050.8720.9230.9121.030

0.8530.9720.9910.9360.9480.9600.9210.8840.9370.9301.0530.8960.9390.9770.9460.9220.9640.8700.990

0.797 *1.0360.9880.9430.9510.9830.9000.9630.9150.9820.9490.9340.944 $0.980 .0.9550.8890.9860.9360.934

MeanNCV

0.935196.3

0.942195.0

0.946195.3

MeanNCV

0.926184.9

0.954173.8

Overall MeanN

sqSECV

0.940540.04420.0064.7

12345678910111213141516171819 T

able 4.

Means of

the patients'

SD

LAB

BS

/96.1846 R

ev. 1

0.28

3.372.522.562.662.572.692.882.342.642.792.322.612.543.222.792.252.682.482.75

2.67

2.942.752.792.782.592.742.662.432.722.862.562.622.432.752.872.412.902.402.62

0.17

2.67

2.823.002.822.792.552.832.652.732.683.042.412.772.292.822.972.372.972.672.54

0.22

2.72

INR

values

3.222.672.702.772.542.712.772.582.742.832.312.792.442.712.802.452.772.582.53

X/95

0.19

2.68

Page

10BC

T/253

ISI=

1.085R

BT

/90I8I=

1.00O

BT

/79IS

I=1.011

ISI=

O.940

BS/96.1846 Rev. 1" "'"'

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Appendix I

Participants (in alphabetical order) of the international collaborative study for the calibrationof a proposed reference preparation for thromboplastin, human, recombinant, plain~

M. Borrell Hospital de la Santa Creu y Sant Pan, Servei d , Hematologia, Barcelona, Spain

A. Bradshaw/R. Manning Department of Haematology, Royal Postgraduate Med. School,Hammersmith Hospital, London, UK

J. Conard Hotel Dieu, Laboratoire Central d'Hematologie, Paris, France

F. Crespo/M. T. Gago Hospital de Santa Cruz, Servico de Patologia Clinica, Carnaxide,Portugal

T. Exner Gradipore Ltd., North Ryde, Australia

C.R. Falcon Departamento de Hematologia, Fundacion Favaloro, Buenos Aires, Argentina

J. Jespersen Ribe County Hospital, Dept. of Clinical Chemistry, Esbjerg, Denmark

M. Johnston Hamilton Civic Hospitals Research Centre, Henderson General Division,Hamilton, Ontario, Canada

S. Kitchen University Dept. of Haematology, Royal Hallamshire Hospital, Sheffield, UK

G. Larson/B. Christenson Dept. of Clinical Chemistry and Transfusion Medicine, Sahlgren' sUniversity Hospital, Goteborg, Sweden

C. Manotti V Divisione Medica, Centro Emostasi, Ospedali Riuniti, Pam1a, Italy

F. Martinez-Brotons Seccion de Hemostasia, Hospital "Princeps d'Espanya", Bracelona,Spain

G. Palareti Servizio di Angiologia e Malattie della Coagulazione, Ospedale S. Orsola,

Bologna, Italy

P.M. Sandset Haematological Research Laboratory, Medical Clinic, Ullevaal Hospital,University of Oslo, Oslo, Norway

BS/95.1846 Rev. 1

'"'.",.

y. Stirling/D.l. Howarth MRC Epidemiology and Medical Care Unit, The Medical Collegeof St. Bartholomew's Hospital, London, UK

D. A. Tabemer Thrombosis Reference Centre, Withington Hospital, Manchester, U.K,

A. Tripodi (Study coordinator) Haemophilia and Thrombosis Centre, Institute of IntetnalMedicine, University and IRCCS Maggiore Hospital, Milano, Italy .ck

Research Centre, Unive~ityA.M.H.P. van den Besselaar Haemostasis & ThrombosisHospital, Leiden, The Netherlands

BS/96.1846 Rev; 1

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ADDendix .II

Aim of the studx

Exoerirnental Drocedu~

Eighteen ampoules of X/95 were reconstituted according to the recommended procedure. Eachampoule was then used to measure the prothrombin time for a normal pooled plasma and a coumarinpooled plasma. The remaining content of each ampoule was then pooled and used for a second seriesof measurements on the same two plasmas. Clotting times were recorded by means of a photoopticalinstrument, ACL 300, IL, Milano, Italy.

Result~

Prothrombin times and CV values are reported in Table 1. The between-ampoules variation was1.32 % for the normal plasma and 1.12 % for the coumarin plasma, whereas the correspondent CVvalues obtained when the ampoules were pooled were 0.86% and 0.88%. It can be concluded thatthe homogeneity and precision of PT measured with X/95 is satisfactory.

BS/9S.1846 Rev. 1

Table 1. Homogeneity and precision of X/95

Prothrombin times (seconds)

Ampoules

normalplasma9.459.609.459.459.459.609.609.309.459.609.459.459.609.459.609.459.309.159.470.1251.32

coumarinplasma21.8021.9022.2021.4021.9021.9022.0021.8021.8022.2021.4021.4021.9021.9022.2021.9021.8021.8021.840.2451.12

normalplasma9.609.459.609.459.459.459.609.609.459.459.459.459.609.459.459.459.309.459.480.0820.86

coumarinplasma22.6022.3022.8022.6022.3022.5022.6022.5022.3022.2022.3022.2022.3022.3022.2022.3022.5022.0022.380.1960.88

123456789101112131415161718MeanSDCV

BS/96.1846 Rev.. 1

--0 _.c """

ADoendix ill

1.

2.

3,

~XDerimental Drocedu~

Results

Tables 1 and 2 show the linear regression lines of PT vs. storage time. Table 3 and Fig 1 show thelinear regression lines of PT -Ratio vs. storage time. Statistical association between PT -Ratio andstorage time was reached only in one series of measurements (Laboratory A at 37°C).

Conclusions

1 PT change as a result of accelerated degradation of the candidate IRP is of minor magnitudel$. 0.04 second/day at 37 OC).

2. Since PT-ratio (coumarin plasma/normal plasma) does not change appreciably also the ISIvalue of the candidate IRP does not change.

References

1 van den Besselaar AMHP, Witteveen E, Schaefer-van Mansfeld H, Meeuwisse-Braun J,Strebus A. Accelerated degradation test of lyophilized recombinant tissue factor-liposomepreparations. Thromb Haemost 1995; 73: 392-7.

BS/96.1846 Rev. 1

-Table 1. Linear regression lines of PT with candidate IRP X/95 and

plasma A vs. storage time (t): PT 2 a + b.10-J.t in which PT is

in seconds and t is in days. The SD of b is given in brackets:n = number of samples; r = Sperman's rank correlation coefficient"

*. p < 0.05.

StorageTemp.

(oC)

LAB A

a bLAB B !.ABCn r n a b r n a b r

-20 27 15.2 -2.7

(1.7)

0.483 30 15.7 11 0.696*(2.7)

27 15.7 -2.5

(1.8)0.266

30 27 14.8 2.4

(2.7)

0.317 30 15.5 9.1 0.442(4.7)

27 15.5 5.7(3.5)

0.133

37 27 14.8 3.7(2.2)

0.766* 30 16.3 0.2 0.333(3.9)

27 15.6 1.1(2.0)

0.383

44 30 15.0 0.7(7.1)

0.067 30 15.7 9.0 0.3.51

(9.9)30 J.6.0 -30

(15)0.697*

Table 2. Linear regression lines of PT with candidate IRP X/95 and

plasma B vs. storage time (t): PT = a + b.1O-J.t

StorageTemp.(oC)

LAB A LABB LABCn b r n b n a b

-20 27 48.7 5.8

(6.0)0.500 30 48.6 -9.6

(14)

0.042 27 50.9 -0.2

(8.5)0.033

30 27 48.1 11.2(7.1)

0.417 30 47.7 -3.5(8.9)

0.030 27 51.1 -29

(9.6)0.633

37 27 418.5 -6.7

(5.9)0.067 30 49.4 2.3

(5.6)0.370 27 50.3 -25

(5.3)0.333

44 30 48.3 -51

(16)

0.709* 30 50.3 -23

(44)0.042 30 48.8 -55

(35)

0.224

BS/96.1846.:.Rev.l

StorageTemp.

(oC)

LAB ALAB B LAB Cn a b r n ba r n a b

-20 27 3.21 1.0(0.4)

0.033 30 3.09 -2.4

(1.1)

0.248 27 3.24 0.5

(0.4)0.666

30 27 3.26 0.2(0.7)

0.033 30 3.09 -1.9

(1.2)0.321 27 3.30 -2.7 0.383

(1.0)37 27 3.21 -1.2

(0.4)0.833* 30 3.05 -0.02 0.345

(0.7)27 3.23 -1.8 0.583

(0.5)44 30 3.21 -3.5

(1.5)0.624 30 3.20 -3.2

(3.8)0.042 30 3.06 2.2 0.309

(3.8)

BS/96.1846 Rev.

~ppendix IY

Table 1. Slopes and Coefficients of Variation (CV) fortion of Y/95 versus IRPs. calibra-

vs.BCT/253Slope CV

vs.RBT/90Slope CV

vs.OBT/79Slope CVLAB

1234567891111111111

1.0090.9350.8280.9480.8770.8750.8770.8840.9760.9050.9010.7640.9281.0650.9240.8460.9190.9151.064

2.1.1.3.2.2.3.

3.3.2.3.2.2.3.2.2.2.2.2.

0.9601.1250.9981.0740.9570.9600.9041.0231.1050.9901.1150.8360.9511.0011.0470.9751.0760.9651.117

2.3.2.3.3.3.4.3.4.2.4.3.3.2.3.3.3.3.3.

0.9001.1620.9801.0730.9310.9670.8691.0961.0621.0250.9710.8700.9570.9901.0450.9091.0550.9951.037

1.2.1.3.

2.2.2.2.3.

2.3.2.8.2.2.2.3.

3.2.

0123456789

19

.6

,0,5,6

33

.3

,0,2

69

.2

,4,6,6

94

,6,0

.0

.7

,3

.0

,1

4.1

.8

.3

,8

,1

.6

.0

.8

.0

.2

.3

84

.7

.0,4

75.9

,0

,5960

.4

.3,9,7

74

BS/96.1846 Rev. 1

Table 2. ISI values for Y/95 vs. IRPs. $, omitted from thecalculation because of poor performance.

final

LAB VS.BCTj253 vs.RBT/90 vs.OBT/79

1111111111

1.0951.0150.8991.0290.9520.9490.9510.9591.0590.9820.9780.8281.0061.1561.0030.9180.9970.9931.155

0.9601.1250.9981.0740.9570.9600.9041.0231.1050.9901.1150.8360.9511.0011.0470.9751.0760.9651.117

0.9091.1750.9911.0850.9410.9780.8781.1081.0741.0360.9810.8800.967 $1.0011.0570.9191.0671.0061.048

0.996198.1

1.009197.8

1.005198.0

MeanNCV

1.007188.1

MeanNCV

Overall MeanNSD

~~ 1\

1.004560.07920.01067.9

1234567890123456789

BS/96.1846 Rev. 1

Appendix V

-";'Comparative evaluation of Xj95 vs. Yj95

AimsTo compare the performance characteristics of X/95 and

and to choose the most suitable candidate IRP.Y/95

DesignThe two candidates were assessed on the basis of the follow-

ing criteria:

1. Within-laboratory precision of the calibration,the CV of the slope.

expressed as

2. Between-laboratory precision of the calibration,the CV of the ISI.

expressed as

3.

Conformity of the calibration model (i.e., (i) 1inearity- ofthe relationship between 10g-PTs obtained with the candidates.vs.existing IRPs and (ii) testing the assumption that the mean -log-PT of normals lies on the orthogonal regression line drawnthrough patient data points.

Resultsfor

which65%

1.

The within-laboratory precision of calibration was betterX/95 than for Y/95 because the percentage of laboratoriesscored a CV value below the recommended 3% were 88% vs.(Fig.s 1-3).

2. The between-laboratory precision of the calibration was betterfor Xj95 than for Yj95 with CV values of 4.7% (see Table 3 of themain report) vs. 7.9%. (see Table 2, Appendix IV).

3. X/95 was more adequate to fulfill the requirements of thecalibration model. Testing for the assumption that the mean 10g-PT of normals lies on the orthogonal regression line drawnthrough patient data points revealed that X/95 deviated from theassumption in 9%, whereas Y/95 in 82% of the cases (Fig.s 4-6).

rnn,..,1,1.Q; I1nROverall, X/95 is the preferred candidate to replace BCT/253.

BS/96.1846 Rev~

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-

ADDenda YI

Characteristics of the candidate X/95

Bulk Material

The lyophilized portion of the candidate material was derived from one bulk lot. The ~orcomponents are: -1. Tissue factor. This is a human recombinant membrane spanning protein, expressed in abaculovirus expression vector and purified using classical ion exchange and size exclusionchromatography. Purity is checked by aminoacid analysis and SDS polyacrilamyde gelelectrophoresis. A densitometer tracing of the candidate lot showed> 90% purity.2. Linids. The individual lipid components are all certified to be greater than 99.9% pure and weremanufactured from soybean and other plant source. They are prepared by transesterification of sn-Glycero-3-Phosphatidylcholine using a single chain fatty acid. No preservatives were used duringsynthesis and purification of the individual components. An antioxidant is included in the fmallipidblends to prevent oxidation.3. Matrix. Liposomes containing the recombinant TF form the matrix of the candidatethromboplastin.Stabilizers. A sugar is used as a stabilizer in the lyophilized product.Preservatives. Sodium azide (0.04%) is used as a preservative in the lyophilized portion of thecandidate. Proxel GXL (0.03%) is used as a preservative in the diluent. This level of preservativepasses Johnson & Johnson and USP requirements for preservative efficacy.Reconstitution of the material. Each ampoules must be reconstituted by adding 1.00 ml of theprovided diluent.

Fillin&. Lyonhilization. Sealin&.1. Reference number of the candidate: DEV 94049 (lyophilized)

DEV 94050 (diluent)

2. Address of laborat aration of the materialnreliminarv testin!!: and stora!!:e: Ortho Diagnostic Systems, Inc. 1001 Route 202, Raritan, NJ

08869, USA.

3. Manufacturing: conditions. The candidate lot was prepared and processed in a non-sterileenvironment according to specification. Temperature was controlled between 15-30 DC and an atmos-phere <20% relative humidity was maintained during the sealing process. Ampoules were heatsealed with an in-line nitrogen flush immediately upon removal from the lyophilizer. Nitrogen was

-

BS/96.1846 Rev. 1-'f. 29

4. Precision of fillig. Fill checks were performed every 400 ampoules for a total of 82measurements during the process and the coefficient of variation was 2.0%. Fill checks wereperformed gravimetrically according to the following protocol: 1. Obtain solution density. 2. Tareampoule. 3. Fill ampoule. 4. Weigh filled ampoule. 5. Apply density conversion factor to determinevolume dispensed. Ampoules of diluent contains on average 1.75 ml solution and were overfIlledfor ease in user sampling.

6. Diluent. The calcium chloride concentration of the diluent stored in glass sealed ampoules waschecked by atomic absorption according to QC protocols. The assay value obtained was 12.04 mM.Residual oxvl!:en and moisture testingAmpoules were sealed under pure nitrogen (99.998%). No residual oxygen was found whenanalyzed by gas chromatography. Moisture levels in the lyophilized product were measured by gaschromatographic analysis of a sample removed from the head space above the lyophilization cake.This method had been developed by Ortho specifically for assay of this product since attempts to usethe Karl-Fisher method gave variable results due problem in sample handling. For samplingampoules, the ampoule was placed in an evacuated tube prior to sampling. An average moisture levelof 0.81 % was observed.The head space moisture analysis by gas chromatography is a measure of free moisture only. Otherclassical methods, Karl Fisher or loss on drying by P20S will measure total moisture (free andbound) in a sample. Thus, there will be a discrepancy in values reported by the different methods.We have found that the amount of free moisture is what is important with respect to productdegradation. Bound moisture is important in maintaining protein integrity, but irrelevant indetermining product robustness.

Sterility testin2

Not applicable. Processing was done according to specification in a non-sterile suite

Stora~e temReratnre

Lyophilized ampoules have been stored in sealed containers from 8/94 to present at -20 DC(specification range: -20 °C or below). Diluent ampoules have been stored in sealed containers overthe same period at 5 °C (specification range: 2-8 °C).

Amnoules offered to WHO10,000-15,000

BS/96.1846 Rev. 1 -

....';

Addendum

The moisture content for X/95 has been independently checked by the NIBSC (potters Bar) andfound to be 2.547% on average.

= - =

expert comm1'l-l'~ on biological standardization …

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