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2010-2

October 2010

PHARMEUROPA BIO

& SCIENTIFIC NOTES

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PHARMEUROPA BIO

& SCIENTIFIC NOTES 2010

-2

Contents

Calibration of Human Coa

gulation Factor VIII Conce

ntrate

Ph. Eur. BRP Batch 4 for U

se in Potency Assays

Collaborative Study for the

Establishment of Replacem

ent Batches

of Heparin Low-Molecular

-Mass for Assay Biologica

l Reference

Preparations

Collaborative Study for Va

lidation of a Serological P

otency Assay

for Rabies Vaccine (inactiv

ated) for Veterinary Use


Establishment of the Secon

d

International Standard for

Gramicidin


Establishment of the Third

International

Standard for Nystatin

Quality Control of Metopr

olol Extended-Release Form

ulations in

the Presence of Ethanol

Possible Ambiguities when

Testing Viscosity in Comp

endial

Monographs - Characteris

ation of Grades of Cellulos

e Ethers

Instructions for Authors

Oct

ober

201

0PH

AR

MEU

RO

PA

BIO

& S

CIE

NTI

FIC

N

OTE

S

201

0-2

The Editor is pleased to consider contributions for publication in Pharmeuropa Bio & Scientific Notes provided they have some relationship with the work of the European Pharmacopoeia. It is assumed that the copyright of material submitted is that of the contributor or that the necessary clearances have been obtained and that material submitted has not been offered simultaneously to another publication. Authors alone are responsible for the views expressed in signed contributions.

Please see instructions for authors published in this issue and available at www.edqm.eu.

Articles published in Pharmeuropa Bio & Scientific Notes are indexed in the PubMed database of the National Library of Medicine, available at www.ncbi.nlm.nih.gov.material submitted has notl

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Printed on acid-free paper by Bialec, Nancy, France

Members of the European Pharmacopoeia Commission: Austria, Belgium, Bosnia and Herzegovina, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Montenegro, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovak Republic, Slovenia, Spain, Sweden, Switzerland,the former Yugoslav Republic of Macedonia, Turkey, United Kingdom and the European Union.Observers to the European Pharmacopoeia Commission: Albania, Algeria, Argentina, Armenia, Australia, Belarus, Brazil, Canada, China, Georgia, Israel, Madagascar, Malaysia, Moldova, Morocco, Republic of Kazakhstan, Russian Federation, Senegal, Syria, Tunisia, Ukraine, United States of America andWHO (World Health Organisation).

Pharmeuropa Bio & Scienti c Notes, 2010-2 i

PHARMEUROPA BIO & SCIENTIFIC NOTES 2010-2

CONTENTS

Calibration of Human Coagulation Factor VIII Concentrate Ph. Eur. BRP Batch 4 for Use in Potency AssaysS. Raut, A. Costanzo, S. Daniels, A. Heath, K.-H. Buchheit .............................................................. 1

Collaborative Study for the Establishment of Replacement Batches of Heparin Low-Molecular-Mass for Assay Biological Reference PreparationsE. Terao, A. Daas, G. Rautmann, K.-H. Buchheit. ........................................................................... 30

Collaborative Study for Validation of a Serological Potency Assay for Rabies Vaccine (inactivated) for Veterinary UseB. Krmer, L. Bruckner, A. Daas, C. Milne....................................................................................... 37

Collaborative Study for the Establishment of the Second International Standard for GramicidinG. Rautmann, A. Daas, K.-H. Buchheit. .......................................................................................... 56

Collaborative Study for the Establishment of the Third International Standard for NystatinG. Rautmann, E. Charton, A. Daas, K.-H. Buchheit ........................................................................ 68

Quality Control of Metoprolol Extended-Release Formulations in the Presence of EthanolA. Amini, A. Dawood, A.-M. Hesselgren, H. Thor, S. Jnsson, T. Arvidsson, G. Ragnarsson, M. Johansson ................................................................................................................................. 86

Possible Ambiguities when Testing Viscosity in Compendial Monographs -Characterisation of Grades of Cellulose EthersE. Doelker ........................................................................................................................................ 92

Instructions for Authors .............................................................................................................. 100

ii Pharmeuropa Bio & Scienti c Notes, 2010-2

:jgdeZVc9^gZXidgViZ[dgi]ZFjVa^ind[BZY^X^cZh=ZVai]8VgZ

Un logiciel ddi lanalyse statistique des rsultats de titrages biologiques selon la Pharmacope Europenne

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CombiStats is a user-friendly computer program for the statistical analysis of data from biological dilution or potency assays. It can perform calculations according to Chapter 5.3 of the European Pharmacopoeia (5th and 6th Edition) including the following models: parallel line models, slope ratio models, probit models, ED50 calculations (and other percentiles), 4-parameter curve models, single dose models, combination of assays,and many more of the family of generalised linear models.

CombiStats is intended for use by those responsible for the analysis of assay data but whose primary training is not in statistics and who have no access to qualified statisticians to assist them with the analysis. The software may be useful in any situation where the analysis of data from dilution assays is required but it is primarily intended for use in the context of monographs of the European Pharmacopoeia.

Much effort has been invested to keep the software easy to use and at the same time versatile enough to cover a wide range of existing potency assays. With the help of the wizard, it only takes a few simple steps to create your own customised templates for routine assays. Just specify: the number of preparations, doses and replicates, the model, the design, a possible transformation, the type of analysis of variance.

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Standard results produced by the software are an analysis of variance, potency estimates, and graphs of the fitted models. The output is formatted in a pleasant and consistent layout. The software also offers more advanced features to allow the more experienced user to access less commonly used options such as weighted regression and export of estimated parameter vectors and associated covariance matrices.

A free demonstration version can be downloaded from www.combistats.eu. This demo-version includes the complete manual and tutorial to help the user to get acquainted with the most important features of the software. Also included is a set of 24 example files offering a wide range of possible configurations for specific assay situations. For more information about system requirements, prices, and how to order a user licence, please consult the website.

Qu'est-ce que CombiStats ?

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Crez vos propres masques

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CombiStats est un logiciel convivial ddi lanalyse statistique des rsultats de titrages biologiques par dilution ou titrages dactivit. Il peut raliser les calculs dcrits dans le chapitre 5.3 de la Pharmacope Europenne (5e et 6e Editions) en utilisant les modles suivants : modle en lignes parallles, modle rapport de pentes, modle des probits, calculs de la DE50 (et autres pourcentiles), modle de distribution 4 paramtres, modle dose unique, combinaison de dosages,ainsi que de nombreux autres modles linaires gnraliss.

CombiStats est loutil de tous ceux qui ont analyser des rsultats de dosages sans tre statisticiens de formation ou avoir accs lassistance dun statisticien. CombiStats peut tre utile dans toute situation impliquant lanalyse de rsultats de titrages par dilution, mais il a t conu en premier lieu pour tre utilis dans le contexte des monographies de la Pharmacope Europenne.

Des efforts considrables ont t faits pour que le logiciel reste simple utiliser tout en tant suffisamment polyvalent pour couvrir une large gamme de titrages rencontrs dans la pratique. En vous laissant guider par le programme, vous pouvez en quelques tapes simples crer vos propres masques de calcul pour les analyses de routine. Il vous suffit de spcifier : le nombre de prparations, doses et rplicats, le modle, le plan dessai, une ventuelle transformation, le type danalyse de variance.

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CombiStats fournit classiquement une analyse de variance, des estimations de lactivit et le graphe des modles ajusts. La prsentation des donnes de sortie est agrable et homogne. Le logiciel offre galement aux utilisateurs expriments des fonctions avances leur permettant daccder des options moins courantes comme la rgression pondre et lexportation de vecteurs de paramtres estims et de matrices de covariance associes.

Une version de dmonstration gratuite est tlchargeable sur le site www.combistats.eu. Elle comprend le manuel intgral de l'utilisateur et un didacticiel permettant de se familiariser avec le fonctionnement du logiciel. Sont galement incluses 24 fiches dexemples offrant un large panorama des configurations possibles dans des situations de dosages spcifiques. Pour de plus amples informations sur la configuration exige, les prix et les modalits de commande de licences d'utilisateurs, rendez-vous sur le site internet !

A programme for the statistical analysis of bio-assays

according to the European Pharmacopoeia

Pharmeuropa Bio & Scientific Notes, 2010-2 1

Calibration of Human Coagulation Factor VIII Concentrate BRP

Calibration of Human Coagulation Factor VIII Concentrate Ph. Eur. BRP Batch 4

for Use in Potency AssaysS. Raut, A. Costanzo, S. Daniels, A. Heath, K.-H. Buchheit

S. Raut. National Institute for Biological Standards and Control (NIBSC), Health Protection Agency, Potters Bar, EN6 3QG, Hertfordshire, UK. A. Costanzo. (Corresponding author: e-mail: [email protected]). European Directorate for the Quality of Medicines & HealthCare (EDQM), 7 alle Kastner, CS 30026, F-67081 Strasbourg, France. S. Daniels. National Institute for Biological Standards and Control (NIBSC), Health Protection Agency, Potters Bar, EN6 3QG, Hertfordshire, UK. A. Heath. National Institute for Biological Standards and Control (NIBSC), Health Protection Agency, Potters Bar, EN6 3QG, Hertfordshire, UK. K.-H. Buchheit. European Directorate for the Quality of Medicines & HealthCare (EDQM), 7 alle Kastner, CS 30026, F-67081, Strasbourg, France.

ABSTRACTThe European Pharmacopoeia Biological Reference Preparation (Ph. Eur. BRP) Batch 4 was established as an international common working standard for potency determination of human coagulation factor VIII (FVIII) preparations to replace the dwindling stocks of the BRP Batch 3, the current European standard. Similarly, stocks of the current World Health Organisation 7th International Standard (WHO 7th IS) were also running low. Therefore a project was jointly organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM, Council of Europe) and the National Institute for Biological Standards and Control (NIBSC, UK) in order to replace both standards concomitantly. The potency of the BRP Batch 4 was assigned during an international collaborative study involving 38laboratories with reference to the WHO 7th IS and the BRP Batch 3. Four candidate materials, 2 plasma-derived (samples A and C) and 2 recombinant (samples B and D) have been evaluated, sample C being the speci c candidate for the replacement of the BRP Batch 3. Participants were instructed to perform 8 independent assays following their own routine validated methods, by either the one-stage clotting assay or the chromogenic assay, or both.Laboratories returned 22 data sets for the clotting assay and 30 data sets for the chromogenic assay. This publication reports the results obtained with both assays but only the results of the chromogenic assay are highlighted in the conclusions, as it is the assay prescribed by the European Pharmacopoeia. Data were analysed separately for both assays. The consensus potency value was calculated as the unweighted geometric mean of the unweighted geometric means of each individual laboratory. For sample C, there was a signi cant difference in potency estimate between the chromogenic and the clotting assay. It was therefore not possible to reconcile both results. The chromogenic potencies however were in very good agreement being 10.4 IU/ampoule (n = 30), when assessed against both standards. The inter-laboratory geometric coef cient of variation (GCV) was 4.8 % and 7.1 % against the WHO 7th IS and the BRP Batch 3 respectively.The Ph. Eur. BRP Batch 4 is a freeze-dried, plasma-derived concentrate. The material was lled in approximately 20000ampoules and lyophilised. The nal residual water content is 0.33 %. Based on accelerated degradation studies, the stability of the material is suitable for a reference preparation. The candidate Ph. Eur. BRP Batch 4 was adopted at the 136thsession of the European Pharmacopoeia Commission in March 2010. The standard will be available from the EDQM with the catalogue number H0920000 upon exhaustion of the current batch.

KEYWORDSHuman coagulation factor VIII, reference standard, European Pharmacopoeia Biological Reference Preparation, collaborative study, chromogenic assay, WHO 8th International Standard.

1. INTRODUCTIONThe current European Pharmacopeia (Ph. Eur.) BRP Batch 3 (BRP3)/Mega 2 (US/FDA) for Factor VIII (FVIII) was established in 2001 by calibration against the World Health Organization (WHO) 6th International Standard (IS) for Factor VIII Concentrate and is used as a working standard in potency estimation of FVIII in plasma-derived and recombinant therapeutic concentrates, which are primarily used in the treatment of haemophilia A (FVIII de ciency). The BRP3/Mega 2 (US/FDA) had been established as working standard by the Ph. Eur. and the FDA. Stocks of the BRP3 were running low and were expected to be exhausted by 2010/11. The Steering Committee of the Biological Standardisation Programme (BSP) thus decided to endorse a project (BSP098) in January 2008 in order to qualify a replacement batch for this BRP, calibrated against the WHO 7th IS for FVIII Concentrate (NIBSC code: 99/678) and against the BRP3.

Concomitantly, the National Institute for Biological Standards and Control (NIBSC) was planning a project to replace the WHO 7th IS for FVIII Concentrate. Therefore, in the interest of harmonisation and continuity, a joint collaborative study between the European Directorate for the Quality of Medicines & HealthCare (EDQM) and the NIBSC was undertaken to replace both standards. Following trial ll studies on a number of therapeutic FVIII concentrates, 4 candidate materials were selected, 2 plasma derived (samples A and C) and 2 recombinant (samples B and D), with sample C designated as speci c candidate for the proposed Ph. Eur. BRP Batch 4. The selection of materials was based on ll characteristics, stability of FVIII potency, minimal inter- and intra-assay variability and minimal discrepancy between assay methodologies, as determined in pre-quali cation studies of the materials. Twenty thousand (20,000) ampoules of each of the 4 candidate materials have been prepared.

2 Pharmeuropa Bio & Scientific Notes, 2010-2


2. AIMThe objective of this collaborative study was to calibrate the candidate Ph. Eur. BRP Batch 4 for Human Coagulation FVIII Concentrate against the WHO 7th IS for FVIII Concentrate and against the BRP3 for FVIII Concentrate by the chromogenic method, although data from the one-stage clotting assays were also assessed for comparison, particularly as this joint study was also intended to assign a calibration value to the replacement candidate for the WHO 7th IS for FVIII Concentrate. A proposal for the establishment of the WHO 8th IS for FVIII Concentrate has been made in a separate report by NIBSC to the 2009 WHO Expert Committee on Biological Standardisation (ECBS) [1]. Although all the data from the joint collaborative study are presented, this report will primarily focus on the calibration of the Ph. Eur. BRP Batch 4 for FVIII Concentrate.

3. PARTICIPANTSThirty eight laboratories participated in the study (from 17 countries world-wide) and returned data for analysis. They are listed in section 9. The participants included 22manufacturers and 16 regulatory authorities. Laboratories were coded for the study and the order of listing in section 9 does not necessarily correspond with the numerical codes. All raw data returned by the participants were analysed at NIBSC.

4. MATERIALS AND METHODS4.1. Sample A (07/350) - plasma derived candidate preparationThe raw material for this candidate was a plasma-derived high purity FVIII concentrate, prepared by chromatography and containing von Willebrand factor. All donations used to prepare this product were tested and found negative for HBsAg, anti-HIV-1 and -2, anti-HCV, HCV-RNA (plasma pools). Manufacturing of this product also included 2 viral inactivation steps, solvent detergent and heat treatment at 81 C for 72 hours. After reconstitution of the product, the concentrate material was pooled and formulated in the following buffer: 0.15M NaCl, 50mM Tris-HCl (pH 7.4), 1mM CaCl2, 10mg/ml trehalose, 10mg/ml human albumin. The formulated material was lled and freeze-dried in sealed glass ampoules at NIBSC, under conditions required for International Standards [2]. One ml of this material was dispensed into each of approximately 20,000 ampoules. The mean lling weight was 1.0054 g (range 1.0015 g to 1.0100g) and the coef cient of variation (CV) was 0.16 % based on 624 check-weight samples. Mean residual moisture after freeze-drying was 0.46 % (CV 20.6 %, n = 12) and mean oxygen headspace was 0.44 % (CV 11.8 %, n = 11).

4.2. Sample B (07/352) - recombinant candidate preparationThe raw material for this candidate was a recombinant FVIII concentrate bulk. After thawing, the bulk concentrate solution was pooled and formulated in the following buffer: 0.15M NaCl, 50mM Tris-HCl (pH 7.4), 1mM CaCl2, 10mg/ml trehalose, 10mg/ml human albumin. The formulated material was lled and freeze-dried in sealed glass ampoules at NIBSC, under conditions required for International Standards [2]. One ml of this material was dispensed into each of approximately 20,000 ampoules. The mean lling weight was 1.0054 g (range 1.0010 g to 1.0100 g) and the coef cient of variation (CV) was 0.11 % based on 689 check-weight samples. Mean residual moisture after freeze-drying was 0.32 % (CV 21.9 %, n = 12) and mean oxygen headspace was 0.37 % (CV 9.1 %, n = 12).

4.3. Sample C (08/106) - plasma derived candidate preparationThe material was procured by the EDQM as the prime candidate material for the proposed Ph. Eur. BRP Batch 4 for FVIII Concentrate from a European manufacturer and is part of a batch that was also used to produce a therapeutic product available on the European market. The raw material for this candidate was a plasma-derived intermediate purity FVIII concentrate, prepared by multiple precipitation method and containing von Willebrand factor (vWF). All donations used to prepare this product were tested and found negative for HBsAg, anti-HIV-1 and -2, anti-HCV, HCV-RNA (plasma pools). Manufacturing of this product also included a viral inactivation pasteurisation process at 60 C for 10 hours in aqueous solution. After reconstitution of the product, the concentrate material was pooled and formulated in the following buffer: 50mM NaCl, 267mM glycine (pH7.3), 18.7mM tri-sodium citrate dihydrate, 10mg/ml human albumin. The formulated material was lled and freeze-dried in sealed glass ampoules at NIBSC, under conditions required for International Standards [2]. One ml of this material was dispensed into each of approximately 20,000ampoules. The mean lling weight was 1.0058 g (range 1.0015 g to 1.0085 g) and the coef cient of variation (CV) was 0.22 % based on 686 check-weight samples. Mean residual moisture after freeze-drying was 0.33% (CV 33.6 %, n = 12) and mean oxygen headspace was 0.28 % (CV 26.7%, n = 11). The material was sent to the EDQM and stored at -20 C immediately upon receipt.

4.4. Sample D (08/104) - recombinant candidate preparationThe raw material for this candidate was a recombinant FVIII concentrate bulk. After thawing, the bulk concentrate solution was pooled and formulated in the following buffer: 0.15M NaCl, 50mM Tris-HCl (pH 7.4), 1mM CaCl2, 10mg/ml trehalose, 10 mg/ml human albumin. The formulated material was lled and freeze-dried in sealed glass ampoules at NIBSC, under conditions required for International Standards [2]. One ml of this material was dispensed into each of approximately 20,000 ampoules. The mean lling weight was 1.0056 g (range 1.0010 g to 1.0085 g) and the coef cient of variation (CV) was 0.12 % based on 739 check-weight samples. Mean residual moisture after freeze-drying was 0.57 % (CV 11.8 %, n=12) and mean oxygen headspace was 0.27 % (CV 13.2 %, n=10).

4.5. Sample E - Ph. Eur. BRP Batch 3 (BRP3) for FVIII ConcentrateThis secondary working standard was supplied by EDQM and is also the same standard as the Mega 2 US secondary working standard. Sample E has been assigned a potency of 8.6 IU/vial [3] for the chromogenic method through an international collaborative study in 2001. During this study, the BRP3 was also tested in the one-stage clotting assay and the mean estimated potency was found to be 11.3 IU/vial for this method. However, since the Ph. Eur. has adopted the chromogenic assay as its sole method of determining potency for FVIII preparations, no of cial value was assigned for the clotting assay to the BRP3. The FDA, on the other hand, calibrated this same material as their Mega 2 US FVIII Concentrate standard with assigned potencies for both the chromogenic method (8.6 IU/vial) and the one-stage clotting method (11.3 IU/vial).

4.6. Sample 7th IS - WHO 7th IS for FVIII ConcentrateThis primary standard is a plasma-derived FVIII concentrate. It was supplied by NIBSC and has a single assigned potency



of 11.0 IU/ampoule for all methods.

4.7. Materials dispatched for the studyEight ampoules/vials of each of the above materials were dispatched by NIBSC. Participants were asked not to use any additional test materials in the assays, such as any internal (in-house) standards.

4.8. Assay methods and study designParticipants were requested to carry out 8 independent assays using their normal routine methodology, either one-stage or chromogenic methods, preferably assays by both methods. They were requested to carry out assays on separate occasions (days) using a fresh ampoule/vial of samples A, B, C, D, E and 7th IS for each assay, according to balanced assay designs recommended in the study protocol. A separate ampoule/vial of each material was provided for each assay. The laboratories that used more than one method were requested to use material from the same ampoules/vials for both methods, provided this could be done within 3 hours of reconstitution.

Participants were also requested to pre-dilute all samples in congenital severe haemophilic plasma (SHP) (or immunodepleted or chemically depleted plasma provided it contained normal levels of vWF), to a nal FVIII concentration of ~1.0 IU/ml. Detailed assay instructions were provided in the study protocol. The details of the methods, instruments and reagents used by the participants are listed in Appendix I.

4.9. Statistical analysisAll assays were analysed as multiple parallel line bioassays comparing log response to log concentration [4]. All assays data were plotted, and in some cases individual data points were excluded as possible data transcription errors or potential technical errors. The statistical validity of the assays was assessed by the usual ANOVA tests. The parallelism of the assays was also assessed by comparing the slopes of the dose-responses across the assays.

For each assay the potency estimates of samples A - E were calculated relative to the concurrently tested WHO 7th IS for FVIII Concentrate (sample 7th IS). Potencies were also calculated relative to the BRP3 for FVIII Concentrate (sample E). Combined potency estimates for each laboratory were obtained by taking unweighted geometric means of results from all assays. Overall combined estimates were obtained by taking unweighted geometric means of the mean results from the different laboratories. Where a laboratory performed more than one assay method, the results for each method were analysed as if from separate laboratories. Intra- and inter-laboratory variability is expressed as the percentage geometric coef cient of variation (%GCV) [5].

The mean potency estimates calculated by the participating laboratories are presented (Appendix II). Differences in potency estimates between laboratories were assessed using a Duncan's multiple range test [6]. Differences in potency estimates between assay method (one-stage or chromogenic) were assessed by 2-sample t-tests, and con rmed by Wilcoxon rank-sum tests.

5. RESULTS5.1. Data receivedData were received from 38 laboratories in total. Laboratory8 performed repeat one-stage assays with two different machines. The results are analysed separately

and coded 8A (ACL Advance) and 8B (Coag-A-Mate X2), see Appendix I. Laboratory 37 performed two different chromogenic assays. They are analysed separately and coded 37A (chromogenic for plasma derived FVIII samples) and 37B (chromogenic for recombinant FVIII samples). Several laboratories performed both one-stage and chromogenic assays. In total there were 52 data sets (22 for one-stage assays and 30 for chromogenic assays). Methods and reagents used by the participants are summarized in Appendix I.

5.2. Assay validityWhen the individual assay data were plotted, all laboratories had dose-responses that appeared to be a good t to the parallel-line model, when the log transform of the response was used. For laboratory 4, the untransformed response was used. Some individual data points were excluded, but all samples and assays were included in subsequent analysis, with the exception of one assay from laboratory 28 (chromogenic) where sample C was excluded as non-linear.

5.3. Potency estimates relative to the 7th ISThe laboratory geometric mean estimates of potency relative to the current 7th IS, the intra-laboratory %GCVs, the overall geometric mean and inter-laboratory %GCVs, for both the one-stage and chromogenic assay methods, and for all assays combined are shown in Tables 1-5.The potency estimates are also shown in the form of stacking histograms (Figures 1-5). Each box represents the laboratory mean, expressed as a percentage of the overall geometric mean, calculated from results from both assay methods combined. The boxes are labelled with the laboratory code number. Results from one-stage assays are shaded in grey, whilst the results from chromogenic assays are unshaded (white).The potency estimates calculated by the participating laboratories are shown in Appendix II.

5.3.1. Sample AFrom the histogram (Fig. 1a) and Table 1, it can be seen that there is very good agreement between laboratories and assay methods. The overall inter-laboratory GCV is below 5 %, and the overall geometric means from the two methods are identical at 9.44 IU/ml by one-stage (n = 22) and chromogenic (n = 30) methods. There are no signi cant differences between methods and the combined mean value is 9.44 IU/ml (n = 52; GCV = 5.0 %). Duncans multiple range test did not indicate the presence of any outliers (laboratories with results signi cantly different from all other laboratories) when applied to the results from both methods combined. When applied to the chromogenic results alone, laboratory 4 was signi cantly higher than other laboratories. Given the overall tight agreement between laboratories, laboratory 4 was not excluded from any subsequent calculations. Intra-laboratory GCVs ranged from 0.64-12.81 % (n = 22) for the one-stage assays and from 0.34-9.49 % (n = 30) for the chromogenic assays.

5.3.2. Sample BFrom the histogram (Fig. 2a) and Table 2, it can be seen that there is poorer agreement between laboratories for sample B compared to sample A, when potency is expressed relative to the current IS. The overall inter-laboratory GCV is 14.48% with a combined geometric mean potency of 8.13 IU/ml (n=52). There were no signi cant differences between methods with geometric mean potencies of 8.26IU/ml (n =22) and 8.03 IU/ml (n = 30) for one-stage and chromogenic assays, respectively. No individual laboratory was found to be an outlier, although laboratories 1, 3 and



26 formed a group that were signi cantly different from the other laboratories. Intra-laboratory GCVs ranged from 2.34-25.81 % (n = 22) for the one-stage assays and from 1.56-29.29 % (n = 30) for the chromogenic assays.

5.3.3. Sample C

From the histogram (Fig. 3a) and Table 3, it can be seen that there is generally good agreement between laboratories, with an overall GCV of 5.86 %. The one-stage assays generally gave lower results than the chromogenic assays, with the overall means being 9.59 (n = 22) and 10.35 IU/ml (n = 30) respectively. The difference between methods was statistically signi cant (p



temperatures (e.g. -20 C) based on the observed loss in samples stored at elevated temperatures (e.g. +4, +20, +37, +45 C) [7]. This is an indirect method used to determine rate of loss based on the relationship between reaction rates and temperature given by an Arrhenius equation and where a rst order reaction rate is assumed [8].

Ampoules of the 4 candidate preparations were placed into elevated temperature storage in June 2008 at NIBSC. An initial accelerated degradation study carried out at NIBSC has indicated very low predicted degradation rates for all 4 candidate preparations. A more extensive accelerated degradation study involving 3 laboratories was further performed after a storage period of 8 or 12 months. Three laboratories assayed samples that had been stored for 8months, and one laboratory also assayed samples

stored for 12 months. Each laboratory carried out 3 repeat assays (either one-stage or chromogenic). All laboratories performed both types of assays except laboratory A which performed only chromogenic assays.The potencies of the samples stored at elevated temperature were expressed relative to the -20 C sample. The gures indicate good stability, with only a drop of around 10 % after 12 months storage at + 37 C for both candidate samples A and B.The Arrhenius model was tted to obtain predictions of the expected loss in potency over time. For candidate standard samples B and D, there was insuf cient degradation to be able to obtain any estimates of expected loss. For candidate standard samples A and C the predicted % loss per year at the different temperatures are shown in the Table I.

The results indicate that the predicted stability of sampleC would allow it be a suitable material as the Ph. Eur. BRP Batch 4 for FVIII Concentrate. Furthermore, the BRP will be monitored regularly, i.e. yearly, throughout its life-time. To this end, a subset of 30 ampoules of the candidate material (sample C) was set aside at -80C, at the EDQM. The ampoules were placed into aluminium bags containing suf cient number of containers for one monitoring assay, i.e. 3 ampoules, according to the internal Standard Opera-ting Procedure. These samples will be used as a reference for real-time stability monitoring of the BRP.

6. DISCUSSION

The current Ph. Eur. BRP Batch 3/Mega 2 (US/FDA) for FVIII Concentrate was established in 2001 through an interna-tional collaborative study and has an assigned potency of 8.6 IU/vial for the chromogenic assay [3]. It is used as a working standard for the determination of FVIII potency in plasma-derived and recombinant therapeutic products by the chromogenic assay method. Stocks of this standard are running low. Similarly, stocks of the WHO 7th IS for FVIII Concentrate were also running low and both standards were expected to be depleted by 2009/10. Therefore, it was decided

to organize a joint international collaborative study between NIBSC and EDQM in order to calibrate replacement batches of both standards.

The current Ph. Eur. BRP Batch 3 for FVIII Concentrate was calibrated against the WHO 6th IS (97/616) [9] and the WHO 5th IS (88/640) [10] for FVIII Concentrate, the Mega 1 US and the Ph. Eur. BRP Batch 2 [11] for FVIII Concentrate in both the one-stage clotting and the chromogenic assays[3]. Similarly, for the sake of harmonisation and continuity of the International Unit (IU), assays on candidate materials in this study were carried out using both types of assays and against the current WHO 7th IS for FVIII Concentrate (99/678) (sample 7th IS) as well as against the current Ph. Eur. BRP Batch 3 for FVIII Concentrate (sample E).

6.1. Intra- and inter-laboratory variabilityThe variability of assays within laboratories differed considerably, with GCVs ranging from 0.34 % to 36.32 % (Tables1-10). There was no obvious trend for one method to give better or worse inter-assay variability; lower GCVs were associated with particular laboratories rather than particular methods, indicating that internal quality control procedures within laboratories are probably the most important factor

Table I - Summary results of the accelerated degradation study

Candidate Material Lab

Mean residual potencies after storage Mean predicted % loss per year

(% vs -20 C ampoules)

+4C +20C +37C +45C

Sample A (07/350)

A 95 93 82

0.01% (at -20C) 0.15% (at + 4C)

B 95 93 81

C 99 95 83

C* 98 98 91 79

Sample B (07/352)

A 104 104 98

Insufficient degradation to obtain estimates.

B 102 102 88

C 101 98 89

C* --- --- ---

Sample C (08/106)

A 90 89 86

0.52% (at -20C) 2.22% (at + 4C)

B 96 95 90

C 99 93 90

C* 101 97 91 88

Sample D (08/104)

A 98 108 98

Insufficient degradation to obtain estimates

B 101 105 94

C 102 104 92

C* --- --- ---

- tested after storage for 8 months; * - tested after storage for 12 months; Results are the combined mean values from 3 independent one-stage and chromogenic assays



in determining reproducibility of FVIII assays, rather than the method used. A summary of intra-laboratory varia-bility for estimates of all samples by all methods is given in Table11. For all candidate samples (A-D) and for both methods, there was a higher percentage of laboratories with GCVs below 5% for estimates relative to the 7th IS than for estimates relative to sample E (BRP3/Mega 2 US). Further-more, intra-laboratory GCVs tended to be greater for the recombinant candidates (sample B and D) compared to the plasma-derived candidates (samples A and C). This may not be so surprising as recombinant concentrates can give in-creased assay variability, as observed in previous studies[12]. Figures generally represented good reproducibility and for candidate samples A and C, majority of the labs (>90 %) gave intra-laboratory GCVs < 10 %.

A summary of inter-laboratory variability for estimates of all samples by all methods is given in Table 12. Relative to the 7th IS, there was very good agreement between laboratories for the plasma-derived candidates, samples A and C, with overall combined method GCVs of 5.0 % and 5.9 % respec-tively. Relative to sample E (BRP3/Mega 2 US), low inter-laboratory variability was similarly obtained for the plasma-derived candidates, samples A and C, with overall combined method GCVs of 8.4 % and 10.1 % respectively. There was poorer agreement between laboratories for the recombinant candidates, samples B and D, when assayed relative to both standards with GCVs between 15-19.7 %. This re ects the known higher variability when assaying recombinant mate-rials. Sample E (BRP 3/Mega 2 US) is known to have a signi- cant one-stage/chromogenic discrepancy, however within methods there is good inter-laboratory agreement (Fig. 5).

6.2. Potency estimates of candidate materialsMean potency estimates by the two methods for all samples relative to both standards are summarized in Table 12. In the study to calibrate the current WHO 7th IS for FVIII Concen-trate (99/678), there was an overall method discrepancy of < 2 % [13]. In this study, when assayed relative to the 7thIS, sample A gave identical mean estimates of potency of 9.44 IU/ml (with an overall inter-laboratory GCV of < 5 %), and with less than 5% discrepancy between methods when assayed relative to sample E (BRP3/Mega 2 US) (Tables 12-13). The 2 recombinant samples B and D also gave low assay discrepancy between methods (



Blessing H., CSL Behring GmbH, GermanyHunfeld A., Paul-Ehrlich Institut, GermanyGambelli D., Cini E., Kedrion SpA, ItalySardelli R., Istituto Superiore di Sanit, ItalyKamimura K., Kaketsuken, JapanTaneichi M., National Institute of Infectious Diseases, JapanWatanabe Y., Baxter Ltd, JapanDa Silva A., INFARMED, Portugal Inder L., National Bio-products Institute, South AfricaAlonso C., Agencia Espanola de Medicamentos y Productos Sanitarios, SpainHosta N., Instituto Grifols, SpainRosen S., Rossix AB, SwedenWermeille M., Chessa J-P., Baxter AG, SwitzerlandYang Y-C., Bureau of Food and Drug Analysis Department of Health, TaiwanBlande A., Bio Products Laboratory, UKRaut S., Daniels S., National Institute for Biological Stan-dards and Control, UKJankowski M., Wyeth BioPharma (CAD), USAKhabbaz N., Allyn T., Bayer Healthcare, USALeerabhandh M., Baxter Thousand Oaks, USAWong L., Baxter Ltd, USAWood L., Lee T., CBER/U.S. FDA, USA

10. ABBREVIATIONSBRP: Biological Reference Preparation; BSP: Biological Stan-dardisation Programme; CI: Con dence Interval; CL:Con -dence Limits; CV: Coef cient of Variation; ECBS:WHO Expert Committee on Biological Standardisation; EDQM: European Directorate for the Quality of Medicines&Heal-thCare; FVIII: Factor VIII; FDA: Food and Drug Administra-tion; GCV: Geometric Coef cient of Variation; GM: Geo-metric Mean; IS: International Standard; IU: International Units; Lab: laboratory; log: logarithm; NIBSC: National Institute for Biological Standards and Control; OMCL: Of cial Medicines Control Laboratory; Ph. Eur.: European Pharmacopoeia; RT: Room temperature; US: United States of America; WHO: World Health Organization.

11. REFERENCES[1] Raut S, Daniels S, Heath A. An international collabora-tive study to assign value to the 8th WHO International Stan-dard for blood coagulation Factor VIII Concentrate (07/350).

Ref: BS/09.2117. WHO Expert Committee on Biological Standardization; 2009.[2] Campbell PJ. International biological standards and reference preparations. 1. Preparation and presentation of materials to serve as standards and reference preparations. JBiol Standardisation 1974;2:249-67.[3] Kirschbaum N, Wood L, Lachenbruch P, Weinstein M, Daas A, Rautmann G, Spieser JM, Buchheit KH. Calibration of the Ph. Eur. BRP Batch 3/Mega 2 (US/FDA) standard for human coagulation factor VIII concentrate for use in potency assay. Pharmeuropa Bio 2002(1):31-60.[4] Finney DJ. Statistical methods in biological assay. 3rdedi-tion. London, UK: Charles Grif n; 1978.[5] Kirkwood TBL. Geometric means and measures of dis-persion. Biometrics 1979;35:908-9.[6] Duncan DB. T-tests and intervals for comparisons sug-gested by the data. Biometrics 1975;31:339-59.[7] Kirkwood TBL, Tydeman MS. Design and analysis of accelerated degradation tests for the stability of biological standards II. A exible computer program for data analysis. J Biol Standardisation 1984;12:207-14. [8] Kirkwood, TBL. Predicting the stability of biological standards and products. Biometrics 1977;33(4):736-42.[9] Raut S, Heath A, Barrowcliffe TW. Establishment of the 6th International Standard for FVIII concentrate. Thromb Haemost 2001;85:1071-78.[10] Barrowcliffe TW, Kemball-Cook G, Heath A. Proposed 5th International Standard for Factor VIII Concentrate. Re-port to participants. An SSC working party study on behalf of the SSC FVIII/IX subcommittee. Unpublished report 1994.[11] Barrowcliffe TW. Establishment of a reference prepara-tion and evaluation of a chromogenic method. Pharmeuropa Bio 1996(1):39-47.[12] Raut S, Sands D, Heath AB, Barrowcliffe TW. Variability in factor VIII concentrate measurement: results from SSC eld collaborative studies. J Thromb Haemost 2003;1:1927-34.[13] Raut S, Bevan S, Hubbard AR, Sands D, Barrowcliffe TW. A collaborative study to establish the 7th International Standard for Factor VIII Concentrate. J Thromb Haemost 2005;3:119-26.[14] Assay of Human Coagulation Factor VIII, general chap-ter 2.7.4. Ph. Eur. 6th edition. Strasbourg, France: Council of Europe; 2008.



Table 1 - Mean laboratory estimates for sample A relative to WHO 7th IS FVIII Concentrate (sample 7th IS - 11.0IU/ml) by one-stage and chromogenic assays together with estimates of intra-laboratory

variability (GCV%) for individual laboratories and inter-laboratory variability for method estimates and combined estimates

Number of Geometric Intra-lab Number of Geometric Intra-labAssays Mean GCV% Assays Mean GCV%

(n) (IU/ml) (n) (IU/ml)1 - - - 8 9.57 4.082 8 9.38 6.15 8 9.32 8.663 - - - 8 9.85 4.164 8 9.76 4.38 8 10.92 3.665 8 9.26 3.29 8 9.15 1.936 8 9.39 3.19 - - -7 - - - 8 9.71 5.67

8A 8 9.76 3.19 - - -8B 8 9.51 5.03 - - -9 8 9.10 4.06 8 9.34 0.34

10 8 9.37 1.55 8 8.97 3.7411 8 9.37 4.39 8 9.15 1.2212 - - - 8 9.18 4.2613 8 11.25 9.67 - - -14 - - - 8 9.63 5.8315 - - - 8 9.52 2.3416 8 9.72 1.53 8 9.51 0.9317 8 9.75 0.64 8 9.54 1.4818 - - - 8 9.35 6.1619 8 9.19 1.62 8 9.43 2.3720 8 9.43 2.73 - - -21 - - - 8 9.33 3.2122 8 9.30 11.49 - - -23 - - - 8 9.24 9.4924 8 8.79 4.97 8 9.59 1.9325 - - - 8 9.73 5.4526 - - - 4 9.12 3.7827 8 8.79 12.81 - - -28 - - - 8 9.76 4.2629 8 8.35 4.20 8 8.60 4.4630 8 9.23 0.77 - - -31 - - - 8 9.57 9.2932 - - - 8 9.38 7.1233 - - - 8 9.47 3.9734 8 9.34 2.99 - - -35 - - - 8 9.86 5.7836 8 10.42 5.41 - - -

37A - - - 8 9.29 1.8037B - - - 8 9.07 3.5038 8 9.54 2.70 8 9.39 3.55

Inter-Lab GCV = 4.11%

O ne-Stage Chromogenic

Lab No.

Combined Geometric Mean = 9.44 IU/ml (n=52)

Combined Inter-Lab GCV = 4.98%

Geometric Mean = 9.44 (n=22) Geometric Mean = 9.44 (n=30)




Table 2 - Mean laboratory estimates for sample B relative to WHO 7th IS FVIII Concentrate (sample 7th IS - 11.0IU/ml) by one-stage and chromogenic assays together with estimates of intra-laboratory

variability (GCV%) for individual laboratories and inter-laboratory variability for method estimates and combined estimates


(n) (IU/ml) (n) (IU/ml)1 - - - 8 5.30 6.572 8 8.70 5.28 8 7.80 13.693 - 8 5.85 10.674 8 9.06 7.26 8 8.81 10.465 8 8.11 25.81 8 7.90 5.306 8 8.22 5.90 - - -7 - - - 8 6.80 4.99

8A 8 8.07 5.12 - - -8B 8 7.72 5.58 - - -9 8 8.05 9.98 8 9.16 4.59

10 8 6.83 3.47 8 8.00 5.6711 8 7.75 7.45 8 7.95 4.2812 - - - 8 8.88 6.4313 8 9.39 4.75 - - -14 - - - 8 8.08 5.8415 - - - 8 9.73 4.3616 8 9.06 2.96 8 8.86 1.5617 8 9.17 4.42 8 9.91 9.0518 - - - 8 7.88 17.7219 8 8.84 4.78 8 8.89 3.1620 8 7.64 16.77 - - -21 - - - 8 8.03 2.9822 8 7.08 10.59 - - -23 - - - 8 8.67 10.0824 8 7.28 2.53 8 8.31 6.3025 - - - 8 9.55 6.1526 - - - 4 5.30 5.7927 8 7.92 10.58 - - -28 - - - 8 6.87 10.7329 8 7.49 3.22 8 8.24 4.2730 8 8.62 2.34 - - -31 - - - 8 8.49 5.8732 - - - 8 9.27 4.6933 - - - 8 8.72 29.2934 8 8.97 6.82 - - -35 - 8 8.05 6.2536 8 10.21 3.06 - - -

37A - - - 8 8.18 15.4637B - - - 8 8.60 10.4638 8 8.34 4.05 8 7.49 11.70

Inter-Lab GCV = 10.48% Inter-Lab GCV = 17.00%




Lab No.




Table 3 - Mean laboratory estimates for sample C relative to WHO 7th IS FVIII Concentrate (sample 7th IS - 11.0IU/ml) by one-stage and chromogenic assays together with estimates of intra-

laboratory variability (GCV%) for individual laboratories and inter-laboratory variability for method estimates and combined estimates


(n) (IU/ml) (n) (IU/ml)1 - - - 8 10.67 6.652 8 9.33 3.66 8 11.12 11.323 - - - 8 10.46 8.904 8 9.41 8.61 8 11.19 6.675 8 9.51 2.32 8 10.21 3.736 8 9.41 3.27 -7 - - - 8 10.47 7.63

8A 8 9.87 2.01 - - -8B 8 9.77 4.57 - - -9 8 9.84 4.93 8 10.10 3.36

10 8 9.67 2.69 8 9.96 6.8411 8 9.47 4.73 8 10.17 2.1512 - - - 8 10.54 4.4613 8 10.43 8.71 - - -14 - - - 8 10.94 5.4315 - - - 8 10.10 4.7616 8 9.85 0.91 8 10.56 1.6917 8 9.92 2.76 8 10.24 0.6318 - - - 8 10.19 4.2119 8 9.37 3.13 8 10.19 2.2920 8 9.72 1.55 - - -21 - - - 8 10.48 2.5822 8 9.18 5.79 - - -23 - - - 8 10.54 5.9124 8 9.49 7.88 8 10.15 2.5225 - - - 8 10.61 4.7526 - - - 4 10.34 3.0327 8 9.17 9.86 - - -28 - - - 7 10.87 6.4929 8 8.84 1.31 8 9.05 6.3530 8 9.55 1.13 - - -31 - - - 8 10.46 8.4132 - - - 8 10.68 3.6233 - - - 8 10.70 8.9334 8 9.57 3.79 - - -35 - - - 8 10.48 3.2036 8 10.14 7.05 - - -

37A - - - 8 10.52 1.8537B - - - 8 9.83 1.3138 8 9.68 3.18 8 9.07 30.31





Lab No.





Table 4 - Mean laboratory estimates for sample D relative to WHO 7th IS FVIII Concentrate (sample 7th IS - 11.0IU/ml) by one-stage and chromogenic assays together with estimates of intra-

laboratory variability (GCV%) for individual laboratories and inter-laboratory variability for method estimates and combined estimates


(n) (IU/ml) (n) (IU/ml)1 - - - 8 3.07 9.392 8 4.89 21.02 8 5.67 10.493 - - - 8 3.68 9.494 8 5.43 7.86 8 5.82 11.245 8 5.12 20.57 8 5.02 7.626 8 5.36 3.08 - - -7 - - - 8 3.81 5.42

8A 8 5.60 6.22 - - -8B 8 5.42 3.47 - - -9 8 4.73 6.05 8 6.01 2.31

10 8 4.62 4.53 8 5.29 1.7511 8 5.08 3.14 8 5.68 2.9012 - - - 8 5.32 7.8413 8 5.78 4.28 - - -14 - - - 8 5.10 8.5315 - - - 8 6.22 3.4716 8 5.48 0.53 8 5.50 2.8317 8 5.75 2.51 8 6.34 4.7518 - - - 8 4.58 3.7919 8 5.42 4.75 8 5.70 4.2820 8 5.74 20.69 - - -21 - - - 8 5.68 8.2622 8 4.63 11.93 - - -23 - - - 8 5.05 10.0124 8 4.51 4.39 8 5.65 10.2625 - - - 8 6.26 2.9926 - - - 4 3.68 6.8827 8 5.06 8.89 - - -28 - - - 8 4.29 9.3329 8 2.92 14.45 8 2.99 10.5030 8 5.47 1.12 - - -31 - - - 8 5.36 6.1632 - - - 8 5.72 7.1933 - - - 8 5.58 23.2634 8 5.60 6.14 - - -35 - - - 8 5.41 4.2636 8 6.43 2.29 - - -

37A - - - 8 5.43 17.1737B - - - 8 5.63 7.8738 8 5.21 4.87 8 4.99 7.42


ChromogenicO ne-Stage


Inter-Lab GCV = 16.54% Inter-Lab GCV = 22.15%


Lab No.



Table 5 - Mean laboratory estimates for sample E (Ph. Eur. BRP Batch 3/Mega 2 US) relative to WHO 7th IS FVIII Concentrate (sample 7th IS - 11.0 IU/ml) by one-stage and chromogenic assays together with estimates of intra-laboratory variability (GCV%) for individual laboratories and inter-laboratory variability for method estimates

and combined estimates


(n) (IU/ml) (n) (IU/ml) 1 - - - 8 8.51 5.522 8 13.59 5.57 8 8.64 8.763 - - - 8 8.55 7.854 8 13.18 8.35 8 9.41 14.735 8 12.65 9.77 8 8.74 3.486 8 11.66 8.02 - - -7 - - - 8 8.62 5.98

8A 8 11.20 5.85 - - -8B 8 11.78 3.84 - - -9 8 10.62 4.49 8 9.05 3.28

10 8 10.94 4.78 8 8.52 2.9911 8 11.51 4.76 8 8.42 5.4012 - - - 8 8.10 2.8113 8 10.87 10.42 - - -14 - - - 8 7.86 11.1015 - - - 8 8.58 1.1116 8 12.76 4.31 8 8.42 3.9817 8 11.32 2.93 8 8.79 4.5618 - - - 8 8.53 3.1019 8 12.65 4.28 8 8.62 2.9320 8 12.89 2.84 - - -21 - - - 8 8.43 5.3222 8 11.88 12.99 - - -23 - - - 8 8.50 5.8424 8 11.06 4.48 8 8.76 2.7625 - - - 8 8.36 7.1526 - - - 8 8.71 3.3927 8 11.25 16.24 - - -28 - - - 8 8.43 6.0829 8 11.31 10.55 8 6.17 10.1030 8 11.58 2.90 - - -


Lab No.Number of Geometric Intra-lab Number of Geometric Intra-lab

Assays Mean GCV% Assays Mean GCV%(n) (IU/ml) (n) (IU/ml)

1 - - - 8 8.51 5.522 8 13.59 5.57 8 8.64 8.763 - - - 8 8.55 7.854 8 13.18 8.35 8 9.41 14.735 8 12.65 9.77 8 8.74 3.486 8 11.66 8.02 - - -7 - - - 8 8.62 5.98

8A 8 11.20 5.85 - - -8B 8 11.78 3.84 - - -9 8 10.62 4.49 8 9.05 3.28

10 8 10.94 4.78 8 8.52 2.9911 8 11.51 4.76 8 8.42 5.4012 - - - 8 8.10 2.8113 8 10.87 10.42 - - -14 - - - 8 7.86 11.1015 - - - 8 8.58 1.1116 8 12.76 4.31 8 8.42 3.9817 8 11.32 2.93 8 8.79 4.5618 - - - 8 8.53 3.1019 8 12.65 4.28 8 8.62 2.9320 8 12.89 2.84 - - -21 - - - 8 8.43 5.3222 8 11.88 12.99 - - -23 - - - 8 8.50 5.8424 8 11.06 4.48 8 8.76 2.7625 - - - 8 8.36 7.1526 - - - 8 8.71 3.3927 8 11.25 16.24 - - -28 - - - 8 8.43 6.0829 8 11.31 10.55 8 6.17 10.1030 8 11.58 2.90 - - -31 - - - 8 8.71 3.3532 - - - 8 8.77 6.7533 - - - 8 7.55 20.0234 8 11.81 7.46 - - -35 - - - 8 8.82 7.7136 8 12.37 7.17 - - -

37A - - - 8 9.11 4.7037B - - - 8 9.12 2.8838 8 11.37 3.28 8 8.57 18.89





Lab No.





Table 6 - Mean laboratory estimates for sample A relative to Ph. Eur. BRP Batch 3/Mega 2 US (sample E: one-stage potency - 11.3 IU/ml; chromogenic potency- 8.6 IU/ml) by one-stage and chromogenic assays together

with estimates of intra-laboratory variability (GCV%) for individual laboratories and inter-laboratoryvariability for method estimates and combined estimates


(n) (IU/ml) (n) (IU/ml) 1 - - - 8 9.50 3.522 8 7.76 6.18 8 8.78 12.723 - - - 8 9.91 7.534 8 8.61 5.67 8 10.03 8.915 8 8.25 1.73 8 9.18 4.166 8 9.22 8.48 - - -7 - - - 8 9.61 2.83

8A 8 9.74 4.55 - - -8B 8 9.05 5.11 - - -9 8 9.65 5.02 8 8.77 3.52

10 8 9.37 2.94 8 8.88 5.0011 8 9.08 2.88 8 9.19 4.7312 - - - 8 9.84 1.9613 8 11.70 8.89 - - -14 - - - 8 10.17 7.4115 - - - 8 9.55 2.5116 8 8.36 1.20 8 9.42 1.0017 8 9.62 2.16 8 9.15 2.5518 - - - 8 9.58 2.5219 8 8.21 4.89 8 9.37 4.6120 8 8.27 4.59 - - -21 - - - 8 9.17 3.9222 8 9.78 9.20 - - -23 - - - 8 9.57 5.5924 8 8.89 7.24 8 9.33 3.9425 - - - 8 10.09 9.9526 - - - 4 9.03 6.7127 8 9.35 9.74 - - -28 - - - 8 10.32 8.8729 8 8.20 13.52 8 12.27 14.3430 8 8.92 2.80 - - -




1 - - - 8 9.50 3.522 8 7.76 6.18 8 8.78 12.723 - - - 8 9.91 7.534 8 8.61 5.67 8 10.03 8.915 8 8.25 1.73 8 9.18 4.166 8 9.22 8.48 - - -7 - - - 8 9.61 2.83

8A 8 9.74 4.55 - - -8B 8 9.05 5.11 - - -9 8 9.65 5.02 8 8.77 3.52

10 8 9.37 2.94 8 8.88 5.0011 8 9.08 2.88 8 9.19 4.7312 - - - 8 9.84 1.9613 8 11.70 8.89 - - -14 - - - 8 10.17 7.4115 - - - 8 9.55 2.5116 8 8.36 1.20 8 9.42 1.0017 8 9.62 2.16 8 9.15 2.5518 - - - 8 9.58 2.5219 8 8.21 4.89 8 9.37 4.6120 8 8.27 4.59 - - -21 - - - 8 9.17 3.9222 8 9.78 9.20 - - -23 - - - 8 9.57 5.5924 8 8.89 7.24 8 9.33 3.9425 - - - 8 10.09 9.9526 - - - 4 9.03 6.7127 8 9.35 9.74 - - -28 - - - 8 10.32 8.8729 8 8.20 13.52 8 12.27 14.3430 8 8.92 2.80 - - -31 - - - 8 9.10 9.6732 - - - 8 9.25 7.7133 - - - 8 10.56 8.9434 8 8.84 5.84 - - -35 - - - 8 9.27 2.7036 8 9.75 12.57 - - -

37A - - - 8 8.99 3.2137B - - - 8 8.57 3.8038 8 9.59 1.93 8 9.51 15.96





Lab No.





Table 7 - Mean laboratory estimates for sample B relative to Ph. Eur. BRP Batch 3/Mega 2 US (sample E: one-stage potency - 11.3 IU/ml; chromogenic potency- 8.6 IU/ml) by one-stage and chromogenic assays

together with estimates of intra-laboratory variability (GCV%) for individual laboratories and inter-laboratory variability for method estimates and combined estimates


(n) (IU/ml) (n) (IU/ml) 1 - - - 8 5.44 6.862 8 7.27 8.36 8 8.2 17.23 - - - 8 5.89 14.574 8 7.55 2.1 8 8.01 13.235 8 7.27 36.32 8 7.62 3.86 8 7.86 4.49 - - -7 - - - 8 6.84 1.81

8A 8 8.24 7.75 - - -8B 8 7.46 7.24 - - -9 8 8.59 10.42 8 8.81 4.36

10 8 7.3 5.68 8 8.25 7.4311 8 7.7 8.48 8 8.24 7.2512 - - - 8 9.35 4.6413 8 9.76 9.67 - - -14 - - - 8 9.16 14.215 - - - 8 9.75 4.8416 8 8.26 4.76 8 9.33 2.6517 8 9.27 4.75 8 9.88 12.9518 - - - 8 7.82 18.7419 8 7.9 4.54 8 8.91 3.7820 8 6.69 20.1 - - -21 - - - 8 8.49 7.1322 8 6.09 8.58 - - -23 - - - 8 8.58 4.5524 8 7.52 2.57 8 8.24 7.5725 - - - 8 9.66 10.7326 - - - 4 5.22 6.4427 8 7.51 6.86 - - -28 - - - 8 6.76 9.8329 8 7.62 5.08 8 11.22 6.0930 8 8 5 0 9 - - -




1 - - - 8 5.44 6.862 8 7.27 8.36 8 8.2 17.23 - - - 8 5.89 14.574 8 7.55 2.1 8 8.01 13.235 8 7.27 36.32 8 7.62 3.86 8 7.86 4.49 - - -7 - - - 8 6.84 1.81

8A 8 8.24 7.75 - - -8B 8 7.46 7.24 - - -9 8 8.59 10.42 8 8.81 4.36

10 8 7.3 5.68 8 8.25 7.4311 8 7.7 8.48 8 8.24 7.2512 - - - 8 9.35 4.6413 8 9.76 9.67 - - -14 - - - 8 9.16 14.215 - - - 8 9.75 4.8416 8 8.26 4.76 8 9.33 2.6517 8 9.27 4.75 8 9.88 12.9518 - - - 8 7.82 18.7419 8 7.9 4.54 8 8.91 3.7820 8 6.69 20.1 - - -21 - - - 8 8.49 7.1322 8 6.09 8.58 - - -23 - - - 8 8.58 4.5524 8 7.52 2.57 8 8.24 7.5725 - - - 8 9.66 10.7326 - - - 4 5.22 6.4427 8 7.51 6.86 - - -28 - - - 8 6.76 9.8329 8 7.62 5.08 8 11.22 6.0930 8 8.5 0.9 - - -31 - - - 8 8.52 5.1532 - - - 8 9.05 14.5233 - - - 8 10.15 11.9234 8 8.67 12.32 - - -35 - - - 8 8.13 7.3836 8 9.11 6.27 - - -

37A - - - 8 7.54 19.3137B - - - 8 8.1 9.9638 8 8.2 5.01 8 7.15 24.65





Lab No.





Table 8 - Mean laboratory estimates for sample C relative to Ph. Eur. BRP Batch 3/Mega 2 US (sample E: one-stage potency - 11.3 IU/ml; chromogenic potency- 8.6 IU/ml) by one-stage and chromogenic assays together

with estimates of intra-laboratory variability (GCV%) for individual laboratories and inter-laboratory variability for method estimates and combined estimates


(n) (IU/ml) (n) (IU/ml) 1 - - - 8 10.60 1.262 8 7.71 8.36 8 10.49 11.953 - - - 8 10.52 8.564 8 8.30 7.83 8 10.28 13.435 8 8.47 3.30 8 10.25 5.176 8 9.23 9.12 - - -7 - - - 8 10.36 2.78

8A 8 9.84 4.32 - - -8B 8 9.29 4.92 - - -9 8 10.44 5.80 8 9.49 4.23

10 8 9.66 3.17 8 9.86 6.2711 8 9.18 5.20 8 10.22 3.5712 - - - 8 11.30 5.3113 8 10.85 2.54 - - -14 - - - 8 11.55 7.6915 - - - 8 10.13 5.5216 8 8.47 1.54 8 10.46 3.2717 8 9.78 3.01 8 9.82 1.7318 - - - 8 10.44 2.0619 8 8.37 4.60 8 10.12 4.3720 8 8.53 2.66 - - -21 - - - 8 10.30 0.9222 8 9.65 6.41 - - -23 - - - 8 10.91 0.7424 8 9.59 5.12 8 9.87 5.3325 - - - 8 10.69 8.7726 - - - 4 10.23 4.0427 8 9.76 10.85 - - -28 - - - 7 11.25 1.6929 8 8.68 16.47 8 12.92 16.9330 8 9 22 2 58 - - -




1 - - - 8 10.60 1.262 8 7.71 8.36 8 10.49 11.953 - - - 8 10.52 8.564 8 8.30 7.83 8 10.28 13.435 8 8.47 3.30 8 10.25 5.176 8 9.23 9.12 - - -7 - - - 8 10.36 2.78

8A 8 9.84 4.32 - - -8B 8 9.29 4.92 - - -9 8 10.44 5.80 8 9.49 4.23

10 8 9.66 3.17 8 9.86 6.2711 8 9.18 5.20 8 10.22 3.5712 - - - 8 11.30 5.3113 8 10.85 2.54 - - -14 - - - 8 11.55 7.6915 - - - 8 10.13 5.5216 8 8.47 1.54 8 10.46 3.2717 8 9.78 3.01 8 9.82 1.7318 - - - 8 10.44 2.0619 8 8.37 4.60 8 10.12 4.3720 8 8.53 2.66 - - -21 - - - 8 10.30 0.9222 8 9.65 6.41 - - -23 - - - 8 10.91 0.7424 8 9.59 5.12 8 9.87 5.3325 - - - 8 10.69 8.7726 - - - 4 10.23 4.0427 8 9.76 10.85 - - -28 - - - 7 11.25 1.6929 8 8.68 16.47 8 12.92 16.9330 8 9.22 2.58 - - -31 - - - 8 10.15 9.2932 - - - 8 10.52 4.5533 - - - 8 11.93 20.2134 8 9.06 8.58 - - -35 - - - 8 9.86 3.0436 8 9.48 10.46 - - -

37A - - - 8 10.18 1.4737B - - - 8 9.28 2.6138 8 9.72 0.77 8 9.59 24.27





Lab No.





Table 9 - Mean laboratory estimates for sample D relative to Ph. Eur. BRP Batch 3/Mega 2 US (sample E: one-stage potency - 11.3 IU/ml; chromogenic potency- 8.6 IU/ml) by one-stage and chromogenic assays together

with estimates of intra-laboratory variability (GCV%) for individual laboratories and inter-laboratoryvariability for method estimates and combined estimates


(n) (IU/ml) (n) (IU/ml) 1 - - - 8 3.16 9.852 8 4.09 26.85 8 5.96 14.533 - - - 8 3.70 14.944 8 4.52 5.29 8 5.29 13.355 8 4.59 34.09 8 4.84 8.016 8 5.13 3.34 - - -7 - - - 8 3.83 5.20

8A 8 5.72 9.93 - - -8B 8 5.24 3.94 - - -9 8 5.05 9.14 8 5.78 1.79

10 8 4.93 4.02 8 5.45 3.7311 8 5.06 3.98 8 5.89 6.7312 - - - 8 5.62 6.8413 8 6.01 10.22 - - -14 - - - 8 5.78 9.9415 - - - 8 6.23 3.8616 8 5.00 3.95 8 5.79 4.6417 8 5.81 3.02 8 6.32 9.0818 - - - 8 4.55 4.8219 8 4.84 6.79 8 5.72 6.6420 8 5.03 17.07 - - -21 - - - 8 6.01 4.4422 8 3.98 11.51 - - -23 - - - 8 4.99 7.7224 8 4.66 4.32 8 5.60 11.0425 - - - 8 6.71 5.7026 - - - 8 3.62 8.1927 8 4.80 7.50 - - -28 - - - 8 4.22 8.14




1 - - - 8 3.16 9.852 8 4.09 26.85 8 5.96 14.533 - - - 8 3.70 14.944 8 4.52 5.29 8 5.29 13.355 8 4.59 34.09 8 4.84 8.016 8 5.13 3.34 - - -7 - - - 8 3.83 5.20

8A 8 5.72 9.93 - - -8B 8 5.24 3.94 - - -9 8 5.05 9.14 8 5.78 1.79

10 8 4.93 4.02 8 5.45 3.7311 8 5.06 3.98 8 5.89 6.7312 - - - 8 5.62 6.8413 8 6.01 10.22 - - -14 - - - 8 5.78 9.9415 - - - 8 6.23 3.8616 8 5.00 3.95 8 5.79 4.6417 8 5.81 3.02 8 6.32 9.0818 - - - 8 4.55 4.8219 8 4.84 6.79 8 5.72 6.6420 8 5.03 17.07 - - -21 - - - 8 6.01 4.4422 8 3.98 11.51 - - -23 - - - 8 4.99 7.7224 8 4.66 4.32 8 5.60 11.0425 - - - 8 6.71 5.7026 - - - 8 3.62 8.1927 8 4.80 7.50 - - -28 - - - 8 4.22 8.1429 8 2.96 9.72 8 4.07 16.0230 8 5.39 1.93 - - -31 - - - 8 5.37 6.5732 - - - 8 5.58 9.8933 - - - 8 6.50 7.0234 8 5.42 13.25 - - -35 - - - 8 5.47 10.4336 8 5.74 4.03 - - -

37A - - - 8 5.01 21.4437B - - - 8 5.30 6.8738 8 5.12 6.43 8 4.76 21.96





Lab No.





Table 10 - Mean laboratory estimates for sample 7th IS (WHO 7th IS - 99/678) relative to Ph. Eur. BRP Batch 3/Mega 2 US (sample E: one-stage potency - 11.3 IU/ml; chromogenic potency- 8.6 IU/ml) by one-stage and

chromogenic assays together with estimates of intra-laboratory variability (GCV%) for individuallaboratories and inter-laboratory variability for method estimates and combined estimates


(n) (IU/ml) (n) (IU/ml) 1 - - - 8 11.11 5.522 8 9.14 5.57 8 10.95 8.763 - - - 8 11.06 7.854 8 9.43 8.35 8 10.05 14.735 8 9.83 9.77 8 10.83 3.486 8 10.66 8.02 - - -7 - - - 8 10.97 5.98

8A 8 11.10 5.85 - - -8B 8 10.55 3.84 - - -9 8 11.70 4.49 8 10.45 3.28

10 8 11.36 4.78 8 11.11 2.9911 8 10.80 4.76 8 11.23 5.4012 - - - 8 11.68 2.8113 8 11.44 10.42 - - -14 - - - 8 12.03 11.1015 - - - 8 11.02 1.1116 8 9.74 4.31 8 11.23 3.9817 8 10.98 2.93 8 10.76 4.5618 - - - 8 11.09 3.1019 8 9.83 4.28 8 10.98 2.9320 8 9.64 2.84 - - -21 - - - 8 11.22 5.3222 8 10.46 12.99 - - -23 - - - 8 11.13 5.8424 8 11.24 4.48 8 10.80 2.7625 - - - 8 11.32 7.1526 - - - 8 10.86 3.3927 8 11.05 16.24 - - -28 - - - 8 11.22 6.0829 8 10.99 10.55 8 15.33 10.1030 8 10 73 2 90 - - -




1 - - - 8 11.11 5.522 8 9.14 5.57 8 10.95 8.763 - - - 8 11.06 7.854 8 9.43 8.35 8 10.05 14.735 8 9.83 9.77 8 10.83 3.486 8 10.66 8.02 - - -7 - - - 8 10.97 5.98

8A 8 11.10 5.85 - - -8B 8 10.55 3.84 - - -9 8 11.70 4.49 8 10.45 3.28

10 8 11.36 4.78 8 11.11 2.9911 8 10.80 4.76 8 11.23 5.4012 - - - 8 11.68 2.8113 8 11.44 10.42 - - -14 - - - 8 12.03 11.1015 - - - 8 11.02 1.1116 8 9.74 4.31 8 11.23 3.9817 8 10.98 2.93 8 10.76 4.5618 - - - 8 11.09 3.1019 8 9.83 4.28 8 10.98 2.9320 8 9.64 2.84 - - -21 - - - 8 11.22 5.3222 8 10.46 12.99 - - -23 - - - 8 11.13 5.8424 8 11.24 4.48 8 10.80 2.7625 - - - 8 11.32 7.1526 - - - 8 10.86 3.3927 8 11.05 16.24 - - -28 - - - 8 11.22 6.0829 8 10.99 10.55 8 15.33 10.1030 8 10.73 2.90 - - -31 - - - 8 10.86 3.3532 - - - 8 10.79 6.7533 - - - 8 12.53 20.0234 8 10.52 7.46 - - -35 - - - 8 10.73 7.7136 8 10.05 7.17 - - -

37A - - - 8 10.39 4.7037B - - - 8 10.37 2.8838 8 10.93 3.28 8 11.04 18.89





Lab No.





Table 11 - Intra-laboratory variability (GCV%) for estimates of samples A, B, C, D, E and 7th IS by all methods. Figures indicate number of laboratories with GCVs < 5% or > 10%

Table 12 - Summary table showing the overall mean potency estimates (IU/ml) for the 2 methods (one-stage and chromogenic assays) and the combined mean potency estimates (IU/ml) of samples A, B, C, D, E and 7th

IS together with the inter-laboratory variability (GCV%)

Inter-laboratory GCVs are given in parenthesis.

*Difference between methods statistically signi cant (p 10% GCVs < 5% GCVs > 10% GCVs < 10%

A 39/52 (75%) 2/52 (4%) 27/52 (52%) 5/52 (10%) 97/104 (93%)

B 20/52 (39%) 14/52 (27%) 15/52 (29%) 15/52 (29%) 75/104 (72%)

C 33/52 (64%) 2/52 (4%) 27/52 (52%) 8/52 (15%) 94/104 (90%)

D 24/52 (46%) 12/52 (23%) 15/52 (29%) 14/52 (27%) 78/104 (75%)

E 27/52 (52%) 11/52 (21%) - - -

7th IS - - 25/48 (75%) 9/52 (17%) -

Samples Assay vs 7th IS Assays vs BRP3 Assays vs both standards

O ne-Stage Chromogenic Combined Potency O ne-Stage ChromogenicCombined

Potency

(IU/ml) (IU/ml) (IU/ml) (IU/ml) (IU/ml) (IU/ml)

(n=22) (n=30) (n=52) (n=22) (n=30) (n=52)

A 9.44 (6.09%) 9.44 (4.11%) 9.44 (4.98%) 9.06 (9.29%) 9.51 (7.19%) 9.32 (8.44%)

B 8.26 (10.48%) 8.03 (17.00%) 8.13 (14.48%) 7.88 (11.39%) 8.16 (19.18%) 8.04 (16.23%)

C 9.59* (3.65%) 10.35 (4.83%) 10.02 (5.86%) 9.21* (8.48%) 10.43 (7.10%) 9.89 (10.08%)

D 5.14 (16.54%) 5.06 (22.15%) 5.09 (19.74%) 4.91 (16.56%) 5.15 (20.73%) 5.05 (19.07%)

E 11.80* (7.04%) 8.49 (7.63%) 9.76 (19.57%) - - -

7th IS - - - 10.53$ (7.04%) 11.14 (7.63%) 10.88 (7.89%)

Samples

Assay vs 7th IS Assays vs BRP3

O ne-Stage Chromogenic O ne-Stage Chromogenic

(n=22) (n=30) (n=22) (n=30)

A 100.0% 100.0% 97.2% 102.0%

B 101.6% 98.8% 98.0% 101.5%

C 95.7% 103.3% 93.1% 105.5%

D 101.0% 99.4% 97.2% 102.0%

E 120.9% 87.0% - -

7th IS - - 96.8% 102.4%

Samples

Assay vs 7th IS Assays vs BRP3



Figure 1 - Stacking histograms showing mean laboratory potency estimates (as a % of Overall Mean) for sampleA relative to: (a) 7th IS and (b) BRP Batch 3. Results from one-stage assays are shaded in grey;

results from chromogenic assays are unshaded (white)

Sample A vs IS

0123456789

101112131415161718192021222324252627282930

% of overall mean potency

50 60 70 80 90 100 110 120 130 140 150

2 9

2 9

0 9

1 9

2 4

2 7

0 5

1 0

1 1

1 2

2 6

3 7 B

0 2

0 5

0 6

0 8 B

1 0

1 1

2 0

2 2

3 0

3 4

3 8

0 1

0 2

0 9

1 4

1 5

1 6

1 7

1 8

1 9

2 1

2 3

2 4

3 1

3 2

3 3

3 7 A

3 8

0 4

0 8 A

1 6

1 7

0 3

0 7

2 5

2 8

3 5

3 6 0 4 1 3

(a)

(b)

Sample A vs E

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50 60 70 80 90 100 110 120 130 140 150

02 04

05

16

19

20

29

37B

08B

11

24

30

34

02

09

10

26

37A

06

10

27

01

05

11

15

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31

32

35

38

08A

09

17

22

36

38

03

07

12

18

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04

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33 13 29



Figure 2 - Stacking histograms showing mean laboratory potency estimates (as a % of Overall Mean) for sampleB relative to: (a) 7th IS and (b) BRP Batch 3. Results from one-stage assays are shaded in grey;


(a)

(b)

Sample B vs IS

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50 60 70 80 90 100 110 120 130 140 150

0 1

2 6

0 3 1 0

2 2

0 7

2 8

2 4

2 9

3 8

0 8 B

1 1

2 0

2 7

0 2

0 5

1 8

0 5

0 6

0 8 A

0 9

1 0

1 1

1 4

2 1

2 4

2 9

3 5

3 7 A

0 2

3 0

3 8

2 3

3 1

3 3

3 7 B

0 4

1 6

1 9

3 4

0 4

1 2

1 6

1 9

1 3

1 7

0 9

2 5

3 2

1 5

1 7

3 6

Sample B vs E

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50 60 70 80 90 100 110 120 130 140 150

2 6 0 1 2 2

0 3

2 0

0 7

2 8

0 2

0 5

1 0

3 8

0 4

0 8 B

1 1

2 4

2 7

2 9

0 5

1 8

3 7 A

0 6

0 8 A

1 9

3 8

0 2

0 4

2 4

3 5

3 7 B

0 9

1 6

3 0

1 0

1 1

2 1

2 3

3 1

3 4

0 9

1 9

1 7

3 6

1 2

1 4

1 6

3 2

1 3

1 5

2 5

1 7

3 3

2 9



Figure 3 - Stacking histograms showing mean laboratory potency estimates (as a % of Overall Mean) for sampleC relative to: (a) 7th IS and (b) BRP Batch 3. Results from one-stage assays are shaded in grey;


(a)

(b)

Sample C vs IS

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50 60 70 80 90 100 110 120 130 140 150

22

27

29

29

38

02

04

05

06

08B

10

11

19

20

24

30

34

38

08A

09

16

17

36

05

09

10

11

15

17

18

19

24

37B

13

01

03

07

12

16

21

23

25

26

31

32

33

35

37A

02

04

14

28

Sample C vs E

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50 60 70 80 90 100 110 120 130 140 150

02 04

05

16

19

20

29

34

06

08B

11

24

30

36

09

37B

38

08A

10

17

22

27

38

10

15

17

19

24

35

09

01

02

03

04

05

07

11

16

18

21

26

31

32

37A

13

23

25

12

14

28

33 29



Figure 4 - Stacking histogram showing mean laboratory potency estimates (as a % of Overall Mean) for sampleD relative to: (a) 7th IS and (b) BRP Batch 3. Results from one-stage assays are shaded in grey;


(a)

(b)

Sample D vs IS

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50 60 70 80 90 100 110 120 130 140 150

29 01

29

03

26

07 28 10

22

24

18

02

09

05

11

27

38

05

14

23

38

04

06

08B

19

30

10

12

31

35

37A

08A

16

34

02

11

16

19

21

24

32

33

37B

13

17

20

04

09

15

36

17

25

Sample D vs E

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50 60 70 80 90 100 110 120 130 140 150

29 01 26 03

07

02

22

29

28 04

05

24

18

19

27

05

38

06

09

10

11

16

20

38

23

37A

08B

30

34

04

31

37B

10

12

24

32

35

08A

17

36

09

11

14

16

19

13

02

21

15

17

33 25



Figure 5 - Stacking histogram showing mean laboratory potency estimates (as a % of Overall Mean) for: (a) sample E (BRP Batch 3) relative to 7th IS and (b) sample 7th IS relative to E (BRP Batch 3). Results from one-

stage assays are shaded in grey; results from chromogenic assays are unshaded (white)

(a)

(b)

Sample E vs IS

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50 60 70 80 90 100 110 120 130 140 150

29 33 14 01

10

11

12

16

18

21

23

25

28

02

03

05

07

15

17

19

24

26

31

32

35

38

04

09

37A

37B

09

10

13

08A

17

24

27

29

38

06

08B

11

22

30

34

36 05

16

19

20

04 02

Sample IS vs E

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50 60 70 80 90 100 110 120 130 140 150

02

04

05

16

19

20

36

04

08B

22

34

09

37A

37B

06

08A

11

17

27

29

30

38

01

02

03

05

07

10

15

17

18

19

23

24

26

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32

35

38

10

13

24

11

12

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28

09

14

33 29



APPENDIX I:

Methods and reagents used by the participants in the study

Lab Method Pre-dilution: Buffer or Def Plasma Source of Factor VIII

Deficient Plasma Analyser APTT Reagent Used/

Chromogenic Kit Used/Kit

1 Chromogenic Def Plasma (P) Diagnostica Stago iEMS Reader COAMATIC FVIII Chromogenix

2 Chromogenic P Dade-Behring Dade-Behring BCS COAMATIC FVIII Chromogenix

2 One-Stage P Dade-Behring Dade-Behring BCS Dade-Behring; Dade Actin FSL

3 Chromogenic P Werfen Austria ACL 3000 COAMATIC FVIII Chromogenix

4 Chromogenic P Dade-Behring Bio-TEK: Synergy HT Reader COAMATIC FVIII

Chromogenix

4 One-Stage P Dade-Behring Trinity Biotechnology: KC-40 MicroTM Dade-Behring; Dade

Actin FSL

5 Chromogenic P Siemens Siemens: Behring Coagulation Timer COAMATIC FVIII

Chromogenix

5 One-Stage P Siemens Schnitger-Gross Siemens; Pathromtin SL

6 One-Stage P Dade-Behring Sysmex: Coagrex-100S Dade-Behring; Dade

Actin Activated Cephaloplastin Reagent

7 Chromogenic Buffer Not Used Not Given COAMATIC FVIII Chromogenix

8A One-Stage P HRF Inc. Beckman Coulter ACL Advance Beckman Coulter

APTT SP lyophilized

8B One-Stage P HRF Inc. General Diagnostics: Coag-A-Mate X2 BioMerieux Automated

APTT

9 Chromogenic P Dade-Behring ACL Advance COAMATIC FVIII Chromogenix

9 One-Stage P Dade-Behring Dade-Behring BCS-XP Dade-Behring; Actin FS

10 Chromogenic P George King Tecan Robotic Processor COATEST SP4 FVIII

Chromogenix

10 One-Stage P George King BioMerieux: Coag-a-Mate MTX BioMerieux Micronized

silica/Platelin L

11 Chromogenic P Helena Biosciences ACL 200/ACL 200 Plus/ACL 3000 Plus COAMATIC FVIII

Chromogenix

11 One-Stage P Helena Biosciences ACL 200/ACL 200 Plus/ACL 3000 Plus IL; Micronized silica +

bovine brain (lyo)

12 Chromogenic P Dade-Behring VersaMax Molecular Devices COAMATIC FVIII

Chromogenix 13 One-Stage P Dade-Behring ACL Elite Pro Ilex APTT reagent

14 Chromogenic P Dade-Behring Spectra Max Plus micro plate reader COAMATIC FVIII

Chromogenix

15 Chromogenic P Dade-Behring ACL Advance COAMATIC FVIII Chromogenix

16 Chromogenic P Dade-Behring Plate Reader COAMATIC FVIII Chromogenix

16 One-Stage P Dade-Behring STAGO Diagnostica STA-R IL; HemosIL APTT-SP

(liquid)

17 Chromogenic P Dade-Behring ACL Futura COATEST SP4 FVIII Chromogenix

17 One-Stage P Dade-Behring ACL Futura IL; HemosIL APTT-SP (liquid)

18 Chromogenic P Dade-Behring Not Given COAMATIC FVIII Chromogenix



Analyser

Used/Kit

19 Chromogenic P Dade-Behring Automate Sysmex CA 540Dade-Behring Chromogenic

Kit

19 One-Stage P Dade-Behring Automate Sysmex CA540Dade-Behring; Dade Actin

FSL

20 One-Stage P Siemens Amelung KC-10A microSiemens; Dade Actin Activated

Cephaloplastin

21 Chromogenic P Technoclone iEMS ReaderCOAMATIC FVIII

Chromogenix22 One-Stage P Dade-Behring ACL 9000 IL; SynthasiL

23 Chromogenic P Dade-Behring iEMS Reader MFCOAMATIC FVIII

Chromogenix

24 Chromogenic P Baxter Plate Reader Baxter; Immunochrom FVIII C

24 One-Stage P Baxter Amelung KC-4A micro Technoclone; DAPTTIN

25 Chromogenic P Dade-Behring ACL ?COAMATIC FVIII

Chromogenix

26 Chromogenic P Diagnostic Grifols ACL 3000 & ACL Elite ProCOAMATIC FVIII

Chromogenix

27 One-Stage P HRF Inc. ACL 7000 BioMerieux Automated APTT

28 Chromogenic P Diagnostic Grifols Amelung: AMAX CS-190COAMATIC FVIII

Chromogenix

29 Chromogenic P HRF Inc. ACL AdvanceCOAMATIC FVIII

Chromogenix29 One-Stage P HRF Inc. ACL Advance Trinity

30 One-Stage P Vital Products Sysmex CA-50 Dade-Behring

31 Chromogenic P Dade-BehringMolecular Devices: T-Max

readerCOATEST SP4 FVIII

Chromogenix

32 Chromogenic P Dade-Behring ACL 3000 PlusCOATEST SP4 FVIII

Chromogenix

33 Chromogenic P Dade-Behring Plate ReaderDade-Behring Chromogenic

Kit

34 One-Stage P HRF Inc. Dade-Behring BCSPacific Hemostasis;

KONTACT

35 Chromogenic P Dade-Behring Dade-Behring BCS-XPDade-Behring Chromogenic

Kit

36 One-Stage P In-house SHP Dade-Behring BCSPacific Hemostasis;

KONTACT37A Chromogenic P Not Given Not Given Not Given

37B Chromogenic P Not Given Not Given Not Given

38 Chromogenic P Baxter Plate ReaderCOATEST SP4 FVIII

Chromogenix38 One-Stage P Baxter Dade-Behring BCS-XP Technoclone; DAPTTIN

Lab MethodPre-dilution: Buffer or Def

Plasma

Source of Factor VIII Deficient Plasma

APTT Reagent Used/ Chromogenic Kit



APPENDIX II:

Laboratories Own Calculations / Potency relative to IS (= 11.0 IU/ml) / One-stage assays

A B C D E

Lab Method

2 9.72 8.70 9.41 4.87 13.61

4 9.76 9.06 9.46 5.44 13.18

5 9.30 7.10 9.50 4.60 12.70

6 9.60 8.30 9.60 5.40 11.60

08A 9.80 8.10 9.90 5.50 12.00

08B 9.50 7.80 9.80 5.50 12.60

9 9.10 8.07 9.82 4.74 10.60

10 9.35 6.83 9.66 4.62 10.94

11 9.37 8.22 9.47 5.08 11.54

13 10.99 9.23 10.24 5.67 10.85

16 9.70 9.10 9.80 5.50 12.70

17 9.54 8.64 9.84 5.57 11.52

19 9.20 8.80 9.40 5.40 12.80

20 9.35 7.62 9.67 5.77 12.80

22 10.71 7.28 10.88 7.97 12.17

24 9.07 7.27 9.51 4.63 10.58

27 8.97 8.15 9.48 5.20 11.67

29 9.20 8.00 9.90 5.60 11.70

30 9.23 8.59 9.56 5.46 11.53

34 9.34 8.97 9.58 5.61 11.84

36 9.55 9.38 9.31 5.91 10.83

38 9.56 8.35 9.69 5.22 11.09

Sample

O ne-Stage



Laboratories Own Calculations / Potency relative to IS (= 11.0 IU/ml) / Chromogenic assays

A B C D E

Lab Method

1 9.4 5.3 10.6 3.1 8.5

2 9.74 7.78 11.25 5.58 8.74

3 9.93 6.46 11.12 3.91 8.66

4 11.01 8.53 11.32 5.67 9.34

5 9.1 7.9 10.2 5 8.7

7 9.5 6.75 10.37 3.82 8.52

9 9.37 9.15 10.11 6 8.98

10 8.97 8.05 10.01 5.29 8.53

11 9.14 8.38 10.14 5.68 8.55

12 9.6 9.2 10.9 5.5 8.9

14 . . . . .

15 9.6 9.9 10.5 6.2 8.7

16 9.5 8.8 10.2 5.5 8.5

17 9.5 9.94 10.19 6.34 8.74

18 9.37 7.71 10.02 4.58 8.43

19 9.6 8.9 10.2 5.9 8.7

21 9.1 8.1 10.2 5.7 8.2

23 9.26 8.59 10.48 5.08 8.5

24 9.65 8.39 10.2 5.74 8.92

25 9.82 9.32 10.35 6.14 8.38

26 9 5.26 10.23 3.62 8.66

28 10.2 7.4 11 4.4 8.5

29 9.4 9.1 9.8 5.4 8.5

31 9.71 8.6 10.6 5.44 8.73

32 9.09 9.11 10.73 6.07 8.98

33 9.9 9.5 10.65 5.43 7.7

35 9.74 8.05 10.48 5.41 8.82

37A 9.42 8.3 10.64 5.53 8.19

37B 9.18 8.76 9.91 5.71 8.16

38 9.66 7.62 10.9 5.07 9.07

Chromogenic

Sample



Laboratories Own Calculations / Potency relative to BRP3 (= 11.3 IU/ml) / One-stage assays

A B C D IS

Lab Method

02 7.90 7.45 7.88 4.03 9.11

4 8.42 7.39 8.15 4.45 9.23

5 9.20 6.80 9.40 4.40 10.80

6 9.30 8.20 9.30 5.30 10.70

08A 9.20 7.60 9.30 5.20 10.30

08B 8.60 6.90 8.80 4.90 9.90

9 9.70 8.62 10.47 5.06 11.74

10 9.16 7.18 9.45 4.84 11.10

11 9.17 8.12 9.25 5.00 10.99

13 11.43 9.66 10.63 5.92 11.23

16 8.50 8.50 8.60 5.10 9.90

17 9.17 8.69 9.46 5.60 10.38

19 8.40 8.10 8.60 5.00 10.00

20 8.27 6.77