who/bs/2015.2261 english only expert committee on
TRANSCRIPT
WHO/BS/2015.2261
ENGLISH ONLY
EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION
Geneva, 12 to 16 October 2015
COLLABORATIVE STUDY REPORT ON BLOOD COAGULATION FACTOR IX A. Value assignments:
1. Addition of Factor IX antigen value to The 4th
International Standard for Blood
Coagulation Factors II, VII, IX, X, Plasma, 09/172
2. The WHO 5th International Standard for Blood Coagulation Factor IX, Concentrate and
The Ph Eur BRP for Human Coagulation Factor IX, Concentrate, Batch 3
B. Investigation of the suitability of the 4th
International Standard for Blood Coagulation Factor
IX, Concentrate as a potency standard for purified full length recombinant FIX
1Elaine Gray, John Hogwood, Thomas Dougall* and Peter Rigsby*
Haemostasis Section and *Biostatistics Section
National Institute for Biological Standards and Control
Potters Bar, Hertfordshire, UK.
Eriko Terao
Department of Biological Standardisation, OMCL network and HealthCare (DBO)
European Directorate for the Quality of Medicines and HealthCare (EDQM)
Council of Europe, Strasbourg, France 1Principal Investigator
NOTE:
This document has been prepared for the purpose of inviting comments and suggestions on the
proposals contained therein, which will then be considered by the Expert Committee on
Biological Standardization (ECBS). Comments MUST be received by 14 September 2015 and
should be addressed to the World Health Organization, 1211 Geneva 27, Switzerland, attention:
Technologies, Standards and Norms (TSN). Comments may also be submitted electronically to
the Responsible Officer: Dr M Nübling at email: [email protected]
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WHO/BS/2015.2261
Page 2
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Summary
There are three main aims for this collaborative study:
1. to add a FIX antigen value to the 4th
International Standard for Blood Coagulation Factors II,
VII, IX and X, Plasma, Human (09/172);
2. to value assign a World Health Organisation (WHO) replacement International Standard (IS)
for Blood Coagulation Factor IX, Concentrate, Human and a replacement Pharmacopoeial (Ph
Eur) Biological Reference Preparation (BRP) for Blood Coagulation FIX, and
3.To investigate the suitability of the 4th
International Standard for Blood Coagulation Factor IX,
Concentrate as a potency standard for purified full length recombinant FIX.
Assignment of antigen value to the 4th International Standard for Blood Coagulation Factors II,
VII, IX and X, Plasma, Human (09/172) relative to local normal plasma pool was carried out by
15 laboratories returning 17 sets of data in total for analysis. Only 5 sets of data gave intra-
laboratory GCVs greater than 10%. There was good inter-laboratory agreement, GCV was 7.9%.
It was clear that local pools or the 4th
IS for FIX Plasma could not be used as an antigen
measurement standard for recombinant products since some reagent kits gave much lower results
than other kits and this is reflected in the high inter-laboratory variation (GCVs A: 26.6%; D;
31.5%). It is recommended that a FIX antigen value of 0.90 IU/ampoule be added to the label of
the 4th
IS for International Standard for Blood coagulation Factors II, VII, IX, X, Plasma.
Fifty laboratories from 18 countries took part for the value assignment of the plasma derived 5th
IS for FIX, Concentrate and Ph Eur BRP for FIX Concentrate, batch 3 relative to the 4th
IS for
FIX Concentrate, with 55 sets of clotting assay and 15 sets of chromogenic assay results returned
for analysis. The intra-laboratory variability was reasonably low, with the majority of geometric
coefficient of variation (GCV) below 10%. Lower inter-laboratory agreement was obtained for
sample B, 14/148 than for sample C, 14/162. Although there were no assay discrepancies
between clotting and chromogenic assays for either samples, a significantly lower (~6%) potency
was obtained for sample C with clotting assays when buffer rather than FIX deficient plasma was
used as pre-diluent. It is recommended that sample B, 14/148 be established as the 5th
International Standard for Blood Coagulation Factor IX, Concentrate and the Ph Eur BRP for
Blood coagulation Factor IX Batch 3, with an assigned value of 10.5 IU/ampoule.
The study also investigated the comparability of the plasma derived concentrate standard with the
full length recombinant FIX products and considered the feasibility of establishing an
International Standard for Recombinant FIX. There are currently 3 full length recombinant FIX
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products licenced and these were all represented in this collaborative study. Data from 49
laboratories (55 sets of clotting, 15 sets of chromogenic results) for two candidate recombinant
samples, A and D were received with additional results for another full length recombinant test
sample (Purple) returned by 6 laboratories. The intra-laboratory variability when samples A, D
and Purple were assayed against the 4th
IS for FIX Concentrate was acceptably low, majority
being less than 10%. In terms of inter-laboratory agreement, the clotting assay variability was
markedly high. For recombinant sample A, the potencies ranged 7.7 - 12.4 IU/ampoule (up to
62% discrepancy), with overall inter-laboratory GCV of 11.6% and for sample D, the potencies
ranged 6.6 – 12.4 IU/ampoule (up to 88% discrepancy), with overall inter-laboratory GCV of
13.4%. There were no major differences in the inter-laboratory GCVs for the chromogenic
assays. The inter-laboratory GCVs for overall potency estimates (including both clotting and
chromogenic assays) relative to the 4th
IS were high for both recombinant samples A (15.5%) and
D (16.8%); however, the GCV was reduced to 5.7% for Sample A when it was reanalysed using
sample D as the putative standard. In addition, reanalysis of clotting and chromogenic data for
sample A relative to sample D also markedly reduced the clotting and chromogenic discrepancy
observed when sample A was assayed against the 4th
IS for FIX Concentrate
(clotting/chromogenic ratios: vs 4th
IS – 1.25 (p <0.001) vs D – 0.98 (p=0.282). Although the full
length recombinant FIX could be assayed against the plasma derived 4th
IS Concentrate and
provided statistically valid results, there was high assay discrepancies amongst the clotting
assays using different APTT reagents and there was also significant clotting and chromogenic
assay discrepancies. The data from the present study indicated that a recombinant standard for
recombinant products will minimise assay discrepancies and improve inter-laboratory agreement.
Except for one laboratory who disagreed with a proposal of establishing an International Standard
for Recombinant FIX on the grounds that this may cause a shift in the amount of proteins in the
final products, all other participants agreed with the proposal. The data have also been evaluated
by 8 experts nominated by the Scientific and Standardisation Committee (SSC) of the
International Society for Thrombosis and Haemsotasis (ISTH). Seven SSC experts agreed with
the proposal, with 2 experts strongly support the establishment of an International Standard for
Recombinant FIX based on the reduction in assay discrepancies. One SSC Expert strongly
opposed and indicated that the use of multiple International Standards for the same coagulation
factor should be avoided whenever possible. Because of these 2 objections and after discussion
with the ISTH/WHO Liaison Group, it is unlikely that SSC will endorse such a proposal and
therefore the ECBS will not be requested to consider the establishment of an International
Standard for Recombinant FIX. The results and analysis for this investigation presented in this
report is for information only.
Proposals for establishment:
Sample P, NIBSC code 09/172: FIX antigen value: 0.90 IU/ampoule
WHO 4th
International Standard for Blood Coagulation Factors II, VII, IX, X, Plasma
Sample B, NIBSC code 14/148: Functional activity - 10.5 IU/ampoule
WHO 5th
International Standard for Blood Coagulation Factor IX, Concentrate
WHO/BS/2015.2261
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Introduction
The 4
th International Standard (IS) for Blood Coagulation Factor IX, Concentrate, Human
(07/182) was established by the Expert Committee on Biological Standardisation (ECBS) of the
World Health Organisation (WHO) in October 20081,2
. Part of this batch of material was also
established as the European Pharmacopoeia Biological Reference Preparation (BRP) Batch 2 and
the US FDA reference standard for Blood Coagulation Factor IX Concentrate. The stock level of
the WHO 4th
IS and the Ph Eur BRP reference standards are now near depletion and replacement
standards are required. The aim of the study is to assay factor IX concentrate candidate
preparations against the 4th
International Standard, 07/182, with a view to establish a new
material as the 5th
International Standard for Blood Coagulation Factor IX, Concentrate, and EP
Human Coagulation Factor IX Concentrate Batch 3. In addition, as there are now three
recombinant FIX products licensed, this study also included these 3 recombinant preparations (2
of which could be candidate International Standard for Recombinant FIX) with a view to assess
the need and possible establishment of an International Standard for recombinant FIX.
This study also serves to value assign a factor IX antigen value to the 4th International Standard
for Blood Coagulation Factors II, VII, IX and X, Human, Plasma, 09/172 and provides an
opportunity to assess the factor IX unitage as defined by the concentrate and plasma standards.
The replacement of 4th
IS for FIX, Concentrate project and the antigen value assignment to the 4th
IS for FII, VII, IX and X, plasma were endorsed by the WHO Expert Committee on Biological
Standardisation in October 2012 and October 2014 respectively.
Participants
Fifty laboratories agreed to participate, with 49 laboratories (6 Austria, 1 Australia, 4 Canada, 1
China, 1 Croatia, 2 Denmark, 5 France, 6 Germany, 1 Italy, 1 Japan, 1 Korea, 3 Netherlands, 1
Portugal, 2 Spain, 1 Sweden, 1 Switzerland, 6 UK, 6 USA) returning data for the study. The
participants included 7 diagnostic reagents manufacturers, 19 therapeutic manufacturers, 14
regulatory authorities and 9 clinical laboratories. A list of participants is given in Appendix I at
the end of this report. Each laboratory is referred to in this report by an arbitrarily assigned
number, not necessarily representing the order of listing in the Appendix.
Samples
Coded samples included in the study were:
S – the 4th
I.S. Factor IX, Concentrate, 07/182, potency 7.9 IU/ampoule
WHO/BS/2015.2261
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P – the 4th
I.S. Blood Coagulation Factor II, VII, IX, X, Plasma, 09/172, FIX potency 0.86
IU/ampoule
A – candidate sample, 07/142, recombinant FIX, nominal potency 8 – 10 IU/ampoule
B – candidate sample, 14/148, plasma derived FIX, nominal potency 10 – 12 IU/ampoule
C – candidate sample, 14/162, plasma derived FIX, nominal potency 8 – 10 IU/ampoule
D – candidate sample, 14/180, recombinant FIX, nominal potency 8 – 10 IU/ampoule
An additional frozen FIX concentrate sample, coded Pr (Purple) was also sent and assayed by 6
consenting participants.
The plasma pools for both the plasma derived candidates, the final products, the excipient human
albumin and the two International Standards have been tested negative for HBsAg, anti-HCV,
anti-HIV 1/2, and HCV RNA by PCR. All the candidates were prepared and freeze-dried
according to recommendations for the preparation, characterization and establishment of
international and other biological reference materials3. The product characteristics of each of the
candidates are summarised in Table 1. In addition, clotting times from activated coagulation
factors test (Non-activated partial Thromboplastin times, NAPTT), carried out in accordance with
Ph Eur method (2.6.22) and levels of activated factor IXa in the candidates are presented in
Tables 13 and 14 respectively.
With the exception of the local normal plasma pools, all samples were provided by NIBSC and
the participants were requested to reconstitute the samples according to protocol provided
(Appendix II).
Study design and assay methods
Details of the assay design were as stated in the protocol which is attached as Appendix II.
Briefly, each participant was requested to carry out 4 independent assays on 4 sets of samples and
to follow one of the suggested balanced assay designs as described in the study protocol.
Each participant was requested to perform their routine in-house method for FIX activity.
Multiple result sets returned by a participating laboratory were treated as results from an
independent laboratory and were given a separate lab code, e.g. Lab 2a, Lab 2b.
Statistical Analysis
Relative potencies of all samples in all assays were calculated by parallel line analysis with a log
transformation of assay response, using a minimum of three dilutions on a linear section of the
dose-response curve. Calculations were performed using the EDQM software CombiStats
Version 5.04. Non-linearity and non-parallelism were considered in the assessment of assay
validity. All dose-response lines showing no significant non-linearity (p>0.01) were accepted for
further analysis. All instances of significant non-linearity (p<0.01) were assessed visually and
those showing clear departures from linearity were excluded from further analysis. Non-
parallelism was assessed by calculation of the ratio of fitted slopes for the test and reference
WHO/BS/2015.2261
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samples under consideration. The samples were concluded to be non-parallel when the slope ratio
was outside of the range 0.80 – 1.25 and no estimates are reported.
Relative potency estimates from all valid assays were combined to generate an unweighted
geometric mean (GM) for each laboratory and these laboratory means were used to calculate
overall unweighted geometric means for each sample. Variability between assays within
laboratories and between laboratories has been expressed using geometric coefficients of
variation (GCV = {10s-1}×100% where s is the standard deviation of the log10 transformed
estimates). Comparisons between assay methods were made by two-tailed t-test of log
transformed laboratory mean estimates (paired or unpaired as appropriate for the comparison
being made).
The relative contents of the accelerated thermal degradation samples were used to fit an
Arrhenius equation relating degradation rate to absolute temperature assuming first-order decay5
and hence predict the degradation rates when stored at -20°C.
Results and Discussions
FIX Antigen value assignment to 4
th IS for FII, VII, IX, X, Plasma relative to
local normal plasma pools
The Candidate, 4th
IS for FII, VII, IX, X, Plasma, 09/172 The candidate 09/172, coded sample P in the study was established by the ECBS/WHO in 2010
as the 4th
IS for FII, VII, IX, X, Plasma, with functional potencies assigned for these 4 factors. It
was produced from a pool of 85 donations of platelet poor normal plasma (The Welsh Blood
Service), each collected in CPD-adenine and buffered with 0.05 M HEPES. The final product
was filled and freeze-dried according to guidelines for production of international biological
standards (WHO TRS, 20063). Each individual plasma donation has been tested and found
negative for anti-HIV 1/2, HBsAg and anti-hepatitis C. The product characteristics are shown in
the following table:
NIBSC Code 09/172
Presentation Sealed, glass 5 ml DIN ampoules
Number of Ampoules available 16,000
Liquid filling weight (g) Mean=1.1078; Range=1.1000 – 1.1140
CV of fill mass (%) 0.20 (n=849)
Mean dry weight (g, n = 5) 0.1034
Mean head space oxygen (%) 0.14 (n =12)
Residual moisture (%) 0.225 (n = 12)
Storage temperature -20°C
Address of processing facility NIBSC, Potters Bar, EN6 3QG, UK
Address of present custodian NIBSC, Potters Bar, EN6 3QG, UK
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Assay methods
The FIX antigen value assignment was relative to the local normal pooled plasmas. Participants
were requested to collect fresh plasma on two separate days to prepare two normal plasma pools.
It was requested that each fresh pool was tested in the study on the day of collection and that a
sample of the same pool should be frozen for use on a second day. The normal pool was to be
used as the standard in the antigen assays. With the exception of Lab 28 and Lab 40, the
laboratories were not able to prepare fresh plasma pools and used in-house frozen pools or
commercial normal plasma pools; the number of donors across all pools was > 1800.
In total, 17 sets of antigen results (66 assays in total) from 15 labs were returned. Five
commercial ELISA kits were used (Table 2C). Laboratory 2 returned three sets of assays coded
2a, 2b and 2c. All laboratories performed four independent assays except Lab 48 which was only
able to perform 2 assays.
This study also offered the opportunity to assess whether a plasma antigen standard would be an
appropriate comparator for therapeutic concentrates as purified plasma derived and recombinant
FIX concentrates (samples A, B, C and D) were included in this study.
Assay validity
The majority of antigen assays gave valid estimates of relative potency. A slightly higher
proportion of non-linear and non-parallel exclusions (both 2-4%) was observed, but this partially
reflects the use of a log transformation of assay response which was consistently applied in all
laboratories and this may not reflect the best transformation choice in a small number of cases.
Individual cases of exclusions due to non-linearity or non-parallelism are indicated in Appendix
III, Table S3.
Results
The antigen value was assessed relative to the local normal plasma pools (L). Individual assay
results are shown in Appendix III, Table S3. Individual laboratory mean and overall potency
estimates for antigen are presented in Tables 8 and 9A.
Antigen relative to local normal plasma pools
o The intra-laboratory variation (%GCV) ranged 1.8 – 27.1%, with 5 laboratories
having %GCV greater than 10% (Table 8, 9A).
o The inter-laboratory variability was 7.9% (Table 8, 9A) and there was no obvious
assay kit bias or any detectable outliers.
o The overall FIX antigen value for the 4th
IS for FII, VII, IX, X, plasma against
local pool was 0.90 u/ml (Table 8, 9A).
Antigen values for samples S, A, B, C, D relative to local normal pooled plasma, L and
sample P, assuming antigen value of 0.90 u/ml
o When samples S, B and C, the plasma derived samples were assayed against L, the
local normal pooled plasma, there was good laboratory agreement of antigen
values for sample S, B and C; the inter-laboratory %GCVs were 9.2%, 9.8% and
WHO/BS/2015.2261
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8.6% respectively (Table 9A) and these were slightly reduced when these samples
were assayed against P, the plasma IS, with an assumed value of 0.90 u/ml.
o The inter-laboratory %GCVs for A and D, the recombinant samples, were
extremely high (28.9% and 30.7% for samples A and D) and they were not
reduced when the samples were reanalysed against sample P (25.2% for A and
30.2% for D). The ranges of potencies against P for samples A and D were found
to be 3.8 – 9.4 u/ml and 3.1 – 8.1 u/ml respectively.
Discussion
Antigen measurements are carried out to assess phenotypes of haemophilia B, expression of
antigen in gene therapy and characterisation of therapeutic products. Since this is the first time a
FIX antigen value is assigned to an International Standard, the calibration is relative to local
normal plasma pool. Ideally, fresh normal plasma pool with large numbers of donors is used for
the comparison. However, for logistical reasons, it is difficult for laboratories to collect and
prepare fresh plasma to be used for assays on the same day. NIBSC have carried out in-house
studies to show that there was no significant difference in the antigen values of fresh plasmas and
their corresponding frozen/thawed samples (data not shown), the study therefore allowed the use
of fresh frozen plasmas as calibrators. With the exception of 2 laboratories (labs 28 and 40), all
laboratories used frozen pools (Table 2C) and although the total number of donors was not
known, it was >1800. Five different commercial reagents/kits were used: Asserachrom (8),
Visulize (5) and Zymutest (2), AssayPro (1) and Cedarlane (1) by 17 laboratories. The following
summarises the essential points for consideration:
Antigen value of sample P relative to local pooled plasma, L
o Intra-laboratory variability: 5 laboratories gave GCVs greater than 10% and only one
laboratory obtained GCV of 27% (Tables 8A, 9A). This indicates the kits and the
laboratories were able to measure antigen in plasma with reasonable precision.
o Inter-laboratory agreement: There was no significant difference in values by different
assay kits and outliers were not detected. Considering that the calibration was against
local pools, good agreement of antigen values was obtained; GCV was 7.9% (Tables
8, 9A). This may be partly aided by the sufficiently large number of donors in each
pool to offset the differences in individual donors. The overall value for sample P was
0.90 u/ampoule.
Antigen values of samples S, A, B, C and D relative to local normal pool plasma, L or
sample P, assuming assigned value of 0.90 u/ampoule
Low intra-laboratory variability was observed with the majority of GCVs less than
10% for all 4 samples (Table 9A) when assayed against L. The GCVs were
slightly reduced when these samples were assayed against P. (Table 9B)
The inter-laboratory GCVs for samples B and C, the plasma derived preparations
was reasonable when assayed against the local pool and were only marginally
reduced when assayed against P (Tables 9A, 9B).
The inter-laboratory GCVs for A and D, the recombinant samples, were extremely
high, being close to 30% when assayed against L (Table 9A) and were not reduced
when the samples were reanalysed against sample P (A: 26.6%; D 31.5%). The
WHO/BS/2015.2261
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ranges of potencies against P for samples A and D were found to be 3.8 – 9.4 U/ml
and 3.1 – 8.1 U/ml respectively. This indicates that antigen measurement of
recombinant products, including FIX expressed by gene therapy vectors, against
normal plasma, using different antigen kits or antibody pairs may give
substantially different results and product specific standards may be required for
accurate measurement of antigen in recombinant FIX products.
Long term and on-bench stability of the 4th
IS for FII, VII, IX and X Plasma
The 4th
IS for FII, VII, IX, X, Plasma, 09/172 was established in 2010. Accelerated degradation
study for FIX antigen at 6 month time point showed no loss of activity. Stability data on other
analytes (functional activities of FII, VII, IX and X) showed that this reference standard is highly
stable. As with all International Standards, the proposed International Standards will also be
under real time stability monitoring throughout the life time of the standard.
Assessment of on-bench stability was carried out at NIBSC by storage of the reconstituted sample on
melting ice. Samples were reconstituted at 0 h, 2 h, and 3 h and stored on melting ice. After storage
of the first sample for 4 hours on melting ice, all samples were assayed relative to freshly
reconstituted ampoules of 09/172. The results indicated that FIX antigen would be stable for up to 4
hours after reconstitution when stored on melting ice.
Time point Potency % fresh ampoule
(95% confidence intervals)
2 h 96.1 (93.3 -100.0)
3 h 101.0 (98.1 – 103.8)
4 h 98.1 (95.2 -101.0)
Conclusions Intra- and inter-laboratory variability were low when P, the 4
th IS for FIX Plasma was
assayed against local normal pooled plasma (L).
Sample P improved antigen estimates agreement for samples S, B and C, the plasma
derived concentrates when used as a putative standard.
Both local pooled plasma (L) and the 4th
IS for plasma FIX (P) gave highly variable
results for samples A and D, the recombinant products.
It is recommended that an antigen value of 0.90 IU/ampoule be included in the labelling of the
4th
International Standard for FII, VII, IX and X, Plasma.
However, it should be noted that the use of this International Standard for measurement of
antigen in recombinant/modified products, post-infusion plasma from patients who have been
treated with recombinant/modified products or gene therapy products is not recommended. If this
standard is used for these purposes, it needs to be qualified and validated by the end-user with
their own reagents and methods. The draft Instruction for Use for this proposed IS is illustrated in
Appendix IV.
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Value assignment of the 5th
IS for IX, Concentrate and Ph Eur BRP for FIX, Batch
3 relative to the 4th
IS for FIX, Concentrate, 07/182 and comparability of full
length recombinant FIX with plasma derived concentrate IS
The Candidates, Samples B (14/148) and C (14/162) The characteristics of the candidates are as described in the Sample Section.
Recombinant FIX, Samples A (07/142), D (14/180) and Purple (Pr) The characteristics of the candidates are as described in the Sample Section.
Assay methods
Each participant was requested to perform their routine in-house method for FIX activity.
Multiple result sets returned by a participating laboratory were treated as results from an
independent laboratory and were given a separate lab code, e.g. Lab 2a, Lab 2b.
FIX functional activity assays
Clotting: The details of the instruments and reagents used by the participants are listed in Table
2A. With the exception of 3 laboratories that returned more than one set of results (Lab 2 – 5 sets;
Lab 8 – 4 sets; Lab 28 – 3 sets with 1 assay for two sets and 2 assays for the third set), all other
laboratories returned one set of results. In total 55 sets of clotting assays were analysed. Of the
clotting assays, 35 sets of results were obtained using FIX deficient plasma as pre-diluent for the
concentrate preparations (samples A, B, C and D), as stated in the European Pharmacopoeia
monograph for assay of human coagulation factor IX (01/2008:20711). Twenty sets of results
were from assays using buffer for pre-dilution (Table 2A). Sample P, the plasma IS did not
require pre-dilution as its activity is less than 1 IU/ml.
Chromogenic: In total, 15 sets of chromogenic assay results from 13 labs were returned. Two
commercial kits, Biophen Factor IX (Hyphen Biomed) and Rox Factor IX (Rossix AB) were
employed (Table 2B). The participants carried out the assays as described by the instruction given
by the manufacturers of the kit and pre-dilutions were carried out using kit buffers recommended.
Purple sample
In total, 10 sets of clotting results were returned from 5 labs (1 lab returned 5 sets), 4 sets of
chromogenic results from 3 labs (1 lab returned 2 sets) and 2 sets of antigen assays from 1 lab.
Assay data returned Clotting assays
Fifty-five sets of results from clotting assays (213 assays in total) were returned. Laboratory 2
performed five sets of assays which are coded 2a, 2b, 2c, 2d and 2e. Laboratory 8 performed four
sets of assay assigned codes 8a, 8b, 8c and 8d. Laboratory 31 performed 2 sets of assays, coded
31a and 31b. All laboratories performed four independent assays, apart from laboratory 33 which
performed 5 assays (extra assay performed using samples supplied for antigen testing).
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Laboratory 28 performed four assays, but with three different APTT agents and results were 28a,
28b and 28c. Sample P was not tested by laboratory 42.
Chromogenic assays
Thirteen laboratories performed chromogenic assays (59 assays in total). Laboratory 2 and 12
both performed two sets of assays, coded 2a & 2b and 12a & 12b respectively. All laboratories
performed four independent assays, except laboratory 48 which was only able to perform 3
assays.
Assay validity
The majority of clotting and chromogenic assays gave valid estimates of relative potency when
assessed as described in the analysis section above. Samples omitted for showing a non-linear
dose-response accounted for around 1% of cases. Reference-test pairs concluded to be non-
parallel accounted for only 1-2% of cases. A high number of slope ratios were in the range 0.90 –
1.11 indicating that more stringent criteria for parallelism could be applied to these assays.
Individual cases of exclusions due to non-linearity or non-parallelism are indicated in Appendix
III. In general, relative to the plasma derived 4th
International Standard for FIX Concentrate,
statistically valid assays by all method types were obtained for all the test samples, including
recombinant preparations.
Results
Samples A, B, C and D relative to the 4th
IS for FIX Concentrate
The potencies of samples A, B, C and D have been calculated relative to sample S, the 4th
IS for
FIX Concentrate. Individual assay results are shown in Appendix III. Laboratory mean and
overall potency estimates for clotting and chromogenic assays are presented in Tables 3A and 3B
respectively and are also illustrated as histograms in Figures 1 – 4.
Clotting assays
o Of the 55 laboratories, <10 laboratories had an intra-laboratory GCV >10% and over
60% of laboratories had intra-laboratory GCV <5% for all 4 samples.
o Inter-laboratory variability for the clotting assays was 4.8% and 8.4% for the plasma
derived samples, B and C and higher at 11.6% and 13.4% for the recombinant
samples, A and D. Figure 5 shows that there was no obvious effect with regards to
APTT reagents on the potencies obtained, but wider ranges of estimates were
observed for the recombinant samples A and D as confirmed by the higher inter-
laboratory variation for these two samples.
o With the exception of sample C, there was no effect of the diluents used for pre-
dilution of the samples on the overall potency estimates by clotting assay (Table 4).
For sample C, there was a 6% (including all results) - 8% (excluding Lab 15) decrease
in potency when the samples were diluted in buffer rather than in FIX deficient
plasma.
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o Tables 5A and 5B show the overall potency estimates by clotting methods were 9.8,
10.5, 8.7 and 9.4 IU/ml for samples A, B, C and D. Results from Lab 15 were
identified as outliers for samples B, C and D. Mean potency estimates excluding
results from Lab 15 were marginally increased for samples A (9.9 IU/ml) and C (8.8
IU/ml), with a reduction in %GCV for samples B, C and D.
Chromogenic assays
o Intra-lab variability for chromogenic assays was higher than for clotting assays, but
GCVs in the majority of cases were still <10%.
o Inter-laboratory variability for the chromogenic assays was 9.3% and 8.6% for the
plasma derived samples, B and C, and only slightly higher at 10.2% and 10.4% for the
recombinant samples, A and D.
o The overall potencies by chromogenic methods were found to be 7.8, 10.6, 9.1 and 7.3
IU/ml for samples A, B, C and D respectively. Results from Lab 21 were identified as
outliers for samples B and C. Potency estimates excluding results from Lab 21 were
marginally reduced for samples A, B and C, with a marked reduction in %GCV also
for samples B and C (Tables 5A and 5B).
o The results from the 2 chromogenic assay kits used in the study showed no significant
differences in the potencies obtained for plasma derived samples B and C, although
the Hyphen kit gave approximately 10% lower potencies for the recombinant samples
A and D.
Comparison of clotting and chromogenic assays (Table 5A and 5B)
o It is clear that there were no clotting and chromogenic assay discrepancies for the
plasma derived samples B and C, including or excluding the outliers.
o For recombinant samples A and D, significantly higher potencies (approximately
30%) were obtained by clotting assays.
Overall potency estimates by clotting and chromogenic assays
o Table 7 presents the overall geometric mean potencies obtained by both clotting and
chromogenic assays for samples A, B, C and D, including and excluding Lab 15 for
clotting assay and Lab 21 for chromogenic assay. The GM estimates were 9.3, 10.5,
8.8, 8.9 IU/ml for samples A, B, C and D when all the assays were included. With the
exception of sample A, which gave a potency estimate of 9.4 IU/ml, the GM potency
did not change when labs 15 (clotting) and 21 (chromogenic) were excluded. The
inter-laboratory variation was markedly lower for samples B and C (5.9% and 8.6%
respectively) than for samples A (15.4%) and D (17.1%). The %GCV for samples A
and D were not changed by the exclusion of labs 15 and 21, but they were reduced by
approximately 2% for samples B and C.
Sample A relative to Sample S, the 4th
IS for FIX Concentrate and sample D, the putative
recombinant standard
Results for sample A were reanalysed relative to sample D with an assumed assigned value of 9.4
IU/ml based on clotting assays (Dcl) only or 8.9 IU/ml based on overall potency estimates
(Dcl+ch).
WHO/BS/2015.2261
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Table 10A shows comparison of potencies for sample A relative to sample S, the 4th
IS
for FIX Concentrate and sample D, the putative recombinant standard. For clotting
assays, similar ranges of intra-laboratory variability as expressed by %GCVs were
obtained when A was assayed against S or D, with 5 labs giving >10% GCV. For
chromogenic assays, the majority of the labs gave intra-laboratory GCVs of <10% (Table
10B).
There was no significant difference between the potency estimates by clotting assays
obtained against sample S and sample Dcl (Table 10A; p = 0.985), but there was
significant difference when compared with results against sample Dcl+ch (Table 10A;
p=0.003). The inter-laboratory variability as expressed by GCVs was markedly reduced
for both cases from 11.6% against sample S to 6.1% against samples Dcl or Dcl+ch.
There was a significant difference between the potency estimates by chromogenic assays
obtained against sample S, sample Dcl or sample Dcl+ch (Table 10B, p<0.001); however,
the inter-laboratory variation was reduced from 10.2% against sample S to 3.6% against
both samples D.
Clotting and chromogenic assays discrepancy (clotting and chromogenic ratio= 1.25, p
<0.001) was apparent when sample A was assayed against sample S. There was no
clotting and chromogenic discrepancy when sample A was assayed against either sample
Dcl or Dcl+ch (clotting and chromogenic ratio of 0.98; p = 0.282).
Sample Purple relative to Sample S, the 4th
IS for FIX Concentrate and using sample A and
sample D as putative recombinant standards
In total, 14 sets of results, 10 clotting and 4 chromogenic assays, were returned for analysis.
Results for sample Pr were analysed relative to sample S and then recalculated against samples A
(Acl) and D (Dcl) using assigned values (based on clotting assays only, 9.8 IU/ml for Acl and 9.4
IU/ml for Dcl)) and against samples A (Acl+ch) and D (Dcl+ch) using assigned values (based on
overall potency estimates, 9.4 IU/ml for Acl and 8.9 IU/ml for Dcl))
Similar ranges of intra-laboratory GCVs were obtained for both clotting and chromogenic
assays when sample Pr was assayed against samples S, A or D (Tables 11A, 11B). Only 1
lab gave GCV >10% for chromogenic assay when Purple was assayed against A; for
clotting assays, 2 labs gave GCVs just over 10% when assayed against sample A and 3
labs gave GCVs over 10% when assayed against sample D (Tables 11A, 11B).
Some significant clotting and chromogenic assay discrepancies were observed (Table
11C: vs S, p < 0.001; vs Acl, p = 0.038; vs Dcl, p = 0.02). However, the clotting and
chromogenic ratio was reduced from 1.26 when sample S was used as the standard to 0.90
and 0.88 when against samples A and D.
The overall potency was around 4.8% higher against Acl and was higher, at 6.0%, when
assayed against Dcl (Table 11A). Good agreement of overall estimates was obtained
when Pr was assayed against samples Dcl+ch and Acl+ch and it was only 1.2% higher
than against sample S (Table 11B). The inter-laboratory variability as expressed by
GCVs were reduced from 14.6% against sample S to 9.5% against A and 10.7% against
sample D.
WHO/BS/2015.2261
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Sample P, the 4th
IS for FII, VII, IX and X Plasma relative to the 4th
IS for FIX Concentrate
The FIX potency of sample P was calculated relative to sample S, the 4th
IS for FIX concentrate.
Individual assay results are shown in Appendix III. Individual laboratory mean and overall
potency estimates for clotting and chromogenic assays are presented in Table 8 and are also
shown in Figure 6.
Clotting and chromogenic assays against 4th
IS for FIX Concentrate
o The intra-laboratory variation (%GCV) ranges were 0.7 – 14.9% for clotting
assays and narrower, 1.6 – 7.9%, for the chromogenic assays. Only 4 out of 54
laboratories had %GCV >10% and 25 laboratories had %GCVs <5% for the
clotting assays (Table 8).
o The inter-laboratory variability was high, at 16.3% for the clotting assays and
lower, at 8.6% for the chromogenic assays (Tables 8, 5A, 5B).
o Overall potency estimates for clotting and chromogenic assays were 0.91 and 0.81
IU/ml and remained the same when Lab 15 for clotting and Lab 21 for
chromogenic were excluded. (Tables 5A, 5B).
o A significant assay discrepancy was observed with estimates by clotting methods
(12% higher than chromogenic assay results) yielding a clotting to chromogenic
ratio of 1.11 (Table 5B).
o The overall potency by both clotting and chromogenic assay was 0.88 IU/ml and
this equates to 2.3% difference to the labelled potency of 0.86 IU/ml. However
this difference is not statistically significant (unpaired t-test p=0.195). The inter-
laboratory GCV, including both clotting and chromogenic assay results was 15.4%
(Table 7)
Long term stability of the candidates and samples
Accelerated degradation studies have been initiated for all four concentrate preparations. Table 12
shows the predicted loss of clotting activity for samples A, B, C and D after being stored at
various elevated temperatures (-70, –20, +4, +20, +37 and +45°C). All samples showed low
predicted loss of activity at storage temperature of -20 °C. However, it should be noted that these
data are preliminary and further results are required to fully evaluate the long term stability of
these samples.
Discussion The most important prerequisite for a potency reference standard is improvement of laboratory
agreement and this is evidenced by its ability to improve agreement of potency estimates within a
method and between method types. This study generated data from 55 laboratories, using
different methods to support the choice of candidates and value assignment of these candidates as
potency standards for FIX. There were two main aims to this part of the study:
a. to assign the blood coagulation factor IX functional activity values to the replacement
WHO International Standard for Blood Coagulation Factor IX, Concentrate and Ph
WHO/BS/2015.2261
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Eur. BRP for Human Blood Coagulation Factor IX, relative the 4th
IS for FIX,
Concentrate
b. To assess the comparability of the plasma derived IS for FIX concentrate with
recombinant FIX preparations and calibrate an IS for recombinant FIX relative to the
4th
IS for FIX, Concentrate
Overall, the majority of the assays were statistically valid even when the recombinant samples (A,
D, Purple) were compared with the plasma derived International Standard for FIX concentrate,
thus supporting potency labelling of recombinant FIX products in IU. The majority of the intra-
laboratory GCVs were under 10% and a high proportion were less than 5% for all assay method
types, thus indicating the laboratories were able to perform the assays with good reproducibility.
Results from only 2 laboratories were identified as outliers, Lab 15 for clotting assays and Lab 21
for chromogenic assays, and these were excluded in the value assignment of the proposed
International Standards and Ph Eur BRP.
Value assignment to the 5th
IS for FIX, Concentrate and the Ph Eur BRP for FIX, Batch 3
The current (4th
IS) and the previous International Standards were value assigned using 1-stage
clotting assays only. In the 2008 collaborative study that value assigned the 4th
IS, the Ph Eur
BRP Batch 2 and the FDA/CBER standard, two chromogenic kits were used, however, since
there was only one set of data for each assay kit returned for analysis, it was decided that
assignment of the three standards should exclude results from chromogenic assays. Since the
2008 study, the 2 commercial chromogenic assay kits have become more widely used, especially
in the development of new generation products and in the estimation of low levels of FIX in
haemophilic patients. In the present study, 15 laboratories returned data for chromogenic assays
and since discussions with manufacturers and regulators indicate that chromogenic assays are
used as part of the characterisation package for FIX products, value assignment for the 5th
IS for
FIX, Concentrate also considered results from chromogenic assays as well as those obtained
using 1-stage clotting assays. For the Ph Eur BRP, it is clear that the calibration should consider
results only from the Monograph method and this is the 1-stage clotting assay, using FIX
deficient plasma as diluent. There are 2 plasma derived candidates, samples B and C, both could
be considered as the replacement IS and Ph Eur BRP. The following summarises the essential
points for consideration:
o Intra-laboratory variability: Both samples gave similar ranges of GCVs for all assay
methods when assayed against sample S, the 4th
IS for FIX, Concentrate (Tables 3A, 3B)
o Effect of pre-diluent: For sample B, the potency estimates were 10.5 IU/ml, using either
FIX deficient plasma or buffer as pre-diluent. However, for sample C, the estimates were
9.0 IU/ml when FIX deficient plasma was used as pre-diluent and 8.5 IU/ml, 6% lower,
when buffer was used (Table 4). The inter-laboratory agreement was better for sample B
(GCVs – FIX deficient plasma: 3.4%, buffer: 4.6%) than sample C (GCVs –FIX deficient
plasma: 4.8%, buffer: 7.8%) regardless of diluent, and the agreement was improved when
FIX deficient plasma was used, variation as expressed by GCVs was lower. Both samples
were prepared from the same FIX therapeutic product and the major difference between
WHO/BS/2015.2261
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samples B and C is the formulation. It is possible that sample B which has a similar
formulation as the current IS, and different formulation of sample C caused this discrepant
result. This interesting effect will be further investigated by NIBSC.
o Effect of APTT reagents: There was no obvious bias with the 15 APTT reagents
employed in this study on the potency estimates obtained using different APTT reagents
for either samples B and C (Figure 5).
o Effect of Chromogenic assay kits: For samples B and C, when assayed against the 4th
IS,
Concentrate , there was no significant difference in potency estimates obtained using
either kits; the Hyphen to Rossix potency ratios were 0.98 and 0.99 for samples B and C
respectively. The Inter-laboratory GCVs were less than 8% for both samples and both
kits (Table 6).
o Stability: Preliminary accelerated degradation studies on both samples B and C predicted
no loss of activity when stored at -20°C.
o Number of ampoules available: For harmonisation purpose, it would be ideal for the IS
and BRP to be the same batch of material, providing there is sufficient stock to support
this. There are approximately 20,000 ampoules of sample B and 24000 ampoules of
sample C. Although more ampoules of sample C are available, there should be sufficient
ampoules of sample B to cover both reference standards.
In summary, given that sample B gave agreement of potencies by all assay method types and that
sample C gave discrepant results in clotting assays using FIX deficient plasma or buffer as
diluents, it is proposed that sample B be the 5th
IS for FIX, Concentrate and the Ph Eur BRP
Batch 3. The assignment of the 5th
IS is based on results from 1-stage clotting with both FIX
deficient plasma and buffer as diluents and chromogenic assays, while the value for the BRP is
assigned with value from 1-stage clotting assays using only FIX deficient plasma as diluent. For
both standards, the unitage proposed is 10.5 IU/ampoule.
Assessment of on-bench stability of sample B was carried out at NIBSC by storage of the
reconstituted sample on melting ice. The potency at 1 h, 2 h, 3 h and 4 h were estimated relative to
freshly reconstituted 14/148 at each time-point. Two assays were carried. No significant difference
was observed between the potency values for the time points. This indicates that the material would
be stable for up to 4 hours storage on melting ice.
Time point Potency % fresh ampoule
(95% confidence intervals)
1 h 100.0 (96.5 – 104.4)
2 h 101.7 (98.2 - 105.3)
3 h 100.0 (94.7 - 105.3)
4 h 98.2 (93.8 – 103.5)
Rationale for route of value assignment to an International Standard for Recombinant FIX
Results from a previous international collaborative study (NIBSC Phase II study 2013) indicated
that although the 3 recombinant FIX products could be assayed validly against the 4th
IS for FIX
Concentrate, there were substantial potency discrepancies within 1-stage clotting assays and
between clotting and chromogenic methods. Agreement of potencies was obtained when the 3
WHO/BS/2015.2261
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recombinant products were assayed against a recombinant reference preparation. The current
study aimed to investigate these discrepancies further and confirm the need for a recombinant
reference standard to harmonise assay methods and improve agreement of potency estimates
within and between laboratories. The present study included 2 recombinant products, samples A
and D. Additionally, a third recombinant product, coded Purple, was also sent to 6 laboratories so
that the 3 recombinant products could be compared.
The current practice for potency labelling of recombinant FIX products is by one-stage clotting
assays against the 4th
IS for FIX Concentrate or a reference standard that is traceable to the 4th
IS.
The impact of value assignment using a recombinant putative standard labelled with overall
potency by both clotting and chromogenic assay results or labelled with estimate by clotting
assays only was evaluated. For sample A, in addition to analysis of data exploring the effect of
using sample D as a putative standard with value assigned from all assays (Dcl+ch), with the
overall potency estimate, 8.9 IU/ml against sample S, the results were also reanalysed using the
clotting assay GM for sample D (Dcl) at 9.4 IU/ml.
Potency estimates for sample A relative to sample S, the 4th
IS for FIX Concentrate and
sample D:
o The intra-laboratory variation was similar when sample A was assayed against
sample S or sample Dcl or sample Dcl+ch (Table 10A, 10B)
o The inter-laboratory variability for both clotting and chromogenic assays were
reduced by approximately two-fold (Table 10C).
o There was significant clotting and chromogenic assay discrepancy when sample A
was assayed against sample S, with chromogenic assays giving 25% lower
potency than the clotting assays (p<0.001) This discrepancy was eliminated when
sample A was reanalysed against either sample Dcl or Dcl+ch (clotting and
chromogenic ratio 0.98, p = 0.282).
o Potency estimates
The clotting potency estimates for sample A against samples S and Dcl
were the same, giving 9.8 IU/ml, but approximately 5% lower, at 9.3 IU/ml
when assayed against Dcl+ch (Table 10C).
The estimates by chromogenic assays against S were over 20% lower than
those obtained against Dcl or Dcl+ch.
It is clear that there is improvement in agreement of potency by both
clotting and chromogenic assays when sample A was assayed against
sample D (compare Figures 1 and 7). While the overall potency estimate
generated against Dcl+ch was not significantly different to values from
assays against S (Table 10C, p =0.956), potency obtained against Dcl was
significantly different (Table 10C, p = 0.003).
WHO/BS/2015.2261
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o This indicates that if sample D is a standard for sample A, it will reduce both inter-
laboratory variability and improve agreement of potencies by both clotting and
chromogenic assays.
o Taking into account that the current recombinant products are labelled against the
4th
IS for FIX concentrate using clotting assays, the clotting potency of sample A
relative to sample S, the 4th
IS for FIX concentrate was compared with the overall
potency estimates obtained against Dcl and Dcl+ch. Table 10C shows that there
was good agreement of potency against samples S and Dcl (A vs S clotting: 9.8
IU/ml; A vs Dcl overall: 9.8 IU/ml; p =0.779). However there was significant
differences in potencies when compared with the overall value obtained with
Dcl+ch (A vs S clotting: 9.8 IU/ml; A vs Dcl+ch overall: 9.3 IU/ml; p =0.003).
Therefore if sample D is to be the standard for recombinant products, value
assigned using clotting assays results only, this will ensure the continuity of the IU
and there should be minimal shift in potency labelling of recombinant products.
The recombinant standard will also help to reduce inter-laboratory variability and
harmonise clotting and chromogenic assay results.
Potency estimates for sample Purple relative to sample S, the 4th
IS for FIX Concentrate,
sample A and sample D:
o The intra-laboratory variation was similarly low when sample Purple was assayed
against samples S, A, or D (Table 11A, 11B), indicating that the laboratories were
able to assay the recombinant products with precision and reproducibility.
o While the inter-laboratory GCVs were slightly reduced for the clotting assay when
Purple was assayed against A or D than against S (Tables 11C, 11D; GCVs vs S:
8.3%; vs A: 7.1%; vs D: 8.0%;), the GCVs for the chromogenic assays were not
reduced (Tables 11C, 11D; GCVs vs S: 10.1%; vs A: 10.6%; vs D: 10.8%;).
o Although some clotting and chromogenic discrepancies were found to be
statistically significant, the clotting to chromogenic ratio was reduced from 1.26
when assayed against S to 0.90 against A and 0.88 against D, (Tables 11C, 11D).
o Although there was no significant difference to the overall potency estimates
against the different standards, using results from all methods (Tables 11A. 11B),
the overall potency agreement was improved when Purple was assayed against
samples A or D as exemplified by the approximately 5% reduction in the inter-
laboratory variability as expressed by GCVs. (Tables 11A, 11B, 11C, 11D).
o These results indicate that if sample A or D was used as a standard for sample
Purple, it will minimise clotting and chromogenic discrepancies and improve
potency agreement by reduction of overall inter-laboratory variability
The reanalysis of data for samples A, D and Purple confirms finding from the previous study that
assaying recombinant product against a recombinant standard helps to reduce assay discrepancies
and increase potency agreement between laboratories.
WHO/BS/2015.2261
Page 19
Conclusions
The 5th
IS for Blood Coagulation Factor IX, Concentrate and Ph Eur BRP batch 3
The high inter-laboratory %GCV (Table 7, 15.4%) obtained when the 4th
IS for
FIX Plasma was assayed against the 4th
IS for FIX Concentrate support the
rationale of a concentrate potency reference standard for plasma derived FIX
products.
Both candidates B, C gave similarly low within laboratory variability.
There was no clotting and chromogenic discrepancy for either candidate.
Preliminary accelerated degradation studies showed that both candidates are
predicted to be equally stable.
Candidate B gave slightly lower inter-laboratory variability
Different pre-diluent gave discrepant clotting assay results for candidate C.
Since candidate B gave agreement of potencies by different methods and pre-diluents, it is
recommended that candidate B, 14/148 be the 5th
International Standard for Blood Coagulation
Factor IX, Concentrate and EP BRP for Human Coagulation Factor IX Concentrate, Batch 3.
Both reference standards are value assigned relative to the 4th
International Standard for Blood
Coagulation Factor IX, Concentrate, with the 5th
International Standard for Blood Coagulation
FIX, Concentrate potency labelled based on all functional activity assays and the Ph Eur BRP
labelled based on one-stage clotting assays using FIX deficient plasma as pre-diluent. In both
cases, the value is 10.5 IU/ampoule. The draft Instruction for Use for this proposed IS is
illustrated in Appendix IV.
Should an International Standard for Recombinant FIX be established?
It is clear that statistically valid assays can be obtained when the current recombinant FIX
products are assayed against the plasma derived IS for FIX Concentrate and that one of these
products has been potency labelled against the IS for the last decade. Unlike FVIII, there is global
agreement that 1-stage clotting assays is the assay of choice for potency labelling and therefore
clotting and chromogenic assay discrepancies have not so far been an issue. Nonetheless,
chromogenic assay kits for FIX are now available and these kits will suit the clinical need for
assays with higher sensitivity for low level of FIX. The results from this study showed that there
are significant chromogenic and clotting assay discrepancies when the recombinant samples were
assayed against the current IS. In addition, the availability of new recombinant and modified
products has brought attention to assay discrepancies within the 1-stage clotting assays. The
package insert from one of the most recently licensed recombinant full-length FIX indicates that
the FIX potency results for this product can be affected by the type of APTT reagent and
reference standard used in the assay and that differences of up to 40% have been observed. The
current study, using a range of APTT reagents routinely used by manufacturers, regulators and
clinical laboratories indicated that up to >60 % and >80% differences in potencies could be seen
when recombinant samples A and D were assayed by clotting assays against the plasma derived
IS. The APTT reagent discrepancy for sample A was reduced to ~20% and was reflected by the
decrease of inter-laboratory GCV from 11.6 to 6.4% when sample D was used as the reference
WHO/BS/2015.2261
Page 20
standard. In addition when sample D was used as the putative standard for samples A or Purple,
apart from lowering the overall inter-laboratory variability, the clotting and chromogenic
discrepancies was eliminated or reduced to an acceptable level.
Although having a standard for recombinant products in addition to the plasma derived
concentrate IS may cause some initial confusion, precedent has been set for a number of
Biotherapeutics (Prolactin: WHO 3rd
International Standard for Prolactin, Human, NIBSC code:
84/500 and WHO 1st International Reference Reagent for Prolactin, Human, recombinant. NIBSC
code: 97/714; Follicle Stimulating Hormone (FSH): WHO 2nd
International Standard for Follicle-
Stimulating Hormone, human, recombinant, for bioassay, NIBSC code: 08/282 and WHO 5th
International Standard for Follicle Stimulating Hormone, Luteinizing Hormone human, urinary
for bioassay, NIBSC code:10/286) which have co-existing International Standards for
plasma/human derived and recombinant proteins and both of these International Standards serve
well to support potency labelling and clinical measurement in patient samples of the different
forms of the same therapeutic product types. The results from the study also support the
continuity of the FIX IU when transferring to the recombinant standard. This is illustrated by the
same clotting potency of A, 9.8 IU/ml against S and sample D (when sample D is assigned using
the clotting potency value; Tables 10C). The replacement strategy for such a standard should also
be considered carefully and it is proposed that successive replacement recombinant standards
should follow the same route as other International Standards i.e. value assignment relative to the
previous recombinant standard but study should include current plasma derived concentrate
standard so that any drift of IUs would be monitored.
While the establishment of an IS for recombinant full length FIX based on the data from the
current study will serve well against the current licensed products, there is a possibility that more
recombinant FIX products will be on market and that the future recombinant FIX products may
not compare well with this recombinant IS. The NIBSC Phase II study has already identified that
the modified recombinant products (e.g. pegylated-FIX) do not compare well against full length
recombinant products and recombinant full length FIX should not be used as potency standard for
these modified products. This study showed that the 3 recombinant full length products compared
better against each other than against the plasma derived IS and the recombinant IS will promote
better comparability of potencies amongst these products. The likelihood that another
recombinant full length FIX will behave substantially different to these 3 products is low, but not
impossible and should be investigated should another recombinant full length FIX becomes
available. There is also another advantage of the availability of an IS for recombinant FIX.
There have been numerous discussions amongst regulators, manufacturers and clinical
laboratories on assay discrepancies related to clinical monitoring. The sensitivity and the
reproducibility of chromogenic assays are being considered alongside the conventional one-stage
clotting assay and is supported by the US National Hemophilia Foundation who urged the US
FDA to accelerate the approval of chromogenic assay kit and clinical laboratories to establish the
chromogenic assays. The clinical laboratories will require support to improve intra- and inter-
laboratory agreement and minimise assay discrepancies whenever they change reagents,
instruments and operators. Since product specific standards are not yet feasible, the availability
WHO/BS/2015.2261
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of a global recombinant International Standard will go a long way to aid the development and
continual validation of assay methods for measurement of FIX activity following replacement
therapy with recombinant full length FIX. The following table summarises the pros and cons of
establishing an International Standard for Recombinant FIX and on balance, having a global
standard for recombinant FIX would be useful to the community as the disadvantages could be
minimised by careful and considered risk management:
Pros Cons
Improve intra-laboratory
agreement, less reliant on
calibration against the plasma
derived IS and stability of
manufacturers’ own in-house
standards
Minimize the substantial assay
discrepancies within and between
clotting and chromogenic assays
Improve inter-laboratory
agreement, giving more
confidence in label potencies and
interchangeability of products if
and when required
Useful independent resource for
clinical labs to develop assays for
measurement of recombinant FIX
in patient samples
Too many standards, can be
confusing to end-users
Future recombinant full length
FIX may not assay so well against
recombinant standard
Complex considerations for route
of value assignment
Replacement strategy require
careful consideration
Possible discontinuity of for
labelling of licensed recombinant
products
In consideration of which candidate should go forward as the 1st International Standard for
Recombinant FIX, both candidates have similar physical characteristics in terms of coefficient of
variation, residual moisture and oxygen headspace and passed all the recommended specifications
for a WHO international biological reference standards. They gave similar intra- and inter-
laboratory variability. Candidate A, 07/142, was ampouled in 2007 with fewer ampoules
available than candidate D, 14/180. In addition, although all the candidates including samples A
and D passed the activated coagulation factors test (all test clotting times >150s; Table 13), the
amount of FIXa estimated in sample A was 5 times higher than that obtained for sample D (Table
14). High levels of FIXa in a FIX reference standard may have some influence on the potency
estimates of test preparations. So it is recommended that candidate D be the 1st International
Standard for Recombinant FIX. In terms of value assignment, since the Ph Eur monograph
method for labelling is the one-stage clotting assay and globally the current licensed products are
labelled by one-stage clotting assay, to ensure good continuity of the IU, it is recommended that
the 1st IS for recombinant FIX be value assigned with overall clotting potency of 9.4 IU/ampoule,
against the 4th
IS for FIX concentrate. The established standard can be used as a reference
WHO/BS/2015.2261
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standard for measurement of recombinant FIX activity by either 1-stage clotting or chromogenic
assays.
Based on the above rationale, it was proposed to the participants that Sample D, 14/180 be
established as the WHO 1st International Standard for Recombinant Human Blood Coagulation
Factor IX, with the assigned value for functional activity of 9.4 IU/ampoule.
Participants Comments and Responses Thirty out of the 49 participating laboratories returned comments on the study. Most of the
comments were associated with typos and queries related to analysis of results for individual
assays. Following investigation of the revised data, the statisticians concluded that there will not
be any major impact on the overall potency estimates or the recommendations for establishment
of the standards and the tables have been updated for the ECBS report. All responded participants
agreed with the proposal of the value assignment of the 5th
International Standard for Blood
Coagulation Factor IX, Concentrate and the addition of the FIX antigen value to the 4th
International standard for Factors II, VII, IX and X, Plasma.
For the proposal of an International Standard for Recombinant FIX, with the exception of one lab
(see comment A), who did not agree with the proposal, all others agree with the proposed
establishment. There were 2 other significant comments (comments B and C).
Comment A
“At this time, we would recommend not supporting the proposal to establish an
International Standard (IS) for recombinant Factor IX (FIX) because this will complicate
the meaning of the International Unit (IU) as it has been understood for decades by users
of FIX products. Although we know that the IU is dependent on many factors, having one
IU for all FIX products provides a reference with which one can gauge the effect of the
variables on the resultant potency values. If we agree to having an IS for recombinant
FIX concentrates, manufacturers of currently licensed recombinant FIX products would,
in practice, need to recalibrate their in-house recombinant product-specific standards,
which may result in adjustment of FIX protein content in the final container. As a result,
we would have to re-assign the potency of all currently licensed recombinant FIX
products (both traditional and long-acting analogues), which would most likely
necessitate changes in dosing regimens. In addition, we can expect that these two FIX IS
will be replaced at different times and experience different drifts, which will further
exacerbate the differences between the two standards.
On the other hand, we know that plasma-derived and recombinant FIX preparations do
behave differently as we have seen in laboratory studies comparing different APTT
reagents. It may be worthwhile to investigate the possibility of harmonizing potency
assignment and product description in the labelling of different recombinant and long-
acting FIX products. To this end, the availability of a recombinant FIX reference reagent
would be helpful in such studies. Thus, we can support establishing the proposed
candidate as a WHO Reference Reagent for recombinant FIX concentrates.”
WHO/BS/2015.2261
Page 23
Response from NIBSC We like to make clear that a recombinant FIX International Standard or
International Reference Reagent should not be used for assays of long-acting
products or any other modified products and this will be explicit in the Instruction
for Use to the end-users.
There is a possibility that a recombinant FIX International Standard may cause a
shift in labelling of the current licensed (or product in development) recombinant
FIX products. The current study data (Table 10A) showed that the overall potency
estimate for sample A (a recombinant product) against the 4th
International
Standard (sample S) was the same as the value against sample D (another
recombinant product). However, due to the variability of the APTT reagents, there
will be discrepancies within individual laboratories. So results on recombinant
products against the proposed International Standard from manufacturers will be
needed to assess the impact of the recombinant standard on the potency labelling
of their products. Even if there is a slight shift of the unit, this one-time transfer
will have the long-lasting advantage that a recombinant standard will diminish the
APTT reagents and the clotting and chromogenic discrepancies.
Comment B “1. The mere fact that only “the OS method” is prescribed by regulatory authorities
as the method to be used for release testing, and the results of which are hence proposed
for Sample D assignment, should not be taken as an argument to prevent introduction of
CS methods for release testing. We also want to state that there is no support for any
tentative view that the OS results for Sample D are more true than the CS results. The
ratio of close to 1.3 for OS/CS (close to 1.2 if only including the seven Rox Factor IX
results) when A and D were value assigned vs the 4th IS may encourage follow-up work to
investigate why the difference appears.
2. The data from the study clearly show a significantly lower inter-lab GCV for CS
methods when Sample A was value assigned vs Sample D, no matter whether Dcl or
Dcl+ch was used for D. Such results strongly indicate that CS methods may well improve
consistency in FIX potency assignments in manufacturers´QC labs. Again, it would we
quite unfortunate if regulatory authorities will not consider CS methods to be allowed for
potency assignments by referring to the Sample D value assignment.
3. The proposed value and use of Sample D (14/180) will not solve the problems with
OS methods on analysis of long-life rFIX concentrates. The data from CS methods,
although so far limited, clearly indicate that CS methods may be a better alternative.”
Response from NIBSC
The value assignment of the proposed standard based on clotting assays only is to
minimize the risk of a shift in potency labelling of current licensed products which were
licensed on potency labelling by the clotting assay. The recombinant standard should
reduce clotting and chromogenic assay discrepancy and allow the development of the
chromogenic assay not only as an alternative for potency labelling but also for clinical
monitoring.
Comment C
WHO/BS/2015.2261
Page 24
“We understand that the European Pharmacopoeia focuses on the one-stage clotting
assay for blood coagulation factor IX. Nevertheless, we think that in light of the current
uncertainties around potency testing of coagulation factor products, alignment of assays
which are based on different assay principles should not be ignored. Thus, the proposed
value assignment may not only be substantiated by a majority driven decision.”
Response from NIBSC Pharmacopoeial standards are conventionally calibrated using only the pharmacopoeial
assay while WHO International Standards are value assigned by multiple methods. Assay
methods for value assignment of an International Standard for Recombinant FIX could be
further discussed.
SSC Experts Comments and Responses
Eight experts nominated by the SSC sent in responses to the SSC report. Two experts agreed with
all the proposals with no comments and 2 experts with minor comments related to the plasma
derived 5th
IS. There were 4 significant comments:
Comment A
“Is the evidence truly convincing that the definition of a factor IX unit is equivalent
between the plasma and recombinant standards?”
Response from NIBSC
The results from this study showed that the IU derived for the recombinant candidates was
directly traceable to the plasma derived 4th
IS for FIX, Concentrate. The potencies of
sample A, a recombinant preparation by clotting assays were similar against the 4th
IS or
against sample D (the other recombinant samples) used as the putative standard (Table
10A). This demonstrates that if one of the recombinant samples in the study is the
International Standard, the IU expressed by this standard is traceable and equivalent to the
IU expressed by the plasma derived IS.
Comment B
“I understand the argument for retaining just 1 concentrate standard, as for FVIII,
especially as potency is assigned currently using the same methodology (1-stage clotting
assay) in both Europe and US. However, the discrepancy between clotting and
chromogenic assay results with recombinant products when compared to a plasma-
derived concentrate standard, and especially the variability encountered within 1-stage
clotting assays with different reagents suggests that future use of plasma-derived
standards to assign potency of current and new recombinant products could lead to
problems. Therefore a recombinant concentrate standard with potency itself assigned
against a plasma-derived concentrate standard but able to reduce variability between
methods is a welcome step.”
WHO/BS/2015.2261
Page 25
Response from NIBSC
The results from this study showed that a recombinant International Standard would
significantly reduce variability between methods whilst maintaining traceability to the
plasma derived concentrate IS.
Comment C
“1. Sample B- proposed plasma derived reference – Many of us expect that chromogenic
IX assays will be increasingly used in future when pegylated FIX and some other extended
half life products. Therefore it is useful that the chromogenic assay data is in agreement
with the one stage (as used for potency assignment. This predicts that labs can use
chromogenic assays for post infusion monitoring of plasma derived products whose
potency has been assigned against sample B in future.
2. Sample D as first recombinant standard. Probably sample A could have also been
suitable but the 3k extra stock of D and lower level of FIXa probably make it the better
candidate. It would be helpful if the failed candidates can be returned to the provider so
that they can take advantage of the collaborative data. This would assist future
standardization. Does this routinely occur?
3. There is an important difference between chromogenic and clotting assay results for
the recombinant materials (as in previous studies). Since potency assignment to
commercial batches of recombinant IX is by one stage I think its really important to have
this recombinant material and for potency assignment to be by the one stage assay (as
proposed). The data predict that post infusion monitoring of these (unmodified )
recombinants should be by one stage (not chromogenic) assays.
4. The GCV of recombinant materials (A and D) against plasma standards is
approximately three times as high as for recombinant against recombinant. This predicts
that potency assignments by manufactures will be less method dependent when assaying
recombinant against recombinant which in turn will help make post infusion monitoring
less dependent on the reagents in local use. Just another example of the like versus like
principle improving the agreement between methods. “
Response from NIBSC
1. Since there was no significant difference between clotting and chromogenic assay
methods for the plasma derived candidates relative to the 4th
IS for FIX Concentrate,
chromogenic assays could be used for potency estimation of purified concentrates.
There was significant clotting and chromogenic assay discrepancy (12% higher by
clotting, Tables 5A and 5B) when the plasma IS was assayed against the concentrate
IS. This indicates that if clinical labs use normal pooled plasma as a standard for
estimation of post infusion of plasma derived therapeutics, clotting and chromogenic
assay discrepancies may still be obtained. However, the high variability of assay
results from clinical labs may mask this assay discrepancy.
WHO/BS/2015.2261
Page 26
2. The provider of donated materials always has an option to obtain part of the batch for
their in-house use should that particular material not be established as an IS.
3. The rationale for potency assignment of a proposed recombinant FIX IS using
clotting assay only was based on all 3 current commercial recombinant FIX are
potency labelled by one-clotting clotting assay. However, one of the participants
comment indicated that a combination of multiple methods i.e including chromogenic
assays should be considered. In terms of post infusion monitoring, if a recombinant IS
is used as the local calibrant for administered recombinant FIX, there should not be
assay discrepancy between clotting and chromogenic and between one-stage clotting
assays using different APTT reagents.
4. Using a recombinant FIX standard will reduce method dependent variability.
Comment D
“This is an important study that has been well performed. Unfortunately however, the
report is confusing because it contains multiple proposals. Some of these raise issues that
need to be clarified before I could recommend any SSC endorsement:
- Sample P (09/172) FIX antigen value: proposal supported
- Sample B (14/148) FIX concentrate: potency proposal supported. However, according
to the report “Sample B degradation data is not yet available” (see report page 12, see
also Table 12 on page 50). In absence of such date I cannot provide support for
recommending this material to WHO as the 5th
IS to the WHO.
The same holds for proposing this as the Ph Eur BRP, but I am not sure whether this
would need any formal SSC endorsement at all.
I would be in favor of proposing preparation B as 5th
IS for FIX provided that stability
data would be available to justify this.
- Sample D (14/180) recombinant FIX: I do not support this proposal because:
The use of multiple International Standards for the same coagulation factor should
be avoided whenever possible (see SSC recommendation by Hubbard et al, JTH
2013, 11(5); 988-9). The proposal for a separate recombinant FIX standard does
not fit into this policy.
This standard is not needed al all because plasma-derived and recombinant FIX
can well be assayed against the proposed 5th
IS.
Apart from these formal points, I fully support the comments from one of the
participants (see participants comment A) relating to the implications for
manufacturers of recombinant FIX products.
In view of these comments, I feel that the section on the establishment of an IS for recFIX
(Page 19-22(top)) is inappropriate and confusing.”
WHO/BS/2015.2261
Page 27
Response from NIBSC
The stability data for sample B is now presented in Table 12 and it shows that this
candidate is highly stable and therefore suitable as a WHO IS.
With reference to Sample D, the recombinant FIX, the SSC publication quoted is a
recommendation and was written when limited data on the comparability of recombinant
and modified products with plasma derived standards were available. The publication
indeed mapped out routes for potency labelling and that if the product can be assayed
validly against the current IS using a particular method type, then it should be assayed
against the IS and labelled in IU. However, the recommendation did not give an
indication on how to deal with assay discrepancy within one method type, for example,
within the clotting method. The study results did show that both plasma derived and
recombinant FIX “can well be assayed against the proposed 5th
IS” i.e. gave statistically
valid assays, however, there is significant potency discrepancies (up to 63 and 88% for
samples A and D respectively) by clotting assays using different APTT reagents when the
recombinant FIX was assayed against the IS and this discrepancy was markedly lower
when the plasma derived FIX was assayed against the IS.
Proposal and Recommendation
In view of the objections raised by one participant and one SSC expert on the proposal to
establish an International Standard for Recombinant FIX, this proposal will not be put forward to
the ECBS and the data and discussion on this topic are for information only. The value
assignments for FIX antigen and the 5th
IS for FIX Concentrate have unanimous support and
therefore it is proposed to the ECBS that:
1. Sample P, the 4th
International Standard for Factors II, VII, IX, X, Plasma,
(09/172)
Addition of FIX antigen value to existing standard
The assigned value for FIX antigen: 0.9 IU/ampoule
2. Sample B, 14/148 be established as the:
WHO 5th
International Standard for Blood Coagulation Factor IX, Concentrate,
Human
The assigned value for functional activity: 10.5 IU/ampoule
WHO/BS/2015.2261
Page 28
References
1. WHO technical report series no 964, Fifty-ninth report;
http://www.who.int/28iological/WHO_TRS_964_web.pdf?ua=1
2. WHO/BS/08.2097. http://whqlibdoc.who.int/hq/2008/WHO_BS_08.2097_eng.pdf
3. WHO Technical Report Series, No. 932, 2006. Annex 2 Recommendations for the
preparation,characterization and establishment of international and other biological
reference standards (revised 2004)
4. CombiStats v5.0, EDQM – Council of Europe, www.combistats.eu.
5. Kirkwood, TBL. Predicting the stability of biological standards and products. Biometrics.
1977; 33: 736-742.
Acknowledgements
We would like to acknowledge:
the participants of the study
Dr Paul Matejtschuk, Technology, Development and Infrastructure (TDI), NIBSC for the
formulation and trial fills of candidates
the staff of the Centre for Biological Reference Material (CBRM) for processing the
candidates
Wyeth BioPharma, USA; CSL Behring, USA; Baxter BioScience AG, Austria; Emergent
Biosolutions, Canada for the kind donation of candidate materials
.
WHO/BS/2015.2261
Page 29
Table 1: Product Summary
Code 07/142
Sample A
14/148
Sample B
14/162
Sample C
14/180
Sample D
Presentation Sealed, glass 3 ml DIN ampoules
Number of Ampoules
available 22,399 20,689 23,300 25,133
Date Filled 24 May 2007 19 Jun 2014 25 Sept 2014 02 Oct 2014
Excipients
50 mM Tris, pH 7.4
150 mM NaCl,
2 mg/ml Trehalose,
5 mg/ml human
albumin,
50 mM Tris, pH 7.4
150 mM NaCl,
2 mg/ml Trehalose,
10 mg/ml human
albumin
10 mM Tris, pH 7.4
1% sucrose,
4% mannitol,
5 mg/ml human
albumin,
10 mM Tris pH 7.4
1% sucrose,
4% mannitol,
5 mg/ml human
albumin,
CV of fill mass (%) 0.134 (n=653) 0.242 (n=869) 0.213 (n=839) 0.126 (n=825)
Mean dry weight (g, n = 6) 0.0242 0.0316 0.0560 (n=5) 0.0613
Mean head space oxygen
(%, n=12) 0.70 0.43 0.30 0.20
Residual moisture (%,
n=12) 0.139 0.220 0.842 0.65
Storage Conditions -20°C
Address of processing
facility NIBSC, Potters Bar, EN6 3QG, UK
Address of present
custodian NIBSC, Potters Bar, EN6 3QG, UK
WHO/BS/2015.2261
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Table 2A: Methods and reagents used by the participants in the study – clotting assays
Lab
No Method
Pre-
dilution in
Plasma (P)
or Buffer
(B)
APTT
Reagent/Chromogenic
Kit source
Source of plasma Instrument
1 Clotting P Actin-FSL Siemens Sysmex CS2100i
2a Clotting P Actin-FS Technoclone ACL-TOP 500
2b Clotting P APTT-SP Technoclone ACL-TOP 500
2c Clotting P Cephascreen Technoclone ACL-TOP 500
2d Clotting P Dapttin Technoclone ACL-TOP 500
2e Clotting P Synthasil Technoclone ACL-TOP 500
3 Clotting P C.K. Prest Stago Coagulometer Start 4
4 Clotting P Dapttin Technoclone BCS-XP
5 Clotting P Pathromtin Siemens BCS-XP
6 Clotting P Actin FS Instrumentation Laboratory BCS-XP
7 Clotting B PTT-A George King STA-R Evolutic
8a Clotting P Actin-FSL Siemens BCS-XP
8b Clotting P Actin-FSL Siemens CA-1500
8c Clotting P Pathromtin Siemens BCS-XP
8d Clotting P Pathromtin Siemens CA-1500
9 Clotting P Actin-FS Siemens ACL-TOP 700
10 Clotting B Actin-FSL Precision Biologics BCS-XP
11 Clotting B CK-Prest Affinity Biologicals STA-R Evolution
13 Clotting B TriniClot aPTT HS Precision Biologics ACL-TOP
14 Clotting P Actin-FS Siemens BCS-XP
15 Clotting B PTT Stago Stago Compact
16 Clotting P Actin Siemens CL 8 (Behnk Electonik)
17 Clotting B TriniClot APTT Precision Biologics STA-R Evolution
18 Clotting B Actin-FS Stago ACL-9000
19 Clotting P Trinity George King ACL-TOP 500
20 Clotting P Actin-FSL HRF BCS-XP
22 Clotting B SynthASil Instrumentation Laboratory ACL-TOP 500
23 Clotting B APTT-SP Helena Biosciences ACL-9000
24 Clotting P PTT-A Precision Biologics STA-R Eveolution
25 Clotting P Pathromtin SL Siemens BCS
27 Clotting P APTT-SP Stago ACL Elite Pro
28 Clotting B Cephascreen Stago STAR
28 Clotting B CK-Prest Stago STAR
28 Clotting B PTTA Stago STAR
29 Clotting P Actin Siemens Sysmex CA-1500
30 Clotting P APTT Synth Grifols Grifols Amelug CS-190 Amax
31 Clotting B Cephascreen Stago STA-R Evolution
WHO/BS/2015.2261
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Lab
No Method
Pre-
dilution in
Plasma (P)
or Buffer
(B)
APTT
Reagent/Chromogenic
Kit source
Source of plasma Instrument
31 Clotting B CK-Prest Stago STA-R Evolution
32 Clotting B CK Prest George King STA-R Evolution
33 Clotting P SynthaFax HRF ACL Elite Pro
34 Clotting P Pathromtin SL Siemens BCS-XP
35 Clotting B Actin Siemens CA-1500
36 Clotting B Dapttin Hyphen BCS-XP
37 Clotting B Cephen Hyphen BCS
39 Clotting P APTT-SP Helena Biosciences ACL 200 & ACL 3000Plus
40 Clotting P Actin-FS Siemens BCS-XP
41 Clotting B Actin-FSL Siemens BCS-XP
42 Clotting P SynthaFax Instrumentation Laboratory ACL Elite Pro
43 Clotting P APTT-SP Instrumentation Laboratory ACL-10000
44 Clotting P SynthaSil Instrumentation Laboratory ACL TOP 300
45 Clotting P Actin-FS Precision Biologic Sysmex CS5100i
46 Clotting P Actin-FSL Siemens Sysmex CA7000
47 Clotting P Actin-FS Technoclone Sysmex CS5100
48 Clotting B SynthaSil Instrumentation Laboratory ACL-TOP 700
49 Clotting P Cephen Hyphen STAR
WHO/BS/2015.2261
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Table 2B: Methods and reagents used by the participants in the study – chromogenic assays
Lab
No
APTT
Reagent/Chromogenic
Kit source
Instrument
2a Hyphen ACL-TOP 500
2b Rossix ACL-TOP 500
4 Rossix Biotek Platereader
6 Rossix BCS-XP
12a Hyphen SpectraMax
12b Rossix SpectraMax
21 Hyphen BCS-XP
26 Rossix ThermoMax
33 Hyphen Biotek Platereader
36 Hyphen Elx808
40 Rossix BCS-XP
41 Rossix BCS-XP
43 Hyphen SpectraMax
*45 Hyphen* Sysmex CA7000
48 Hyphen ACL-TOP 700
49 Hyphen STAR
*Single point results, not included in analysis
WHO/BS/2015.2261
Page 33
Table 2C: Methods and reagents used by the participants in the study – antigen assays
Lab
No ELISA Kit used Pool used
2a Asserchrom (Stago) Frozen pool, n = 10
2b Visulize (Affinity
Biologicals) Frozen pool, n = 10
2c Zymatest (Hyphen
Biomed) Frozen pool, n = 10
10 Visulize (Affinity
Biologicals) Local pool, n = 12
11 Visulize (Affinity
Biologicals) Two Local pools n = 20 (total)
21 Cedarane Commercial Pool, n > 240
23 AssayMax ELISA
(AssayPro) Frozen pool, n = 1134
24 Asserchrom (Stago) Two commercial pool, n = 21 & n = 26
28 Asserchrom (Stago) Two Local pools (2 days fresh and 2 days frozen), n =
20 (total)
31 Asserchrom (Stago) Two Local pools, n = 37 (total)
33 Visulize (Affinity
Biologicals) Two commercial pools, n > 50
34 Asserchrom (Stago) Commercial pool
38 Affinity Biologicals Commercial pool n > 30
40 Asserchrom (Stago) Two local pools (2 days fresh and 2 days frozen), n = 18
(total)
43 Asserchrom (Stago) Kit standard
48 Visulize (Affinity
Biologicals) Two commercial pools, n = 200 (total)
49 Zymutest (Hyphen) Two commercial pools, n > 40 (total)
WHO/BS/2015.2261
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Table 3A: Clotting assays results for samples A, B, C and D calculated relative to S:
Laboratory mean (GM), geometric coefficient of variation (GCV) and overall GM and
GCV. Results using buffer as pre-diluent are denoted in red, all other laboratories used
haemophilia plasma as diluent.
Lab APTT reagent
A B C D
GM
IU/ml GCV n
GM
IU/ml GCV n
GM
IU/ml GCV n
GM
IU/ml GCV n
1 Actin-FSL 9.8 6.1% 4 10.8 7.2% 4 9.8 4.2% 4 9.5 4.3% 4
2a Actin-FS 12.3 3.1% 4 10.5 6.5% 4 9.0 5.0% 4 11.9 6.3% 4
2b APTT-SP 9.4 2.9% 4 10.5 5.3% 4 8.8 3.9% 4 8.9 5.4% 4
2c Cephascreen 12.4 4.2% 4 10.8 4.3% 4 8.8 3.7% 4 11.5 4.7% 4
2d Dapttin 11.2 6.4% 4 11.0 0.5% 3 9.2 2.9% 4 10.1
2
2e Synthasil 10.9 4.3% 4 10.4 7.0% 4 8.9 3.1% 4 10.6 5.6% 4
3 C.K. Prest 9.0 7.3% 4 10.9 6.6% 4 8.7 5.5% 4 8.3 7.0% 4
4 Dapttin 8.1 6.5% 4 10.5 4.3% 4 8.5 7.0% 4 7.6 3.0% 4
5 Pathromtin SL 9.5 1.5% 4 11.1 0.8% 4 8.8 2.2% 4 8.5 1.9% 4
6 ActinFS 12.4 7.1% 4 10.4 4.0% 4 9.2 6.7% 4 11.7 16.6% 4
7 PTT-A 10.4 3.4% 4 10.4 21.9% 4 9.4 7.0% 4 12.4 19.2% 4
8a ActinFSL-BSCXP 9.2 1.7% 4 10.3 4.1% 4 8.4 5.1% 4 9.2 4.3% 4
8b ActinFSL-CA1500 10.0 3.2% 4 10.3 1.9% 4 8.8 4.7% 4 10.2 2.1% 4
8c Pathromtin SL-
BSCXP 9.1 3.1% 4 10.4 4.3% 4 8.6 6.4% 4 8.7 5.3% 4
8d Pathromtin SL-
CA1500 8.7 5.1% 4 10.2 5.0% 4 8.7 8.0% 4 8.6 2.8% 4
9 ActinFS 9.2 5.8% 4 10.8 2.9% 4 8.9 4.2% 4 8.8 3.1% 4
10 ActinFSL 9.9 11.5% 4 9.3 17.7% 4 7.9 6.3% 4 9.7 9.1% 4
11 CK-Prest 10.1 3.8% 4 10.4 3.5% 4 8.7 4.3% 4 9.5 5.9% 4
13 TriniClot 10.3 2.9% 4 11.4 6.0% 4 8.2 3.8% 4 9.1
2
14 ActinFS 11.4 3.5% 4 10.5 1.4% 4 9.0 4.1% 4 11.2 3.7% 4
15 PTT-A 7.7
2 8.5 2.8% 3 6.0 4.6% 4 6.6
2
16 Actin 10.2 4.5% 4 10.4 4.7% 4 8.7 3.5% 4 9.5 2.8% 4
17 TriniClot 9.5 2.8% 4 10.5 5.3% 4 8.5 3.0% 4 9.1 1.6% 4
18 ActinFS 10.1 3.7% 4 10.4 5.5% 4 8.9 5.5% 4 9.8 3.8% 4
19 TriniClot 10.2 1.9% 4 10.7 4.1% 4 9.2 1.3% 4 9.3 2.6% 4
20 ActinFSL 11.1 4.4% 3 11.3 5.9% 4 9.2 3.9% 4 10.8 2.9% 4
22 SynthaSil 10.6 4.2% 4 10.9 10.4% 4 8.8 2.3% 4 10.9 13.0% 4
23 APPT-SP 11.0 8.4% 4 10.4 5.9% 4 8.5 10.3% 3 10.4 10.2% 4
24 PTT-A 10.2 13.1% 4 10.3 11.5% 4 8.5 10.4% 4 10.1 7.4% 4
25 Pathromtin SL 9.4 3.7% 4 10.4 1.4% 4 8.5 3.3% 4 8.8 3.8% 4
27 APTT-SP 10.6 2.7% 4 10.8 2.3% 4 8.9 3.9% 4 10.0 4.0% 4
28a Cephascreen 9.8
1 10.3
1 7.3
1 9.0
1
28b CK-Prest 10.0
2 10.7
2 7.8
2 8.6
2
28c PTT-A 10.8
1 11.2
1 9.4
1 11.0
1
29 Actin 8.8 8.5% 4 10.3 2.6% 4 8.6 3.7% 4 8.4 11.7% 4
30 APTT-synth Grifol 9.7 2.0% 4 10.6 1.7% 4 8.7 0.5% 4 9.2 0.5% 4
31a Cephascreen 9.4 16.1% 4 11.3 25.6% 3 8.6 9.6% 3 9.1 13.4% 3
31b CK-Prest 9.5 7.1% 4 9.9 6.4% 4 7.1 6.6% 3 8.0 13.9% 4
32 CK Prest 10.0 6.8% 4 10.8 5.1% 4 8.5 11.5% 4 9.3 5.9% 4
33 SynthaFax 7.6 9.3% 5 11.2 17.9% 5 9.5 16.5% 5 7.2 11.4% 5
34 Pathromtin SL 10.1 9.2% 4 10.9 8.4% 4 8.9 10.2% 3 9.0 3.7% 4
35 Actin 10.2 2.6% 4 10.4 1.5% 4 8.7 1.8% 4 10.1 2.4% 4
36 Dapttin 8.0 4.7% 4 10.6 4.7% 4 9.0 2.4% 4 7.6 4.1% 4
WHO/BS/2015.2261
Page 35
Lab APTT reagent
A B C D
GM
IU/ml GCV n
GM
IU/ml GCV n
GM
IU/ml GCV n
GM
IU/ml GCV n
37 Cephen 9.9 1.5% 4 10.5 1.6% 4 9.0 1.7% 4 9.6 1.0% 4
39 APTT-SP 9.1 3.1% 4 10.4 2.3% 4 9.2 6.1% 4 9.2 4.5% 4
40 ActinFS 9.8 4.7% 4 9.7 7.2% 4 9.2 5.4% 4 9.5 9.9% 4
41 ActinFSL 10.1 4.3% 4 10.6 3.1% 4 8.2 1.9% 4 9.3 3.8% 3
42 SynthaFax 7.6 13.3% 4 10.3 19.5% 4 10.6 17.7% 4 7.5 23.6% 4
43 APTT-SP 9.7 2.3% 4 10.4 2.5% 4 9.0 2.4% 4 9.0 11.9% 4
44 Synthasil 10.5 2.9% 4 10.6 3.3% 4 9.1 2.5% 4 9.6 11.4% 4
45 ActinFS 9.0 8.2% 4 9.7 6.6% 4 8.6 6.9% 4 10.4 31.8% 4
46 ActinFSL 8.9 3.6% 4 10.4 2.3% 4 8.4 5.3% 3 8.4 0.6% 4
47 ActinFS 11.0 2.5% 4 10.5 2.6% 4 9.6 1.0% 4 10.5 1.6% 4
48 SynthaSil 9.8 4.8% 4 10.2 3.3% 4 8.6 5.5% 4 9.5 3.3% 4
49 Cephen 10.9 1.7% 4 10.5 1.2% 4 9.1 1.1% 3 10.2 1.0% 4
Overall GM / GCV/ n 9.8 11.6% 55 10.5 4.8% 55 8.7 8.4% 55 9.4 13.4% 55
Table 3B: Chromogenic assays results for samples A, B, C and D calculated relative to S:
Laboratory mean (GM), geometric coefficient of variation (GCV) and overall GM and
GCV.
Lab Chromogenic
Kit
A B C D
GM GCV n GM GCV n GM GCV n GM GCV n
12a Hyphen 7.2 4.3% 4 10.1 6.7% 4 8.5 5.5% 4 6.7 5.8% 4
12b Rossix 7.5 6.4% 4 10.3 2.0% 3 8.8 4.6% 4 7.5 7.9% 4
2a Hyphen 7.0 11.6% 4 9.5 32.5% 4 9.0 12.1% 4 6.5 11.9% 4
2b Rossix 7.9 5.9% 4 11.0 6.2% 4 8.6 8.0% 4 7.3 5.2% 4
21 Hyphen 9.3 8.8% 4 13.7 4.3% 4 11.4 7.4% 4 8.4 4.0% 3
26 Rossix 8.8 6.1% 4 10.4 2.8% 4 8.8 1.8% 4 8.6 4.0% 4
33 Hyphen 8.5 5.5% 4 11.8 3.5% 4 10.0 3.2% 4 8.3 4.4% 4
36 Hyphen 7.5 6.4% 4 10.5 6.4% 4 9.1 4.0% 4 7.1 3.9% 4
4 Rossix 7.9 7.9% 4 10.6 5.7% 4 8.9 6.1% 4 7.5 5.3% 4
40 Rossix 8.3 5.3% 3 10.4 4.8% 3 10.1 12.3% 3 7.8 10.3% 3
41 Rossix 7.9 7.9% 4 10.3 1.2% 4 8.7 4.6% 4 7.3 0.9% 4
43 Hyphen 7.5 12.8% 3 9.5 11.2% 4 8.4 7.3% 3 7.2 22.3% 3
48 Hyphen 6.7 6.3% 3 10.2 7.2% 3 8.8 7.0% 3 6.3 2.2% 3
49 Hyphen 6.7 4.7% 4 10.4 4.3% 4 8.7 1.8% 4 6.2 4.6% 4
6 Rossix 8.5 13.5% 4 10.7 12.6% 4 8.9 6.4% 4 7.5 8.3% 4
Overall GM / GCV/ n 7.8 10.2% 15 10.6 9.3% 15 9.1 8.6% 15 7.3 10.4% 15
WHO/BS/2015.2261
Page 36
Table 4: Effect of pre-diluent on potency estimates by clotting assay for samples A, B, C
and D
Samples
FIX deficient plasma Buffer (without/with outlier
excluded)
P-value
(without/with
outlier
excluded) GM
IU/ml GCV n GM IU/ml GCV n
A 9.8 13.0% 35 9.8/9.9 9.0/6.9% 20/19 0.927/0.747
B 10.5 3.3% 35 10.4/10.5 6.7/4.6% 20/19 0.435/0.977
C 8.9 4.8% 35 8.3/8.5 11.0/7.8% 20/19 0.001/0.001
D 9.4 12.9% 35 9.3/9.5 14.6/11.9% 20/19 0.872/0.713
Table 5A: Overall potency estimates by clotting or chromogenic methods
Samples
Clotting Chromogenic Ratio
Clotting/Chromogenic P-value GM
IU/ml GCV n
GM
IU/ml GCV n
A 9.8 11.6% 55 7.8 10.2% 15 1.26 <0.001
B 10.5 4.8% 55 10.6 9.3% 15 0.99 0.546
C 8.7 8.4% 55 9.1 8.6% 15 0.96 0.087
D 9.4 13.4% 55 7.3 10.4% 15 1.29 <0.001
P 0.90 16.1% 54 0.81 8.6% 15 1.10 0.012
Table 5B: Overall potency estimates by clotting or chromogenic methods. Excluding
Lab 15 (Clotting) and Lab 21 (Chromogenic)
Samples
Clotting Chromogenic Ratio
Clotting/Chromogenic
P-value
GM
IU/ml GCV n
GM
IU/ml GCV n
A 9.9 11.1% 54 7.7 9.0% 14 1.29 <0.001
B 10.5 3.8% 54 10.4 5.6% 14 1.01 0.330
C 8.8 6.6% 54 8.9 5.7% 14 0.99 0.337
D 9.4 12.5% 54 7.3 10.0% 14 1.29 <0.001
P 0.90 16.2% 53 0.81 8.1% 14 1.11 0.009
Table 6: Comparison of potency estimates obtained using Hyphen and Rossix
chromogenic kits (excluding Lab 21)
Samples
Hyphen Rossix Ratio
Hyphen/Rossix P-value GM
IU/ml GCV n
GM
IU/ml GCV n
A 7.3 8.5% 7 8.1 5.6% 7 0.90 0.014
B 10.3 7.7% 7 10.5 2.4% 7 0.98 0.415
C 8.9 6.1% 7 9.0 5.6% 7 0.99 0.810
D 6.9 10.7% 7 7.6 5.9% 7 0.91 0.032
P 0.78 9.4% 7 0.83 5.0% 7 0.94 0.133
WHO/BS/2015.2261
Page 37
Table 7: Summary of overall potency estimates (GM) for functional activity (clotting
and chromogenic assays) and coefficient of variation expressed as %GCV
Samples All Excluding outliers
GM IU/ml GCV n GM IU/ml GCV n
A 9.4 15.4% 70 9.4 15.5% 68
B 10.5 5.9% 70 10.5 4.2% 68
C 8.8 8.6% 70 8.8 6.4% 68
D 8.9 17.1% 70 8.9 16.9% 68
P 0.88 15.4% 69 0.88 15.7% 68
WHO/BS/2015.2261
Page 38
Table 8: Clotting, chromogenic and antigen results for sample P, the 4th
IS for FII, VII,
IX and X Plasma: Laboratory mean (GM), geometric coefficient of variation (GCV)
and overall GM and GCV. For clotting assays, results using buffer as pre-diluent are
denoted in red, all other laboratories used haemophilia plasma as diluent.
Clotting vs S Chromogenic vs S Antigen vs Local Pool
Lab APTT reagent GM
IU/ml GCV n Kit
GM
IU/m
l
GCV n Kit GM
u/ml GCV n
1 Actin-FSL 0.90 3.3% 4
2a Actin-FS 0.78 6.8% 4 Hyphen 0.77 7.8% 4 Asserachrom 0.94 3.8% 4
2b APTT-SP 0.90 2.6% 4 Rossix 0.81 4.4% 4 Visulize 1.02 10.5% 4
2c Cephascreen 0.82 5.5% 4 Zymutest 0.98 7.9% 4
2d Dapttin 0.85 3.3% 3
2e Synthasil 0.78 4.0% 4
3 C.K. Prest 0.68 4.5% 4
4 Clotting 0.81 6.9% 3 Rossix 0.84 4.6% 4
5 Pathromtin 1.15 1.2% 4
6 ActinFS 0.80 7.8% 4 Rossix 0.83 6.0% 3
7 PTT 1.10 14.9% 3
8a ActinFSL-BSCXP 0.81 4.4% 4
8b ActinFSL-
CA1500 0.77 2.3% 4
8c Pathromtin SL-
BSCXP 0.86 4.4% 4
8d Pathromtin SL-
CA1500 0.79 3.3% 4
9 ActinFS 0.88 3.3% 4
10 ActinFSL 1.09 9.0% 4 Visulize 0.87 2.6% 4
11 CK-Prest 1.02 3.0% 4 Visulize 0.85 8.3% 4
12a Hyphen 0.79 3.4% 4
12b Rossix 0.77 7.7% 4
13 TriniClot 1.22 1
14 ActinFS 0.87 2.9% 4
15 PTT 0.86 9.9% 4
16 Actin 0.94 5.7% 4
17 APTT 1.06 1.8% 4
18 ActinFS 0.92 5.2% 4
19 Trinity 0.80 0.7% 4
20 ActinFSL 0.79 8.0% 4
21 Hyphen 0.92 2.4% 4 Cedarlane 0.81 17.7% 4
22 SynthaSil 0.91 1.7% 4
23 APPT 0.94 14.4% 3 AssayPro 0.93 27.1% 4
WHO/BS/2015.2261
Page 39
Clotting vs S Chromogenic vs S Antigen vs Local Pool
Lab APTT reagent GM
IU/ml GCV n Kit
GM
IU/m
l
GCV n Kit GM
u/ml GCV n
24 PTT-A 0.84 7.1% 4 Asserachrom 0.87 4.8% 4
25 Pathromtin SL 0.98 5.1% 4
26 Rossix 0.86 7.1% 4
27 APTT 0.84 2.9% 4
28a Cephascreen 1.07 1 Asserachrom 0.83 7.5% 4
28b CK-Prest 1.07 2
28c PTTA 1.17 1
29 Actin 0.82 4.5% 4
30 APTT 0.82 2.7% 4
31a Cephascreen 1.20 9.9% 4 Asserachrom 0.88 7.7% 4
31b CK-Prest 0.96 4.5% 4
32 CK Prest 1.09 5.6% 4
33 SynthaFax 1.00 12.5% 5 Hyphen 0.93 3.0% 4 Visulize 0.96 5.2% 4
34 Pathromtin SL 0.92 12.0% 4 Asserachrom 0.96 1.8% 4
35 Actin 1.10 3.7% 4
36 Dapttin 0.95 5.3% 4 Hyphen 0.80 3.3% 4
37 Cephen 0.84 3.0% 4
38 Affinity Bio’ 0.97 2.3% 4
39 APTT-SP 0.87 5.5% 3
40 ActinFS 0.81 7.8% 4 Rossix 0.83 7.9% 3 Asserachrom 0.96 1
41 ActinFSL 1.31 1 Rossix 0.90 1.6% 3
42 SynthaFax
43 APTT-SP 0.88 5.4% 3 Hyphen 0.73 2 Asserachrom 0.79 10.1% 4
44 SynthaSil 0.84 3.2% 3
45 ActinFS 0.70 5.2% 4
46 ActinFSL 0.71 3.2% 4
47 ActinFS 0.74 2.3% 4
48 SynthaSil 1.00 5.3% 4 Hyphen 0.71 5.9% 3 Visulize 0.83 2
49 Hyphen 0.80 4.1% 4 Hyphen 0.74 5.5% 4 Zymutest 0.88 11.1% 3
Overall GM / GCV / n 0.90 16.1% 54 0.81 8.6% 15 0.90 7.9% 17
Overall potency (clotting
& chromogenic) 0.88 IU/ml, GCV 15.4%, n = 69
WHO/BS/2015.2261
Page 40
Table 9A: Antigen values for samples S, A, B, C, D, and P, assuming local plasma pool (L) value is 1.0 U/ml
S vs L A vs L B vs L C vs L D vs L P vs L
Lab Method GM
IU/ml GCV n
GM
IU/ml GCV n
GM
IU/ml GCV n
GM
IU/ml GCV n
GM
IU/ml GCV n
GM
IU/ml GCV n
2 Asserachrom 10.7 10.2% 4 8.2 7.6% 4 12.4 16.4% 4 10.6 13.3% 4 7.2 6.6% 4 0.94 3.8% 4
2 Visulize 11.4 8.7% 4 7.0 4.6% 3 12.4 4.8% 4 10.4 7.1% 4 6.1 5.4% 3 1.02 10.5% 4
2 Zymutest 9.4 3.8% 4 9.5 4.2% 4 10.8 2.5% 4 9.2 3.3% 4 8.0 3.3% 4 0.98 7.9% 4
10 Visulize 9.7 4.0% 4 6.0 4.3% 4 10.7 1.9% 4 9.1 3.9% 4 5.5 3.3% 4 0.87 2.6% 4
11 Visulize 9.5 10.5% 4 6.1 8.1% 4 10.6 6.8% 4 9.0 8.5% 4 5.2 6.8% 4 0.85 8.3% 4
21 Cedarlane 11.3 17.6% 4 3.4 11.5% 4 10.5 23.7% 4 8.6 13.4% 3 2.9 15.1% 4 0.81 17.7% 4
23 AssayPro 9.8
2 6.9 14.8% 3 11.4 11.4% 4 10.4 15.3% 3 6.3
2 0.93 27.1% 4
24 Asserachrom 9.7 8.7% 4 7.9 8.4% 4 11.4 8.1% 4 9.6 4.9% 4 6.2 10.2% 4 0.87 4.8% 4
28 Asserachrom 9.3 10.6% 4 7.2 11.9% 4 10.3 9.6% 4 8.8 13.0% 4 6.0 13.2% 4 0.83 7.5% 4
31 Asserachrom 9.7 10.1% 4 7.6 9.3% 4 10.9 10.5% 4 9.3 10.8% 4 6.4 7.7% 4 0.88 7.7% 4
33 Visulize 9.4 1.3% 3 5.3 4.9% 4 9.3 12.9% 3 9.3 2.5% 3 4.6 2.6% 3 0.96 5.2% 4
34 Asserachrom 10.6 3.2% 4 8.0 3.0% 3 12.0 1.6% 4 10.5 4.4% 4 6.5 2.4% 4 0.96 1.8% 4
38 Affinity BIologicals 11.8 3.3% 4 6.8 3.2% 4 12.3 1.1% 4 10.4 2.9% 4 5.8 2.3% 4 0.97 2.3% 4
40 Asserachrom 9.5
1 9.9
1 12.3
2 11.2
2 7.7
2 0.96
1
43 Asserachrom 9.4 9.0% 4 6.4 7.8% 3 9.2
1 9.0 6.0% 4 5.0 28.0% 3 0.79 10.1% 4
48 Visulize 11.5
2 8.2
1 11.0
2 10.0
2 3.6
1 0.83
2
49 Zymutest 8.8 10.6% 3 9.0 9.4% 3 10.2 4.5% 3 8.7 9.2% 3 7.8 9.1% 3 0.88 11.1% 3
GM / GCV / n 10.0 9.2% 17 7.1 28.9% 17 11.0 9.8% 17 9.6 8.6% 17 5.7 30.7% 17 0.90 7.9% 17
WHO/BS/2015.2261
Page 41
Table 9B: Antigen values for samples S, A, B, C and D relative to sample P, the 4th
IS for FII, VII, IX and X Plasma, assuming antigen
value of 0.9 U/ml
Lab Method
S vs P A vs P B vs P C vs P D vs P
GM
IU/ml GCV n
GM
IU/ml GCV n
GM
IU/ml GCV n
GM
IU/ml GCV n
GM
IU/ml GCV n
2 Asserachrom 10.2 11.7% 4 7.8 9.1% 4 11.9 15.2% 4 10.2 17.0% 4 6.8 9.4% 4
2 Visulize 10.1 12.1% 4 6.1 10.6% 3 10.9 12.3% 4 9.2 10.1% 4 5.3 7.5% 3
2 Zymutest 8.6 11.3% 4 8.7 5.4% 4 9.8 6.5% 4 8.4 6.9% 4 7.3 5.9% 4
10 Visulize 10.0 4.4% 4 6.2 3.7% 4 11.1 3.4% 4 9.4 4.3% 4 5.7 4.2% 4
11 Visulize 10.0 3.2% 4 6.5 5.9% 4 11.2 6.2% 4 9.5 4.6% 4 5.6 5.2% 3
21 Cedarlane 12.4 4.7% 4 3.8 8.5% 4 11.6 9.4% 4 9.3 8.8% 4 3.1 15.5% 3
23 AssayPro 9.2 18.0% 4 6.4
2 9.9 14.2% 3 9.5 16.6% 4 5.7 21.0% 3
24 Asserachrom 9.8 10.8% 3 8.1 7.0% 4 11.7 8.7% 4 9.9 7.3% 4 6.5 9.5% 4
28 Asserachrom 10.0 5.1% 4 7.8 6.0% 4 11.1 3.2% 4 9.5 8.3% 4 6.5 7.1% 4
31 Asserachrom 9.9 2.6% 4 7.8 2.6% 4 11.2 2.8% 4 9.5 3.5% 4 6.6 0.6% 4
33 Visulize 8.9 5.4% 3 4.9 7.4% 4 8.7 11.4% 3 8.8 9.0% 3 4.4 8.6% 4
34 Asserachrom 9.9 2.6% 4 7.6 2.9% 3 11.3 1.7% 4 9.9 4.6% 4 6.1 3.4% 4
38 Affinity Biologicals 10.9 4.8% 4 6.3 1.5% 4 11.4 3.2% 4 9.6 5.1% 4 5.4 1.4% 4
40 Asserachrom 8.9
1
11.1
1 10.1
1 6.5
1
43 Asserachrom 10.8 6.9% 4 7.2 8.7% 3 11.4 9.7% 3 10.3 10.6% 4 5.6 19.9% 3
48 Visulize 10.7
2 7.4
1 10.3
2 9.3
2 3.1
1
49 Zymutest 9.2 7.6% 4 9.4 3.5% 4 10.4 5.6% 4 8.9 7.6% 4 8.1 3.8% 4
GM / GCV / n 9.9 9.1% 17 6.8 26.6% 16 10.9 8.2% 17 9.5 5.4% 17 5.6 31.5% 17
WHO/BS/2015.2261
Page 42
Table 10A: Comparison of potency estimates by clotting assays for sample A, relative to
sample S, the 4th
IS for FIX concentrate and sample D with assigned potency of 9.4 IU/ml
based on clotting assays or value of 8.9 IU/ml based on overall potency estimate.
A vs D
Lab APTT reagent
A vs S
Assigned value of D
by clotting only: 9.4
IU/ml
Assigned value of D by
clotting and chromogenic:
8.9 IU/ml
GM
IU/ml GCV n
GM
IU/ml GCV n
GM
IU/ml GCV n
1 Actin-FSL 9.8 6.1% 4 9.8 10.1% 4 9.3 10.1% 4
2a Actin-FS 12.3 3.1% 4 9.7 3.2% 4 9.2 3.2% 4
2b APTT-SP 9.4 2.9% 4 10.0 7.5% 4 9.4 7.5% 4
2c Cephascreen 12.4 4.2% 4 10.1 3.3% 4 9.6 3.3% 4
2d Dapttin 11.2 6.4% 4 10.2
2 9.7 2
2e Synthasil 10.9 4.3% 4 9.7 6.6% 4 9.2 6.6% 4
3 C.K. Prest 9.0 7.3% 4 10.2 3.3% 4 9.6 3.3% 4
4 Dapttin 8.1 6.5% 4 10.1 3.7% 4 9.5 3.7% 4
5 Pathromtin SL 9.5 1.5% 4 10.4 2.1% 4 9.9 2.1% 4
6 ActinFS 12.4 7.1% 4 9.9 10.0% 4 9.4 10.0% 4
7 PTT-A 10.4 3.4% 4 7.9 17.5% 4 7.4 17.5% 4
8a ActinFSL-BSCXP 9.2 1.7% 4 9.5 4.1% 4 9.0 4.1% 4
8b ActinFSL-CA1500 10.0 3.2% 4 9.2 2.3% 4 8.7 2.3% 4
8c Pathromtin SL-
BSCXP 9.1 3.1% 4 9.9 5.2% 4 9.3 5.2% 4
8d Pathromtin SL-
CA1500 8.7 5.1% 4 9.4 2.6% 4 8.9 2.6% 4
9 ActinFS 9.2 5.8% 4 9.9 7.4% 4 9.4 7.4% 4
10 ActinFSL 9.9 11.5% 4 9.6 7.1% 4 9.1 7.1% 4
11 CK-Prest 10.1 3.8% 4 10.0 6.1% 4 9.5 6.1% 4
13 TriniClot 10.3 2.9% 4 10.7 3.1% 4 10.1 3.1% 4
14 ActinFS 11.4 3.5% 4 9.64 1.5% 4 9.1 1.5% 4
15 PTT-A 7.7
2 10.6
1 10.1 1
16 Actin 10.2 4.5% 4 10.1 4.1% 4 9.6 4.1% 4
17 TriniClot 9.5 2.8% 4 9.81 3.3% 4 9.3 3.3% 4
18 ActinFS 10.1 3.7% 4 9.4
1 8.9 1
19 TriniClot 10.2 1.9% 4 10.3 2.9% 4 9.8 2.9% 4
20 ActinFSL 11.1 4.4% 3 8.8 3.4% 3 8.3 3.4% 3
22 SynthaSil 10.6 4.2% 4 9.1 11.0% 4 8.6 11.0% 4
23 APPT-SP 11.0 8.4% 4 9.9 7.2% 4 9.4 7.2% 4
24 PTT-A 10.2 13.1% 4 9.5 5.8% 4 9.0 5.8% 4
25 Pathromtin SL 9.4 3.7% 4 10.1 2.7% 4 9.6 2.7% 4
27 APTT-SP 10.6 2.7% 4 10.0 2.6% 4 9.5 2.6% 4
WHO/BS/2015.2261
Page 43
A vs D
Lab APTT reagent
A vs S
Assigned value of D
by clotting only: 9.4
IU/ml
Assigned value of D by
clotting and chromogenic:
8.9 IU/ml
GM
IU/ml GCV n
GM
IU/ml GCV n
GM
IU/ml GCV n
28a Cephascreen 9.8
1 10.2
1 9.7 1
28b CK-Prest 10.0
2 11.0 8.5% 2 10.4 8.5% 2
28c PTT-A 10.8
1 9.3
1 8.8 1
29 Actin 8.8 8.5% 4 9.8 2.9% 4 9.2 2.9% 4
30 APTT-synth Grifol 9.7 2.0% 4 9.9 2.2% 4 9.4 2.2% 4
31a Cephascreen 9.4 16.1% 4 9.9 9.2% 4 9.4 9.2% 4
31b CK-Prest 9.5 7.1% 4 11.2 7.7% 4 10.6 7.7% 4
32 CK Prest 10.0 6.8% 4 10.1 6.9% 4 9.6 6.9% 4
33 SynthaFax 7.6 9.3% 5 10.0 8.2% 5 9.5 8.2% 5
34 Pathromtin SL 10.1 9.2% 4 10.5 7.1% 4 9.9 7.1% 4
35 Actin 10.2 2.6% 4 9.5 1.0% 4 9.0 1.0% 4
36 Dapttin 8.0 4.7% 4 10.0 0.8% 4 9.4 0.8% 4
37 Cephen 9.9 1.5% 4 9.7 1.0% 4 9.2 1.0% 4
39 APTT-SP 9.1 3.1% 4 9.6
1 9.1 1
40 ActinFS 9.8 4.7% 4 9.7 7.3% 4 9.2 7.3% 4
41 ActinFSL 10.1 4.3% 4 10.4 6.8% 4 9.8 6.8% 4
42 SynthaFax 7.6 13.3% 4 9.5 9.3% 4 9.0 9.3% 4
43 APTT-SP 9.7 2.3% 4 10.1 14.2% 4 9.6 14.2% 4
44 Synthasil 10.5 2.9% 4 10.3 8.5% 4 9.7 8.5% 4
45 ActinFS 9.0 8.2% 4 8.2 33.7% 4 7.7 33.7% 4
46 ActinFSL 8.9 3.6% 4 10.0 3.0% 4 9.4 3.0% 4
47 ActinFS 11.0 2.5% 4 9.9 1.7% 4 9.3 1.7% 4
48 SynthaSil 9.8 4.8% 4 9.7 3.2% 4 9.1 3.2% 4
49 Cephen 10.9 1.7% 4 10.1 2.6% 4 9.6 2.6% 4
Overall GM / GCV / n 9.8 11.6% 55 9.8 6.1% 55 9.3 6.1% 55
Paired t-test p-value vs S 0.985 0.002
WHO/BS/2015.2261
Page 44
Table 10B: Comparison of potency estimates by chromogenic assays for sample A, relative
to sample S, the 4th
IS for FIX concentrate and sample D with assigned potency of 9.4
IU/ml based on clotting assays or value of 8.9 IU/ml based on overall potency estimate.
A vs D
Lab Kit
A vs S Assigned value of D by
clotting only: 9.4 IU/ml
Assigned value of D by
clotting and chromogenic:
8.9 IU/ml
GM
IU/ml GCV n
GM
IU/ml GCV n
GM
IU/ml GCV n
12 Hyphen 7.2 4.3% 4 10.1 4.8% 4 9.6 4.8% 4
12 Rossix 7.5 6.4% 4 10.1 3.4% 4 9.5 3.4% 4
2 Hyphen 7.0 11.6% 4 9.8 4.7% 4 9.3 4.7% 4
2 Rossix 7.9 5.9% 4 10.7 9.4% 4 10.1 9.4% 4
21 Hyphen 9.3 8.8% 4 10.1 7.4% 4 9.5 7.4% 4
26 Rossix 8.8 6.1% 4 9.4 11.8% 4 8.9 11.8% 4
33 Hyphen 8.5 5.5% 4 10.7 8.0% 3 10.2 8.0% 3
36 Hyphen 7.5 6.4% 4 9.7 8.4% 4 9.2 8.4% 4
4 Rossix 7.9 7.9% 4 9.6 1.1% 4 9.1 1.1% 4
40 Rossix 8.3 5.3% 3 9.9 4.6% 4 9.4 4.6% 4
41 Rossix 7.9 7.9% 4 9.9 11.3% 3 9.4 11.3% 3
43 Hyphen 7.5 12.8% 3 10.2 8.2% 4 9.6 8.2% 4
48 Hyphen 6.7 6.3% 3 9.8 8.8% 3 9.3 8.8% 3
49 Hyphen 6.7 4.7% 4 10.0 4.2% 3 9.4 4.2% 3
6 Rossix 8.5 13.5% 4 10.2 4.5% 4 9.7 4.5% 4
GM / GCV/n 7.8 10.2% 15 10.0 3.6% 15 9.5 3.6% 15
Paired t-test p-value vs S <0.001 <0.001
WHO/BS/2015.2261
Page 45
Table 10C: Summary of sample A potencies against sample S, the 4th
IS for FIX
concentrate and sample D, assuming (assuming the assigned potency of 9.4 IU/ml based on
clotting assays).
Method
A v S A v D (9.4 IU/ml) A v D (8.9 IU/ml)
GM
IU/ml GCV n
GM
IU/ml GCV n
GM
IU/ml GCV n
Clotting 9.8 11.6% 55 9.8 6.1% 55 9.3 6.1% 55
Chromogenic 7.8 10.2% 15 10.0 3.6% 15 9.5 3.6% 15
Clotting /Chromogenic ratio (unpaired
t test) 1.25 (p <0.001) 0.98 ( p = 0.282)
Overall potency (incl clotting and
chromogenic) 9.4 15.4% 70 9.9 5.7% 70 9.3 5.7% 70
paired t-test p value for A vs S against
A vsD 0.007 0.956
unpaired t-test p value for A vs S
Clotting only against overall potency
for A vsD
0.779 0.003
WHO/BS/2015.2261
Page 46
Table 11A: Comparison of potency estimates for sample Purple against sample S, the 4th
IS for FIX concentrate, sample A (Acl), assuming the assigned potency of 9.8 IU/ml based
on clotting assays, and sample D (Dcl), assuming the assigned potency of 9.4 IU/ml based
on clotting assays.
Lab Type Method Purple v S Purple v Acl Purple v Dcl
GM
IU/ml GCV n
GM
IU/ml GCV n
GM
IU/ml GCV n
2 Clotting Actin-FS 9.6 6.3% 4 7.7 3.3% 4 7.6 1.9% 4
2 Clotting APTT-SP 8.3 8.9% 4 8.6 10.5% 4 8.7 11.3% 4
2 Clotting Cephascreen 10.3 7.4% 4 8.1 5.1% 4 8.4 5.6% 4
2 Clotting Dapttin 9.3 7.4% 3 8.0 9.5% 3 9.2 2.9% 2
2 Clotting Synthasil 9.2 4.5% 4 8.3 8.1% 4 8.2 6.8% 4
36 Clotting Dapttin 7.7 1.9% 4 9.5 4.3% 4 9.6 3.5% 4
37 Clotting Cephen 8.7 7.5% 4 9.0 4.9% 4 8.9 6.2% 4
44 Clotting SynthaSil 9.0 1.1% 4 8.5 2.3% 4 8.9 11.0% 4
45 Clotting ActinFS 8.5 7.3% 4 9.2 10.2% 4 7.7 26.3% 4
48 Clotting SynthaSil 9.1 2.7% 4 9.2 5.4% 4 9.0 5.2% 4
2 Chromo Hyphen 8.0 25.2% 4 11.1 24.6% 4 11.4 20.9% 4
2 Chromo Rossix 7.2 5.8% 4 9.0 3.7% 4 9.3 5.6% 4
36 Chromo Hyphen 7.0 4.2% 4 9.0 1.6% 4 9.3 4.8% 4
48 Chromo Hyphen 6.3 4.1% 3 9.2 2.8% 3 9.4 1.8% 3
GM / GCV / n 8.4 14.6% 14 8.8 9.4% 14 8.9 10.7% 14
paired t-test, S v A; S v D; p-
value 0.338 0.273
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Table 11B: Comparison of potency estimates for sample Purple against sample S, the 4th
IS for FIX concentrate, sample A (Acl+ch),assuming the assigned potency of 9.4 IU/ml
based on overall potency estimate, and sample D (Dcl+ch) assuming the assigned potency of
8.9 IU/ml based on overall potency estimate.
Lab Type Method
Purple v S Purple v Acl+ch Purple v Dcl+ch
GM
IU/ml GCV n
GM
IU/ml GCV n
GM
IU/ml GCV n
2 Clotting Actin-FS 9.6 6.3% 4 7.3 3.3% 4 7.2 1.9% 4
2 Clotting APTT-SP 8.3 8.9% 4 8.2 10.5% 4 8.3 11.3% 4
2 Clotting Cephascreen 10.3 7.4% 4 7.8 5.1% 4 8.0 5.6% 4
2 Clotting Dapttin 9.3 7.4% 3 7.7 9.5% 3 8.7 2.9% 2
2 Clotting Synthasil 9.2 4.5% 4 8.0 8.1% 4 7.8 6.8% 4
36 Clotting Dapttin 7.7 1.9% 4 9.1 4.3% 4 9.1 3.5% 4
37 Clotting Cephen 8.7 7.5% 4 8.6 4.9% 4 8.6 6.2% 4
44 Clotting SynthaSil 9.0 1.1% 4 8.1 2.3% 4 8.4 11.0% 4
45 Clotting ActinFS 8.5 7.3% 4 8.8 10.2% 4 7.3 26.3% 4
48 Clotting SynthaSil 9.1 2.7% 4 8.8 5.4% 4 8.5 5.2% 4
2 Chromo Hyphen 8.0 25.2% 4 10.7 24.6% 4 10.8 20.9% 4
2 Chromo Rossix 7.2 5.8% 4 8.6 3.7% 4 8.8 5.6% 4
36 Chromo Hyphen 7.0 4.2% 4 8.7 1.6% 4 8.8 4.8% 4
48 Chromo Hyphen 6.3 4.1% 3 8.8 2.8% 3 8.9 1.8% 3
GM / GCV / n 8.4 14.6% 14 8.5 9.4% 14 8.5 10.7% 14
paired t-test, S v A; S v D; p-
value 0.832 0.847
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Table 11C: Summary of potency estimates for sample Purple against sample S, the 4th
IS
for FIX concentrate, sample Acl (assuming the assigned potency of 9.9 IU/ml based on
clotting assays), and sample Dcl (assuming the assigned potency of 9.4 IU/ml based on
clotting assays).
Table 11D: Summary of potency estimates for sample Purple against sample S, the 4th
IS
for FIX concentrate, sample A (Acl+ch),assuming the assigned potency of 9.4 IU/ml based
on overall potency estimate, and sample D (Dcl+ch) assuming the assigned potency of 8.9
IU/ml based on overall potency estimate.
Method Purple v S Purple v Acl Purple v Dcl
GM
IU/ml GCV n
GM
IU/ml GCV n
GM
IU/ml GCV n
Clotting 9.0 8.3% 10 8.8 7.1% 10 9.1 8.0% 10
Chromogenic 7.1 10.1% 4 9.4 10.6% 4 10.4 10.8% 4
Overall (incl clotting and chromogenic) 8.4 14.6% 14 9.0 9.4% 14 9.5 10.7% 14
Clotting /Chromogenic ratio
(unpaired t-test p-value) 1.26 ( p < 0.001) 0.90 (p =0.038) 0.88 (p = 0.020)
Method
Purple v S Purple v Acl+ch Purple v Dcl+ch
GM
IU/ml GCV n
GM
IU/ml GCV n
GM
IU/ml GCV n
Clotting 9.0 8.3% 10 8.2 7.1% 10 8.1 8.0% 10
Chromogenic 7.1 10.1% 4 9.2 10.6% 4 9.3 10.8% 4
Overall (incl clotting and chromogenic) 8.4 14.6% 14 8.8 9.4% 14 8.5 10.7% 14
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Figure 1: Histogram showing estimated potency of sample A relative to sample S, the 4th
IS
for Blood Coagulation Factor IX, Concentrate, 07/182. Each box denotes overall geometric
mean potency (GM) from one laboratory
Figure 2: Histogram showing estimated potency of sample B relative to sample S, the 4
th IS
for Blood Coagulation Factor IX, Concentrate, 07/182. Each box denotes overall geometric
mean potency (GM) from one laboratory
GM= 9.4 IU/ml; GCV= 15.5%, n = 68
Outliers: clotting, Lab15; Chromogenic , Lab 21
GM= 10.5 IU/ml; GCV= 4.2%, n = 68
Outliers: clotting, Lab15; Chromogenic , Lab 21
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Figure 3: Histogram showing estimated potency of sample C relative to sample S, the 4th
IS
for Blood Coagulation Factor IX, Concentrate, 07/182. Each box denotes overall geometric
mean potency (GM) from one laboratory
Figure 4: Histogram showing estimated potency of sample D relative to sample S, the 4
th IS
for Blood Coagulation Factor IX, Concentrate, 07/182. Each box denotes overall geometric
mean potency (GM) from one laboratory
GM= 8.8 IU/ml; GCV= 6.4%, n = 68
Outliers: clotting, Lab15; Chromogenic ,
Lab 21
GM= 8.9 IU/ml; GCV= 16.9%, n = 68
Outliers: clotting, Lab15; Chromogenic ,
Lab 21
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Figure 5: Laboratories’ potency estimates for samples A, B, C and D relative to S, the 4th
IS
for FIX concentrate, obtained using different APTT reagents
Potency range: 7.6 – 12.4 IU/ml
Potency range: 8.5 – 11.4 IU/ml
Potency range: 6.6 – 12.4 IU/ml
Potency range: 6.0 – 10.6 IU/ml
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Figure 6: Histogram showing estimated potency of sample P relative to sample S, the 4th
IS
for Blood Coagulation Factor IX, Concentrate, 07/182. Each box denotes overall geometric
mean potency (GM) from one laboratory
Figure 7: Histogram showing estimated potency of sample A relative to sample D,
assuming potency of 9.4 IU/ml. Each box denotes overall geometric mean potency (GM)
from one laboratory. For comparison - Inset: histogram of sample A relative to 4th
IS for
FIX concentrate.
Table 12: Accelerated Degradation Study
GM= 0.88 IU/ml; GCV= 15.7%, n = 68
Outliers: clotting, Lab15; Chromogenic, Lab 21
GM= 9.9 IU/ml; GCV= 5.7%, n = 68
Outliers: clotting, Lab15; Chromogenic, Lab 21
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Sample A, 07/142
(7.5 years)
Sample B, 14/148
(6 and 8 months)
Sample C, 14/162
(6 months)
Sample D, 14/180
(6 months)
Temp °C Potency
Loss
%/year
95%
Upper
CL %Loss
/year
Potency
Loss
%/year
95%
Upper
CL %Loss
/year
Potency
Loss
%/year
95%
Upper
CL %Loss
/year
Potency
Loss
%/year
95%
Upper
CL %Loss
/year
-20 0.000 0.000 0.000 0.000 0.001 0.002 0.000 0.000
+4 0.002 0.005 0.018 0.028 0.068 0.160 0.000 0.001
+20 0.105 0.164 0.547 0.715 1.088 2.031 0.003 0.186
+37 3.745 4.584 12.884 14.315 14.136 19.614 0.431 13.184
Table 13: Mean NAPTT clotting times carried out in accordance with EP method for
activated coagulation factors. Dilutions carried out based on estimated potency against
sample S by clotting assays (Table 3A)
Mean Blank (s)
N = 4
Mean 1/10 (s)
N = 2
Mean 1/100 (s)
N = 2
S 316 238 269
A 315 232 272
B 315 243 275
C 312 245 271
D 288 239 273
Table 14: Estimated level of activated factor IX (FIXa) (by chromogenic assays, n = 2,
against the 1st International Standard for FIXa) in the 4
th IS for FIX, concentrate (S) and
the candidates.
FIXa mIU/ampoule
(95% CL)
IU FIXa/1000
IU FIX*
S 3.5
(2.9 – 4.1) 0.44
A 12.3
(11..8 – 12.9) 1.26
B 4.3
(3.9 – 4.8) 0.41
C 3.7
(3.4 – 4.0) 0.43
D 2.3
(2.0 – 2.6) 0.24
*Based on estimated FIX against sample S (clotting)
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Appendix I: List of Participants
Kirsten Villadsen, Aarhus Universitets Hospital, Denmark
Denise Foulon, Affinity Biologicals, Canada
Stephanie Eichmeir, Christoph Kefeder & Heidemarie Schindl, AGES MEA OMCL, Austria
Natalija Kerestes, Baxter AG (IBC/Coagulation & Biochemical Methods), Austria
Peter Gaertner, Baxter AG (QC Vienna), Austria
Martin Blum, Baxter AG, Austria
Herbert Gritsch, Baxter Innovations GmbH, Austria
Amanda Blande, Bio Products Laboratory (QC), UK
Ruth Archer & Sarah Kingsland, Bio Products Laboratory (R&D), UK
Buyue Yang, Jurg Sommer & Sara Bardan, Biogen (Cambridge Campus), USA
Maureen Shreve, Biogen Idec, USA
Kerstin Dohme, Biotest AG (QC), Germany
Steffen Kistner, Karin Fuchs & Jens Daufenbach, Biotest AG, Germany
Yideng Liang, Tseday Tegegn & Mikhail Ovanesov, CBER/FDA, USA
Wendy Hollon & Meera Chitlur, Children's Hospital of Michigan, USA
Annette Feusser, CSL Behring, Germany
Klara Howard & Martina Treutlein, CSL Behring, Germany
Nathalie Barat, François Nicham & Jérôme Beltran, Diagnostica Stago (R&D Department), France
Celine Aime, Juliette Grenet, Isabelle Regairaz & Jessica Lelansans, Diagnostica Stago, France
Joy Bosica & Derek Toth, Emergent Biosolutions, Canada
Dot Adcock, Stephanie Stubert & Mike Taylor, Esoterix Coagulation, Laboratory Corporation of
America Holdings, USA
Brigitte Wild & Chantal Raphalen, European Directorate for the Quality of Medicines and
Healthcare, France
Sharon Flan & Barb Young, George King BioMedical, USA
Will Stevens & Albert Cheung, Health Canada, Canada
Joanne McGrath, Hemotasis Reference Laboratory, Canada
Jean Amiral, Hyphen Biomed, France
Luis Soares & Mjoao Portela, Infarmed (DCQ), Portugal
Nuria Hosta, Instituto Grifols, Spain
Stefania Menga, Paola Rossi & Nicola Luchesi, Kedrion, Italy
Claudia van Rijn & Jeroen Eikenboom, Leiden University Medical Center, Netherlands
Francois Hemery, LFB, France
Chi Young Ahn, Ki Kyung Jung & Sang Mi Park, Ministry of Food and Drug Safety, Korea
Stella Williams & Luis Figerido, National Institute for Biological Standards and Control, UK
Wang Quigzhou, National Institute for Food and Drug Control China
Madoka Kuramitsu, National Institute of Infectious Diseases, Japan
Anne Fuglsang Barslund, Novo Nordisk A/S, Denmark
Vibeke Sønder, Novo Nordisk Park, Denmark
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Monika Stadler, Katharina Pock, Susanna Huber and Sandra Janisch, Octapharma Pharmazeutika
Produktions GesmBH, Austria
Peter Baker, Oxford Haemophilia and Thrombosis Centre, UK
Andreas Hunfeld, Sylvia Rosenkranz & Andrea Schroda, Paul-Ehrlich-Institut, Germany
Yolanda Samino Garcia, Jose Alberto Martinez-Marin, Juan Solis-Fernandez, Esther Otero
Rodriguez, Emma Rodriguez Vignote, Helena Holguin Asensio & Pedro Paredes-Martinez, Pfizer
Algete (Wyeth Farma SA), Spain
Robin Verhaar, RIVM-GZB, Netherlands
Pia Bryngelhed & Steffen Rosen, Rossix AB, Sweden
Anne Riddell, Royal Free Hospital, UK
Annette Bowyer, Royal Hallamshire Hospital, UK
Jeannette Rentenaar & Carel Eckmann, Sanquin Blood Supply Foundation, Netherlands
Regina Gebauer & Michael Timme, Siemens Healthcare Diagnostics Products GmbH, Germany
Katja Boegli, Michael Gilgen, Alice Leuenberger, Swissmedic, Switzerland
Samuel Ling, Alison Jones, Lu Liu & Chong Loh, Therapeutics Goods Administration (OLSS
Biochemistry), Australia
Renata Zadro, University Hospital Center Zagreb, Croatia
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Appendix II – Study Protocol
Collaborative study to value assign potencies to International Standard for Factor IX
Aims of study:
1. To value assign the 5th
International Standard for Blood Coagulation Factor IX,
Concentrate/European Pharmacopoeia (EP) Factor IX, BRP
2. To value assign an International Standard for Recombinant Factor IX/EP Recombinant
Factor IX, BRP
3. To value assign Factor IX antigen to the 4th
International Standard for Blood Coagulation
Factor II, VII, IX, X, Plasma
Samples
S – the 4th
I.S. Factor IX, 07/182 potency 7.9 IU/ampoule
P – the 4th
I.S. Blood Coagulation Factor II, VII, IX, X, Plasma, potency 0.86 IU/ampoule
A – candidate sample, potency 8 – 10 IU/ampoule
B – test sample, potency 10 – 12 IU/ampoule
C – candidate sample, potency 8 – 10 IU/ampoule
D – candidate sample, potency 8 – 10 IU/ampoule
Pr – purple capped samples (sent on dry ice) – store at -80°C, and thaw in 37°C waterbath
The samples should be handled as follows:
1. Store all unopened ampoules below -20°C.
2. Allow ampoules to warm to room temperature (~15 minutes) prior to reconstitution.
3. Open ampoule after ensuring all the contents are in the lower half and reconstitute with
1.0 ml distilled water. Allow the ampoule to stand for 10 minutes at room temperature
and aid reconstitution by gentle swirling, ensuring the contents has completely dissolved.
Transfer the entire contents to a plastic tube.
4. Once reconstituted, the materials should be kept on melting ice and assayed within 3
hours.
Design of study – functional activity (FIX:C):
Please read this protocol thoroughly before commencing the testing of samples. If there are
any queries please contact John Hogwood (email address at the end of the protocol)
WHO/BS/2015.2261
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Samples: Samples A, B, C, D, P and S are coded ampoules dispatched from NIBSC. Four
ampoules of each sample have been provided. For laboratories performing the functional and
antigen methods, an extra set of 4 ampoules will be provided.
All participants should carry out 4 independent assays using their routine method for measuring
factor IX, an independent assay being defined as a completely fresh set of dilutions. For each
sample at least 3 dilutions should be used, with each sample tested in replicate. The following is
an example assay design for testing all samples:
Assay design (7/14 place)
Assay 1 S1 A1 B1 C1 D1 P1 Pr1 Pr2 P1 D1 C1 B1 A1 S1
Assay 2 A2 B2 C2 P1 Pr1 P2 S2 S2 P2 D2 Pr2 C2 B2 A2
Assay 3 C3 Pr1 D3 S3 B3 P3 A3 A3 P3 B3 S3 D3 Pr2 C3
Assay 4 P4 D4 Pr1 C4 B4 A4 S4 S4 A4 B4 C4 Pr2 D4 P4
If the above design cannot be carried out in your laboratory, please contact John Hogwood
indicating the number of samples/dilutions that can be tested, and an alternative assay design will
be provided.
The calibration of all samples for functional potency should be against the 4th
International
Standard for FIX, Concentrate. Record clotting times or absorbances and calculated potencies in
the results sheet provide by email with this protocol.
Design of study – antigen (FIX:Ag):
Samples: Samples A, B, C, D, P, S and frozen Pr (Purple capped)are coded ampoules
dispatched from NIBSC. Four ampoules of each sample have been provided. For laboratories
performing the functional and antigen methods, an extra set of 4 ampoules will be provided.
Additional samples required
L1 – locally collected normal pool (day 1)
L2 – locally collected normal pool (day 2)
Please see appendix for pool suggested preparation.
All participants should carry out 4 independent assays for each method used, an independent
assay being defined as a completely fresh set of dilutions. For each sample at least 3 dilutions
should be used, with each sample tested in replicate.
For the assignment of antigen value please used the following design
Assay design (8/16place)
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Assay 1 S1 A1 B1 C1 D1 P1 Pr1 L1 L1 Pr2 P1 D1 C1 B1 A1 S1
Assay 2 A2 B2 C2 D2 L2 Pr1 P2 S2 S2 P2 Pr2 L2 D2 C2 B2 A2
Assay 3 C3 Pr1 L1* D3 S3 B3 P3 A3 A3 P3 B3 S3 D3 L1* Pr2 C3
Assay 4 L2* P4 D4 Pr1 C4 B4 A4 S4 S4 A4 B4 C4 Pr2 D4 P4 L2*
Where L1* is frozen pool of L1 and L2* is frozen pool of L2
It is appreciated that not all laboratories will be able to prepare plasma pools for testing. If frozen
pooled plasma is used, please use more than one batch, and provide details in the worksheet for
the return of results.
If the above design cannot be carried out in your laboratory, please John Hogwood indicating the
number of samples/dilutions that can be tested, and an alternative assay design will be provided.
The calibration of all samples should be against the locally collected pools (fresh or frozen)
assigned with an arbitrary 1 unit/ml. Record absorbances and calculated potencies in the results
sheet provide by email with this protocol. If a local standard or control is included in the assay
please return this data included the expected/assigned value to the material.
Return of data:
If there are any questions or you require additional samples please contact via the email below.
Raw data and estimated potencies should be recorded on the appropriate result sheets, and
returned by email to John Hogwood: [email protected] before 13 February 2015.
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Appendix: PREPARATION OF FRESH NORMAL PLASMA POOLS
Collect fresh normal plasma as described below, on two separate days, giving pools N1 and N2.
The method of collection of the fresh normal plasma is an important part of the study and should
be standardised as far as possible, according to the following protocol.
Donors
Normal healthy volunteers, excluding women who are pregnant or taking oral contraceptives.
Take blood from as many different individuals as possible, on two separate days. If possible, use
a minimum of eight different donors for each pool; if this is not possible, some of the same
individuals can be used again, but the aim is to have a total of at least 12 different donors for
each laboratory.
Anticoagulant
0.109 M tri-sodium citrate, i.e. 3.2% w/v of the dihydrate (or a mixture of tri-sodium citrate and
citric acid with a total citrate concentration of 0.109 M). Ratio of 9 volumes blood to 1 volume
of anticoagulant.
Centrifugation
Blood should be centrifuged at 4 oC as soon as possible after collection either at 50,000 g for 5
minutes or at 2000 g for 20 minutes.
Pooling and Storage
Pool equal volumes of plasma from the different donors and mix gently. Keep the plasma pool
in a plastic stoppered tube at 4 oC during the assay session. Snap-freeze aliquots of each pool
(L1*, L2*) for assays 2 and 4.
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Appendix III: Individual assay results
Table S1 – Functional Activity by clotting: Potency values (IU/ampoule) calculated relative
to S, 4th
IS Factor IX Concentrate for each sample within each assay set returned
Lab Method Sample Assay 1 Assay 2 Assay 3 Assay 4 Assay 5
01 Actin-FSL
A 9.1 10.0 9.9 10.4 B 10.7 9.9 10.8 11.7 C 9.7 9.4 9.7 10.3 D 10.0 9.0 9.4 9.3 P 0.88 0.87 0.93 0.91
02 Actin-FS
A 12.7 11.9 12.1 12.6 B 10.9 9.8 10.1 11.2 C 9.3 8.4 9.0 9.2 D 12.5 11.0 11.8 12.5 P 0.84 0.74 0.73 0.80 Purple 10.3 8.9 9.4 9.8
02 APTT-SP
A 9.3 9.2 9.8 9.6 B 9.8 10.6 10.5 11.1 C 9.2 8.7 8.4 8.8 D 9.6 8.8 8.5 8.8 P 0.93 0.88 0.89 0.90 Purple 7.8 8.8 7.6 9.0
02 Cephascreen
A 12.2 11.8 12.4 13.1 B 10.9 10.9 10.1 11.2 C 8.8 8.8 8.4 9.2 D 11.5 11.2 11.0 12.2 P 0.81 0.82 0.77 0.88 Purple 9.5 10.2 10.1 11.3
02 Dapttin
A 10.8 12.3 11.2 10.7 B 11.0 11.0 nl 10.9 C 9.5 9.4 8.9 9.0 D nl nl 10.1 10.0 P 0.86 nl 0.87 0.82 Purple 8.8 9.2 10.1 nl
02 Synthasil
A 11.6 10.9 10.7 10.5 B 11.0 10.6 9.4 10.7 C 9.2 9.0 8.5 8.9 D 10.9 10.2 10.0 11.2 P 0.82 0.77 0.76 0.76 Purple 8.8 9.6 9.0 9.6
03 C.K. Prest
A 8.8 10.0 8.4 8.9 B 11.8 11.2 10.4 10.3 C 8.3 9.4 8.7 8.6 D 8.2 8.9 7.7 8.7 P 0.71 0.68 0.64 0.67
04 Clotting
A 8.0 7.9 8.9 7.7 B 10.1 10.4 11.2 10.4 C 8.3 8.3 9.5 8.2 D 7.5 7.4 7.9 7.5 P 0.77 nl 0.88 0.80
05 Pathromtin
A 9.6 9.3 9.4 9.6 B 11.1 10.9 11.1 11.1 C 8.9 8.6 8.8 9.0 D 8.3 8.4 8.6 8.7 P 1.14 1.14 1.16 1.16
06 ActinFS
A 11.4 12.1 13.3 12.8 B 10.5 10.8 10.3 9.9 C 9.1 9.1 10.1 8.7 D 9.5 12.1 13.7 12.0
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Lab Method Sample Assay 1 Assay 2 Assay 3 Assay 4 Assay 5
P 0.78 0.77 0.90 0.77
07 PTT
A 9.9 10.7 10.6 10.3 B 10.1 8.3 13.4 10.3 C 9.9 np np 8.6 D 10.5 np 15.5 13.2 P 0.97 np 1.28 1.08
08 ActinFSL-BSCXP
A 9.2 9.4 9.1 9.3 B 10.0 10.5 9.8 10.7 C 8.2 8.2 8.2 9.0 D 9.3 9.0 8.7 9.6 P 0.80 0.83 0.77 0.84
08 ActinFSL-CA-1500
A 10.4 10.0 9.7 9.8 B 10.4 10.2 10.0 10.4 C 9.2 8.2 8.9 8.9 D 10.4 9.9 10.1 10.2 P 0.79 0.78 0.75 0.76
08 Pathromtin-BSCXP
A 8.8 9.2 9.2 9.4 B 9.8 10.3 10.5 10.9 C 7.9 8.5 8.8 9.1 D 8.7 8.1 8.8 9.2 P 0.80 0.88 0.86 0.88
08 Pathromtin-CA-1500
A 8.8 8.2 8.5 9.2 B 10.2 9.6 10.4 10.8 C 8.9 7.8 8.8 9.4 D 8.7 8.3 8.7 8.9 P 0.78 0.76 0.78 0.82
09 ActinFS
A 10.0 9.0 9.2 8.7 B 10.7 11.2 10.5 10.7 C 8.9 9.2 9.1 8.4 D 8.7 9.1 8.4 8.8 P 0.91 0.91 0.86 0.85
10 ActinFSL
A 10.2 8.4 10.7 10.3 B 11.7 8.4 9.5 8.2 C 8.5 7.4 8.1 7.7 D 10.7 8.7 9.9 9.6 P 1.19 0.97 1.14 1.08
11 CK-Prest
A 10.0 9.7 10.2 10.6 B 10.3 10.2 10.1 10.9 C 8.2 8.7 8.7 9.1 D 8.7 9.8 9.6 9.8 P 0.98 1.03 1.02 1.05
13 TriniClot
A 10.2 9.9 10.5 10.5 B 11.0 10.8 12.3 11.5 C 8.1 8.0 8.6 7.9 D np 8.6 np 9.6 P 1.22 np np np
14 ActinFS
A 11.4 11.9 11.5 11.0 B 10.5 10.5 10.7 10.3 C 9.1 9.2 9.1 8.5 D 11.3 11.6 11.1 10.7 P 0.86 0.91 0.86 0.86
15 PTT
A 7.7 7.8 np nl B 8.8 8.4 nl 8.4 C 5.8 6.4 5.9 6.2 D 6.8 6.4 nl nl P 0.80 0.81 0.99 0.85
16 Actin
A 9.7 10.7 10.5 10.0 B 10.0 10.1 10.9 10.8 C 8.4 8.8 8.4 9.1 D 9.1 9.4 9.7 9.7
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Lab Method Sample Assay 1 Assay 2 Assay 3 Assay 4 Assay 5
P 0.95 0.88 0.94 1.01
17 APTT
A 9.5 9.1 9.6 9.7 B 10.5 9.9 11.2 10.3 C 8.5 8.2 8.5 8.8 D 9.3 9.1 8.9 9.1 P 1.03 1.05 1.06 1.08
18 ActinFS
A 10.1 10.2 10.5 9.6 B 10.7 9.7 10.4 10.9 C 8.6 8.4 9.3 9.3 D 9.3 10.1 10.0 9.8 P 0.9 0.9 1.0 0.9 BRP 0.88 0.92 0.99 0.89
19 Trinity
A 10.1 10.1 10.0 10.4 B 10.4 10.4 11.0 11.3 C 9.1 9.2 9.2 9.4 D 9.6 9.1 9.1 9.3 P 0.80 0.81 0.80 0.81
20 ActinFSL
A 10.9 11.6 10.7 nl B 11.0 11.2 10.7 12.2 C 8.9 9.5 9.4 8.9 D 10.4 11.0 10.8 11.0 P 0.78 0.76 0.75 0.89
22 SynthaSil
A 11.0 10.1 10.9 10.3 B 10.8 10.3 12.6 10.2 C 9.0 8.6 9.0 8.8 D 10.3 10.1 13.1 10.4 P 0.91 0.90 0.90 0.94
23 APTT-SP
A 10.5 10.5 12.4 10.8 B 10.7 9.8 11.1 10.1 C 9.4 8.5 nl 7.7 D 11.0 9.4 11.6 9.9 P nl 0.90 1.09 0.84
24 PTT-A
A 11.5 8.7 9.9 10.8 B 11.6 8.9 10.2 10.8 C 9.8 8.1 8.6 7.8 D 10.8 9.1 10.2 10.3 P 0.93 0.81 0.84 0.79
25 Pathromtin SL
A 9.1 9.9 9.5 9.2 B 10.2 10.6 10.4 10.3 C 8.5 8.9 8.4 8.3 D 8.3 8.9 9.0 8.8 P 0.95 1.06 0.98 0.95
27 APTT-SP
A 10.7 11.0 10.4 10.3 B 10.8 11.1 10.5 10.8 C 9.1 9.0 8.4 9.1 D 10.3 10.2 9.5 9.8 P 0.87 0.83 0.82 0.84
28 Cephascreen
A 9.8
B 10.3
C 7.3
D 9.0
P 1.07
28 CK-Prest
A 9.8 10.2
B 10.9 10.4
C 7.4 8.3
D 7.9 9.3
P 1.5 1.08
28 PTT-A A 10.8
B 11.2
C 9.4
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Lab Method Sample Assay 1 Assay 2 Assay 3 Assay 4 Assay 5
D 11.0
P 1.17
29 Actin
A 9.6 8.8 8.9 7.9 B 10.1 10.3 10.1 10.7 C 8.7 8.6 8.2 8.9 D 9.5 8.6 8.6 7.3 P 0.82 0.78 0.80 0.87
30 APTT Synth Grifols
A 9.4 9.6 9.8 9.9 B 10.6 10.4 10.8 10.7 C 8.7 8.6 8.7 8.6 D 9.2 9.1 9.2 9.2 P 0.79 0.81 0.83 0.84
31 Cephascreen
A 8.4 8.3 9.8 11.4 B 9.9 9.9 np 14.7 C 9.2 7.7 np 8.9 D 8.8 8.3 np 10.5 P 1.19 1.05 1.27 1.30
31 CK-Prest
A 8.8 9.2 9.6 10.4 B 9.1 9.9 10.5 10.2 C 6.6 7.3 7.4 nl D 6.7 8.2 7.9 9.2 P 0.91 0.93 1.00 0.98
32 CK Prest
A 9.8 11.0 9.5 9.8 B 10.8 11.5 10.7 10.2 C 8.9 9.4 7.3 8.6 D 9.9 9.5 8.6 9.3 P 1.08 1.18 1.10 1.03
33 SynthaFax
A 7.7 6.6 8.0 8.3 7.8 B 10.3 9.5 10.6 11.9 14.6 C 9.3 7.7 10.0 9.1 11.7
D 6.6 6.5 7.0 8.2 7.9
P 0.94 0.91 0.99 0.99 1.2
34 Pathromtin SL
A 11.2 10.2 9.0 10.0 B 12.0 11.2 10.2 10.2 C 9.9 nl 8.7 8.2 D 9.2 9.4 8.7 8.8 P 0.99 1.00 0.79 0.90
35 Actin
A 10.3 10.4 10.3 9.8 B 10.5 10.4 10.6 10.2 C 8.8 8.7 8.5 8.9 D 10.2 10.4 10.1 9.8 P 0.90 0.87 0.84 0.91
36 Dapttin
A 7.9 8.5 8.1 7.7 B 10.6 11.2 10.7 10.0 C 9.0 9.2 9.2 8.8 D 7.5 8.0 7.6 7.2 P 0.96 1.02 0.93 0.91 Purple 7.9 7.7 7.8 7.5
37 Cephen
A 10.1 9.9 9.7 9.8 B 10.6 10.6 10.5 10.3 C 8.9 9.2 8.8 9.0 D 9.6 9.7 9.5 9.6 P 0.85 0.87 0.82 0.83 Purple 9.1 9.1 9.3 8.8
39 APTT-SP
A 9.2 8.9 9.0 9.5 B 10.6 10.5 10.3 10.1 C 9.1 9.2 8.5 9.8 D 9.7 9.2 8.8 8.9 P np 0.87 0.92 0.82 40 Actin-FS A 10.0 9.2 10.3 9.9
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Lab Method Sample Assay 1 Assay 2 Assay 3 Assay 4 Assay 5
B 10.7 9.5 9.6 9.1 C 9.2 8.6 9.3 9.7 D 10.1 8.4 9.5 10.3 P 0.86 0.73 0.84 0.80
41 Actin-FSL
A 10.1 10.0 9.7 10.7 B 10.7 10.1 10.8 10.8 C 8.3 8.0 8.1 8.3 D 9.0 np 9.6 9.5 P nl 1.31 np np
42 SynthaFax
A 7.5 7.3 6.6 9.0 B 11.2 10.4 8.0 12.1 C 9.6 9.4 10.3 13.4 D 7.1 7.1 6.1 10.1
43 APTT-SP
A 10.0 9.7 9.5 9.5 B 10.5 10.7 10.5 10.1 C 9.1 8.7 9.2 9.1 D 7.7 9.5 9.9 9.2 P 0.94 0.83 0.89 0.87
44 SynthaSil
A 10.4 10.1 10.6 10.8 B 10.6 10.2 10.5 11.1 C 8.9 9.1 8.8 9.3 D 9.5 8.3 10.5 10.3 P np 0.84 0.81 0.87 Purple 9.0 9.0 9.0 9.2
45 Actin-FS
A 8.6 8.6 8.8 10.1 B 9.4 9.1 9.9 10.5 C 7.9 8.8 9.2 8.9 D 8.5 8.9 15.5 9.8 P 0.69 0.74 0.72 0.66 Purple 7.7 8.7 9.0 8.6
46 Actin-FSL
A 9.4 9.0 8.7 8.7 B 10.5 10.5 10.1 10.5 C 8.5 8.8 7.9 nl D 8.5 8.4 8.4 8.4 P 0.69 0.70 0.70 0.74
47 ActinFS
A 11.1 11.2 10.8 10.7 B 10.8 10.6 10.2 10.3 C 9.7 9.5 9.6 9.7 D 10.7 10.5 10.3 10.4 P 0.72 0.76 0.75 0.74
48 SynthaSil
A 9.1 10.1 9.9 9.9 B 9.8 10.1 10.5 10.5 C 8.0 8.6 8.8 9.1 D 9.1 9.5 9.5 9.9 P 0.93 1.05 1.02 1.00 Purple 9.2 9.4 9.0 8.9
49 Cephen
A 11.2 10.9 10.8 10.9 B 10.6 10.6 10.4 10.4 C np 9.2 9.0 9.1 D 10.0 10.3 10.2 10.1 P 0.76 0.83 0.81 0.79 nl = non-linear, np = non-parallel
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Table S2 – Functional Activity by chromogenic: Potency values (IU/ampoule) calculated
relative to S, 4th
IS Factor IX Concentrate for each sample within each assay set returned
Lab Method Sample Assay 1 Assay 2 Assay 3 Assay 4
02 Hyphen
A 7.0 8.2 6.5 6.5 B 10.2 12.0 10.5 6.3 C 9.4 10.3 8.1 8.2
D 6.1 7.7 6.2 6.3 P 0.75 0.86 0.76 0.72
Purple 6.7 9.2 6.4 10.1
02 Rossix
A 8.2 7.8 8.2 7.3 B 11.2 11.1 11.6 10.1
C 8.1 9.2 9.2 8.0 D 7.5 7.6 7.5 6.8
P 0.84 0.83 0.79 0.76
Purple 7.3 7.0 7.8 6.9
04 Rossix
A 7.9 7.7 8.7 7.3 B 10.7 10.0 11.4 10.4 C 9.0 8.5 9.6 8.5
D 7.7 7.1 8.0 7.3 P 0.87 0.81 0.89 0.82
06 Rossix
A 8.8 7.2 9.8 8.4 B 10.8 9.6 12.6 10.2 C 9.7 8.8 8.9 8.4
D 7.0 7.1 8.3 7.6 P np 0.79 0.88 0.82
12 Hyphen
A 7.5 7.3 6.8 7.2 B 9.5 11.0 9.9 10.0 C 8.2 9.2 8.2 8.5
D 6.3 7.2 6.6 6.8 P 0.77 0.83 0.78 0.77
12 Rossix
A 8.2 7.1 7.3 7.6 B 10.4 10.0 10.3 nt
C 9.0 8.6 9.2 8.3
D 7.5 7.6 8.2 6.8 P 0.81 0.76 0.81 0.69
21 Hyphen
A 9.3 10.5 9.0 8.6 B 14.1 14.3 13.4 13.1
C 12.0 10.3 11.5 11.9
D 8.7 8.4 8.0 nl P 0.96 0.91 0.91 0.91
26 Rossix
A 9.1 8.3 9.4 8.5 B 10.2 10.2 10.8 10.5
C 8.9 8.8 9.0 8.6 D 8.3 9.0 8.7 8.3
P 0.91 0.89 0.85 0.78
33 Hyphen
A 8.3 8.8 8.0 8.9 B 11.6 12.1 11.4 12.3
C 9.8 10.4 9.7 10.1 D 8.2 8.6 7.9 8.7
P 0.89 0.95 0.93 0.96
36 Hyphen
A 6.9 7.6 7.8 7.9 B 9.6 10.4 10.9 11.0
C 9.0 8.7 9.5 9.3 D 6.8 7.0 7.5 7.1
P 0.83 0.77 0.83 0.79 Purple 6.6 7.1 7.0 7.3
40 Rossix A 8.1 S nl 8.8 8.0 B 10.6 S nl 10.8 9.9 C 8.9 S nl 10.3 11.2
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D 7.1 S nl 8.0 8.6 P 0.76 S nl 0.87 0.87
41 Rossix
A 8.1 8.6 7.2 7.9 B 10.4 10.2 10.4 10.1
C 8.7 8.6 9.2 8.2 D 7.3 7.4 7.4 7.3
P nl 0.91 0.88 0.89
43 Hyphen
A 6.6 np 7.6 8.4 B 8.9 8.6 10.5 10.5
C 7.8 np 8.4 9.0 D 5.8 np 7.7 8.5
P 0.67 np 0.79 nt
48 Hyphen
A 6.5 7.2 6.5
B 10.2 11.0 9.6
C 8.8 9.4 8.2
D 6.2 6.5 6.3
P 0.68 0.76 0.70
Purple 6.1 6.6 6.3
49 Hyphen
A 7.0 6.8 6.3 6.8
B 10.9 10.0 10.1 10.7 C 8.5 8.6 8.7 8.9
D 6.0 6.4 5.9 6.4 P 0.72 0.78 0.70 0.78
nl = non-linear, np = non-parallel; nt = not tested
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Table S3 – Antigen activity: Potency values (u/ampoule) calculated relative to local normal
plasma pool (assumes value of 1 u/ml) for each sample within each assay set returned
Lab Method Sample Assay 1 Assay 2 Assay 3 Assay 4
02 Asserachrom
A 8.8 8.2 8.3 7.4 B 15.5 12.2 11.5 11.0 C 10.8 10.3 12.5 9.2
D 7.5 6.7 7.6 6.8 P 0.96 0.95 0.89 0.96
Purple 8.8 8.2 8.0 7.0
S 11.4 11.1 11.0 9.2
02 Visulize
A 6.7 np 7.1 7.2 B 12.0 13.3 12.2 12.3 C 10.8 10.9 9.4 10.7
D 5.8 np 6.0 6.4
P 0.99 0.98 0.95 1.19 Purple 6.4 6.7 6.6 6.5
S 10.8 12.7 10.6 11.7
02 Zymutest
A 9.8 9.0 9.7 9.7 B 11.1 10.5 10.6 10.9 C 9.1 8.8 9.5 9.2
D 8.3 7.7 8.0 8.1
P 0.99 0.90 0.98 1.08 Purple 9.0 8.2 8.5 8.7
S 9.7 9.6 9.4 8.9
10 Visulize
A 6.1 6.4 5.8 5.9 B 10.9 10.9 10.7 10.4
C 9.4 9.4 8.7 9.0 D 5.5 5.6 5.6 5.2
P 0.85 0.90 0.87 0.88 S 9.9 10.0 9.3 9.4
11 Visulize
A 6.4 6.1 5.5 6.6 B 10.9 10.4 9.8 11.4
C 9.6 8.5 8.3 9.7
D 5.6 5.3 4.8 5.2 P 0.94 0.81 0.78 0.86
S 10.5 9.0 8.5 10.0
21 Cedarlane
A 3.7 2.9 3.3 3.7 B 13.9 8.9 8.9 11.2
C 9.3 7.5 np 9.3 D 2.9 2.4 3.1 3.3
P 0.99 0.67 0.78 0.84 S 13.2 9.1 10.8 12.4
23 AssayPro
A 6.4 8.1 6.4 np B 12.5 12.6 10.8 10.1
C 10.5 11.8 np 8.9
D np 7.2 5.5 np P 0.80 1.33 0.84 0.83
S np 11.2 np 8.6
24 Asserachrom
A 8.3 8.4 7.0 7.9 B 12.5 11.6 10.4 11.0
C 10.2 9.7 9.2 9.3 D 7.0 6.3 5.5 6.2
P 0.87 0.87 0.83 0.93 S 10.6 8.6 9.7 9.9
28 Asserachrom
A 7.0 8.5 6.5 7.0 B 9.7 11.3 9.3 10.9
C 8.7 10.5 8.0 8.1
D 5.8 7.2 5.4 5.8 P 0.82 0.90 0.76 0.85
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Lab Method Sample Assay 1 Assay 2 Assay 3 Assay 4
S 8.9 10.7 8.5 9.1
31 Asserachrom
A 7.4 7.6 7.0 8.6 B 10.9 11.3 9.6 12.2 C 9.4 9.6 8.1 10.3
D 6.4 6.5 5.9 7.0 P 0.87 0.90 0.80 0.96
S 9.4 10.1 8.7 10.9
33 Visulize
A 5.5 5.3 5.3 4.9 B 10.6 8.4 9.0 nl
C 9.3 9.6 9.1 nl D np 4.7 4.5 4.5
P 0.97 0.89 1.01 0.96 S 9.5 9.3 9.5 nl
34 Asserachrom
A 7.8 8.2 np 8.1 B 12.0 12.0 12.2 11.7 C 10.5 11.0 10.7 10.0
D 6.4 6.7 6.5 6.5 P 0.95 0.95 0.98 0.95
S 10.1 10.6 10.8 10.8
38 Affinity
BIologicals
A 6.9 6.9 7.1 6.5 B 12.4 12.2 12.3 12.5
C 10.7 10.4 10.0 10.5 D 5.9 5.9 5.9 5.6
P 0.96 0.98 1.00 0.95 S 12.3 11.7 11.4 11.6
40 Asserachrom
A 9.9 nl L nl L nl B 12.8 11.9 L nl L nl C 11.6 10.8 L nl L nl
D 8.44 6.94 L nl L nl P nl 0.96 L nl L nl
S np 9.5 L nl L nl
43 Asserachrom
A 5.9 6.9 2 points 6.6 B 9.2 np np np
C 8.5 9.2 8.7 9.6 D 3.8 6.1 5.4 nl
P 0.75 0.91 0.75 0.75 S 8.5 10.4 9.2 9.7
48 Visulize
A 8.2 nl B 11.7 10.4 C 10.3 9.6
D np 3.6 P 0.76 0.91
S 12.7 10.5
49 Zymutest
A np 8.8 8.4 10.0 B np 10.2 9.7 10.6
C np 7.9 8.9 9.4 D np 7.4 7.3 8.6
P np 0.85 0.81 0.99
S np 8.0 8.9 9.8
nl = non-linear, np = non-parallel;
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Appendix IV: Draft Instruction for Use
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