-
WHO/BS/2014.2245
ENGLISH ONLY
EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION
Geneva, 13 to 17 October 2014
VALUE ASSIGNMENT OF THE CANDIDATE 1
ST INTERNATIONAL STANDARD FOR
ACTIVATED BLOOD COAGULATION FACTOR XI (FXIa), HUMAN, NIBSC CODE
13/100
Elaine Gray
1, John Hogwood, Helen Wilmot, Craig Thelwell, Thomas Dougall
and Peter Rigsby
National Institute for Biological Standards and Control
Potters Bar, Hertfordshire, EN6 3QG, UK 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
4 October 2014 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 David Wood at email:
[email protected].
© World Health Organization 2014
All rights reserved. Publications of the World Health
Organization are available on the WHO web site (www.who.int)
or can be purchased from WHO Press, World Health Organization,
20 Avenue Appia, 1211 Geneva 27, Switzerland
(tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail:
[email protected]).
Requests for permission to reproduce or translate WHO
publications – whether for sale or for noncommercial
distribution – should be addressed to WHO Press through the WHO
web site:
(http://www.who.int/about/licensing/copyright_form/en/index.html).
The designations employed and the presentation of the material
in this publication do not imply the expression of any
opinion whatsoever on the part of the World Health Organization
concerning the legal status of any country, territory,
city or area or of its authorities, or concerning the
delimitation of its frontiers or boundaries. Dotted lines on
maps
represent approximate border lines for which there may not yet
be full agreement.
The mention of specific companies or of certain manufacturers’
products does not imply that they are endorsed or
recommended by the World Health Organization in preference to
others of a similar nature that are not mentioned.
Errors and omissions excepted, the names of proprietary products
are distinguished by initial capital letters.
All reasonable precautions have been taken by the World Health
Organization to verify the information contained in
this publication. However, the published material is being
distributed without warranty of any kind, either expressed or
implied. The responsibility for the interpretation and use of
the material lies with the reader. In no event shall the
World Health Organization be liable for damages arising from its
use. The named authors alone are responsible for the
views expressed in this publication.
mailto:[email protected]://www.who.int/mailto:[email protected]://www.who.int/about/licensing/copyright_form/en/index.html
-
WHO/BS/2014.2245
Page 2
Summary Seventeen laboratories from eleven different countries
participated in a value assignment
collaborative study for the proposed 1st International Standard
for Activated Blood Coagulation
Factor XI (FXIa), 13/100. Coded duplicates of the candidate
together with 4 immunoglobulin
preparations from 2 manufacturers were included in the study. In
general the intra-laboratory
variation was low indicating the laboratories were able to carry
out FXIa quantitative assays with
good precision. The inter-laboratory agreement was excellent for
the candidate preparation with
or without the inclusion of clot-based assay results; with GCV
of 3.1 and 3.3% respectively.
There was no assay discrepancy between the chromogenic and
clotting assays. It is
recommended that the value assignment should be based on
functional chromogenic assays only.
It is therefore proposed that the candidate FXIa preparation,
13/100 be assigned as the WHO 1st
International Standard for Activated Blood Coagulation FXI
(FXIa), Human, with an assigned
value of 9.8 International Units (IU) per ampoule.
Introduction The International Reference Reagent for Activated
Blood Coagulation Factor XI (FXIa), Human
was established by the Expert Committee on Biological
Standardization (ECBS) of the World
Health Organization (WHO) in October 2012 to support the
measurement of FXIa in
immunoglobulin products. This Reference Reagent was assigned
with an arbitrary unit of 10
u/ampoule by NIBSC and the “fit for purpose” study (1)
demonstrated good improvement of
intra- and inter-laboratory agreement when it was used as a
standard for measurement of FXIa
in IVIG products. Over 1500 ampoules have now been issued and
the stock will be depleted by
the end of 2014. A candidate preparation of FXIa with similar
characteristics to the International
Reference Reagent (IRR) has been prepared and this study serves
to value assign this candidate
against the IRR, thereby ensuring the continuity of the FXIa
unit as defined by the International
Reference Reagent. Discussions with regulators and manufacturers
indicated that in addition to
being used as a calibrant in the FXIa functional activity
assays, the IRR has also been used as a
“standard” in the non-activated partial thromboplastin time
(NAPTT) and the thrombin
generation assays (TGA). For value assignment purposes, however,
only results from specific
FXIa functional activity assays will be considered, as NAPTT and
TGA assays are not specific
for FXIa activity and these methods are not sufficiently robust
to provide accurate potency
estimates (Note: The results for NAPTT and TGA will be addressed
in a separate report to the
participants). This study also provides the opportunity to
examine the performance of assay
methods for detection of procoagulant activity in
immunoglobulins (IgG) and therefore a
selection of IgG samples were included to investigate the
usefulness of a FXIa reference
standard in these assays.
Candidate, NIBSC code 13/100 The bulk for the candidate was
purchased. The specific activity estimated by NIBSC using a
FXIa functional chromogenic activity assay relative to the
International Reference Reagent for
FXIa, 11/236, was approximately 11500 u/mg . The starting
material was certified by the
manufacturer as being negative for anti-HIV1/2, HBsAg and
hepatitis C. The material was
prepared by activating purified human FXI with FXIIa and
subsequently purified to homogeneity
by a combination of affinity chromographic methods. The single
batch of material was diluted
1/3100 in 50 mM Tris, 150 mM NaCl, 5 mg/ml trehalose and 0.5%
human serum albumin to
give a final concentration of approximately 10 u/ml . The
batches of human serum albumin used
contained minimal protease activities as indicated by lack of
colour development following
incubation with chromogenic substrates (S2765, S2302 and S2251)
for 90 minutes at 37◦C. The
material was distributed in glass ampoules, filled and
freeze-dried according to guidelines for
-
WHO/BS/2014.2245
Page 3
production of international standards. The product
characteristics are listed in the following
table.
NIBSC Code 13/100
Presentation Sealed, siliconised glass 3 ml ampoules
Filling date 11th April 2013
Number of Ampoules available 18925
Liquid filling weight (g) (n=698, measurements taken from
all
3 pumps throughout the duration of the fill) 1.0078
CV of fill mass (%) 0.15
Homogeneity of the fill by activity: 3 ampoules selected
from
the start, 1st quarter, middle, 3rd quarters and end of the
fill;
were assayed against the IRR 11/236 using a FXIa functional
activity method(Biophen FXIa kit). 2 assays per ampoule
were carried out. Effect of fill position was assessed by
ANOVA of log potencies and expressed as geometric
coefficient of variation (GCV).
GCV p
2.2% 0.52
Mean dry weight (g) (n=6) 0.0258 (CV 0.83%)
Mean head space oxygen (%) (n=12) 0.19 (CV 47.3%)
Residual moisture (%) (n=12) 0.150 (CV 18.1%)
Storage temperature -20°C
Address of processing facility NIBSC, Potters Bar, EN6 3QG,
UK
Address of present custodian NIBSC, Potters Bar, EN6 3QG, UK
Determination of Molar Concentration The protein concentration
was determined by the vendor, based on the absorbance value at
280nm and an extinction coefficient (E1%
) of 13.4 and molecular weight of 160000 Da for FXIa.
Active-site titration was carried out at NIBSC on the bulk
preparation against 4
methylumbelliferyl 4-guanidinobenzoate hydrochloride hydrate
(MUGB). Details of the method
and results are summarised in Appendix 1. The molar
concentration of 8.8 nM in the final
ampoules was extrapolated from the estimation of the bulk which
was found to be 27.22µM.
This information will NOT be provided on the label, but will be
given in the Instruction for Use.
Participants Twenty four laboratories agreed to participate,
with 17 laboratories (2 Austria, 2 Australia, 1
Belgium, 2 France, 1 Germany, 1 Israel, 1 Spain, 1 Sweden, 1
Switzerland, 2 UK, 3 USA)
returning data for Part I of the study. The participants
included 3 diagnostics manufacturers, 11
therapeutic manufacturers and 3 regulatory authorities. A list
of participants is given in
Appendix 2 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
CODE PREPARATION
S The International Reference Reagent for Factor XIa (11/236),
10 units/ampoule - 4
ampoules supplied.
A Factor XIa preparation, NIBSC code 13/100; approx. 10
units/ampoule - 4 ampoules
supplied
-
WHO/BS/2014.2245
Page 4
B Factor XIa preparation, NIBSC code 13/100, coded duplicate of
sample A – approx. 10
units/ampoule - 4 ampoules supplied
C IVIG preparation containing medium procoagulant activity, 5%
protein; approx. 0.06
FXIa u/ampoule - 4 ampoules supplied.
D IVIG preparation containing high procoagulant activity, 5%
protein; approx. 0.3 FXIa
u/ampoule - 4 ampoules supplied.
E IVIG preparation containing low procoagulant activity, 5%
protein; approx. 0.01 FXIa
u/ampoule - 4 ampoules supplied.
F IVIG preparation containing high procoagulant activity, 5%
protein; approx. 0.6 FXIa
u/ampoule - 4 ampoules supplied.
Samples A and B were coded duplicates of the candidate
standard.
Study design and assay methods The details of the protocol are
given in Appendix 3. The participants were provided with 4 sets
of samples. Each laboratory was requested to determine the
appropriate dilution ranges for
sample S, the WHO IRR, 11/236, using their FXIa quantitation
test. Once the appropriate
dilutions of sample S had been determined, one independent assay
was to be carried out on each
of the 4 sets, preferably on 4 separate days. An example of
balanced order of testing was
provided.
Participants were requested to return raw data together with
their own calculated estimates for all
of the samples relative to sample S (the WHO IRR, 11/236) from
each individual assay to
NIBSC.
Each participant was requested to perform their routine in-house
functional method(s) for FXIa.
Some laboratories performed more than one method and in this
case the data from each method
were treated as separate sets of results and referred to as Lab
3a and Lab 3b, for example. A list
of reagents, methods and instruments, together with their
in-house FXIa standard (if used) by the
participants is given in Appendix 4. Sixteen sets of results
were returned for functional
chromogenic assays based on the conversion of FIX to FIXa by
FXIa (12 used Hyphen Biomed
kit, 3 used Rossix kit, 1 in house method). Two laboratories
returned data for clot-based assays;
Lab 1 method was based on NAPTT using phospholipid without
activator and FXI deficient
plasma, while Lab 2 used one-stage clotting assay based on
APTT.
Analysis of data
The potencies of all samples were calculated relative to the
International Reference Reagent
11/236 (coded S) or the candidate standard (coded A) by parallel
line analysis of the raw assay
data at NIBSC (2). The majority of assays were analysed with a
log transformation of assay
response; no transformation was used for laboratories 01
(clotting), 02 (clotting), 13, 15, 19 and
22. Analysis was based on a linear section of the response range
using a minimum of three
dilutions for all samples. The majority of samples and assays
from Lab 14 gave non-linear dose
-
WHO/BS/2014.2245
Page 5
response curves and were not analysed further. Calculations were
performed using the EDQM
software CombiStats Version 5.0 (3).
Non-linearity and non-parallelism were considered in the
assessment of assay validity. For
chromogenic assays, samples with a high non-linearity mean
square were excluded for not being
linear, with cut-off values determined through visual assessment
of the plotted data. A cut-off
value of 0.002 was used for assays with a log transformation of
assay response and 0.001 for
those using no transformation. These values were chosen to allow
a consistent approach to
assessment of assay validity within this study and are not
intended to reflect values that may be
appropriate for use within all laboratories. Due to the low
quantity of clotting assays, a visual
assessment was used to determine linearity. Non-parallelism was
assessed by calculation of the
ratio of fitted slopes for the test and reference 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 in
these cases, no estimates are reported.
The final assay result for the candidate standard was taken as
the unweighted geometric mean
(GM) of the results obtained for samples A and B within each
assay. Relative potency estimates
from all valid assays were combined to generate an unweighted
geometric mean for each
laboratory and these laboratory means were used to calculate an
overall unweighted geometric
mean 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).
The agreement between duplicate samples within each assay was
assessed by calculating the
difference in log potency estimates (relative to sample S)
between the duplicates, calculating the
mean of the squared difference for each laboratory, taking the
square root to give a root mean
square (RMS) value, and expressing this as an average percentage
difference.
Results and Discussions The main aim of this study was to value
assign the candidate International Standard relative to
the International Reference Reagent for Activated Coagulation
Factor XI, Human, NIBSC code
11/236. Only results from functional activity assays specific
for FXIa were considered for value
assignment since the inclusion of other semi-quantitative and
non-specific assay methods such as
the NAPTT or the TGA, especially when performed using normal
plasma, may bias the
consensus mean potency.
The individual assay results from each lab are shown in Appendix
5. The majority of the assays
were statistically valid, with invalidity arising mostly from
non-linearity of dose-response curves.
Samples A and B were coded duplicates and tables 1 and 2 show
potency estimates of samples A
and B relative to S, the IRR. With the exception of Lab 3, Lab
12 and Lab 15, the intra-
laboratory GCV were all less than 10% indicating that the
laboratories were able to carry out
these assays with reasonable precision. The overall GMs for
samples A and B were 9.79 and
9.80 U/ampoule respectively. The agreement between laboratories
was good as indicated by the
4% inter-laboratory GCV for both samples A and B. The exclusion
of clotting data did not alter
the overall GM or %GCV. Laboratories 1 and 2 used clot-based
methods and the potency
estimates for samples A and B were not different to those
obtained using chromogenic assay
method. The intra-assay variability (between coded duplicate
samples) is shown in Table 3. The
figures represent an average percentage within-assay difference
in estimates between the
duplicate samples. The majority of laboratories have average %
differences of less than 10%,
which represents good assay repeatability. The combined potency
estimates of samples A and B,
-
WHO/BS/2014.2245
Page 6
the coded duplicates are shown in Table 4 and also illustrated
as a histogram in Figure 1. The
intra-laboratory variability was low, with the majority of GCV
being less than 10%. The overall
GMs with or without the clotting assay results were 9.78
IU/ampoule, with an inter-laboratory
GCV of 3.1% and 3.3% respectively. Although there was no
difference between the clotting and
chromogenic assay results, in principle, the clotting assays are
not sufficiently specific for FXIa
activity and therefore it is recommended to value assign the
proposed IS with potency estimates
obtained by functional chromogenic assays only.
Samples C, D, E and F were immunoglobulin preparations from
different manufacturers, each
with varying degrees of procoagulant activity. Samples C and D
were also included in the 2012
collaborative study (1) for the IRR and were found to have 0.15
U/ampoule (GCV 69.6%) and
0.49 U/ampoule (GCV 54.6%) when assayed against the IRR,
assuming the value of the IRR as
10 U/ampoule. The level of FXIa in sample E was found to be
below the limit of quantification
in the current study. The results from the chromogenic assay
kits showed that FXIa could be
detected, but the ranges of the responses from the standard and
test curves were such that valid
value quantification could not be obtained. It is possible that
a standard curve covering low
concentrations could give statistically valid potency for sample
E, however, the results from the
current study does not allow for this comparison. Tables 5 and 6
and Figures 2 and 3 present
data for samples C and D respectively. The GM potency estimates
of FXIa contents for samples
C and D were found to be 0.07 and 0.32 U/ampoule, excluding the
clotting assay results. These
values were lower than the estimates obtained from the previous
study, however, the inter-
laboratory variation of 13.9 and 6.9% indicated an improvement
in between laboratory
agreement for the measurement of FXIa in these two samples.
Table 7 and Figure 4 show
potency estimates obtained for sample F. When the clotting assay
results were excluded, the
overall GM was 0.65 U/ampoule and inter-laboratory variation was
low, with a GCV of 5.7%. It
is interesting that the results for samples D and F, the higher
procoagulant activity samples, from
the two laboratories carrying out clot-based assays were
different, with Lab 1 obtaining estimates
close to the functional chromogenic assay estimates, while Lab 2
obtaining 7 to 8 fold higher
estimates. This discrepancy was not observed for samples A, B
and C. This could be explained
by the differences in the principles of the clotting assays
carried out by these two labs. Lab 1
used a modified NAPTT assay in combination with FXI deficient
plasma while Lab 2 employed
an APTT based assay. It is possible that samples D and F
contained zymogen and this was
detected by the APTT based assay. This shows the importance of
using a specific assay for
measurement of FXIa in IgG preparations.
Table 8 shows results for laboratory’s own in house controls
(standards). With the exception of
Lab 12, intra-laboratory variation was low indicating the
laboratories are able to carry out these
assays with good reproducibility.
Two functional chromogenic assay kits were used by the
participants. Twelve labs used the
Hyphen Biomed kit, while 3 labs used the Rossix kit. Tables 9 –
13 summarise the results
obtained by these 2 kits. It is clear that there is no
significant difference between the potency
estimates obtained and that the values from the Rossix kit were
all within the range of those
obtained using the Hyphen kit.
The IRR has been used by regulators and manufacturers to develop
their in-house testing
methods and also to accumulate batch data. It is therefore of
paramount importance that the unit
as defined by the IRR could be continued. Appendix 6 presents
the individual laboratories’
overall potency estimates for each sample relative to sample A,
the proposed International
Standard, assuming the assigned value to be 9.8 U/ampoule. Table
14 compares the recalculated
potencies for samples B, C, D and F relative to sample A with
the estimates obtained against the
-
WHO/BS/2014.2245
Page 7
IRR. The estimates were not significantly different to those
obtained against the IRR indicating
that there would be minimal drift in the unit of FXIa when the
IS is established.
Stability studies Accelerated degradation studies have been
performed after 3 and 12 months storage at low and
high temperatures. The predicted loss per year at each
temperature is shown below, based on
cumulative results from both time-points. The predicted
percentage loss at the normal storage
temperature of -20 °C is
-
WHO/BS/2014.2245
Page 8
Proposal and Recommendation The proposed International Standard
for Activated Coagulation Factor XI (FXIa), Human,
13/100 (samples A and B, the coded duplicates), assayed well
against the International Reference
Reagent for FXIa, with an overall GM of 9.8 U/ampoule, excluding
the clotting assays and inter-
laboratory GCV of 3.3%. In general, the intra-laboratory
variability was low, indicating the
participants were able to measure FXIa with precision. There was
also good inter-laboratory
agreement, giving confidence in the overall potency value
obtained for the candidate. In terms
of measurement of FXIa in IgG preparations, the inter-laboratory
GCVs were much lower than
those found in the previous study, demonstrating an improvement
in the capability of the
laboratories to carry out FXIa assays.
It is recommended that the candidate preparation, 13/100 be
established as the WHO 1st
International Standard for Activated Coagulation Factor XI
(FXIa), Human, with a labelled value
of 9.8 IU/ampoule.
A draft Instruction For Use (IFU) is shown in Appendix 6.
Each participant was asked to review the participants report and
whether they agreed with the
proposal. All participants agreed with the proposal to establish
the preparation 13/100 as the 1st
International Standard for Activated Coagulation Factor XI
(FXIa), Human. The experts
nominated by the Factor XI and the Contact System Subcommittee
of the Scientific and
Standardisation Committee (SSC) of the International Society on
Thrombosis and Haemostasis
have also reviewed the study. The SSC has now endorsed the
proposal to go forward for
establishment by the ECBS.
References 1. Gray E, Wilmot H, Hogwood J and Rigsby P.
Evaluation of the proposed WHO 1st
Reference Reagent for Activated Blood Coagulation Factor XI
(FXIa), Human. WHO
technical Report 2012.
http://apps.who.int/iris/bitstream/10665/78047/1/WHO_BS_2012.2206_eng.pdf?ua=
1
2. Finney DJ. Statistical Methods in Biological Assay. 3rd
Edition. London: Charles Griffin 1978.
3. CombiStats v5.0, EDQM – Council of Europe,
www.combistats.eu.
Acknowledgements We would like to thank the participants of the
collaborative study, many of whom completed the
testing under tight timescales and the kind donation of IgG
preparations by Octapharma and
Omrix Biopharmaceuticals.
http://apps.who.int/iris/bitstream/10665/78047/1/WHO_BS_2012.2206_eng.pdf?ua=1http://apps.who.int/iris/bitstream/10665/78047/1/WHO_BS_2012.2206_eng.pdf?ua=1http://www.combistats.eu/
-
WHO/BS/2014.2245
Page 9
Table 1: Potency estimates for Sample A, coded duplicate of the
candidate IS, relative to S,
the International Reference Reagent for FXIa
Lab Method N GM
U/ampoule
GCV
%
1 Clotting 2 9.90 .
2 Clotting 4 9.90 2.4
3 Chromogenic 2 9.34 .
4 Chromogenic 4 9.25 5.2
6 Chromogenic 4 10.37 8.7
7 Chromogenic 2 9.29 .
9 Chromogenic 4 10.07 6.1
10 Chromogenic 4 9.82 5.8
11 Chromogenic 4 9.75 5.2
12 Chromogenic 5 9.28 14.5
13a Chromogenic 4 10.09 5.4
13b Chromogenic 4 9.65 2.8
15 Chromogenic 3 10.56 22.4
16 Chromogenic 1 9.89 .
19 Chromogenic 4 10.22 1.8
22 Chromogenic 3 9.64 2.2
23 Chromogenic 3 9.58 3.5
Overall 17 9.79 4.0
Excl. CL 15 9.78 4.3
GM: geometric mean; GCV: geometric coefficient of variation; N:
number of assays; CL:
clotting
-
WHO/BS/2014.2245
Page 10
Table 2: Potency estimates for Sample B, coded duplicate of the
candidate IS, relative to S,
the International Reference Reagent for FXIa
Lab Method N GM
U/ampoule
GCV
%
1 Clotting 3 9.93 7.8
2 Clotting 4 9.68 3.3
3 Chromogenic 3 9.80 11.0
4 Chromogenic 4 9.69 5.6
6 Chromogenic 3 9.71 3.7
7 Chromogenic 2 9.12 .
9 Chromogenic 2 9.84 .
10 Chromogenic 4 9.97 3.9
11 Chromogenic 4 9.82 4.5
12 Chromogenic 5 9.45 8.2
13a Chromogenic 4 10.02 5.7
13b Chromogenic 4 9.65 3.5
15 Chromogenic 1 11.11 .
16 Chromogenic 1 9.55 .
19 Chromogenic 4 9.66 6.2
22 Chromogenic 4 9.72 2.2
23 Chromogenic 2 9.93 .
Overall 17 9.80 4.0
Excl. CL 15 9.80 4.3
GM: geometric mean; GCV: geometric coefficient of variation; N:
number of assays; CL:
clotting
-
WHO/BS/2014.2245
Page 11
Table 3: Average differences of potencies between coded
duplicate samples A and B within
each assay for each laboratory. The majority of laboratories
gave differences of less than
10% indicating good agreement of potencies for the coded
duplicates
Lab Method Average % difference between A and B
1 Clotting 5.3%
2 Clotting 4.1%
3 Chromogenic 10.6%
4 Chromogenic 6.1%
6 Chromogenic 6.8%
7 Chromogenic 6.8%
9 Chromogenic 3.0%
10 Chromogenic 3.0%
11 Chromogenic 6.3%
12 Chromogenic 9.8%
13a Chromogenic 3.3%
13b Chromogenic 4.3%
15 Chromogenic .
16 Chromogenic .
19 Chromogenic 7.4%
22 Chromogenic 0.6%
23 Chromogenic 10.2%
-
WHO/BS/2014.2245
Page 12
Table 4: Combined potency estimates for the coded duplicates,
samples A and B, relative to
sample S, the International Reference Reagent for FXIa. The
combined potencies were the
unweighted GM of the results obtained for samples A and B within
each assay
Lab Method N GM
U/ampoule
GCV
%
1 Clotting 3 9.79 6.2
2 Clotting 4 9.79 2.1
3 Chromogenic 3 9.51 7.4
4 Chromogenic 4 9.47 5.0
6 Chromogenic 4 10.19 7.4
7 Chromogenic 2 9.20 .
9 Chromogenic 4 10.11 5.8
10 Chromogenic 4 9.89 4.7
11 Chromogenic 4 9.79 3.3
12 Chromogenic 5 9.36 10.3
13a Chromogenic 4 10.05 5.2
13b Chromogenic 4 9.65 2.0
15 Chromogenic 3 10.37 19.9
16 Chromogenic 1 9.72 .
19 Chromogenic 4 9.94 3.7
22 Chromogenic 4 9.71 2.1
23 Chromogenic 3 9.75 3.2
Overall 17 9.78 3.1
Excl. CL 15 9.78 3.3
GM: geometric mean; GCV: geometric coefficient of variation; N:
number of assays; CL:
clotting
-
WHO/BS/2014.2245
Page 13
Table 5: Potency estimates for Sample C relative to S, the
International Reference Reagent
for FXIa
Lab Method N GM
U/ampoule
GCV
%
1 Clotting 3 0.06 19.3
3 Chromogenic 3 0.08 5.1
4 Chromogenic 4 0.07 3.8
6 Chromogenic 4 0.07 9.6
7 Chromogenic 2 0.06 .
9 Chromogenic 1 0.10 .
10 Chromogenic 4 0.08 31.4
11 Chromogenic 3 0.06 6.0
12 Chromogenic 5 0.07 31.5
13a Chromogenic 4 0.06 5.6
13b Chromogenic 4 0.07 6.5
15 Chromogenic 2 0.08 .
16 Chromogenic 1 0.07 .
19 Chromogenic 4 0.08 2.7
22 Chromogenic 3 0.06 3.5
23 Chromogenic 2 0.08 .
overall 16 0.07 16.0
Excl. CL 15 0.07 13.9
GM: geometric mean; GCV: geometric coefficient of variation; N:
number of assays; CL:
clotting
-
WHO/BS/2014.2245
Page 14
Table 6: Potency estimates for Sample D relative to S, the
International Reference Reagent
for FXIa
Lab Method N GM
U/ampoule
GCV
%
1 Clotting 3 0.40 2.2
2 Clotting 4 3.14 2.7
3 Chromogenic 4 0.34 7.1
4 Chromogenic 4 0.31 2.4
6 Chromogenic 3 0.33 7.1
7 Chromogenic 1 0.31 .
9 Chromogenic 3 0.32 7.3
10 Chromogenic 3 0.34 5.0
11 Chromogenic 4 0.28 10.9
12 Chromogenic 4 0.30 8.9
13a Chromogenic 4 0.34 5.3
13b Chromogenic 4 0.32 5.0
15 Chromogenic 2 0.31 .
16 Chromogenic 1 0.32 .
19 Chromogenic 3 0.28 18.0
22 Chromogenic 3 0.32 5.1
23 Chromogenic 2 0.35 .
Overall 17 0.37 74.8
Excl.CL 15 0.32 6.9
GM: geometric mean; GCV: geometric coefficient of variation; N:
number of assays; CL:
clotting
-
WHO/BS/2014.2245
Page 15
Table 7: Potency estimates for Sample F relative to S, the
International Reference Reagent
for FXIa
Lab Method N GM
U/ampoule
GCV
%
1 Clotting 3 0.66 15.1
2 Clotting 4 4.83 2.7
3 Chromogenic 4 0.68 11.4
4 Chromogenic 4 0.61 4.7
6 Chromogenic 4 0.72 10.3
7 Chromogenic 1 0.63 .
9 Chromogenic 4 0.62 7.0
10 Chromogenic 4 0.69 5.5
11 Chromogenic 1 0.64 .
12 Chromogenic 5 0.64 13.1
13a Chromogenic 4 0.65 1.8
13b Chromogenic 4 0.62 6.2
15 Chromogenic 2 0.59 .
16 Chromogenic 1 0.69 .
19 Chromogenic 1 0.61 .
22 Chromogenic 4 0.65 2.1
23 Chromogenic 3 0.68 6.9
Overall 17 0.73 63.3
Excl.CL 15 0.65 5.7
GM: geometric mean; GCV: geometric coefficient of variation; N:
number of assays; CL:
clotting
-
WHO/BS/2014.2245
Page 16
Table 8: Potency estimates for in-house controls relative to S,
the International Reference
Reagent for FXIa
Lab Method N GM
U/ml
GCV
%
2 Clotting 4 6.39 4.5
3 Chromogenic 2 0.04 .
4 Chromogenic 4 0.03 2.9
6 Chromogenic 4 10.01 4.9
9 Chromogenic 3 0.35 7.4
10 Chromogenic 4 0.08 4.2
11 Chromogenic 4 0.15 10.7
12 Chromogenic 4 8.69 21.1
13a Chromogenic 4 1.60 3.2
13b Chromogenic 4 1.57 3.5
15 Chromogenic 2 0.04 .
16 Chromogenic 2 0.11 .
22 Chromogenic 4 0.08 3.2
23 Chromogenic 3 0.05 6.9
GM: geometric mean; GCV: geometric coefficient of variation; N:
number of assays
-
WHO/BS/2014.2245
Page 17
Table 9: Comparison of Potency estimates by Hyphen and Rossix
chromogenic assay kits
for Sample A relative to S, the International Reference Reagent
for FXIa
Method Lab N GM
U/ampoule
GCV
%
Hyphen
3 2 9.34 .
4 4 9.25 5.2
6 4 10.37 8.7
7 2 9.29 .
9 4 10.07 6.1
11 4 9.75 5.2
12 5 9.28 14.5
13b 4 9.65 2.8
15 3 10.56 22.4
16 1 9.89 .
19 4 10.22 1.8
23 3 9.58 3.5
Overall 12 9.76 4.7
Rossix
10 4 9.82 5.8
13a 4 10.09 5.4
22 3 9.64 2.2
Overall 3 9.85 2.3
GM: geometric mean; GCV: geometric coefficient of variation; N:
number of assays
-
WHO/BS/2014.2245
Page 18
Table 10: Comparison of Potency estimates by Hyphen and Rossix
chromogenic assay kits
for Sample B relative to S, the International Reference Reagent
for FXIa
Method Lab N GM
U/ampoule
GCV
%
Hyphen
3 3 9.80 11.0
4 4 9.69 5.6
6 3 9.71 3.7
7 2 9.12 .
9 2 9.84 .
11 4 9.82 4.5
12 5 9.45 8.2
13b 4 9.65 3.5
15 1 11.11 .
16 1 9.55 .
19 4 9.66 6.2
23 2 9.93 .
Overall 12 9.77 4.7
Rossix
10 4 9.97 3.9
13a 4 10.02 5.7
22 4 9.72 2.2
Overall 3 9.90 1.6
GM: geometric mean; GCV: geometric coefficient of variation; N:
number of assays
-
WHO/BS/2014.2245
Page 19
Table 11: Comparison of Potency estimates by Hyphen and Rossix
chromogenic assay kits
for Sample C relative to S, the International Reference Reagent
for FXIa
Method Lab N GM
U/ampoule
GCV
%
Hyphen
3 3 0.08 5.1
4 4 0.07 3.8
6 4 0.07 9.6
7 2 0.06 .
9 1 0.10 .
11 3 0.06 6.0
12 5 0.07 31.5
13b 4 0.07 6.5
15 2 0.08 .
16 1 0.07 .
19 4 0.08 2.7
23 2 0.08 .
Overall 12 0.07 14.0
Rossix
10 4 0.08 31.4
13a 4 0.06 5.6
Overall 2 0.07 -
GM: geometric mean; GCV: geometric coefficient of variation; N:
number of assays
-
WHO/BS/2014.2245
Page 20
Table 12: Comparison of Potency estimates by Hyphen and Rossix
chromogenic assay kits
for Sample D relative to S, the International Reference Reagent
for FXIa
Method Lab N GM
U/ampoule
GCV
%
Hyphen
3 4 0.34 7.1
4 4 0.31 2.4
6 3 0.33 7.1
7 1 0.31 .
9 3 0.32 7.3
11 4 0.28 10.9
12 4 0.30 8.9
13b 4 0.32 5.0
15 2 0.31 .
16 1 0.32 .
19 3 0.28 18.0
23 2 0.35 .
Overall 12 0.31 7.1
Rossix
10 3 0.34 5.0
13a 4 0.34 5.3
22 3 0.32 5.1
Overall 3 0.33 2.8
GM: geometric mean; GCV: geometric coefficient of variation; N:
number of assays
-
WHO/BS/2014.2245
Page 21
Table 13: Comparison of Potency estimates by Hyphen and Rossix
chromogenic assay kits
for Sample F relative to S, the International Reference Reagent
for FXIa
Method Lab N GM
U/ampoule
GCV
%
Hyphen
3 4 0.68 11.4
4 4 0.61 4.7
6 4 0.72 10.3
7 1 0.63 .
9 4 0.62 7.0
11 1 0.64 .
12 5 0.64 13.1
13b 4 0.62 6.2
15 2 0.59 .
16 1 0.69 .
19 1 0.61 .
23 3 0.68 6.9
Overall 12 0.64 6.1
Rossix
10 4 0.69 5.5
13a 4 0.65 1.8
22 4 0.65 2.1
Overall 3 0.66 3.6
GM: geometric mean; GCV: geometric coefficient of variation; N:
number of assays
Table 14: Comparison of potency estimates relative to the
International Reference Reagent
for FXIa or the sample A, the proposed IS (assuming potency of
9.8 U/ampoule)
Samples
All assays
U/ampoule
Excl Clotting
U/ampoule
Vs IRR Vs sample A Vs IRR Vs sample A
B 9.8 9.8 9.8 9.77
C 0.07 0.07 0.07 0.07
D 0.37 0.33 0.32 0.37
F 0.73 0.74 0.65 0.65
Pair t-test: p>0.5 for all samples
-
WHO/BS/2014.2245
Page 22
Figure 1: Potency estimates from each assay for the candidate
standard. Each box depicts
the geometric mean potency of samples A and B in each assay.
Figure 2: Potency estimates from each assay for sample C.
Results from Lab 2 were outside
the range of the histogram.
-
WHO/BS/2014.2245
Page 23
Figure 3: Potency estimates from each assay for sample D.
Results from Lab 2 were outside
the range of the histogram.
Figure 4: Potency estimates from each assay for sample F.
Results from Lab 2 were outside
the range of the histogram.
-
WHO/BS/2014.2245
Page 24
Appendix 1: Molar concentration
Method Active-site titration is a method for calculating the
active molar concentration of a proteolytic
enzyme for which a suitable titrant is available. When an
active-site titrant is added to an
enzyme there is an initial burst of activity followed by a lower
rate as the substrate is turned over
slowly. The magnitude of this initial burst is equivalent to the
molar concentration of active
enzyme, which can be quantified relative to a standard curve of
the released product.
Active-site titration was performed on the bulk FXIa material,
prior to formulation for the fill,
against the fluorimetric titrant 4-Methylumbelliferyl
4-guanidinobenzoate hydrochloride hydrate
(MUGB). A stopped flow unit was used to achieve rapid mixing of
FXIa with MUGB. This
was coupled to a fluorimetric spectrometer to simultaneously
provide an output trigger to start
kinetic measurements to capture the required burst of product
release from MUGB mixed with
FXIa. Kinetic measurements were taken by the fluorimeter at Ex.
355 nm Em. 465 nm (5 nm
slits, 0.1 s) over 200 s.
Three individual assays were performed on three separate
samples. For each assay six dilutions
of FXIa were made in 0.01 M HEPES (pH 7.4) with 0.15 M NaCl
within an approximate range
0.1–2 µM. Each FXIa dilution was titrated against MUGB (4 µM) in
replicate (n>4). For each
assay a standard curve of 4-methylumbelliferone (4-MU, the
fluorophore released form MUGB)
was generated under identical assay conditions.
Results The magnitude of each ‘burst’ was calculated in GraphPad
Prism, based on curve fitting over
120s, and corrected for the blank reading (MUGB titrated against
buffer only). For each assay
the active molar concentration of FXIa was calculated relative
to the 4-MU standard using a
parallel line model. The results for each assay are presented in
the table below, including 95%
confidence intervals (CI), with an overall concentration based
on the unweighted geometric
mean of the three assay results.
Assay number Active FXIa concentration µM
(95% CI)
Geometric mean
(95% CI)
1
26.55
(24.62 – 28.64)
27.22
(24.45 – 30.32) 2
29.64
(27.37 – 32.11)
3
26.33
(24.22 – 28.63)
-
WHO/BS/2014.2245
Page 25
Appendix 2: List of Participants
Gerald Schrenk, Baxter AG, Austria
Iris Timmermans, Baxter Bioscience, Belgium
John More, BioProducts Laboratory Ltd., UK
Steffen Kistner, Karin Fuchs & Karen Martens-Weigand,
Biotest AG, Germany
Innocent Bekard, CSL Behring, Australia
Patrick Schütz, CSL Behring AG, Switzerland
Katherine Tull, Grifols Therapeutics Inc., USA
Ryan Dorfman, Haematologic Technologies, Inc., USA
Jean Amiral, Hyphen Biomed, France
Marta José, Instituto Grifols, S.A. R&D, Spain
Catherine Michalski, LFB-Biomédicaments, France
Luis Figueiredo, NIBSC, UK
Martina Schwarz, Octapharma SAS, Austria
Roni Mintz, Omrix, Israel
Steffen Rosén, Rossix AB, Sweden
Samuel Ling, Alison Jones, Renate Jones & Lu Liu,
Therapeutic Goods Administration/OLSS,
Australia
Mikhail Ovanesov, Yideng Liang & Samuel Woodle, US Food
& Drug Administration (CBER),
USA
-
WHO/BS/2014.2245
Page 26 Appendix 3: Protocols for Collaborative Study
Value Assignment of the 1st International Standard for Activated
Factor XI
December 2013
CS504
Study Protocol
1 INTRODUCTION
In response to the urgent need of a reference preparation for
activated factor XI (FXIa) to aid
development of assay methods and harmonization of assay results
for measurement FXIa in
immunoglobulin (IgG) therapeutics, the World Health Organization
established the International
Reference Reagent for Activated Blood Coagulation Factor XI
(FXIa), Human, in 2012. This
reference reagent has proven to be very helpful, but the supply
of this preparation is now
running low. A collaborative study is now required to establish
an international standard to
ensure the availability of a reference standard and continuity
of the unit defined by the
International Reference Reagent.
There are two objectives to this study:
Primary: The collaborative study will value assign the
functional activity of the proposed 1st
International Standard for Activated Blood Coagulation Factor
XI, Human, relative to the
International Reference Reagent for Activated Blood Coagulation
Factor XI (FXIa), Human.
The performance of the candidate materials relative to several
procoagulant IVIG samples will
also be assessed. Please note that only results from functional
activity methods specific for
FXIa will be used for value assignment.
Secondary: This study also provides an opportunity to explore
the performance of the FXIa
candidates and their use in assessment of procoagulant activity
by Non-activated Partial
Thromboplastin Time (NAPTT) and Thrombin Generation Assay (TGA).
For this purpose, a
panel of IgG preparation will be included and the participants
are requested to carry out
either/both NAPTT and TGA on the FXIa candidates alongside the
panel of IgG. The results
will give an insight into the feasibility of using FXIa as a
control for these procoagulant activity
assays. This part of the study is optional, and additional
samples have been included for this
objective if the participant has agreed to take part.
Please read through this protocol before carrying out the study,
if you are unclear on any
aspect of the study please do get in contact (email address at
the end).
2 SAMPLES FOR STUDY – PRIMARY OBJECTIVE
-
WHO/BS/2014.2245
Page 27
CODE PREPARATION
S Proposed 1st International Reference Reagent for Factor XIa
(11/236), 10
units/ampoule - 4 ampoules supplied.
A Factor XIa preparation – approx. 10 units/ampoule - 4 ampoules
supplied
B Factor XIa preparation – approx. 10 units/ampoule - 4 ampoules
supplied
C IVIG preparation containing medium procoagulant activity, 5%
protein;
approx. 0.06 FXIa u/ampoule - 4 ampoules supplied.
D IVIG preparation containing high procoagulant activity, 5%
protein;
approx. 0.3 FXIa u/ampoule - 4 ampoules supplied.
E IVIG preparation containing low procoagulant activity, 5%
protein; approx.
0.01 FXIa u/ampoule - 4 ampoules supplied.
F IVIG preparation containing high procoagulant activity, 5%
protein;
approx. 0.6 FXIa u/ampoule - 4 ampoules supplied.
Please also include any in-house FXIa reference reagent that you
routinely use in your assay
method. Note – the FXIa activity of the IVIG samples is an
approximate value only
NB one set of samples have been provided for the primary
objective.
SAMPLES FOR STUDY – SECONDARY OBJECTIVE
An additional set of samples, quantity as above, have been
provided if you are participating in
the study for the secondary objective. Due to the limit number
of samples available only one set
of samples can be provided even if you are carrying out both
NAPTT and TGT. If available,
please also include your own in-house FXIa reference
reagent.
3 STORAGE AND RECONSTITUTION OF AMPOULES OF S, A, B, C, D, E and
F
Store all unopened ampoules at -20oC or below. Ampoules should
be allowed to warm to room
temperature before reconstitution.
Directions for opening DIN ampoules
DIN ampoules have an ‘easy-open’ coloured stress point, where
the narrow ampoule stem joins
the wider ampoule body. Tap the ampoule gently to collect the
material at the bottom (labelled)
end. Ensure that the disposable ampoule safety breaker provided
is pushed down on the stem
of the ampoule and against the shoulder of the ampoule body.
Hold the body of the ampoule in
one hand and the disposable ampoule breaker covering the ampoule
stem between the thumb
-
WHO/BS/2014.2245
Page 28 and first finger of the other hand. Apply a bending
force to open the ampoule at the coloured
stress point, primarily using the hand holding the plastic
collar.
Care should be taken to avoid cuts and projectile glass
fragments that might enter the eyes, for
example, by the use of suitable gloves and an eye shield. Take
care that no material is lost from
the ampoule and no glass falls into the ampoule. Within the
ampoule is dry nitrogen gas at
slightly less than atmospheric pressure. A new disposable
ampoule breaker is provided with
each DIN ampoule.
Reconstitute the ampoule contents by adding 1 ml of distilled
water. Allow the ampoule to stand
for 10 minutes at room temperature and aid reconstitution by
gentle swirling. Transfer contents
to a plastic tube and store on melting ice prior to the
assays.
4 ASSAY DESIGN
Primary Objective
The FXIa activity indicated in this protocol is an approximate
value, and each laboratory should
determine the appropriate dilution ranges for their FXIa
quantitation test.
Once the appropriate dilutions of sample S have been determined,
Assays for factor XIa should
be carried out on each of the 4 sets. Please use your own
in-house method. Four ampoules of
each sample are provided for this. Each set should be tested on
a different day (see schedule
below). A balanced order of testing should be used. Please
include your own in-house XIa
reference, if available.
Day
1 S
1 A
1 B
1 C
1 D
1 E
1 F
1 XIa
1 XIa
2 F
2 E
2 D
2 C
2 B
2 A
2 S
2
Day
2 XIa
1 S
1 A
1 B
1 C
1 D
1 E
1 F
1 F
2 E
2 D
2 C
2 B
2 A
2 S
2 XIa
2
Day
3 F
1 XIa
1 S
1 A
1 B
1 C
1 D
1 E
1 E
2 D
2 C
2 B
2 A
2 S
2 XIa
2 F
2
Day
4 E
1 F
1 XIa
1 S
1 A
1 B
1 C
1 D
1 D
2 C
2 B
2 A
2 S
2 XIa
2 F
2 E
2
Each letter refers to a set of three or more different dilutions
(e.g. 1/10, 1/20, 1/40) and S1, S2
and A1, A2 etc. refer to separate sets of dilutions (replicates)
made independently from the
same ampoule. XIa refers to your own in-house reference for XIa.
The range of dilutions
should be chosen to lie on the most linear portion of the
dose-response curve, and dilutions
used should ensure that the responses (raw data points) from the
standard and test
preparatiuons overlap for allow for accurate potency
estimation.
The assays should be completed within two hours of
reconstitution of the samples. It is
preferable to assay one group of samples per day.
For each sample please input dilutions, corresponding raw data
and your estimated potency
values against sample S into the provided excel workbook. Please
also provide the expected
activity of your in-house standard, if included.
Secondary Objective
-
WHO/BS/2014.2245
Page 29 Carried out with the second set of samples
The FXIa potency estimation obtained from the primary objective
can be used as a guide for
dilution to use for the secondary objective. For your chosen
method, TGT and/or NAPTT, we
request that multiple dilutions are carried out for each sample,
where feasible. Each dilution
chosen for each sample will be individual and should ensure that
the raw data points for each
sample overlap with the data points for the standard (and
preferably within a linear range). All
dilutions made should be tested in replicate.
For example for S 1/400, 1/800, 1/1600, 1/3200 assuming 10
units/ampoule
for C 1/2, 1/4, 1/8, 1/16 assuming 0.06 units/ampoule
(Please note this is an example and not a suggestion for
dilution of S or C)
Testing by balanced order should be carried out, please use the
same design as indicated in
the primary objective of the study. The assays should be
completed within two hours of
reconstitution. It is preferable to assay one group of samples
per day.
For each sample please input dilutions, corresponding raw data
and your estimated potency
values against sample S into the provided excel workbook. For
the TGT results please fill in a
separate workbook for each parameter (e.g. Lagtime, ETP, Peak
Thrombin etc.). Please also
provide the expected activity of your in-house standard, if
included.
5 RESULTS
Please return completed excel results sheets by 28th Feb 2014,
and send via email to:
[email protected]
mailto:[email protected]
-
WHO/BS/2014.2245
Page 30
Appendix 4: Reagents, Methods and Instruments used by the
Participants
Lab Assay Method
Machine In house FXIa
01 Clotting Sysmex CS2100i Based on NAPTT with Platelet
substitute and FXI deficient plasma
n/a
02 Clotting ACL-TOP using APTT-SS
HTI
03 Hyphen Plate Reader Kit
04 Hyphen Plate Reader Kit
06 Hyphen Plate Reader Kit
07 Hyphen Plate Reader n/a
09 Hyphen Plate Reader IVIG
10 Rossix Plate Reader Kit
11 Hyphen BSC/XP Kit
12 Hyphen Plate Reader Spiked IVIG
13a Rossix Plate Reader HTI
13b Hyphen Plate Reader HTI
14 In house Plate Reader HTI
15 Hyphen Plate Reader Kit
16 Hyphen BCS/XP HTI
19 Hyphen ACL TOP n/a
22 Rossix Plate Reader Kit
23 Hyphen STAR Kit
-
WHO/BS/2014.2245
Page 31
Appendix 5: Individual assay results - potencies relative to S,
the International Reference Reagent for FXIa
Lab Sample Assay 1 Assay 2 Assay 3 Assay 4 Assay 5
1 A S nl np 9.92 9.88 . 1 B S nl 9.15 10.11 10.60 . 1 C S nl
0.05 0.07 0.07 . 1 D S nl 0.40 0.41 0.40 . 1 F S nl 0.57 0.74 0.70
.
2 A 10.19 9.98 9.76 9.67 . 2 B 9.82 9.62 9.28 10.01 . 2 C Nl nl
nl nl . 2 D 3.10 3.05 3.15 3.25 . 2 F 4.70 4.98 4.89 4.76 .
3 A 9.63 nl np 9.06 . 3 B 11.01 nl 8.99 9.51 . 3 C 0.08 np 0.08
0.07 . 3 D 0.37 0.33 0.36 0.31 . 3 F 0.76 0.65 0.71 0.59 .
4 A 9.04 9.72 9.54 8.71 . 4 B 9.17 10.45 9.66 9.55 . 4 C 0.07
0.07 0.07 0.07 . 4 D 0.30 0.32 0.31 0.31 . 4 F 0.62 0.59 0.59 0.65
.
6 A 9.29 10.97 10.15 11.15 . 6 B 9.31 nl 9.82 9.99 . 6 C 0.06
0.07 0.06 0.08 . 6 D 0.31 0.33 0.36 nl . 6 F 0.62 0.74 0.73 0.78
.
7 A 8.99 np np 9.59 . 7 B 9.40 np np 8.84 . 7 C Nr 0.05 . 0.07 .
7 D Nr . . 0.31 . 7 F . nl nl 0.63 .
9 A 9.70 9.68 9.99 10.97 . 9 B 9.60 10.09 nl nl . 9 C 0.10 nl nl
nl . 9 D 0.30 nl 0.35 0.31 . 9 F 0.66 0.61 0.57 0.66 .
10 A 9.14 9.74 9.94 10.49 . 10 B 9.69 9.71 9.98 10.52 . 10 C
0.06 0.11 0.06 0.07 . 10 D Nl 0.33 0.34 0.36 . 10 F 0.64 0.69 0.73
0.70 .
11 A 9.50 10.11 9.20 10.23 . 11 B 10.42 9.50 9.51 9.89 . 11 C
0.06 np 0.06 0.07 . 11 D 0.24 0.28 0.30 0.29 . 11 F Nl nl 0.64 np
.
12 A 8.97 10.07 10.40 7.44 9.84
-
WHO/BS/2014.2245
Page 32
Lab Sample Assay 1 Assay 2 Assay 3 Assay 4 Assay 5
12 B 9.56 9.37 10.72 8.74 8.99 12 C 0.05 0.07 0.10 0.07 0.06 12
D . 0.31 0.34 0.28 0.29 12 F 0.59 0.65 0.67 0.77 0.55
13a A 10.61 10.29 9.38 10.12 . 13a B 10.86 9.78 9.58 9.90 . 13a
C 0.07 0.07 0.06 0.06 . 13a D 0.36 0.34 0.32 0.33 . 13a F 0.66 0.65
0.63 0.65 .
13b A 9.55 9.71 9.35 9.99 . 13b B 10.15 9.60 9.42 9.44 . 13b C
0.07 0.07 0.06 0.07 . 13b D 0.34 0.33 0.30 0.32 . 13b F 0.58 0.67
0.60 0.62 .
15 A 12.41 8.41 11.27 S nl . 15 B 11.11 nl nl S nl . 15 C 0.08
0.07 nl S nl . 15 D 0.34 0.28 nl S nl . 15 F 0.66 0.53 nl S nl
.
16 A 9.89 S nl nl S nl . 16 B 9.55 S nl nl S nl . 16 C 0.07 S nl
nl S nl . 16 D Nl S nl 0.32 S nl . 16 F Nl S nl 0.69 S nl .
19 A 10.45 10.19 10.21 10.02 . 19 B 10.09 10.27 9.20 9.16 . 19 C
0.08 0.08 0.08 0.08 . 19 D Np 0.32 0.29 0.23 . 19 F Np nl 0.61 nl
.
22 A 9.89 9.53 9.50 nl . 22 B 9.97 9.58 9.52 9.84 . 22 C 0.06
0.06 nl 0.06 . 22 D 0.34 0.32 . 0.31 . 22 F 0.66 0.64 0.63 0.66
.
23 A 9.52 np 9.93 9.28 . 23 B 9.28 nl nl 10.63 . 23 C Np 0.09 nl
0.08 . 23 D 0.35 nl nl 0.34 . 23 F 0.72 np 0.68 0.63 .
nl: non-linear; S nl: sample S non-linear: np: non-parallel; nr:
not in range of standard Lab 14 - the majority of samples and
assays from this lab gave non-linear dose response curves and were
not analysed further.
-
WHO/BS/2014.2245
Page 33
Appendix 6: Potency estimates relative to Sample A, the proposed
International Standard, assuming an assigned value of 9.8 U/ampoule
Sample B
Lab Method N GM GCV
U/ampoule %
1 Clotting 3 10.37 3.3
2 Clotting 4 9.58 3.9
3 Chromogenic 1 10.29 .
4 Chromogenic 4 10.28 4.1
6 Chromogenic 3 9.35 5.9
7 Chromogenic 4 9.79 10.7
9 Chromogenic 2 9.95 .
10 Chromogenic 4 9.95 2.9
11 Chromogenic 4 9.88 7.2
12 Chromogenic 5 9.99 10.8
13a Chromogenic 4 9.73 3.7
13b Chromogenic 4 9.80 5
15 Chromogenic 1 8.77 .
16 Chromogenic 1 9.46 .
19 Chromogenic 4 9.27 5.3
22 Chromogenic 3 9.85 0.3
23 Chromogenic 2 10.36 .
Overall 17 9.80 4.5
Excl.CL 15 9.77 4.5
GM: geometric mean; GCV: geometric coefficient of variation; N:
number of assays; CL: clotting
-
WHO/BS/2014.2245
Page 34
Sample C
Lab Method N GM GCV
U/ampoule %
1 Clotting 2 0.07 -
3 Chromogenic 3 0.08 3.3
4 Chromogenic 4 0.07 3.4
6 Chromogenic 4 0.06 3.3
7 Chromogenic 1 0.04 .
9 Chromogenic 1 0.10 .
10 Chromogenic 4 0.08 32.2
11 Chromogenic 3 0.06 2.9
12 Chromogenic 5 0.07 31.7
13a Chromogenic 4 0.06 2.9
13b Chromogenic 4 0.07 6.1
15 Chromogenic 2 0.07 .
16 Chromogenic 1 0.07 .
19 Chromogenic 4 0.08 2.1
22 Chromogenic 2 0.06 .
Overall 15 0.07 18.7
Excl. CL 14 0.07 19.5
GM: geometric mean; GCV: geometric coefficient of variation; N:
number of assays; CL: clotting
-
WHO/BS/2014.2245
Page 35
Sample D
Lab Method N GM GCV
U/ampoule %
1 Clotting 3 0.42 6.8
2 Clotting 4 3.14 4.8
3 Chromogenic 3 0.36 4.5
4 Chromogenic 4 0.33 3.8
6 Chromogenic 3 0.32 9.4
7 Chromogenic 1 0.32 .
9 Chromogenic 3 0.31 10.6
10 Chromogenic 3 0.33 1.2
11 Chromogenic 4 0.28 11.2
12 Chromogenic 4 0.32 11.5
13a Chromogenic 4 0.33 0.9
13b Chromogenic 4 0.33 4.8
15 Chromogenic 2 0.29 .
19 Chromogenic 3 0.27 16.9
22 Chromogenic 2 0.33 .
23 Chromogenic 2 0.36 .
Overall
16 0.37 78.0
Excl.CL
14 0.32 8.4
GM: geometric mean; GCV: geometric coefficient of variation; N:
number of assays; CL: clotting
-
WHO/BS/2014.2245
Page 36
Sample F
Lab Method N GM GCV
U/ampoule %
1 Clotting 3 0.69 5.5
2 Clotting 4 4.80 3.9
3 Chromogenic 2 0.70 .
4 Chromogenic 4 0.65 10.1
6 Chromogenic 4 0.68 3.3
7 Chromogenic 1 0.64 .
9 Chromogenic 3 0.60 9.2
10 Chromogenic 4 0.69 3.9
11 Chromogenic 1 0.68 .
12 Chromogenic 5 0.68 25.9
13a Chromogenic 4 0.63 3.7
13b Chromogenic 4 0.63 5.5
15 Chromogenic 2 0.57 .
19 Chromogenic 1 0.58 .
22 Chromogenic 3 0.66 0.7
23 Chromogenic 3 0.69 5.3
Overall
16 0.74 65.5
Excl.CL
14 0.65 6.9
GM: geometric mean; GCV: geometric coefficient of variation; N:
number of assays; CL: clotting
-
WHO/BS/2014.2245
Page 37
Appendix 6: Draft IFU
-
WHO/BS/2014.2245
Page 38