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WHO/BS/09.2124

ENGLISH ONLY

EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION

Geneva, 19 to 23 October 2009

An International Collaborative Study to Value Assign

the 6th International Standard for Unfractionated Heparin and

the US Pharmacopeial Heparin Reference Standard for Assay Lot F

Elaine Gray

1,3, John Hogwood

1, Peter Rigsby

1, Barbara Mulloy

1, Anita Y. Szajek

2,

Michael R. Ambrose2, Fabian Jameison

2, Walter Hauck

2 and Tina S. Morris

2

1National Institute for Biological Standards and Control,

Blanche Lane, South Mimms, Potters Bar, Herts EN6 3QG, UK

2United States Pharmacopeial Convention,

12601 Twinbrook Parkway, Rockville, MD 20852, USA

3Principal Investigator

© World Health Organization 2009

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WHO/BS/09.2124

Summary Thirty-three laboratories from 18 countries contributed data obtained from 12 different assay

methods to value assign the 6th International Standard for Unfractionated Heparin against the 5th

International Standard for Unfractionated heparin, 97/578. Six candidates were included in the

study. Candidate W, 07/328, gave the lowest range of intra-laboratory variation (%GCV: 2.2 – 6.4)

for the different assay methods and the lowest inter-laboratory variation by all methods

(%GCV = 3.6). Candidate W, 07/328, is therefore recommended to be the WHO 6th International

Standard for Unfractionated Heparin with an assigned value of 2145 IU/ampoule by all methods.

This collaborative study also served to harmonise the International Unit and the United States

Pharmacopoeia (USP) unit for unfractionated heparin. A new (USP) Heparin Sodium reference

standard (RS) Lot F for assay has been selected and value assigned by sub-group analysis of data

obtained using the proposed new USP monograph potency method (anti-factor IIa antithrombin

dependent assay; aIIa:AT). Nine participants carried out this assay. Candidate X, 07/330, gave low

intra- and inter-laboratory variation for the USP anti-IIa chromogenic assay and it also gave the best

agreement of potency estimates by the three present and future USP monograph methods. Candidate X, 07/330, was accepted by the USP as the USP heparin standard for assay, Lot F, with

an assigned value of 2144 USP unit/ampoule.

As both the 6th International Standard for Unfractionated Heparin and the USP heparin standard for

assay, Lot F, are traceable and value assigned against the 5th International Standard for

Unfractionated Heparin, the long-standing disparity between the USP unit and the International Unit

for Unfractionated Heparin is resolved.

Introduction The 5th International Standard for Unfractionated Heparin, 97/578 was established in 1998 (1) and

it was calibrated against the 4th International Standard. This standard has been widely used for

calibration of clinical products and controls for diagnostic kits for heparin. Due to the depletion of

stock, a replacement for 97/578 is now required. The programme for the replacement of the WHO

IS for Unfractionated Heparin was endorsed by the WHO Expert Committee on Biological

Standardisation in October 2006 and was introduced to the SSC/ISTH Subcommittee on Control

of Anticoagulation at the 53rd SSC meeting in July 2007 (Geneva).

For the last 30 years or more, there has been a 7–13% disparity between the United States

Pharmacopoeia (USP) unit and the International Unit (IU) for unfractionated heparin (1). It is

therefore desirable to harmonize these two units for the measurement of heparin. The need to

replace the 5th International Standard for Unfractionated Heparin and to establish the USP heparin

standard for assay, presents an ideal opportunity for harmonization. The USP is currently

undertaking a revision of the Heparin Sodium anticoagulant potency monograph assay, with the

intention to establish a new USP heparin standard for assay that will enable the harmonization of

the USP unit and the IU for unfractionated heparin. This harmonization will require the calibration

of the new USP heparin standard for assay, Lot F, against the current International Standard (IS)

for Unfractionated Heparin and as the new USP potency assay, the anti-factor IIa chromogenic

assay using purified antithrombin (aIIa:AT) will also be implemented at the same time, the

potency of Lot F would be value assigned using this new aIIa:AT potency assay.

Because of the recent contaminated heparin events, heparin manufacturers have been encouraged

by regulatory authorities to replace clotting assays that can be influenced by contaminants and

process related impurities to methods that are specific for heparin, for example, anti-IIa and anti-

WHO/BS/09.2124

Xa assays using purified antithrombin, for measurement of heparin potency. This study also

allowed us to examine the robustness of these chromogenic methods.

This report describes two important findings of this collaborative study that involved 6

candidates:

1. The analysis of data and conclusions that lead to the value assignment of the proposed

6th International Standard for Unfractionated Heparin

2. Information on the calibration of the USP heparin standard for assay, Lot F and the

performance of anti-IIa and anti-Xa chromogenic assays

Candidates The coding of the candidates is as indicated in Table 1. Candidates T, W, X, Y and Z are all

typical clinical heparin Active Pharmaceutical Ingredients (APIs) from different European and

North American manufacturers. Candidate V is not a routine API and has been manufactured for

research studies only. Preliminary investigations including specific activity, molecular weight

profiles and NMR spectroscopy were carried out at NIBSC. Specific activities of the candidates

were measured against the 5th IS, using antithrombin dependent anti-IIa and anti-Xa assays, on

candidates weighed out at ambient temperature and humidity. With the exception of candidate V,

which has a specific activity close to 300 IU/mg by both anti-IIa and anti-Xa assays, all other

candidates have specific activities between 170 – 230 IU/mg. The higher specific activity

exhibited by candidate V could be due to the higher proportions of high molecular weight

material in this sample (Figure 1). With the exception of 08/254, candidate T, all the other

candidates were filled and freeze-dried at NIBSC in accordance with the conditions normally

used for international biological standards (2). Table 1 shows the fill characteristics of the

candidates. The excipient for all the candidates was sterile water.

The CV of the fills showed that all the candidates were filled with acceptable precision.

Homogeneity of the fill by functional activity was also investigated by assay of ampoules

sampled across the fill. For each candidate filled at NIBSC, 4 ampoules (2 independent assay

per ampoule) at every 5000 ampoules filled were assayed by the Activated Partial

Thromboplastin Time (APTT) against the 5th IS and results were compared by analysis of

variance. Table 2 shows that the variabilities by functional assay for candidates V, W, X, Y and Z

were all low with GCV below 3% and analysis of variance showed that there were no significant

differences between the activities of the ampoules across the fill.

Molecular weight determination of the candidates was carried out according to Mulloy et al. (3).

The profiles and measurements are shown in Figure 1 and in Table 3. Candidate V is noticeably

different from the others, having the highest values for peak molecular weight (Mp), number

average molecular weight (Mn) and weight average molecular weight (Mw). The other candidates

fall within a relatively narrow range, similar to that of the 5th International Standard for

Unfractionated Heparin.

1H NMR spectra at 500 MHz were recorded for each of the candidates in accordance with McEwen

et al. (4) and spectra of the candidates are shown in Figures 2 - 7. The NMR spectra confirmed that

the candidates are all free of oversulphated chondroitin sulphate, a contaminant which has been

found in some recent heparin batches. A small signal in the spectrum of sample W at 2.18 ppm can

be seen, but a spiking experiment showed that this does not originate from oversulphated

chondroitin sulphate (data not shown) .

WHO/BS/09.2124

Participants Thirty-six laboratories from 18 countries (1 Australia, 4 Austria, 1 Bosnia, 1 Brazil, 1 Canada, 1

China, 2 Denmark, 4 France, 5 Germany, 1 India, 2 Italy, 1 Japan, 1 The Netherlands, 1

Portugal, 3 Spain, 3 Switzerland, 1 UK, 3 USA) agreed to take part in the study. Thirty-three

laboratories returned results, each of which is referred to in this report by an arbitrarily assigned

number, not necessarily representing the order of listing in Appendix 1. Lab 20 (Austria) withdrew

from the study, while results from Lab 4 and 14 were not received in time for analysis.

Assay methods All the methods used by the participants are listed in Appendix 2. In total, 12 different methods

were used in the study:

• Anti-IIa chromogenic assay, purified antithrombin (AT), n = 18

• Anti-Xa chromogenic assay, purified AT, n = 23

• Anti-IIa chromogenic assay, human plasma, n =1

• Anti-Xa chromogenic assay, human plasma, n = 3

• Anti-Xa clotting assay, n = 1

• US Pharmacopoeial assay (USP), clot-based sheep plasma assay n = 9

• European Pharmacopoeial assay (EP), clot-based sheep plasma assay n = 12

• Japanese Pharmacopoeial assay (JP), n = 1

• Chinese Pharmacopoeial assay (CP), n = 1

• Human plasma APTT, n = 12

• Thrombin Time (TT), n = 2

• Prothrombinase induced clotting time (PiCT), n = 1

Some laboratories requested assay protocols for anti-IIa and anti-Xa chromogenic assay using

purified antithrombin. NIBSC provided these laboratories with methods for both of these assays

(Appendix 3).

Study design The detail of the study protocol is shown in Appendix 4. Each laboratory was asked to perform

their chosen methods. Where a laboratory performed more than one assay method, each method is

treated as if performed by different laboratories.

Participants were requested to perform four independent assays for each method, an independent

assay being defined as one with a completely fresh set of ampoules and dilutions. To allow for day

to day variation, the participants were asked to carry out the four assays, for each method, on four

separate days. For those laboratories that performed two or more methods, an assay for each

method could be carried out on the same or different days, from one set of reconstituted solutions,

but making fresh dilutions for each method.

Participants were asked to assay concurrently a series of at least three, preferably four dilutions of

each of the five coded samples. The assay order of the materials (including replicates) was to be

varied to give a balanced order overall. Some examples of different assay order schemes were given

in the protocol for participants to choose from.

WHO/BS/09.2124

Raw assay data were to be returned together with a summary of their estimates for the potency of

materials T, V, W, X, Y and Z using S, the 5th IS for Unfractionated Heparin, as the standard.

Statistical analysis An independent statistical analysis of raw data was performed at NIBSC. Potencies were

calculated relative to the 5th International Standard for Unfractionated Heparin, 97/578 (Study

code: S), for each candidate sample using a parallel-line or slope-ratio model (5). All data were

plotted and the assay validity was assessed both visually and by analysis of variance. When

doses did not appear to lie on the linear section of the dose-response curve they were excluded

from analysis. All assays that showed no significant deviations from the model at the 1% level

have been included in this report. Where non-parallelism was found to be significant at the 5%

level in parallel-line models (0.01<p<0.05) the results are included and highlighted. This is also

the case when the intercept difference was found to be significant at the 5% level in slope-ratio

models (0.01<p<0.05). In a small number of cases the results reported by the laboratory have

been used directly and these are indicated below. All mean potencies given in this report are

unweighted geometric mean potencies. Variability between assays and laboratories has been

expressed using geometric coefficients of variation (%GCV) (6). Grubbs’ Test (7) was applied to

the log transformed laboratory mean estimates in order to detect any significant outliers (p<0.05)

and analysis of variance (with Tukey’s test) was used to make comparisons between assay

methods.

Due to the late submission of results by Lab 4 and Lab 14, their data obtained by the European

Pharmacopoeia method and APTT method respectively could not be analysed and was therefore

excluded from the overall potency estimates. For comparison purpose, the potency estimates

from Lab 4 and 14 are shown in Appendix 5.

Anti-IIa chromogenic assays using purified AT A parallel-line model comparing untransformed assay response to log concentration was used in

all laboratories except laboratory 05 where a log transformation appeared to be more suitable.

Anti-Xa chromogenic assays using purified AT A slope-ratio model comparing log transformed assay response to concentration was used for

almost all laboratories. For laboratory 10 a parallel-line model comparing untransformed assay

response to log concentration appeared to be more suitable. For laboratory 36 the assay

responses were transformed to percentages relative to the estimated upper and lower limits of the

dose-response curve and a weighted regression of logit response on log dose was used to

calculate potency estimates.

USP assays No further analysis was carried out as the USP sheep plasma assay is based on a single point

estimate. The results reported by the laboratories are shown and used for overall potency

estimates.

EP assays A parallel-line model comparing log transformed assay response to log concentration was used

in all laboratories. For laboratory 17 deviations from parallelism were compared to deviations

from linearity when assessing the validity of the model due to the low level of residual error

observed.

WHO/BS/09.2124

APTT assays A slope-ratio model comparing log transformed assay response to concentration was used for

almost all laboratories. For laboratory 10 a parallel-line model comparing untransformed assay

response to log concentration appeared to be more suitable. For laboratories 34a and 34b, neither

model appeared to provide a good fit and the reported estimates (calculated using a third order

polynomial calibration curve) are shown.

Anti-Xa chromogenic assay using plasma, Thrombin Time and other methods For laboratories 18 (Japanese Pharmacopoeial method), 30 (anti-Xa assay, using plasma as

source of antithrombin) and 34 (Thrombin Time), the reported estimates are shown as neither

model appeared to fit the data (labs 30 and 34) or only single dilutions of each test sample were

reported (lab 18). In laboratory 34 (anti-Xa Clotting) the untransformed responses were used.

Results

Assay data The 33 participants contributed data from a total of 690 assays; with 642 assays analysed by NIBSC.

For 48 assays, no further analysis was carried out and the potency estimates reported for each of

these assays were used to calculate the overall potency estimates and the intra- and inter-laboratory

variability. Table 4 shows the breakdown of the number of assays per assay method.

Assay validity The assumptions of linearity and parallelism of the log dose-transformed/untransformed response

lines of samples T, V, W, X, Y, Z and S held in 79 % of the assays analysed. In some of the assays,

certain candidates were found to be non-linear and/or non-parallel to S and the potency estimates for

these samples were not included in the overall potency estimation. Tables 6 - 11 show the validity

and potency estimates of all the assays. Some of the invalidities may have been due to inadequate

replication in the assay design, as well as from real deviations from the model assumptions. There

was no evidence of any overall non-parallelism when the 6 candidates were compared with the 5th

IS, however, candidate V did yield the highest number of assays that were non-parallel or had

significantly different intercepts and this was especially true for the EP sheep plasma assay (Table

5).

Potencies relative to the 5th International Standard for Unfractionated Heparin,

97/578 The individual assay potency estimates of candidates T, V, W, X, Y and Z taken relative to sample

S (5th IS for Unfractionated Heparin) by different methods are shown in Tables 6 - 11. Detailed

values of the individual laboratory mean potencies and overall mean potencies of samples T, V, W,

X, Y and Z relative to sample S, the 5th IS for Unfractionated Heparin for each method are listed in

Tables 12 – 18. As there was an insufficient number of data sets returned for the anti-IIa

chromogenic assay using human plasma, the anti-Xa clotting assay, Japanese Pharmacopoeial assay,

Chinese Pharmacopoeial assay and Prothrombinase induced Clotting Time, overall mean potency

estimates were not calculated for these methods and the laboratories’ mean potencies for each

candidate are classified as “other methods” and shown in Table 19. A summary of the potency

estimates by the different methods is also presented in Table 21. The data are also shown in

histogram forms in Figures 7 - 12. Only 9 outliers were identified by Grubb’s test. These were

excluded from the overall mean potency estimates as indicated in Tables 12 -19 and Figures 1 – 6.

Although the Japanese Pharmacopoeial assay produced 5 outliers, only one laboratory carried out

this assay method. It was not possible to conclude whether the problem is related to this method or

WHO/BS/09.2124

with the laboratory’s performance. There was no correlation between assay methods and outliers

identified. The histograms (Fig 7 – 12) illustrate good agreement between both the laboratories

and the assay methods for candidates T, W, X, Y and Z. Analysis of variance on the log potencies

to compare methods shows no significant differences for W (p=0.832) and X (p=0.999). Sample

Y shows significant differences (p=0.036) due to the US Pharmacopoeial assay results being

significantly higher than those for anti-IIa and anti-Xa chromogenic assays using purified AT

(p<0.05 in Tukey’s test). Sample T shows significant differences (p=0.008) due to the anti-IIa

chromogenic assays using purified AT results being significantly lower than the USP results

(p<0.05 in Tukey’s test). Sample V shows significant differences (p<0.001) due to the anti-IIa

chromogenic assays using purified AT results being significantly higher than all other methods

and the anti-Xa chromogenic assays using purified AT results being significantly higher than the

APTT, the European Pharmacopoeial and the US Pharmacopoeial assays results (p<0.05 in

Tukey’s test). Sample Z shows significant differences (p<0.001) due to the anti-IIa chromogenic

assays using purified AT results being significantly higher than the APTT, European

Pharmacopoeial assay and US Pharmacopoeial assay results and the anti-Xa chromogenic assays

using purified AT results being significantly higher than the APTT results (p<0.05 in Tukey’s

test).

Table 21 shows the overall potency estimates by all methods relative to the 5th IS for

Unfractionated Heparin, together with the respective 95% confidence limits for all the candidates.

Figure 13 shows the potency estimates by each assay method expressed as the % of the overall

mean potency estimates for each candidate. It is clear that with the exception of the potency

estimates for candidate V which varied with different methods (PiCT = 82.6% to anti-IIa AT =

123.7%), potency estimates by the different types of assays for the other candidates were close to

the overall mean potency estimates for that candidate; the maximum difference being 6% for

sample X by the Chinese Pharmacopoeial assay and sample T by the Prothrombinase induced

Clotting Time. Potency estimates for candidate W, followed by sample X when assayed against the

5th IS, were the least influenced by the method type.

Intra-laboratory variability: all methods The variability within each laboratory, expressed as geometric coefficients of variation (% GCV's)

for each sample, is given in Tables 12 - 19. Table 20 summarises the %GCV showing the number

under 5%, 7% and 10%. In general, the laboratories were able to perform their chosen methods with

good precision and reproducibility with more than half of the GCVs less than 5% and only a small

percentage of laboratory potency estimates gave over 10%. The clot-based assays, especially the

pharmacopoeial assays, gave lower ranges of intra-laboratory %GCVs than the chromogenic assays.

This may be because the pharmacopoeial assays are well-established methods and the laboratories

are more experienced at carrying out these assays. Nonetheless, the chromogenic assays performed

well and only a few participants produced GCVs over 10%.

Inter-laboratory variability: all methods Variability between laboratories of the potency estimates of samples T, V, W, X, Y and Z relative to

sample S, the 5th IS for Unfractionated Heparin, is shown in Tables 12 – 18 for anti-IIa

chromogenic assay with AT, anti-Xa chromogenic assay with AT, European Pharmacopoeial assay,

US Pharmacopoeial assay, APTT, Anti-Xa chromogenic assay with plasma and Thrombin Time

and is also summarised as % GCV's in Table 19. Inter-laboratory variability is not given for

Thrombin Time as only two laboratories carried out this method and for anti-IIa chromogenic assay

with plasma, anti-Xa clotting assay, Chinese Pharmacopoeial assay, Japanese Pharmacopoeial assay

and Prothrombinase induced Clotting Time as these assays were carried out by one laboratory only.

WHO/BS/09.2124

Variability between laboratories ranged from 0.1 to 18.0% and was consistently higher for

sample V and consistently lower for sample W. The clot based methods (European Pharmacopoeial

assay, US Pharmacopoeial assay and the APTT) tended to give lower inter-laboratory variability,

but the variability found with the chromogenic assays was acceptable, with the majority of the inter-

laboratory GCVs being less than 10%. Pooling the different assay methods (Table 22), the

variability between laboratories is the highest for sample V (GCV = 20.7%) and the lowest for

candidate W (GCV = 3.6%). The variability for samples T, X, Y and Z was also relatively low,

with respective GCVs of 6.6, 4.8, 5.7 and 5.5%.

Comparison of the candidates as putative standards Potencies of candidates were recalculated relative to each other, assuming each sample has an

assigned unitage equivalent to the overall mean potency by all methods, relative to sample S, shown

in Table 22. The overall mean potencies, 95% confidence limits and inter-laboratory variation

expressed as % geometric coefficient of variation (%GCV) for each candidate relative to each other

are shown in Tables 23 – 28 and the potency estimates are summarised in Table 29. These were

similar to those relative to the 5th IS for Unfractionated Heparin, which is not surprising since the

assumed assigned potencies of each sample were calculated relative to the 5th IS for Unfractionated

Heparin.

Variability between laboratories of the potency estimates of Candidates T, V, W, X, Y and Z

relative to each other, for all assay methods, is summarized as % GCVs in Table 30. High

variability was observed when Candidate V was used as the putative standard and as the test

sample against all the other putative standards. Amongst the other candidates, the differences in

inter-laboratory variation were marginal. Generally, variability between laboratories was lowest

when Candidates W or X were used as the putative standard and they could be considered as the

potential replacement international standard.

Sub-group analysis to select and value assign USP RS Lot F Sub-group analyses were carried out to assess the performance of the USP aIIa:AT, USP aXa:AT

and the USP sheep plasma clot based assays and to select and value assign the USP heparin

standard for assay.

Three different USP assay methods related to potency were carried out by the participants: a. the

current sheep plasma clot based assay, b. the current aXa:AT assay and c. the new (to be

implemented in October, 2009) aIIa:AT assay. Appendix 2b lists the laboratories by number and

the USP assay methods that they have performed. For anti-IIa and anti-Xa chromogenic assays,

laboratories 02, 19 and 25 declared that they have performed the USP anti-Xa and the proposed

anti-IIa chromogenic assays. The remainder of the laboratories used protocols provided by

NIBSC. The NIBSC protocols were simplified versions of the USP methods, with the reagents

and assay conditions as described in the USP assay methods (Appendix 3). In total,

9 laboratories carried out the USP anti-IIa, anti-Xa and sheep plasma clot-based assays. For

information, the laboratories that carried out antithrombin dependent anti-IIa and anti-Xa assays

based on commercial kits, in-house or European Pharmacopeoial Low Molecular Mass

Monograph methods are listed in Appendix 2c.

Due to the nature of the USP sheep plasma clot based assay, it was not possible to analyse the raw

data and the laboratory’s own potency estimates were used for comparison. Only statistically valid

USP aIIa:AT assays were used for estimation of potency for the USP RS Lot F.

WHO/BS/09.2124

Intra-laboratory variation: USP sheep plasma clot based, aIIa:AT and aXa:AT

assays Tables 31, 32 and 34 show the intra-laboratory variation, expressed as %GCVs for the USP sheep

plasma clot based, aIIa:AT, aXa:AT assays respectively. Tables 36 and 37 summarise the %GCV

showing the number under 5%, 7% and 10% for the all the antithrombin dependent anti-IIa and

anti-Xa assays. The intra-laboratory variation was low for the USP sheep plasma clot based assay,

GCVs ranging from 0 – 11.5%. The higher %GCVs were found with results from 1 laboratory (Lab

19); the GCVs for the other participants using this method were below 5% indicating that the

laboratories were able to carry out this assay with good precision. The intra-laboratory variation for

the USP aIIa:AT method was also reasonably low, GCV ranging from 1.4 – 29.1%, with the higher

GCVs coming from 2 laboratories (Lab 03 and 19) and the majority of the GCVs being < 7%.

These results were in line with data from other antithrombin dependent anti-IIa chromogenic assays

(Tables 33, 36). The antithrombin dependent anti-Xa assays showed similar reproducibility to the

anti-IIa assay; for both the USP aXa:AT and other chromogenic assays, the majority of the GCVs

were below 7% (Tables 34, 35, 37). These results indicate that the majority of the laboratories

carrying out these chromogenic assays were able to perform these methods with good precision and

reproducibility. There was no obvious correlation between the degree of intra-laboratory variation

and the different candidates. The sheep plasma clot based assay did give lower ranges of intra-

laboratory variation and this may be because this pharmacopoeial assay has been in use for more

than 30 years so that some laboratories are more experienced at carrying out this assay than the new

chromogenic assays.

Inter-laboratory variation: USP sheep plasma clot based, aIIa:AT and aXa:AT

assays Variability between laboratories of the potency estimates of candidates T, V, W, X, Y and Z relative

to sample S, the 5th IS for Unfractionated Heparin, is shown in Table 38. For both the USP aIIa:AT

and aXa:AT methods, it is clear that the variability between laboratories is the highest for candidate

V, the lowest for candidate W, with candidate X having GCVs close to those obtained for candidate

W. The higher inter-laboratory variation exhibited by V could be explained by the molecular

weight differences between V and the 5th IS. It is also important to note that the inter-laboratory

variation for both the USP antithrombin dependent anti-IIa and anti-Xa assays are not too different

to those obtained by the sheep plasma clot based assay.

Potency estimates by USP sheep plasma clot based, aIIa:AT and aXa:AT assays

relative to the 5th IS for unfractionated heparin The individual laboratory potency estimates of candidates T, V, W, X, Y and Z taken relative to

sample S (5th IS for Unfractionated Heparin) by the USP sheep plasma clot based, aIIa:AT,

aXa:AT and all methods are shown in Tables 39 – 41. A summary of the potency estimates by the

different methods is also presented in Table 42. Analysis of variance of log potency estimates

indicated that there were no significant differences between the potencies obtained using the USP

anti-IIa and anti-Xa chromogenic methods and other chromogenic assays for any of the candidates

(p>0.05).

Stability study Preliminary accelerated degradation study, of all the candidates, monitored using both anti-IIa and

anti-Xa chromogenic assays, showed no sign of change after 9 months storage at temperatures of -

150, -70, -20, +4, +20, +37, +45 and +56°C. Continual real time degradation study of the –20°C

against ampoules stored at –70°C and further accelerated degradation study at elevated temperature

will be carried out to monitor the stability of the replacement standard.

WHO/BS/09.2124

Discussion The main aims of this collaborative study were to select from the six candidate materials, firstly a

replacement for the 5th International Standard for Unfractionated heparin and secondly a new USP

heparin standard for assay. It also provided an opportunity to examine performance of the anti-IIa

and anti-Xa chromogenic assays.

Selection of the proposed 6th International Standard for Unfractionated

Heparin

Intra-laboratory (between assay) variability for all 6 candidate materials was good when assayed

against the 5th IS, with the majority of the %GCVs well under 10% and more than half of the

%GCVs being 5% or less (Tables 12 - 19). This indicates that the participants of the study were able

to perform their choice of assay with precision. The clot based assays (European Pharmacopoeial

assay, US Pharmacopoeial assay and APTT) generally gave lower between assay %GCVs and the

chromogenic methods gave slightly higher. The higher %GCVs for anti-IIa chromogenic assay

with AT came mostly from Lab 03 and Lab 19 (Table 12), while the higher GCV for anti-Xa

chromogenic assay with AT were from Lab 16 and Lab 36 (Table 13). Taking into account that the

higher variability was from 4 laboratories only, the %GCVs for other laboratories were excellent for

these two chromogenic assays indicating that the majority of the participants were able to perform

these chromogenic assays reproducibly. There were no other obvious correlations between

performance and parameters such as methods or reagents.

With the exception of candidate V, the inter-laboratory variations were excellent for all the other

candidates when assayed against the 5th International Standard for Unfractionated Heparin (Tables

12 – 19, 21, 22); the %GCVs were under 11% by any of the assay methods used, with at least half

under 4% (range 0.1 – 10.7%). Of all the candidates, W gave marginally lower variability than the

other samples (Tables 21, 22). These data suggest good reproducibility of results by the various

methods amongst the different laboratories. The higher variability (range 5.9 – 18.0%) obtained

with candidate V could be due to the differences between the molecular weight profile of the 5th

International Standard and sample V, with sample V having a higher proportion of high molecular

weight material than the 5th International Standard and the other candidates in the study (data not

shown). The inter-laboratory variation when all methods were taken into account indicated that W

gave the lowest and V gave the highest %GCV (W, GCV = 3.6%; V, GCV = 20.7%). The other

candidates only gave marginally higher %GCVs than sample W (6.6, 4.8, 5.7 and 5.5 for T, X, Y

and Z respectively).

In terms of assay methods, there were no significant differences amongst the potency estimates

obtained using the different methods for candidates W and X against the 5th International Standard

for Unfractionated Heparin. The best agreement was obtained for candidate W where the potency

estimates by various methods were between 96.7 to 101.5 % of the overall mean potency estimate

by all methods (Fig. 14). Potency estimates for candidate T by anti-IIa chromogenic assay using

purified AT were significantly lower than the US Pharmacopoeial assay results. For sample V,

potency estimates were significantly higher by anti-IIa chromogenic assay with purified AT than

results from all other methods and potencies by anti-Xa chromogenic assay with purified AT were

significantly higher than the data from the APTT and the European Pharmacopoeial assay. For

sample Y, the US Pharmacopoeial assay was significantly higher than both the anti-IIa and anti-

Xa chromogenic assay using purified AT. For sample Z, the anti-IIa chromogenic assays using

purified AT results were significantly higher than the APTT, European Pharmacopoeial assay

and US Pharmacopoeial assay results, with the anti-Xa chromogenic assays using purified AT

results being significantly higher than the APTT results. Although not all the assay methods

gave complete agreement of potency estimates for all the candidates, with the exception of

WHO/BS/09.2124

sample V (range of potency estimates by different assay methods = 82.6 – 123.7 of the overall mean

potency), the overall differences between potency estimates obtained with the various methods were

not large and were probably found to be statistically significant because of the good precision of the

assays. This is confirmed by the close agreement in the potency estimates of the different

preparations (except V) by all assay methods, with all except four of the average percentages of

combined potency estimates for each method within 5% of the overall combined potency estimates

from all assay methods (Fig 14).

When each candidate was used as the putative standard, candidate V gave the highest inter-

laboratory variation (GCV range 17.8 – 24.8 %). Although high %GCVs were obtained when V

was assayed against W and X, these two candidates gave low GCVs for all the other samples (GCV

range 4.0 – 6.0%, Table 30). Since candidate W also gave the lowest range of intra- and inter-

laboratory variation when assayed against the 5th IS, W would be the best candidate to go forward

as the 6th IS for Unfractionated Heparin.

Selection of the USP heparin standard for assay As 9 laboratories carried out the USP aIIa:AT, there were sufficient data to carry out sub-group

analysis and obtain estimation of potency by this proposed USP monograph potency method for

heparin sodium. Comparison of log potency estimates obtained using the USP sheep plasma, USP

aIIa:AT and USP aXa:AT assays by one-way analysis of variance (ANOVA) and Tukey’s test

showed that there were no significant differences amongst the potency estimates for candidates W

(p = 0.277), X (p = 0.979) and Z (p = 0.06). Candidate T shows significant differences due to the

USP aIIa:AT assay results being significantly lower than the USP sheep plasma assay results

(p<0.05 in Tukey’s test). Candidate V shows significant differences (p<0.001) due to the USP

aIIa:AT and aXa:AT results being significantly higher than the USP sheep plasma assay data.

Candidate Y shows significant differences due to the USP aIIa;AT assay results being

significantly lower than the USP sheep plasma assay results (p<0.05).

Potency estimates for candidates W and X when assayed against the 5th IS, were the least

influenced by the method type (Figures 14, 15). Table 43 also shows that candidates W and X gave

an anti-Xa to anti-IIa ratio of 1.01 and that both candidates are within the USP specification for anti-

Xa to anti-IIa ratio of 0.9 – 1.1 for Heparin Sodium. Figure 15 illustrates the potency estimates by

the different USP methods expressed as the % of the overall mean potency estimates obtained by all

methods employed in the study. It is clear that for candidate X, the three USP methods gave

excellent agreement in potency estimates. So in considering continuity and traceability of the

international unit and that Lot F will be the RS that unites the international and USP units, candidate

X would be a good choice.

Conclusions and Recommendation • Although the clot based assays gave lower intra- and inter- laboratory variation, the

performance of the anti-IIa and anti-Xa chromogenic assay was good. Some laboratories

performed better than others with intra-laboratory GCV of less than 1%. In general, the

intra-laboratory GCVs were around or less than 5%. Good laboratory agreement was

also obtained by the anti-IIa and anti-Xa chromogenic assays for all candidates except V.

However sample V is very different in physico-chemical characteristics to the current

International Standard and to the other candidates in the study and poor laboratory

agreement was also obtained for V when other assay methods were used. This illustrates

the importance of assaying “like against like” in obtaining good agreement between

laboratories.

WHO/BS/09.2124

• Currently there are insufficient data to assess and compare the stability of these

candidates. However, all the physical parameters such as moisture and oxygen content

are within the specifications set out in the WHO guidelines for the preparation of

international standards and reference materials. Stability data on preparations with

similar characteristics e.g. the 5th International Standard for Unfractionated Heparin and

the 2nd International Standard for Low Molecular Weight Heparin have shown no loss of

measurable activity after more than 10 years storage at -20°C. So these preparations are

expected to be very stable over the lifetime of reference material.

• All candidates except V performed well in the study, with the majority of the intra- and

inter- laboratory GCVs below 10%. Overall, candidate W gave the lowest range of intra-

and inter-laboratory variation whether as a test sample or as the putative standard. It is

therefore recommended that Candidate W, 07/328, to be the 6th International Standard

for Unfractionated Heparin with an assigned value of 2145 IU/ampoule obtained from all

assay methods against the 5th International Standard for Unfractionated Heparin, 97/578.

• The sub-group analysis presented in this report shows that both candidates W and X are

also suitable materials for the next USP heparin standard for assay, Lot F. Both

candidates gave comparably low intra- and inter-laboratory variation for the USP anti-IIa

chromogenic assay, but candidate X is marginally better than candidate W in that it gave

the best agreement of potency estimates by the three present and future USP monograph

methods (figure 15). The molecular weight distribution of candidate X is similar to that

of other typical clinical APIs in the study. The 1H NMR spectrum also indicates that

candidate X is a clean heparin with no detectable over-sulphated chondroitin sulphate and

extremely low levels of other process related impurities such as dermatan sulphate,

acetate or ethanol.

• It was therefore recommended that candidate X, 07/330, be the USP heparin

standard for assay, Lot F, with a value of 2144 USP unit/ampoule as assigned by

USP proposed aIIa:AT assays. The USP considered the recommendation and agreed

with this proposal, and candidate X has been established as the USP heparin standard

for assay Lot F.

• As the calibration of both the proposed 6th IS and USP heparin standard for assay,

Lot F was against the 5th IS, the USP unit and the International Unit for

Unfractionated Heparin are now harmonized.

• A consequence of this harmonization is a discontinuous change in the USP unit. The

effects of this change on users are being monitored by the USP. The US FDA is aware of

this change as well.

Responses from the participants and Scientific and Standardization

Committee (SSC) of the international Society on Thrombosis and

Haemostasis All submitted responses from participants agreed with the recommendations for the proposed 6th

International Standard for Unfractionated heparin. There was a comment from a participant with

regards to the classifications of the method used by their laboratory and the error has been corrected

in this current report. This error has no impact on the recommended replacement IS and its value

assignment. One of the SSC experts required clarification of the rationale for the use of slope ratio

and parallel line analysis and abbreviations used in the report. These comments have also been

considered and amended as required. At the SSC Annual Business Meeting (the 55th SSC

Meeting in Boston, 15 July 2009), the SSC endorsed the proposal that Candidate W, 07/328, be the

6th International Standard for Unfractionated Heparin.

WHO/BS/09.2124

Proposal and recommendation to the ECBS • Candidate W, 07/328, be the 6

th International Standard for

Unfractionated heparin : 2145 IU/ampoule

The proposed Instruction for Use for the proposed 6th International Standard for Unfractionated

Heparin, 07/328 is illustrated in Appendix 6.

References (1) Gray E, Walker AD, Mulloy B, Barrowcliffe TW. A collaborative study to establish the 5th

International Standard for Unfractionated Heparin. Thromb Haemost. 2000 Dec;84(6):1017-

22.

(2) Campbell PJ. Procedures used for the production of biological standards and reference

preparations. J Biol Standardisation. 1974, 2, 259-267

(3) Mulloy B, Gray E, Barrowcliffe TW. Characterisation of unfractionated heparin samples:

Comparison of samples from the last 50 years Thromb Haemostas 2000 84:1052-1056.

(4) McEwen I, Mulloy B, Hellwig E, Kozerski L, Beyer T, Holzgrabe U, Rodomonte A,

Wanko R, Spieser J-M. Determination of oversulphated chondroitin sulphate and

dermatan sulphate in unfractionated heparin by 1H-NMR” Pharmeuropa Bio 2008-1, 31-

39.

(5) Finney DJ. Statistical Method in Biological Assay. 3rd Edition. London: Charles Griffin

1978.

(6) Kirkwood TBL. Geometric means and measures of dispersion. Biometrics 1979; 35:

908-909.

(7) Grubbs F. Procedures for Detecting Outlying Observations in Samples. Technometrics,

1969; 11: 1-21.

Acknowledgments We would like to thank the participants of the study, the support of the Control of Anticoagulation

Subcommittee of the SSC/ISTH and the following manufacturers for their kind donation of

candidate samples:

Leo Pharmaceutical Products Ltd, 55 Industriparken, DK-2750 Ballerup, Denmark

Scientific Protein Laboratories, 700 E Main Street, Waunakee, 53597-0158 USA

Bioiberica, SA, Plaza Francesc Macià , 7 Barcelona 08029 , Spain

Opocrin SPA, 3, V. Pacinotti 41043 Corlo di Formaigine (MO), Italy

NV Organon (Schering-Plough), P O Box 20 5340 BH Oss, Kloosterstraat 6 5349 AB Oss, The

Netherlands

WHO/BS/09.2124

Table 1: Summary of fill characteristics of the candidates. Candidate T was not processed

at NIBSC

Code 08/254

T

07/332

V

07/328

W

07/330

X

07/334

Y

07/324

Z

Presentation Sealed, glass 3 ml DIN ampoules

No of ampoules

available 8000 25000 24000 24500 25000 24000

CV of fill mass (%) 0.24

(n=200)

0.098

(n=857)

0.119

(n=1057)

0.100

(n=888)

0.08

(n= 879)

0.121

(n=970)

Mean dry weight (g,

n = 6) 0.0097 0.007 0.0107 0.0095 0.0104 0.0116

Mean head space

oxygen (%)

3.7

(n =6)

0.329

(n = 12)

0.60

(n = 13)

0.50

(n=12)

0.39

(n=12)

0.72

(n=12)

Residual moisture

(%)

0.70

(n =3)

0.78

(n = 12)

0.76

(n =12)

0.51

(n=12)

0.18

(n=13)

0.60

(n=12)

Storage

Conditions -20°C

Address of

processing facility NIBSC, Potters Bar, EN6 3QG, UK

Address of present

custodian NIBSC, Potters Bar, EN6 3QG, UK

Table 2: Homogeneity of the fill by APTT

NIBSC Code Study Code Geometric Coefficient of

Variation (% GCV)

Analysis of Variance

(ANOVA)

07/332 V 0.97 p = 0.994

07/328 W 1.17 p = 0.252

07/330 X 1.62 p = 0.344

07/334 Y 2.60 p = 0.066

07/324 Z 0.59 p = 0.878

WHO/BS/09.2124

Table 3: Summary of molecular weight measurements on the candidates.

Candidates Mp Mn Mw PD

T 14817 11992 15617 1.40

V 19210 18188 24278 1.33

W 15640 12772 16760 1.31

X 15929 13016 15945 1.23

Y 14646 11231 14236 1.27

Z 15708 12385 17595 1.42

Mp=peak molecular weight

Mn=Number average molecular weight

Mw=Weight average molecular weight

PD=Polydispersity (Mw/Mn)

Table 4: Number of Assays Performed for Each Method

No of Assays Method

Submitted Analysed and Assessed

Anti-IIa chromogenic assay, purified AT 126 126

Anti-Xa chromogenic assay, purified AT 196 196

Anti-IIa chromogenic assay, human plasma 4 4

Anti-Xa chromogenic assay, human plasma 12 8

USP sheep plasma anticoagulant assay 36 0

EP sheep plasma APTT 167 167

Japanese Pharmacopoeia assay 4 0

Chinese Pharmacopoeia assay 24 24

Human plasma APTT 109 109

Thrombin Time 8 4

Prothrombinase induced Clotting Time (PiCT) 4 4

Total 690 642

Table 5: Invalid Potency Estimates Due to Non-parallelism (NP) in Parallel Line or

Significantly Different Intercepts (NI) in Slope Ratio Analysis. Methods where all assays were

valid are not shown in the table

Method T V W X Y Z

Anti-Xa chromogenic

assay, purified AT 3NI 4NI 0 0 3NI 1NI

EP sheep plasma APTT 3NP 8NP 0 1NP 4NP 1NP

Human plasma APTT 1NI 1NI 2NI 0 1NI 0

Thrombin Time 2NP 2NP 0 2NP 1NP 1NP

Prothrombinase

induced clotting Time

(PiCT)

1NP 0 0 0 1NP 0

Total 10 15 2 3 10 3

WHO/BS/09.2124

Table 6: Individual Assay Potency (IU/ampoule) Estimates by Anti-IIa Chromogenic

Assays with Purified AT

Lab Model Transformation Assay T V W X Y Z

02 Parallel None 1 1528 2859 2117 2138 1731 2151

02 Parallel None 2 1606 2939 2071 2112 1780 2274

02 Parallel None 3 1723 3064 2177 2170 1791 2306

03 Parallel None 1 1676 2102 2098 2146 1796 1725

03 Parallel None 2 1445 2092 1714 2169 1928 1939

03 Parallel None 3 1663 2277 2271 2169 1679 1930

03 Parallel None 4 1677 1690 2116 1598 1859 1710

05 Parallel Log 1 1849 2860 2251 2234 1950 2197

05 Parallel Log 2 1697 2613 2217 2095 1811 2171

05 Parallel Log 3 1976 2894 2542 2363 2064 2370

05 Parallel Log 4 1772 2594 2166 2175 1831 2174

06 Parallel None 1.1 1881 . . 2176 1957 .

06 Parallel None 1.2 . 3033 2337 . . 2544

06 Parallel None 2.1 1665 . . 2329 1949 .

06 Parallel None 2.2 . 2912 2234 . . 2246

06 Parallel None 3.1 1903 . . . 1940 2312

06 Parallel None 3.2 . 2932 2260 2340 . .

06 Parallel None 4.1 1720 . . . 1858 2144

06 Parallel None 4.2 . 2791 2099 2155 . .

08 Parallel None 1 1590 2954 2092 2099 1815 2181

08 Parallel None 2 1686 3046 2161 1978 2065 2248

08 Parallel None 3 1618 3054 2030 2051 1759 2122

08 Parallel None 4 1610 2861 2052 2012 1727 2073

10 Parallel None 1.1 . 2505 2210 . . 2054

10 Parallel None 1.2 1892 . . NL 1997 .

10 Parallel None 2.1 . NL 2182 . . 2021

10 Parallel None 2.2 NL . . 1964 NL .

10 Parallel None 3.1 . NL 2220 . . 2069

10 Parallel None 3.2 1887 . . 2015 1990 .

10 Parallel None 4.1 . NL NL . . NL

10 Parallel None 4.2 NL . . NL NL .

11 Parallel None 1 1739 2508 2090 2115 1819 2058

11 Parallel None 2 1894 2496 2071 2127 1805 2066

11 Parallel None 3 1706 2494 2053 2080 1768 2039

11 Parallel None 4 1724 2517 2071 2094 1776 2070

12 Parallel None 1.1 . 2724 1903 . . 2134

12 Parallel None 1.2 2078 . . 2077 1954 .

12 Parallel None 2.1 . 2821 NL . . 2247

12 Parallel None 2.2 2122 . . 2382 1956 .

12 Parallel None 3.1 . 2707 2096 2192 . .

12 Parallel None 3.2 2072 . . . 1971 NL

12 Parallel None 4.1 . 2764 2155 2274 . .

12 Parallel None 4.2 2136 . . . 2036 2256

13 Parallel None 1 1732 2457 2281 2124 2079 2136

13 Parallel None 2 2060 2837 2228 2261 1826 2108

13 Parallel None 3 2057 2931 2372 2417 2071 2384

13 Parallel None 4 1924 3138 2303 2488 1901 2391

15 Parallel None 1 1924 2209 2263 2264 2002 2126

15 Parallel None 2 1961 2215 2328 2412 2146 2183

15 Parallel None 3 1945 2177 2211 2242 2019 2180

16 Parallel None 1 1636 3618 2637 2320 2148 2593

16 Parallel None 2 1963 3957 2502 2347 1800 2505

WHO/BS/09.2124


16 Parallel None 3 1884 3663 2266 2205 1971 2284

16 Parallel None 4 1647 3448 2164 2186 1801 2425

18 Parallel None 1.1 1520 . . . . .

18 Parallel None 1.2 . 2300 . . . .

18 Parallel None 1.3 . . 1993 . . .

18 Parallel None 1.4 . . . 1954 . .

18 Parallel None 1.5 . . . . 1840 .

18 Parallel None 1.6 . . . . . 2114

18 Parallel None 2.1 1658 . . . . .

18 Parallel None 2.2 . 2401 . . . .

18 Parallel None 2.3 . . 2116 . . .

18 Parallel None 2.4 . . . 2130 . .

18 Parallel None 2.5 . . . . 1879 .

18 Parallel None 2.6 . . . . . 2082

18 Parallel None 3.1 1645 . . . . .

18 Parallel None 3.2 . 2422 . . . .

18 Parallel None 3.3 . . 2108 . . .

18 Parallel None 3.4 . . . 2155 . .

18 Parallel None 3.5 . . . . 1828 .

18 Parallel None 3.6 . . . . . 2091

18 Parallel None 4.1 1633 . . . . .

18 Parallel None 4.2 . 2374 . . . .

18 Parallel None 4.3 . . 2120 . . .

18 Parallel None 4.4 . . . 2104 . .

18 Parallel None 4.5 . . . . 1835 .

18 Parallel None 4.6 . . . . . 2014

19 Parallel None 1 NL NL NL NL NL NL

19 Parallel None 2 1602 3916 2571 NP 2105 2335

19 Parallel None 3 NL 2729 2911 1226 1563 2060

19 Parallel None 4 NL NL NL NL NL NL

24 Parallel None 1.1 NL . . . . .

24 Parallel None 1.2 . NL . . . .

24 Parallel None 1.3 . . NL . . .

24 Parallel None 1.4 . . . 2297 . .

24 Parallel None 1.5 . . . . 2076 .

24 Parallel None 1.6 . . . . . 2182




24 Parallel None 2.4 . . . 2314 . .

24 Parallel None 2.5 . . . . 2040 .

24 Parallel None 2.6 . . . . . 2193




24 Parallel None 3.4 . . . 2280 . .

24 Parallel None 3.5 . . . . 2079 .

24 Parallel None 3.6 . . . . . 2175




24 Parallel None 4.4 . . . 2318 . .

24 Parallel None 4.5 . . . . 2058 .

24 Parallel None 4.6 . . . . . 2178

25 Parallel None 1.1 1562 2551 1982 . . .

25 Parallel None 1.2 . . . 2069 1846 2266

WHO/BS/09.2124


25 Parallel None 2.1 1682 2574 2023 . . .

25 Parallel None 2.2 . . . 2142 1732 1978

25 Parallel None 3.1 1710 2651 2011 . . .

25 Parallel None 3.2 . . . 2056 1839 2148

25 Parallel None 4.1 1764 2601 1937 . . .

25 Parallel None 4.2 . . . 1995 1654 1890

27 Parallel None 1 1704 2715 2132 2255 1897 2104

27 Parallel None 2 1812 2355 2180 2065 1980 1968

27 Parallel None 3 1793 2261 2124 2116 1926 1857

27 Parallel None 4 1725 NL 2124 2064 1895 1954

32 Parallel None 1 1624 2233 2055 2139 1951 2167

32 Parallel None 2 1810 2401 2143 2182 2020 2174

32 Parallel None 3 1740 2659 2102 2103 1896 2238

32 Parallel None 4 1743 2581 2117 2096 1910 2300

35 Parallel None 1 1674 2841 2135 2160 1864 2173

35 Parallel None 2 1622 2955 2140 2145 1778 2154

35 Parallel None 3 1847 3038 2132 2121 1986 2353

35 Parallel None 4 1762 2899 1963 2265 1951 2189

Deviations from model

p<0.01, not included in analysis NL = Non-linear

0.01<p<0.05, included in analysis

Table 7: Individual Assay Potency (IU/ampoule) Estimates by Anti-Xa Chromogenic

Assays with Purified AT


02 Slope Log 1 1639 2735 2176 2172 1741 2103

02 Slope Log 2 1785 2709 2165 2065 1792 2085

02 Slope Log 3 1708 2651 2163 2079 1718 2200

03 Slope Log 1 NL NL NL NL NL NL


03 Slope Log 3 1604 NL 2026 2068 1681 1917

03 Slope Log 4 1596 NL 1944 1710 NL NL

05 Slope Log 1 1696 3153 2088 2099 1714 2166

05 Slope Log 2 1611 3097 2169 2098 1701 2238

05 Slope Log 3 1626 3144 2111 1941 1721 2206

05 Slope Log 4 1642 3146 2086 2047 1682 2170

06 Slope Log 1.1 1749 . . 2280 1886 .

06 Slope Log 1.2 . 2782 2255 . . 2236

06 Slope Log 2.1 1807 . . . . .

06 Slope Log 2.2 . . . 2229 1869 .

06 Slope Log 2.3 . 2586 2078 . . 2148

06 Slope Log 3.1 1884 . . . 1905 2389

06 Slope Log 3.2 . 2769 . . . .

06 Slope Log 3.3 . . 2234 2334 . .

06 Slope Log 4.1 1982 . . . 2003 2191

06 Slope Log 4.2 . 2538 2229 2155 . .

07 Slope Log 1.1 1565 . . . . .

07 Slope Log 1.2 . NI . . . .

07 Slope Log 1.3 . . 2144 . . .

07 Slope Log 1.4 . . . 1944 . .

WHO/BS/09.2124


07 Slope Log 1.5 . . . . 1711 .

07 Slope Log 1.6 . . . . . 2141

07 Slope Log 2.1 1782 . . . . .

07 Slope Log 2.2 . 2211 . . . .

07 Slope Log 2.3 . . 2095 . . .

07 Slope Log 2.4 . . . 2324 . .

07 Slope Log 2.5 . . . . 1746 .

07 Slope Log 2.6 . . . . . 2256

07 Slope Log 3.1 1817 . . . . .

07 Slope Log 3.2 . 2409 . . . .

07 Slope Log 3.3 . . 2043 . . .

07 Slope Log 3.4 . . . 2282 . .

07 Slope Log 3.5 . . . . 1760 .

07 Slope Log 3.6 . . . . . 2300

07 Slope Log 4.1 1692 . . . . .

07 Slope Log 4.2 . 2497 . . . .

07 Slope Log 4.3 . . 2212 . . .

07 Slope Log 4.4 . . . 2060 . .

07 Slope Log 4.5 . . . . 1801 .

07 Slope Log 4.6 . . . . . 2075

08a Slope Log 1 1802 2313 2238 2370 1879 2113

08a Slope Log 2 1789 2341 2105 2190 1870 2095

08a Slope Log 3 1711 2282 2155 2147 1792 2057

08a Slope Log 4 1748 2523 2141 2050 1842 2073

08b Slope Log 1 1830 1804 1983 2094 NL 2027

08b Slope Log 2 1785 2266 2115 2083 1831 1953

08b Slope Log 3.1 1859 . 2029 2140 2012 2065

08b Slope Log 3.2 . 1776 . . . .

08b Slope Log 4 1901 1908 2190 2190 1990 2086

10 Parallel None 1.1 . 2626 2308 . . 2153

10 Parallel None 1.2 NL . . NL 1974 .

10 Parallel None 2.1 . 2553 2210 . . 2134

10 Parallel None 2.2 1869 . . 2034 1964 .

10 Parallel None 3.1 . 2582 2253 . . 2135

10 Parallel None 3.2 1855 . . NL 1992 .

10 Parallel None 4.1 . NL NL . . NL

10 Parallel None 4.2 NL . . NL NL .

11 Slope Log 1 1759 2617 2144 2126 1796 2141

11 Slope Log 2 1836 2680 2113 2109 1766 2098

11 Slope Log 3 1692 2659 2099 2089 1732 2089

11 Slope Log 4 1738 2787 2160 2133 1797 2161

12 Slope Log 1.1 . 2413 2106 . . 2103

12 Slope Log 1.2 2171 . . 2292 2016 .

12 Slope Log 2.1 . 2337 2083 . . 2061

12 Slope Log 2.2 2137 . . 2293 2018 .

12 Slope Log 3.1 . 2488 2153 2311 . .

12 Slope Log 3.2 2161 . . . 2039 2033

12 Slope Log 4.1 . 2449 2191 2269 . .

12 Slope Log 4.2 2114 . . . 2012 2144

13 Slope Log 1 1843 2359 2229 2236 2121 2159

13 Slope Log 2 2070 2217 2011 2087 1834 2035

13 Slope Log 3 1883 2532 2399 2441 2070 2342

13 Slope Log 4 1744 2623 2356 2384 2059 2362

15 Slope Log 1 1987 NI 2276 2242 2023 2152

15 Slope Log 2 2043 2206 2299 2409 2085 2149

15 Slope Log 3 2010 2202 2346 2388 2114 2211

WHO/BS/09.2124


15 Slope Log 4 2168 NI 2416 2438 2180 2330

16 Slope Log 1 1793 1746 2046 2073 2000 1912

16 Slope Log 2 2390 2632 2887 2597 2318 2099

16 Slope Log 3 1936 1995 1940 2297 1978 1856

16 Slope Log 4 1913 1779 1922 2106 1864 1846

18 Slope Log 1.1 1857 . . . . .

18 Slope Log 1.2 . 2094 . . . .

18 Slope Log 1.3 . . 2077 . . .

18 Slope Log 1.4 . . . 2152 . .

18 Slope Log 1.5 . . . . 2042 .

18 Slope Log 1.6 . . . . . 2082

18 Slope Log 2.1 1774 . . . . .

18 Slope Log 2.2 . 2021 . . . .

18 Slope Log 2.3 . . 2102 . . .

18 Slope Log 2.4 . . . 2126 . .

18 Slope Log 2.5 . . . . 1886 .

18 Slope Log 2.6 . . . . . 2101

18 Slope Log 3.1 NI . . . . .

18 Slope Log 3.2 . 1856 . . . .

18 Slope Log 3.3 . . 2057 . . .

18 Slope Log 3.4 . . . 2123 . .

18 Slope Log 3.5 . . . . 1843 .

18 Slope Log 3.6 . . . . . 2026

18 Slope Log 4.1 1783 . . . . .

18 Slope Log 4.2 . 2042 . . . .

18 Slope Log 4.3 . . NL . . .

18 Slope Log 4.4 . . . 2142 . .

18 Slope Log 4.5 . . . . 1840 .

18 Slope Log 4.6 . . . . . 2003

19 Slope Log 1 1906 2858 2367 2483 1984 2301

19 Slope Log 2 1838 2756 2182 2133 2250 2139

19 Slope Log 3 1823 2329 2109 2193 1829 2094

19 Slope Log 4 1597 2358 2216 2111 1781 2026

22 Slope Log 1 1782 1883 2030 2163 1922 NL

22 Slope Log 2 1793 1834 2005 2164 1903 1924

22 Slope Log 3 NL 1866 2043 2168 1925 1946

22 Slope Log 4 1798 1856 2008 2142 1899 1900

23 Slope Log 1 1631 3018 2180 2105 NI 2185

23 Slope Log 2 1599 2836 2136 2067 1697 2158

23 Slope Log 3 1742 2991 2208 2112 1807 2181

23 Slope Log 4 1607 2913 2138 2061 NI 2101

25 Slope Log 1.1 1683 . . . . .

25 Slope Log 1.2 . 2597 . . . .

25 Slope Log 1.3 . . 2187 . . .

25 Slope Log 1.4 . . . 2069 . .

25 Slope Log 1.5 . . . . 1793 .

25 Slope Log 1.6 . . . . . 1962

25 Slope Log 2.1 1708 . . . . .

25 Slope Log 2.2 . 2415 . . . .

25 Slope Log 2.3 . . 1867 . . .

25 Slope Log 2.4 . . . 2180 . .

25 Slope Log 2.5 . . . . 1890 .

25 Slope Log 2.6 . . . . . 2114

25 Slope Log 3.1 1707 . . . . .

25 Slope Log 3.2 . 2524 . . . .

25 Slope Log 3.3 . . 2091 . . .

WHO/BS/09.2124


25 Slope Log 3.4 . . . 1934 . .

25 Slope Log 3.5 . . . . 1811 .

25 Slope Log 3.6 . . . . . 2142

25 Slope Log 4.1 1734 . . . . .

25 Slope Log 4.2 . 2429 . . . .

25 Slope Log 4.3 . . 2186 . . .

25 Slope Log 4.4 . . . 2093 . .

25 Slope Log 4.5 . . . . 1867 .

25 Slope Log 4.6 . . . . . 2228

25 Slope Log 1.1r 1674 . . . . .

25 Slope Log 1.2r . 2592 . . . .

25 Slope Log 1.3r . . 2145 . . .

25 Slope Log 1.4r . . . 1915 . .

25 Slope Log 1.5r . . . . 1784 .

25 Slope Log 1.6r . . . . . 2199

25 Slope Log 2.1r 1709 . . . . .

25 Slope Log 2.2r . 2470 . . . .

25 Slope Log 2.3r . . 2270 . . .

25 Slope Log 2.4r . . . 2194 . .

25 Slope Log 2.5r . . . . 2261 .

25 Slope Log 2.6r . . . . . 2127

25 Slope Log 3.1r 1719 . . . . .

25 Slope Log 3.2r . 2178 . . . .

25 Slope Log 3.3r . . 2027 . . .

25 Slope Log 3.4r . . . 1963 . .

25 Slope Log 3.5r . . . . 1815 .

25 Slope Log 3.6r . . . . . 2121

25 Slope Log 4.1r 1855 . . . . .

25 Slope Log 4.2r . 2425 . . . .

25 Slope Log 4.3r . . 2204 . . .

25 Slope Log 4.4r . . . 1987 . .

25 Slope Log 4.5r . . . . 1919 .

25 Slope Log 4.6r . . . . . 2195

27 Slope Log 1 1898 2298 2198 2193 2029 NI

27 Slope Log 2 1757 2127 2153 2115 1943 2074

27 Slope Log 3 NL NL NL NL 1911 2038

27 Slope Log 4 1903 2193 NL 2109 NL NL

32 Slope Log 1 1755 2013 2098 2141 1908 2045

32 Slope Log 2 1812 2108 2114 2193 1934 2021

32 Slope Log 3 NL 2217 2161 2218 1952 2173

32 Slope Log 4 1891 2133 2080 2242 2034 NL

33 Slope Log 1 1798 2133 2277 2232 1950 2161

33 Slope Log 2 1711 NL 2033 2130 2011 2070

33 Slope Log 3 1623 NI 2015 2034 NL 1914

33 Slope Log 4 1693 2013 2188 2142 1810 2003

35 Slope Log 1 2000 1924 2207 2194 2055 2304

35 Slope Log 2 2021 2010 2296 2292 2265 2206

35 Slope Log 3 2116 2224 2135 2222 1921 2076

35 Slope Log 4 1854 1982 2112 2194 1826 2048

36 Parallel Sigmoid 1.1 . 1294 2362 . . 2096

36 Parallel Sigmoid 1.2 1929 . . 2149 2272 .

36 Parallel Sigmoid 2.1 . 1650 2822 2632 . .

36 Parallel Sigmoid 2.2 2080 . . . 2024 2168

36 Parallel Sigmoid 3.1 . 2067 3054 2248 . .

36 Parallel Sigmoid 3.2 1997 . . . 2366 2522

WHO/BS/09.2124

Deviations from model:


0.01<p<0.05, included in analysis NI = Significant intersection difference

WHO/BS/09.2124

Table 8: Individual Assay Potency (IU/ampoule) Estimates by the EP Pharmacopoeial

Assay


01 Parallel Log 1.1 1888 2035 . 2164 1929 .

01 Parallel Log 1.2 . . 2170 . . 2070

01 Parallel Log 2.1 1777 . . 2194 1916 .

01 Parallel Log 2.2 . 2019 2047 . . 2049

01 Parallel Log 3.1 1792 . . . 1961 2043

01 Parallel Log 3.2 . 2109 2108 2223 . .

01 Parallel Log 4.1 1840 . . . 1922 2119

01 Parallel Log 4.2 . NP 2240 2140 . .

06 Parallel Log 1.1 1858 . . . . .

06 Parallel Log 1.1r 1827 . . . . .

06 Parallel Log 1.1r 1896 . . . . .

06 Parallel Log 1.2 . 2455 . . . .

06 Parallel Log 1.2r . 2436 . . . .

06 Parallel Log 1.2r . 2524 . . . .

06 Parallel Log 1.3 . . 2146 . . .

06 Parallel Log 1.3r . . 2166 . . .

06 Parallel Log 1.3r . . 2165 . . .

06 Parallel Log 1.4 . . . 2163 . .

06 Parallel Log 1.4r . . . 2161 . .

06 Parallel Log 1.4r . . . 2243 . .

06 Parallel Log 1.5 . . . . 2077 .

06 Parallel Log 1.5r . . . . 2100 .

06 Parallel Log 1.5r . . . . 2049 .

06 Parallel Log 1.6 . . . . . 2079

06 Parallel Log 1.6r . . . . . 2041

06 Parallel Log 1.6r . . . . . 2109

06 Parallel Log 2.1 1876 . . . . .

06 Parallel Log 2.1r 1925 . . . . .

06 Parallel Log 2.1r 1893 . . . . .

06 Parallel Log 2.2 . 2573 . . . .

06 Parallel Log 2.2r . 2476 . . . .

06 Parallel Log 2.2r . 2610 . . . .

06 Parallel Log 2.3 . . 2202 . . .

06 Parallel Log 2.3r . . 2257 . . .

06 Parallel Log 2.3r . . 2234 . . .

06 Parallel Log 2.4 . . . 2093 . .

06 Parallel Log 2.4r . . . 2044 . .

06 Parallel Log 2.4r . . . 2130 . .

06 Parallel Log 2.5 . . . . 1878 .

06 Parallel Log 2.5r . . . . 1904 .

06 Parallel Log 2.5r . . . . 1932 .

06 Parallel Log 2.6 . . . . . 2103

06 Parallel Log 2.6r . . . . . 2026

06 Parallel Log 2.6r . . . . . 2048

06 Parallel Log 3.1 1898 . . . . .

06 Parallel Log 3.1r 1999 . . . . .

06 Parallel Log 3.1r 1926 . . . . .

06 Parallel Log 3.2 . 2418 . . . .

06 Parallel Log 3.2r . 2540 . . . .

06 Parallel Log 3.2r . 2496 . . . .

06 Parallel Log 3.3 . . 2153 . . .

06 Parallel Log 3.3r . . 2158 . . .

06 Parallel Log 3.3r . . 2222 . . .

WHO/BS/09.2124


06 Parallel Log 3.4 . . . 2226 . .

06 Parallel Log 3.4r . . . 2174 . .

06 Parallel Log 3.4r . . . 2254 . .

06 Parallel Log 3.5 . . . . 2018 .

06 Parallel Log 3.5r . . . . 2040 .

06 Parallel Log 3.5r . . . . 2084 .

06 Parallel Log 3.6 . . . . . 1998

06 Parallel Log 3.6r . . . . . 2051

06 Parallel Log 3.6r . . . . . 2083

06 Parallel Log 4.1 1863 . . . . .

06 Parallel Log 4.1r 1890 . . . . .

06 Parallel Log 4.1r 1885 . . . . .

06 Parallel Log 4.2 . 2504 . . . .

06 Parallel Log 4.2r . 2437 . . . .

06 Parallel Log 4.2r . 2526 . . . .

06 Parallel Log 4.3 . . 2229 . . .

06 Parallel Log 4.3r . . 2219 . . .

06 Parallel Log 4.3r . . 2225 . . .

06 Parallel Log 4.4 . . . 2251 . .

06 Parallel Log 4.4r . . . 2159 . .

06 Parallel Log 4.4r . . . 2191 . .

06 Parallel Log 4.5 . . . . 1995 .

06 Parallel Log 4.5r . . . . 2040 .

06 Parallel Log 4.5r . . . . 2024 .

06 Parallel Log 4.6 . . . . . 2057

06 Parallel Log 4.6r . . . . . 2070

06 Parallel Log 4.6r . . . . . 2040

07 Parallel Log 1.1 1931 . . . . .

07 Parallel Log 1.2 . 1869 . . . .

07 Parallel Log 1.3 . . 2195 . . .

07 Parallel Log 1.4 . . . 2201 . .

07 Parallel Log 1.5 . . . . 1968 .

07 Parallel Log 1.6 . . . . . 2042

07 Parallel Log 2.1 1857 . . . . .

07 Parallel Log 2.2 . 1805 . . . .

07 Parallel Log 2.3 . . 2124 . . .

07 Parallel Log 2.4 . . . 2169 . .

07 Parallel Log 2.5 . . . . 1957 .

07 Parallel Log 2.6 . . . . . 2009

07 Parallel Log 3.1 1881 . . . . .

07 Parallel Log 3.2 . 1787 . . . .

07 Parallel Log 3.3 . . 2179 . . .

07 Parallel Log 3.4 . . . 2171 . .

07 Parallel Log 3.5 . . . . 1936 .

07 Parallel Log 3.6 . . . . . 2029

07 Parallel Log 4.1 1867 . . . . .

07 Parallel Log 4.2 . 1817 . . . .

07 Parallel Log 4.3 . . 2211 . . .

07 Parallel Log 4.4 . . . 2185 . .

07 Parallel Log 4.5 . . . . 1957 .

07 Parallel Log 4.6 . . . . . 2038

08 Parallel Log 1.1 . NL 2114 . . NL

08 Parallel Log 1.2 1802 . . 2124 1858 .

08 Parallel Log 2.1 . 1897 2084 . . 1937

08 Parallel Log 2.2 1849 . . 2223 1903 .

08 Parallel Log 3.1 . 1787 2139 2103 . .

08 Parallel Log 3.2 1693 . . . 1724 1931

WHO/BS/09.2124


08 Parallel Log 4.1 . 1793 2111 2094 . .

08 Parallel Log 4.2 1872 . . . 1942 2102

09 Parallel Log 1.1 NP . . 2109 . .

09 Parallel Log 1.2 . . 2122 . 2054 .

09 Parallel Log 1.3 . NP . . . 2092

09 Parallel Log 2.1 1918 NP . . . .

09 Parallel Log 2.2 . . 2091 2104 . .

09 Parallel Log 2.3 . . . . NP NP

09 Parallel Log 3.1 1913 . 2125 . . .

09 Parallel Log 3.2 . . . 2111 NP .

09 Parallel Log 3.3 . NP . . . 2059

09 Parallel Log 4.1 NP NP . . . .

09 Parallel Log 4.2 . . . 2168 2076 .

09 Parallel Log 4.3 . . 2144 . . 2092

13 Parallel Log 1 1823 NP 2213 2171 1960 2024

13 Parallel Log 2 1923 2488 2121 2140 2077 2177

13 Parallel Log 3 2079 2526 2176 2105 2095 2119

13 Parallel Log 4 1919 2564 2135 2149 1986 2103

17 Parallel Log 1 1802 2038 2086 2106 1892 1998

17 Parallel Log 2 1772 2000 2100 2105 1825 1998

17 Parallel Log 3 1730 2042 2122 2115 1830 2002

17 Parallel Log 4 1747 2040 2108 2100 1838 2012

18 Parallel Log 1.1 1707 . . . . .

18 Parallel Log 1.2 . 1954 . 2120 1870 .

18 Parallel Log 1.3 . . 2080 . . 1949

18 Parallel Log 2.1 1991 . . . . .

18 Parallel Log 2.2 . 2023 2075 . . 1968

18 Parallel Log 2.3 . . . 2108 1819 .

18 Parallel Log 3.1 1604 . . . . .

18 Parallel Log 3.2 . 1897 2022 . . 1903

18 Parallel Log 3.3 . . . 2111 1813 .

18 Parallel Log 4.1 1703 . . 2111 1821 .

18 Parallel Log 4.2 . NP 2055 . . 1930

21 Parallel Log 1.1 1753 . . 2237 1963 .

21 Parallel Log 1.2 . 1752 2227 . . 2006

21 Parallel Log 2.1 1890 . . 2338 2073 .

21 Parallel Log 2.2 . NP 1993 . . 1878

21 Parallel Log 3.1 1841 . . . 2002 2172

21 Parallel Log 3.2 . 1743 2110 2189 . .

21 Parallel Log 4.1 1999 . . 2253 2085 .

21 Parallel Log 4.2 . 1812 2115 . . 1883

25 Parallel Log 1.1 1825 . . NL NP .

25 Parallel Log 1.2 . NP 2061 . . 1956

25 Parallel Log 2.1 1780 . . 2131 1910 .

25 Parallel Log 2.2 . 1766 2107 . . NL

25 Parallel Log 3.1 NL . . . NL NL

25 Parallel Log 3.2 . NL NL NL . .

25 Parallel Log 4.1 NL . . . NL NL

25 Parallel Log 4.2 . 1751 2087 2024 . .

27 Parallel Log 1 NL 1916 2268 2298 2021 2118

27 Parallel Log 2 NL NL NL NL NL NL

27 Parallel Log 3 1873 1834 2231 NP 1989 2053

27 Parallel Log 4 2014 1996 NL 2295 2140 2184

31 Parallel Log 1 1825 1844 2086 2059 1863 1985

31 Parallel Log 2 NP 1852 2106 2160 1976 2171

WHO/BS/09.2124


31 Parallel Log 3 1807 1911 2121 2121 NP 2078

31 Parallel Log 4 1873 1933 2227 2190 1944 2142



0.01<p<0.05, included in analysis NP = Non-parallel

WHO/BS/09.2124

Table 9: Individual Assay Potency Estimates (IU/ampoule) by the USP Pharmacopoeial

Assay. Laboratory’s own potency estimates were used for overall calculation of potency

estimates of the candidates.

Lab Assay T V W X Y Z

06 1 1970 1875 2136 2183 2000 2041

06 2 1967 1779 2136 2136 2000 2017

06 3 1956 1898 2150 2102 2065 2000

06 4 1946 1826 2131 2165 1993 2022

10 1 1976 2088 2031 1976 2120 2031

10 2 1976 2088 2031 2031 2088 2003

10 3 2031 2090 2031 2031 2122 2031

10 4 2031 2088 2088 2031 2088 2031

12 1 1918 1792 2240 2240 2097 2109

12 2 1921 1737 2110 2136 2034 2034

12 3 1903 1758 2198 2185 2070 2070

12 4 1977 1722 2158 2158 2020 2065

18 1 1931 1801 2203 2229 2031 2040

18 2 1895 1789 2153 2265 2031 1999

18 3 1823 1685 2183 2183 2031 2132

18 4 1823 1681 2183 2183 2008 1949

19 1 1923 1742 2250 2261 1965 1965

19 2 1780 1664 2149 2050 2010 1990

19 3 1700 1910 2250 1953 1634 2084

19 4 2044 1783 2356 2285 2085 2084

24 1 1986 1830 2249 2237 2063 2070

24 2 2000 1804 2236 2240 2024 2029

24 3 1981 1804 2244 2232 2006 2030

24 4 2030 1837 2261 2241 2026 2016

25 1 1842 1837 2077 2077 1939 1979

25 2 1871 1754 2135 2234 2019 2060

25 3 1901 1820 2170 2077 2005 2003

25 4 1821 1708 2172 2123 1878 1917

26 1 2000 1905 2210 2210 2000 2000

26 2 2000 1952 2210 2210 2099 2048

26 3 2024 1905 2210 2210 2074 2074

26 4 2000 1905 2210 2100 2000 2050

28 1 1939 1777 2168 2036 2064 2064

28 2 1939 1777 2236 2168 2129 2064

28 3 1939 1777 2202 2170 2097 2131

28 4 1939 1833 2271 2263 2097 2086

WHO/BS/09.2124

Table 10: Individual Assay Potency Estimates (IU/ampoule) by APTT. Results from Lab

34 were not analysed by NIBSC and the laboratory’s own potency estimates were used for

calculation of overall potency estimates of the candidates.


03 Slope Log 1 1633 2116 2055 2029 1711 1873

03 Slope Log 2 1417 2204 2101 2068 1716 1779


03 Slope Log 4 1638 2077 2035 1852 NL 1693

05 Slope Log 1 1802 1702 2062 2104 1934 1908

05 Slope Log 2 1778 1696 2050 2181 1881 1917

05 Slope Log 3 1888 1727 2091 2256 1994 NL

05 Slope Log 4 1934 1711 2127 2217 2022 2033

08 Slope Log 1 1786 1869 2185 2138 1874 1994

08 Slope Log 2 1791 1852 2239 2284 2011 2107

08 Slope Log 3 NI 1919 2102 2215 NI 1995


10 Parallel None 1.1 1920 . . . . .

10 Parallel None 1.2 . 2428 . . . .

10 Parallel None 1.3 . . 2235 . . .

10 Parallel None 1.4 . . . 1964 . .

10 Parallel None 1.5 . . . . 1943 .

10 Parallel None 1.6 . . . . . 1955

10 Parallel None 2.1 1837 . . . . .

10 Parallel None 2.2 . 2399 . . . .

10 Parallel None 2.3 . . 2253 . . .

10 Parallel None 2.4 . . . 1925 . .

10 Parallel None 2.5 . . . . 1927 .

10 Parallel None 2.6 . . . . . 1993

10 Parallel None 3.1 1859 . . . . .

10 Parallel None 3.2 . 2389 . . . .

10 Parallel None 3.3 . . 2161 . . .

10 Parallel None 3.4 . . . 1959 . .

10 Parallel None 3.5 . . . . 1922 .

10 Parallel None 3.6 . . . . . 1932

10 Parallel None 4.1 1867 . . . . .

10 Parallel None 4.2 . 2403 . . . .

10 Parallel None 4.3 . . 2210 . . .

10 Parallel None 4.4 . . . 1924 . .

10 Parallel None 4.5 . . . . 1950 .

10 Parallel None 4.6 . . . . . 2036

13 Slope Log 1 1785 NI 2199 2188 1938 2019

13 Slope Log 2 1766 1926 2178 2194 2048 2166

13 Slope Log 3 1903 2002 2263 2296 2036 2103

13 Slope Log 4 1815 2019 2220 2234 1931 2093

16 Slope Log 1 1772 1823 2245 2360 2064 2050

16 Slope Log 2 1950 1891 2200 2225 1929 2060

16 Slope Log 3 1968 1975 2265 2356 2016 2129

16 Slope Log 4 1847 1935 2193 2293 1985 1946

22 Slope Log 1 1781 NL 2091 2140 1904 1924

22 Slope Log 2 1799 1799 2011 2257 1932 1925

22 Slope Log 3 1833 1837 2138 2192 1977 1993

22 Slope Log 4 1744 1747 1992 2108 1832 1927

28 Slope Log 1.1 . . NL . . .

28 Slope Log 1.2 . . . . 1950 2032

28 Slope Log 1.3 1751 1933 . . . .

WHO/BS/09.2124


28 Slope Log 1.4 . . . 2106 . .

28 Slope Log 2.1 1785 1868 . . . .

28 Slope Log 2.2 . . 2107 2177 . .

28 Slope Log 2.3 . . . . 1914 1993

28 Slope Log 3.1 1833 1904 . . . .

28 Slope Log 3.2 . . NI 2213 . .

28 Slope Log 3.3 . . . . 1956 2027

28 Slope Log 4.1 1850 1916 . . . .

28 Slope Log 4.2 . . 2103 2224 . .

28 Slope Log 4.3 . . . . 1962 2036

29 Slope Log 1.1 1879 1849 2064 . . .

29 Slope Log 1.2 1958 . . 2020 . 1877

29 Slope Log 1.3 . . NL . 1865 1783

29 Slope Log 1.4 . 1635 . 2109 1866 .

29 Slope Log 1.5 . . . 2010 1910 1882

29 Slope Log 1.6 . 1718 2004 . 1803 .

29 Slope Log 1.7 1800 1710 . 1864 . .

29 Slope Log 1.8 1844 . 2167 . . 1886

29 Slope Log 2.1 1873 1588 2156 . . .

29 Slope Log 2.2 1949 . . 2130 . 1908

29 Slope Log 2.3 . . NI . 2010 2178

29 Slope Log 2.4 . 1603 . 2065 1847 .

29 Slope Log 2.5 . . . 2007 1884 1885

29 Slope Log 2.6 . 1529 2157 . 1976 .

29 Slope Log 2.7 1867 1621 . 2032 . .

29 Slope Log 2.8 1847 . 2057 . . 1970

29 Slope Log 3.1 1786 1917 2044 . . .

29 Slope Log 3.2 1805 . . 1908 . 1941

29 Slope Log 3.3 . . 2136 . 1786 1938

29 Slope Log 3.4 . 1751 . 1876 1738 .

29 Slope Log 3.5 . . . 2179 1911 2097

29 Slope Log 3.6 . 1918 2056 . 1860 .

29 Slope Log 3.7 NL 1809 . 1993 . .

29 Slope Log 3.8 1720 . 1996 . . 2038

29 Slope Log 4.1 1939 2052 2220 . . .

29 Slope Log 4.2 2013 . . 2404 . 2218

29 Slope Log 4.3 . . 2334 . 2145 1972

29 Slope Log 4.4 . 2089 . 2393 2214 .

29 Slope Log 4.5 . . . 2278 2043 1907

29 Slope Log 4.6 . 2041 2311 . 2090 .

29 Slope Log 4.7 2067 2076 . 2376 . .

29 Slope Log 4.8 1994 . 2273 . . 2035

34 As reported None 1 1782 2043 2160 2244 1852 2017

34 As reported None 2 1710 2445 2234 2253 1758 2041

34 As reported None 3 1755 2283 2179 2240 1832 2186

34 As reported None 4 1705 2119 2174 2139 1799 2107

34 As reported None 1 1896 1817 2186 2260 1965 2068

34 As reported None 2 1829 2047 2265 2299 1864 2012

34 As reported None 3 1850 1939 2197 2283 2002 2111

34 As reported None 4 1826 1840 2180 2192 1935 2049

36 Slope Log 1.1 . 2194 2181 . . 1872

36 Slope Log 1.2 1809 . . 2003 1888 .

36 Slope Log 2.1 . 1563 2048 . . 2061

36 Slope Log 2.2 1881 . . 2185 1884 .

36 Slope Log 3.1 . 1625 2062 2491 . .

36 Slope Log 3.2 1877 . . . 1993 1893

WHO/BS/09.2124


36 Slope Log 4.1 . 1141 2001 1671 . .

36 Slope Log 4.2 1832 . . . 2304 2329



0.01<p<0.05, included in analysis NI = Significant intersection difference

WHO/BS/09.2124

Table 11: Individual Assay Potency Estimates (IU/ampoule) by Anti-Xa chromogenic assay

with plasma (A/Xa P), Anti-IIa chromogenic assay with plasma (A/IIa P), Thrombin Time

(TT), Chinese Pharmacopoeial Assay (CP), Japanese Pharmacopoeial Assay (JP) and

Prothrombinase induced Clotting time (PiCT). Results from Lab 18 (JP assays), Lab 30

(TT) and Lab 34(A/Xa P) could not be analysed by NIBSC and the laboratory’s own

potency estimates were used for calculation of overall potency estimates of the candidates

Lab Method Model Transformation Assay T V W X Y Z

03 A/Xa P Slope Log 1 NL NL 1952 1960 NL NL

03 Slope Log 2 1596 2462 NL NL NL NL

03 Slope Log 3 1697 2314 2054 2123 1829 1879

03 Slope Log 4 1693 2226 2127 1683 1694 1848

03 A/IIa P Parallel None 1 1825 2470 2072 2587 2412 2330

03 Parallel None 2 1535 2493 1962 2514 2021 2481

03 Parallel None 3 2130 2139 2034 1859 1670 1882

03 Parallel None 4 1721 2186 2300 1900 2118 1740

03 TT Parallel Log 1 1792 1539 2036 NP 1746 1954

03 Parallel Log 2 NP NP 2142 NP NP 1969

03 Parallel Log 3 NP NP 2013 NP 1835 NP

03 Parallel Log 4 1834 1663 2159 1657 1883 1869

06 CP Parallel Log 1.1 1789 . . . . .

06 Parallel Log 1.2 . 2392 . . . .

06 Parallel Log 1.3 . . 2016 . . .

06 Parallel Log 1.4 . . . 2009 . .

06 Parallel Log 1.5 . . . . 2011 .

06 Parallel Log 1.6 . . . . . 2021

06 Parallel Log 2.1 1884 . . . . .

06 Parallel Log 2.2 . 2412 . . . .

06 Parallel Log 2.3 . . 2129 . . .

06 Parallel Log 2.4 . . . 1995 . .

06 Parallel Log 2.5 . . . . 1960 .

06 Parallel Log 2.6 . . . . . 1909

06 Parallel Log 3.1 1729 . . . . .

06 Parallel Log 3.2 . 2361 . . . .

06 Parallel Log 3.3 . . 2068 . . .

06 Parallel Log 3.4 . . . 2018 . .

06 Parallel Log 3.5 . . . . 2028 .

06 Parallel Log 3.6 . . . . . 1976

06 Parallel Log 4.1 1924 . . . . .

06 Parallel Log 4.2 . 2381 . . . .

06 Parallel Log 4.3 . . 2084 . . .

06 Parallel Log 4.4 . . . 2073 . .

WHO/BS/09.2124

Lab Method Model Transformation Assay T V W X Y Z

06 Parallel Log 4.5 . . . . 1934 .

06 Parallel Log 4.6 . . . . . 2077

18 JP As reported None 1 2835 1980 3011 2971 2937 3036

18 As reported None 2 2658 1887 2848 2835 2823 2851

18 As reported None 3 2495 1750 2797 2784 2725 2916

18 As reported None 4 2583 1772 2759 2721 2739 2797

22 PiCT Parallel None 1 NP 1839 2223 2231 NP 2079

22 Parallel None 2 1763 1813 2106 2009 1775 2003

22 Parallel None 3 1900 1776 2111 2154 1901 2089

22 Parallel None 4 1706 1749 2036 2097 1827 1998

30 A/Xa P © As reported None 1 2148 2029 2177 2858 2095 2422

30 As reported None 2 1577 1811 1886 2229 1777 2013

30 As reported None 3 1836 1990 2414 2084 1974 2803

30 As reported None 4 2039 1721 2018 2259 2081 1962

34 TT As reported None 1 1671 1674 1960 2164 1751 1910

34 As reported None 2 1757 1932 2190 2230 1751 1910

34 As reported None 3 1823 1916 2310 2424 1958 2145

34 As reported None 4 1740 1806 2163 2291 1823 1990

34 A/Xa P CL Slope Log 1 2071 2201 2209 2257 1930 NL

34 Slope Log 2 1937 2135 2176 2105 1803 2105

34 Slope Log 3 1863 NL NL 2099 NL 2145

34 Slope Log 4 1918 2098 1972 1865 1892 1994

34 A/Xa P CH Slope None 1 1904 NL 2126 2200 1992 2101

34 Slope None 2 1961 1712 2172 2239 1978 2028

34 Slope None 3 NI 1747 2185 2217 NI 2158

34 Slope None 4 NI 1809 2162 2217 2026 NI



0.01<p<0.05, included in analysis NP = Non-parallel; NI = Significant

intersection difference

A/IIa P: Anti-IIa chromogenic assay with plasma

A/Xa P CH: Anti-Xa chromogenic assay with plasma

A/Xa P:CL: Anti-Xa clotting assay with plasma

TT: Thrombin Time

CP: Chinese Pharmacopoeial Assay

JP: Japanese Pharmacopoeial Assay

PiCT: Prothrombinase induced Clotting Time

WHO/BS/09.2124

Table 12: Laboratory Mean Potency estimates, Intra- and Inter-Laboratory Variation,

expressed as % Geometric Coefficient of Variation (%GCV) for Anti-IIa Chromogenic

Assays with AT

T V W X Y Z Lab GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

02 1617 6.2 2953 3.5 2121 2.5 2140 1.4 1767 1.8 2243 3.7

03 1612 7.6 2028 13.6 2039 12.9 2004 16.3 1813 6.1 1823 7.1

05 1821 6.7 2737 6.0 2290 7.4 2215 5.2 1911 6.3 2227 4.3

06 1789 6.8 2916 3.5 2231 4.6 2248 4.5 1926 2.4 2307 7.5

08 1626 2.6 2978 3.1 2083 2.8 2035 2.6 1837 8.4 2155 3.6

10 1889 0.2 2505 - 2204 0.9 1989 1.8 1993 0.2 2048 1.2

11 1764 4.9 2504 0.4 2071 0.7 2104 1.0 1792 1.3 2058 0.7

12 2102 1.5 2754 1.8 2048 6.7 2228 6.0 1979 2.0 2212 3.1

13 1938 8.5 2830 10.9 2295 2.6 2318 7.3 1966 6.6 2251 7.1

15 1943 1.0 2200 0.9 2267 2.6 2305 4.0 2055 3.9 2163 1.5

16 1777 9.7 3667 5.9 2385 9.4 2263 3.6 1925 8.8 2449 5.5

18 1613 4.1 2374 2.3 2084 3.0 2084 4.5 1845 1.2 2075 2.1

19 1602 - 3269 29.1 2736 9.2 1226 - 1814 23.4 2193 9.3

24 . - - - - - 2302 0.8 2063 0.9 2182 0.4

25 1678 5.3 2594 1.7 1988 2.0 2065 3.0 1766 5.4 2065 8.5

27 1758 3.0 2436 10.1 2140 1.3 2124 4.3 1924 2.1 1969 5.3

32 1728 4.6 2463 8.1 2104 1.8 2130 1.9 1944 2.9 2219 2.8

35 1724 5.9 2932 2.9 2091 4.3 2172 2.9 1893 5.1 2216 4.1

Overall

GM 1758 2687 2150 2158 1899 2154

Lower

95% CL 1689 2495 2092 2104 1854 2085

Upper

95% CL 1831 2895 2210 2214 1945 2225

Between-

lab

%GCV

8.1 15.6 5.3 5.1 4.9 6.7

Overall GM excludes outlier

Outlier

WHO/BS/09.2124


expressed as % Geometric Coefficient of Variation (%GCV) for Anti-Xa Chromogenic

Assays with AT

T V W X Y Z

Lab GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

02 1710 4.4 2698 1.6 2168 0.3 2105 2.8 1750 2.2 2129 2.9

03 1600 0.4 - - 1985 3.0 1880 14.4 1681 - 1917 -

05 1643 2.3 3135 0.8 2113 1.8 2045 3.7 1704 1.0 2195 1.5

06 1853 5.6 2667 4.8 2198 3.8 2249 3.5 1915 3.1 2239 4.7

07 1711 6.9 2369 6.4 2123 3.4 2147 8.8 1754 2.1 2191 4.8

08a 1762 2.4 2363 4.6 2159 2.6 2186 6.2 1845 2.2 2084 1.2

08b 1843 2.7 1929 11.8 2078 4.5 2126 2.3 1943 5.3 2032 2.9

10 1862 0.5 2587 1.4 2257 2.2 2034 - 1977 0.7 2141 0.5

11 1755 3.5 2685 2.7 2129 1.3 2114 0.9 1773 1.8 2122 1.6

12 2146 1.2 2421 2.7 2133 2.3 2291 0.8 2021 0.6 2085 2.4

13 1881 7.4 2428 7.8 2244 8.3 2283 7.3 2018 6.7 2220 7.3

15 2051 4.0 2204 0.1 2334 2.7 2368 3.8 2100 3.2 2209 3.9

16 1996 13.3 2010 20.8 2166 21.3 2259 10.9 2033 9.7 1926 6.1

18 1804 2.5 2001 5.4 2079 1.1 2136 0.6 1901 5.0 2053 2.3

19 1787 8.0 2565 11.1 2217 5.0 2225 7.8 1953 11.1 2138 5.5

22 1791 0.5 1860 1.1 2021 0.9 2159 0.5 1912 0.7 1923 1.2

23 1644 4.0 2939 2.8 2165 1.6 2086 1.3 1751 4.5 2156 1.8

25 1723 2.9 2450 4.3 2119 3.4 2039 5.1 1887 6.8 2135 2.6

27 1851 4.6 2205 4.0 2175 1.5 2139 2.2 1960 3.1 2056 1.2

32 1818 3.8 2116 4.1 2113 1.6 2198 2.0 1956 2.8 2079 4.0

33 1705 4.3 2072 4.2 2125 6.1 2133 3.9 1922 5.6 2035 5.3

35 1996 5.6 2032 6.5 2186 3.8 2225 2.1 2010 9.8 2156 5.6

36 2001 3.8 1640 26.4 2730 14.1 2334 11.2 2216 8.4 2255 10.4

Overall GM 1818 2308 2148 2161 1908 2105

Lower 95%

CL 1760 2151 2114 2113 1852 2063

Upper 95%

CL 1879 2476 2182 2210 1966 2148

Between-lab

% GCV 7.8 17.2 3.6 5.3 7.2 4.8

Overall GM excludes outlier Outlier

WHO/BS/09.2124


expressed as % Geometric Coefficient of Variation (%GCV) for the European

Pharmacopoeial Assay

T V W X Y Z Lab

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

01 1824 2.8 2054 2.4 2140 3.9 2180 1.7 1932 1.0 2070 1.7

06 1894 1.8 2499 1.5 2198 1.6 2173 2.7 2011 3.9 2059 0.7

07 1884 1.7 1819 1.9 2177 1.8 2181 0.7 1954 0.7 2029 0.7

08 1803 4.6 1825 3.4 2112 1.1 2135 2.8 1855 5.3 1988 4.9

09 1915 0.2 - - 2120 1.0 2123 1.4 2065 0.8 2081 0.9

13 1934 5.6 2526 1.5 2161 1.9 2141 1.3 2029 3.3 2105 3.1

17 1763 1.8 2030 1.0 2104 0.7 2106 0.3 1846 1.7 2002 0.3

18 1746 9.7 1957 3.3 2058 1.3 2112 0.2 1831 1.4 1937 1.4

21 1869 5.6 1769 2.1 2110 4.6 2254 2.8 2030 2.9 1981 7.1

25 1802 1.8 1758 0.6 2085 1.1 2077 3.7 1910 - 1956 -

27 1942 5.3 1914 4.3 2249 1.2 2296 0.1 2049 3.9 2118 3.1

31 1835 1.9 1885 2.3 2134 3.0 2132 2.7 1927 3.1 2093 4.1

Overall GM

1850 1988 2137 2158 1952 2034

Lower

95% CL 1808 1826 2104 2119 1899 1996

Upper

95% CL 1892 2164 2170 2198 2005 2073

Between-lab

% GCV

3.6 13.5 2.5 2.9 4.4 3.1

WHO/BS/09.2124


expressed as % Geometric Coefficient of Variation (%GCV) for the US Pharmacopoeial

Assay

T V W X Y Z

Lab GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

06 1960 0.6 1844 2.9 2138 0.4 2146 1.7 2014 1.7 2020 0.8

10 2003 1.6 2088 0.0 2045 1.4 2017 1.4 2104 0.9 2024 0.7

12 1930 1.7 1752 1.7 2176 2.6 2179 2.1 2055 1.7 2069 1.5

18 1867 2.9 1738 3.8 2180 1.0 2215 1.8 2025 0.6 2029 3.9

19 1857 8.5 1773 5.9 2250 3.8 2133 7.9 1915 11.5 2030 3.1

24 1999 1.1 1819 1.0 2247 0.5 2237 0.2 2030 1.2 2036 1.2

25 1859 1.9 1779 3.4 2138 2.1 2127 3.5 1959 3.4 1989 3.0

26 2006 0.6 1917 1.2 2210 0.0 2182 2.6 2043 2.5 2043 1.5

28 1939 0.0 1791 1.6 2219 2.0 2158 4.5 2097 1.3 2086 1.5

Overall

GM 1935 1831 2177 2154 2026 2036

Lower 95% CL

1887 1751 2128 2105 1980 2015

Upper 95% CL

1983 1913 2228 2204 2073 2058

Between-lab

%GCV

3.3 5.9 3.1 3.0 3.0 1.4

WHO/BS/09.2124


expressed as % Geometric Coefficient of Variation (%GCV) for the APTT

T V W X Y Z

Lab GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

03 1559 8.6 2132 3.1 2063 1.6 1981 6.1 1713 0.2 1780 5.2

05 1849 4.0 1709 0.8 2082 1.7 2189 3.0 1957 3.3 1952 3.6

08 1788 0.2 1880 1.9 2175 3.2 2212 3.4 1941 5.1 2031 3.2

10 1871 1.9 2405 0.7 2214 1.8 1943 1.1 1935 0.7 1979 2.3

13 1817 3.4 1982 2.5 2215 1.6 2228 2.2 1988 3.2 2095 2.9

16 1883 5.0 1905 2226 1.6 2308 2.8 1998 2.9 2045 3.8

22 1789 2.1 1794 2.6 2057 3.4 2174 3.0 1911 3.2 1942 1.7

28 1804 2.5 1905 1.5 2105 0.1 2180 2.5 1945 1.1 2022 1.0

29 1880 5.2 1797 11.8 2134 4.8 2096 8.5 1930 7.0 1967 4.0

34a 1738 2.1 2217 8.3 2187 1.5 2219 2.5 1810 2.3 2087 3.7

34b 1850 1.8 1909 5.6 2207 1.8 2258 2.1 1941 3.1 2060 2.0

36 1849 1.9 1588 30.7 2072 3.7 2066 18.2 2010 9.9 2031 10.6

Overall GM

1828 1924 2144 2152 1922 1997

Lower

95% CL 1798 1789 2102 2081 1867 1942

Upper

95% CL 1859 2068 2186 2225 1977 2054

Between-

lab

%GCV

2.5

12.1

3.1

5.4

4.6 4.5


Table 17: Laboratory Mean Potency estimates, Intra- Laboratory Variation, expressed as %

Geometric Coefficient of Variation (%GCV) for Anti-Xa chromogenic assay with plasma

(A/Xa P)

T V W X Y Z

Lab GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

03 1661 3.5 2332 5.2 2043 4.4 1913 12.5 1760 5.6 1863 1.2

30 1887 14.6 1883 8.1 2115 11.2 2340 14.8 1978 7.9 2276 18.3

34e 1932 2.1 1756 2.8 2161 1.2 2218 0.7 1999 1.2 2095 3.2

Overall

GM 1827 2112 2091 2103 1869 2066

Lower

95% CL 1491 1372 1964 1661 1604 1616

Upper

95% CL 2228 2845 2258 2780 2273 2654

Between-

lab

%GCV

8.5 15.9 2.9 11.0 7.3 10.6

Outlier

WHO/BS/09.2124


Geometric Coefficient of Variation (%GCV) for Thrombin Time (TT)

T V W X Y Z

Lab GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

03 1813 1.7 1600 5.6 2087 3.6 1657 - 1820 3.9 1930 2.9

34 1747 3.6 1829 6.8 2152 7.1 2275 4.9 1819 5.4 1986 5.6

Overall

GM 1780 1711 2119 2275 1820 1958



Geometric Coefficient of Variation (%GCV) for Anti-IIa chromogenic assay with plasma

(A/IIa P), Chinese Pharmacopoeial Assay (CP), Japanese Pharmacopoeial Assay (JP),

Prothrombinase induced Clotting time (PiCT) and Anti-Xa Clotting assay (A/Xa Cl.

T V W X Y Z Lab

Method

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

03 A/IIa P 1790 14.6 2316 8.3 2088 7.1 2189 19.3 2038 16.5 2086 18.4

06 CP 1830 5.0 2386 0.9 2074 2.3 2024 1.7 1983 2.2 1995 3.6

18 JP 2640 5.6 1845 5.9 2852 3.9 2826 3.8 2805 3.5 2899 3.6

22 PiCT 1788 5.7 1794 2.2 2118 3.7 2121 4.5 1834 3.5 2042 2.4

34d A/Xa

Cl 1946 4.6 2144 2.5 2116 6.4 2077 8.2 1874 3.6 2080 3.9

Overall GM exclude outlier

Table 20: Summary of Intra-Laboratory Variability by Different Assay Methods, Expressed

as the Number of GCVs Less than 5, 7 and 10%

No of GCV/total number of GCV

Intra-lab

variability

(GCV) Anti-IIa

AT

Anti-Xa

AT EP USP APTT

Anti-Xa

Plasma

Thrombi

n Time

Anti-IIa

Plasma CP PiCT

<5% 63/102 99/139 63/69 50/54 57/71 9/18 6/11 0/6 6/6 5/6

<7% 84/102 117/139 67/69 51/54 63/71 11/18 10/11 0/6 - 6/6

<10% 95/102 128/139 69/69 53/54 67/71 13/18 11/11 2/6 - -

>10% 7/102 11/139 - 1/54 4/71 5/18 - 4/6 - -

Range (% GCV)

0.2 – 29.1 0.1 – 26.4 0.1 – 9.7 0.0 – 11.5 0.1 – 30.7 1.2 – 18.3 1.7 – 7.1 8.3 – 19.3 0.9 – 5.0 2.2 – 5.7

Outlier

Outlier

WHO/BS/09.2124

Table 21: Summary of Potency Estimates Relative to the 5th International Standard for

Unfractionated Heparin and Inter-Laboratory Variation expressed as Geometric Coefficient

of Variation (% GCV) by Different Assay Methods

T V W X Y Z Method

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

Anti-IIa AT

n = 18 1758 8.1 2687 15.6 2150 5.3 2158 5.1 1899 4.9 2154 6.7

Anti-Xa AT

n = 23 1823 7.8 2280 18.0 2149 3.5 2163 5.2 1912 7.1 2105 4.7

EP

n = 12 1850 3.6 1988 13.5 2137 2.5 2158 2.9 1952 4.4 2034 3.1

USP

n = 9 1935 3.3 1831 5.9 2177 3.1 2154 3.0 2026 3.0 2036 1.4

APTT

n = 12 1828 2.5 1924 12.1 2144 3.1 2152 5.4 1922 4.6 1997 4.5

Anti-Xa P

CH

n = 3

1827 8.5 2112 15.9 2091 2.9 2103 11.0 1869 7.3 2066 10.6

Thrombin

Time

n = 2

1780 2.7 1711 9.9 2119 2.2 2275 - 1820 0.1 1958 2.1

CP

n = 1 1830 - 2386 - 2074 - 2024 - 1983 - 1995 -

PiCT

n = 1 1788 - 1794 - 2118 - 2121 - 1834 - 2042 -

JP

n = 1 - - 1845 - - - - - - - - -

A/Xa P Cl

n = 1 1946 4.6 2144 2.5 2116 6.4 2077 8.2 1874 3.6 2080 3.9

A/Xa P CH: anti-Xa plasma chromogenic assay; A/Xa P Cl: anti-Xa clotting assay

Table 22:

Overall Potency Estimates and Inter-laboratory Variation Expressed as % Geometric

Coefficient of Variation (%GCV) Relative to the 5th International Standard for

Unfractionated Heparin

ALL METHODS T V W X Y Z

Overall GM 1824 2172 2145 2156 1926 2074

Lower 95% CL 1799 2084 2128 2134 1903 2050

Upper 95% CL 1850 2265 2161 2178 1949 2099

Between-lab %GCV 6.6 20.7 3.6 4.8 5.7 5.5

WHO/BS/09.2124

Table 23: Overall Potency Estimates and Inter-laboratory Variation Expressed as %

Geometric Coefficient of Variation (%GCV) Relative to Candidate T, Assuming an Assigned

Potency of 1824 IU/ampoule


Overall GM - 2175 2144 2152 1927 2077

Lower 95% CL - 2070 2117 2126 1911 2041

Upper 95% CL - 2285 2172 2179 1942 2113

Between-lab %GCV - 24.8 6.0 5.7 3.8 8.2


Geometric Coefficient of Variation (%GCV) Relative to Candidate V, Assuming an Assigned



Overall GM 1822 - 2146 2156 1921 2070

Lower 95% CL 1734 - 2061 2064 1832 1996

Upper 95% CL 1914 - 2234 2252 2015 2147

Between-lab %GCV 24.8 - 19.6 21.4 23.9 17.8


Geometric Coefficient of Variation (%GCV) Relative to Candidate W, Assuming an

Assigned Potency of 2145 IU/ampoule


Overall GM 1825 2171 - 2152 1922 2070

Lower 95% CL 1801 2085 - 2133 1901 2049

Upper 95% CL 1848 2261 - 2171 1944 2091

Between-lab %GCV 6.0 19.6 - 4.0 5.0 4.7

WHO/BS/09.2124


Geometric Coefficient of Variation (%GCV) Relative to Candidate X, Assuming an Assigned



Overall GM 1827 2172 2149 - 1929 2075

Lower 95% CL 1804 2079 2131 - 1908 2052

Upper 95% CL 1850 2269 2168 - 1950 2098

Between-lab %GCV 5.7 21.4 4.0 - 5.1 5.2


Geometric Coefficient of Variation (%GCV) Relative to Candidate Y, Assuming an Assigned



Overall GM 1824 2177 2149 2153 - 2075

Lower 95% CL 1809 2076 2126 2130 - 2044

Upper 95% CL 1838 2284 2173 2177 - 2106

Between-lab %GCV 3.8 23.9 5.0 5.1 - 7.2


Geometric Coefficient of Variation (%GCV) Relative to Candidate Z, Assuming an Assigned

Potencyof 2074 IU/ampoule


Overall GM 1822 2176 2149 2155 1925 -

Lower 95% CL 1790 2098 2128 2131 1896 -

Upper 95% CL 1854 2256 2171 2179 1955 -

Between-lab %GCV 8.2 17.8 4.7 5.2 7.2 -

WHO/BS/09.2124

Table 29: Summary of Overall Potency Estimates Using Each Candidate as the Putative

Standard

Potency Estimates (IU/ampoule) Putative

standard T V W X Y Z

5thIS 1824 2172 2145 2156 1926 2074

T - 2175 2144 2152 1927 2077

V 1822 - 2146 2156 1921 2070

W 1825 2171 - 2152 1922 2070

X 1827 2172 2149 - 1929 2075

Y 1824 2177 2149 2153 - 2075

Z 1822 2176 2149 2155 1925 -

Table 30: Summary of Inter-Laboratory Variation Expressed as % Geometric Coefficient of

Variation (GCV) Using Each Candidate as the Putative Standard

GCV Putative

standard T V W X Y Z

5thIS 6.6% 20.7% 3.6% 4.8% 5.7% 5.5%

T . 24.8% 6.0% 5.7% 3.8% 8.2%

V 24.8% . 19.6% 21.4% 23.9% 17.8%

W 6.0% 19.6% . 4.0% 5.0% 4.7%

X 5.7% 21.4% 4.0% . 5.1% 5.2%

Y 3.8% 23.9% 5.0% 5.1% . 7.2%

Z 8.2% 17.8% 4.7% 5.2% 7.2% .

WHO/BS/09.2124

Table 31: USP sheep plasma clot based assays - Intra-laboratory variation (% GCV) based

on laboratories’ own potency estimates

Lab T V W X Y Z

06 0.6 2.9 0.4 1.7 1.7 0.8

10 1.6 0.0 1.4 1.4 0.9 0.7

12 1.7 1.7 2.6 2.1 1.7 1.5

18 2.9 3.8 1.0 1.8 0.6 3.9

19 8.5 5.9 3.8 7.9 11.5 3.1

24 1.1 1.0 0.5 0.2 1.2 1.2

25 1.9 3.4 2.1 3.5 3.4 3.0

26 0.6 1.2 0.0 2.6 2.5 1.5

28 0.0 1.6 2.0 4.5 1.3 1.5

Table 32: USP aIIa:AT Method: Intra-Laboratory Variation (%GCV)

Lab T V W X Y Z

02 6.2 3.5 2.5 1.4 1.8 3.7

03 7.6 13.6 12.9 16.3 6.1 7.1

06 6.8 3.5 4.6 4.5 2.4 7.5

08 2.6 3.1 2.8 2.6 8.4 3.6

12 1.5 1.8 6.7 6.0 2.0 3.1

13 8.5 10.9 2.6 7.3 6.6 7.1

19 . 29.1 9.2 . 23.4 9.3

25 5.3 1.7 2.0 3.0 5.4 8.5

32 4.6 8.1 1.8 1.9 2.9 2.8

Table 33: Other antithrombin dependent anti-IIa chromogenic methods - Intra-Laboratory

Variation (%GCV)

Lab T V W X Y Z

05 6.7 6.0 7.4 5.2 6.3 4.3

10 0.2 . 0.9 1.8 0.2 1.2

11 4.9 0.4 0.7 1.0 1.3 0.7

15 1.0 0.9 2.6 4.0 3.9 1.5

16 9.7 5.9 9.4 3.6 8.8 5.5

18 4.1 2.3 3.0 4.5 1.2 2.1

24 . . . 0.8 0.9 0.4

27 3.0 10.1 1.3 4.3 2.1 5.3

35 5.9 2.9 4.3 2.9 5.1 4.1

WHO/BS/09.2124

Table 34: USP aXa:AT Method: Intra-Laboratory Variation (%GCV)

Lab T V W X Y Z

02 4.4 1.6 0.3 2.8 2.2 2.9

03 0.4 - 3.0 14.4 - -

06 5.6 4.8 3.8 3.5 3.1 4.7

08b 2.4 4.6 2.6 6.2 2.2 1.2

12 1.2 2.7 2.3 0.8 0.6 2.4

13 7.4 7.8 8.3 7.3 6.7 7.3

19 8.0 11.1 5.0 7.8 11.1 5.5

25 2.9 4.3 3.4 5.1 6.8 2.6

32 3.8 4.1 1.6 2.0 2.8 4.0

Table 35: Other antithrombin dependent anti-Xa chromogenic methods - Intra-Laboratory

Variation (%GCV)

Lab T V W X Y Z

05 2.3 0.8 1.8 3.7 1.0 1.5

07 6.9 6.4 3.4 8.8 2.1 4.8

08e 2.7 11.8 4.5 2.3 5.3 2.9

10 0.5 1.4 2.2 - 0.7 0.5

11 3.5 2.7 1.3 0.9 1.8 1.6

15 4.0 0.1 2.7 3.8 3.2 3.9

16 13.3 20.8 21.3 10.9 9.7 6.1

18 2.5 5.4 1.1 0.6 5.0 2.3

22 0.5 1.1 0.9 0.5 0.7 1.2

23 4.0 2.8 1.6 1.3 4.5 1.8

27 4.6 4.0 1.5 2.2 3.1 1.2

33 4.3 4.2 6.1 3.9 5.6 5.3

35 5.6 6.5 3.8 2.1 9.8 5.6

36 3.8 26.4 - 11.2 8.4 10.4

Table 36: Intra-Laboratory Variation - Comparison of anti-IIa chromogenic methods.

<5% <7% <10%

USP aIIa:AT

Other

methods USP aIIa:AT

Other

methods USP aIIa:AT

Other

methods

No of GCVs 27/52 36/50 44/52 40/50 46/52 49/50

% of GCVs 52 72 85 80 88 98

For the USP aIIa:AT method: 3 GCVs 10 - 15%; 3 GCVs >15%

WHO/BS/09.2124

Table 37: Intra-Laboratory Variation - Comparison of anti-Xa chromogenic methods

<5% <7% <10%

USP aXa:AT

Other

methods USP aXa:AT

Other

methods USP aXa:AT

Other

methods

No of GCVs 35/51 60/82 41/51 70/82 48/51 74/82

% of GCVs 69 73 80 85 94 90

For other chromogenic methods: 5 GCVs 10 - 15%; 3 GCVs >15%

Table 38: Comparison of inter-laboratory variation

Inter-laboratory variation (%GCV)

Candidates

USP

Sheep

Plasma

(n=9)

USP

IIa:AT

method

(n=9)

Anti-IIa

Other AT

methods

(n=9)

Anti-IIa

All AT

methods

(n=18)

USP

aXa:AT

Method

(n=9)

Anti-Xa

Other AT

methods

(n=14)

Anti-Xa

All AT

methods

(n=23)

All

Methods in

the study

(n=86)

T 3.3 10.0 5.9 8.1 8.4 7.7 7.8 6.6

V 5.9 14.9 17.2 15.6 7.2 20.1 17.2 20.7

W 3.1 4.9 5.3 5.3 3.5 3.8 3.6 3.6

X 3.0 5.3 5.1 5.1 6.5 4.6 5.3 4.8

Y 3.0 4.7 4.8 4.9 6.5 7.8 7.2 5.7

Z 1.4 7.3 6.6 6.7 4.1 5.0 4.8 5.5

Table 39: Laboratory Mean Potency estimates (GM), Intra- and Inter-Laboratory Variation,

expressed as % Geometric Coefficient of Variation (%GCV) for the US Pharmacopoeial

Assay (sheep plasma)

T V W X Y Z

Lab GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

06 1960 0.6 1844 2.9 2138 0.4 2146 1.7 2014 1.7 2020 0.8

10 2003 1.6 2088 0.0 2045 1.4 2017 1.4 2104 0.9 2024 0.7

12 1930 1.7 1752 1.7 2176 2.6 2179 2.1 2055 1.7 2069 1.5

18 1867 2.9 1738 3.8 2180 1.0 2215 1.8 2025 0.6 2029 3.9

19 1857 8.5 1773 5.9 2250 3.8 2133 7.9 1915 11.5 2030 3.1

24 1999 1.1 1819 1.0 2247 0.5 2237 0.2 2030 1.2 2036 1.2

25 1859 1.9 1779 3.4 2138 2.1 2127 3.5 1959 3.4 1989 3.0

26 2006 0.6 1917 1.2 2210 0.0 2182 2.6 2043 2.5 2043 1.5

28 1939 0.0 1791 1.6 2219 2.0 2158 4.5 2097 1.3 2086 1.5

Overall GM 1935 1831 2177 2154 2026 2036

Lower 95%

CL 1887 1751 2128 2105 1980 2015

Upper 95% CL 1983 1913 2228 2204 2073 2058

Between-lab % GCV

3.3 5.9 3.1 3.0 3.0 1.4

WHO/BS/09.2124

Table 40: Laboratory Mean Potency estimates (GM), Intra- and Inter-Laboratory

Variation, expressed as % Geometric Coefficient of Variation (%GCV) for USP aIIa:AT

Outlier


Table 41: Laboratory Mean Potency estimates (GM), Intra- and Inter-Laboratory

Variation, expressed as % Geometric Coefficient of Variation (%GCV) for USP aXa:AT

T V W X Y Z

Lab GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

GM

(IU/amp)

GCV

(%)

02 1710 4.4 2698 1.6 2168 0.3 2105 2.8 1750 2.2 2129 2.9

03 1600 0.4 - - 1985 3.0 1880 14.4 1681 - 1917 -

06 1853 5.6 2667 4.8 2198 3.8 2249 3.5 1915 3.1 2239 4.7

08b 1762 2.4 2363 4.6 2159 2.6 2186 6.2 1845 2.2 2084 1.2

12 2146 1.2 2421 2.7 2133 2.3 2291 0.8 2021 0.6 2085 2.4

13 1881 7.4 2428 7.8 2244 8.3 2283 7.3 2018 6.7 2220 7.3

19 1787 8.0 2565 11.1 2217 5.0 2225 7.8 1953 11.1 2138 5.5

25 1723 2.9 2450 4.3 2119 3.4 2039 5.1 1887 6.8 2135 2.6

32 1818 3.8 2116 4.1 2113 1.6 2198 2.0 1956 2.8 2079 4.0

Overall GM 1804 2457 2147 2158 1889 2112

Lower 95%

CL 1694 2304 2089 2055 1800 2040

Upper 95%

CL 1920 2620 2207 2267 1982 2186

Between- lab % GCV

8.5 8.0 3.7 6.6 6.5 4.6

T V W X Y Z

Lab GM (IU/amp)

GCV (%)

GM (IU/amp)

GCV (%)

GM (IU/amp)

GCV (%)

GM (IU/amp)

GCV (%)

GM (IU/amp)

GCV (%)

GM (IU/amp)

GCV (%)

02 1617 6.2 2953 3.5 2121 2.5 2140 1.4 1767 1.8 2243 3.7

03 1612 7.6 2028 13.6 2039 12.9 2004 16.3 1813 6.1 1823 7.1

06 1789 6.8 2916 3.5 2231 4.6 2248 4.5 1926 2.4 2307 7.5

08 1626 2.6 2978 3.1 2083 2.8 2035 2.6 1837 8.4 2155 3.6

12 2102 1.5 2754 1.8 2048 6.7 2228 6.0 1979 2.0 2212 3.1

13 1938 8.5 2830 10.9 2295 2.6 2318 7.3 1966 6.6 2251 7.1

19 1602 - 3269 29.1 2736 9.2 1226 - 1814 23.4 2193 9.3

25 1678 5.3 2594 1.7 1988 2.0 2065 3.0 1766 5.4 2065 8.5

32 1728 4.6 2463 8.1 2104 1.8 2130 1.9 1944 2.9 2219 2.8

Overall

GM 1736 2731 2112 2144 1866 2158

Lower 95%

CL 1614 2456 2029 2053 1802 2045

Upper 95%

CL 1868 3038 2198 2238 1933 2279

Between-lab

%GCV 10.0 14.9 4.9 5.3 4.7 7.3

WHO/BS/09.2124

Table 42: A summary of estimated potencies of the candidates by USP sheep plasma, USP

aIIa:AT, other antithrombin dependent anti-IIa chromogenic, all antithrombin dependent

anti-IIa chromogenic, USP aXa:AT, other antithrombin dependent anti-Xa chromogenic

and all antithrombin dependent anti-Xa chromogenic assays relative to the 5th

International Standard for Unfractionated Heparin

Estimated Potency: Geometric Mean IU/ampoule Candidate

USP Sheep

Plasma

(n=9)

Anti-IIa

USP method

(n=9)

Anti-IIa

Other

methods

(n=9)

Anti-IIa

All

methods

(n=18)

Anti-Xa

USP Method

(n=9)

Anti-Xa

Other

methods

(n=14)

Anti-Xa

All methods

(n=23)

T 1935 1736 1784 1758 1804 1828 1818

V 1831 2731 2638 2687 2457 2226 2308

W 2177 2112 2189 2150 2147 2149 2148

X 2154 2144 2171 2158 2158 2163 2161

Y 2026 1866 1932 1899 1889 1920 1908

Z 2036 2158 2150 2154 2112 2101 2105

Table 43: Anti-Xa to anti-IIa ratio of the candidates by USP aIIa:AT and aXa:AT methods

Candidate

Anti-Xa USP

Method IU/ampoule

(n=9)

Anti-IIa USP

Method IU/ampoule

(n=9)

Anti-Xa to anti-IIa

ratio

T 1804 1736 1.04

V 2457 2731 0.89

W 2147 2112 1.02

X 2158 2144 1.01

Y 1889 1866 1.01

Z 2112 2158 0.98

WHO/BS/09.2124

Figure 1: Molecular weight distributions of the candidates

Figure 2: 1H NMR spectrum at 500 MHz of candidate T

WHO/BS/09.2124

Figure 3: 1H NMR spectrum at 500 MHz of candidate V

Figure 4: 1H NMR spectrum at 500 MHz of candidate W

WHO/BS/09.2124

Figure 5: 1H NMR spectrum at 500 MHz of candidate X

Figure 6: 1H NMR spectrum at 500 MHz of candidate Y

WHO/BS/09.2124

Figure 7: 1H NMR spectrum at 500 MHz of candidate Z

Nu

mb

er o

f la

bs

0

5

10

15

20

25

30

35

40

Potency (IU/ampoule)

10

00

20

00

30

00

40

00

Method: aIIa aXa APTT EP USP Other

Figure 8: Potency Estimates for Candidate T relative to the 5th IS

Geometric mean potency = 1824 IU/ampoule

Inter-lab GCV = 6.6%

Outlier, Lab 18; JP

WHO/BS/09.2124

Nu

mb

er o

f la

bs

0

5

10

15

20

25

30

35

40


100

0

2000

3000

4000


Figure 9: Potency Estimates for Candidate V relative to the 5th IS



Nu

mb

er o

f la

bs

0

5

10

15

20

25

30

35

40


10

00

20

00

30

00

40

00


Figure 10: Potency Estimates for Candidate W relative to the 5th IS



Outliers, Lab 19: aIIa;

Lab 36: aXa; Lab 18: JP

WHO/BS/09.2124

Nu

mb

er o

f la

bs

0

5

10

15

20

25

30

35

40


10

00

20

00

30

00

40

00


Figure 11: Potency Estimates for Candidate X relative to the 5th IS



Outlier, Lab 18; JP

Outliers, Lab 19: aIIa;

Lab 03: Thrombin Time

Nu

mb

er o

f la

bs

0

5

10

15

20

25

30

35

40


100

0

20

00

30

00

40

00


Figure 12: Potency Estimates for Candidate Y relative to the 5th IS



Outlier, Lab 18; JP

WHO/BS/09.2124

Nu

mb

er o

f la

bs

0

5

10

15

20

25

30

35

40


1000

2000

3000

4000


Figure 13: Potency Estimates for Candidate Z relative to the 5th IS



Outlier, Lab 18; JP

WHO/BS/09.2124

Fig 14: Potency Estimates by Each Assay Method Expressed as the % of the Overall Mean

Potency Estimates for Each Candidate. Japanese Pharmacopoeial assay results are not

presented as only one laboratory returned data for this method and 5 out of the 6 estimates

were outliers.

WHO/BS/09.2124

Figure 15: Potency estimates by USP current and proposed monograph assay method

expressed as the % of the overall mean potency estimates by all methods in the study for each

candidate

WHO/BS/09.2124

Appendix 1: List of Participants

Ginés Diaz and Maria Mercedes Lopez, B Braun Medical S.A., SPAIN

Peter Gärtner, Baxter AG, AUSTRIA

Patrick Shaklee, Biocascade Incorporated, USA

Jesus Cabañas, Bioiberica, S.A, SPAIN

Maik Gehrke and Gerrit Oldekamp, CKW Pharma-Extrakt, GERMANY

Eva Sandberg, Danish Medical Agency, DENMARK

François Nicham and Nathalie Barat, Diagnostic Stago, FRANCE

Guy Rautmann, European Directorate for the Quality of Medicines, FRANCE

C.S. Venkatesan, Gland Pharma Ltd., INDIA

Dean Smith and Hing Chong, Health Canada, CANADA

Luis Soares, Inframed, PORTUGAL

Ljiljana Kudra and Dženita Softić, Institue for Quality Control of Medicines, BOSNIA AND

HERZEGOVINA

Donata Bensi, Laboratori Derivati Organici, ITALY

Pilar Carrasco, Laboratorios Farmacéuticos Povi S.A., SPAIN

Bjørn Fehrmann, LEO Pharma A/S, DENMARK

Oliver Rädler, Merckle GmbH, GERMANY

Fan Huihong and Li Jing, National Institute for the Control of Pharmaceutical & Biological

Products, P.R. CHINA

John Hogwood, National Institute for Biological Standards and Control, UK

Hinrich Nagel, Nordmark Arzeimittel – Quality Control, GERMANY

Peter Bruegger, Novartis Pharma A.G. SWITZERLAND

Bruna Pharma, Opocrin SpA, ITALY

Karin Stoll- Rubin, DSM Nutritional Products Ltd - Branch Pentapharma, SWITZERLAND

Susanne Alban,Pharmazeutisches Institut Alteilung Pharmazeutishce Biologe, GERMANY

WHO/BS/09.2124

Thomas Freudemann, Sandoz GmbH, AUSTIA

Cécile Auguste and Céline Houiste, Sanofi-Aventis, FRANCE

Hester Hasper, Schering-Plough, THE NETHERLANDS

Hitendra Mehta and Anisha Akula, Scientific Protein Labs LLC, USA

Wolfgang Klein, Siemens Healthcare Diagnostics Products GmbH, GERMANY

Stephanie Berrier, Stago, FRANCE

Roger Meier, Swissmedic, SWITZERLAND

Barbara Kerbl, Technoclone GmbH, AUSTRIA

Toshimi Murai and Yukari Nakagawa, The Society of Japanese Pharmacopoeia, JAPAN

Chong Loh and Allison Jones, Therapeutic Goods Administration, AUSTRALIA

Sérgio Luiz Dalmora, Universidade Federal de Santa Maria, BRASIL

Michael Ambrose and Jeanne Fringer, US Pharmacopeia, USA

WHO/BS/09.2124

Appendix 2a: Methods Used by the Participants

Lab Methods No. Methods

1 EP Assay 1

2 Proposed USP anti-IIa and USP anti-Xa on STA Compact 2

3 NIBSC anti-IIa, Modified NIBSC anti-Xa, in-house anti-IIa and anti-Xa plasma AT, APTT (Pathrombin SL) and Thrombin Time

5

4 EP assay 1

5 LMWH based anti-IIa, LMWH based anti-Xa and APTT 3

6 NIBSC anti-IIa and anti-Xa, EP assay, USP assay and CP assay 5

7 In-house anti-Xa assay on Stago Analyser and EP assay on BCT 2

8 NIBSC anti-IIa and anti-Xa assay on ACL-7000, APTT (APTT-SP) assay, EP assay and Coatest anti-Xa

assay 5

9 EP assay 1

10 In-house anti-IIa and anti-Xa assay, APTT assay and USP assay 4

11 In-house anti-IIa and anti-Xa assay 2

12 NIBSC anti-IIa and anti-Xa assay and USP assay 3

13 NIBSC anti-IIa and anti-Xa assay, APTT (APTT-SP) assay and EP assay using ACL-TOP-500 4

14 APTT Assay 1

15 In-house anti-IIa and anti-Xa assay 2

16 In-house anti-IIa and anti-Xa assay and APTT assay 3

17 EP assay 1

18 EP LMWH anti-IIa and anti-Xa assays, EP assay, USP assay and JP assay 5

19 Proposed USP anti-IIa assay, USP anti-Xa assay and USP assay 3

21 EP assay 1

22 Biophen anti-Xa assay, APTT (Actin-FC) assay and PiCT assay 3

23 Berichrom anti-Xa assay 1

24 Actichrom anti-IIa assay and USP assay 2

25 Proposed USP anti-IIa assay, USP anti-Xa assay, EP assay and USP assay 4

26 USP assay 1

27 Chromogenix’s anti-IIa and anti-Xa kits and EP assay 3

28 APTT (Pathrombin SL) assay and USP assay 2

29 APTT assay 1

30 Comatic plasma AT anti-Xa 1

31 EP assay 1

32 In-house anti-IIa and anti-Xa assays 2

33 Stachrom anti-Xa assay 1

34 Rotachrom anti-Xa assay, Staclot plasma AT anti-Xa, APTT (STA-C.K.Prest and STA-PTTA) assays and thrombin Time

5

35 In-house anti-IIa assay and Coatest anti-Xa 2

36 Coatest anti-Xa assay and APTT (Thrombotest) assay 2

Total 35 Participants returning results 87

NB – Lab 20 withdrew from the study, Lab 4 and 14 not included in analysis due to late return.

WHO/BS/09.2124

Appendix 2b: Laboratories using USP Monograph Assay Methods

Assay methods

Lab Anti-IIa

(antithrombin)

Anti-Xa

(antithrombin) Sheep Plasma

Participants

02 USP USP Reagent manufacturer

03 NIBSC NIBSC Clinical Lab

06 NIBSC NIBSC � Pharmacopoeia

08 NIBSC NIBSC Heparin manufacturer

10 � Regulator

12 NIBSC NIBSC � Heparin manufacturer

13 NIBSC NIBSC Regulator

18 � Heparin manufacturer

19 USP USP � Pharmacopoeia


25 USP USP � Heparin manufacturer



32 NIBSC NIBSC Regulator

Appendix 2c: Laboratories using other antithrombin dependent

anti-IIa and anti-Xa assays

Assay methods Lab

Anti-IIa (antithrombin) Anti-Xa (antithrombin)

05 � �

07 �

08 �

10 � �

11 � �

15 � �

16 � �

18 � �

22 �

23 �

24 �

27 � �

33 �

34 �

35 � �

36 �

WHO/BS/09.2124

Appendix 3: NIBSC Protocols for Anti-IIa and Anti-Xa

Chromogenic Assay Using Purified Antithrombin

NIBSC Protocol for:

Anti-IIa Antithrombin Dependant Assay for Unfractionated Heparin

Materials:

Buffer - 7.5 mM Na2EDTA, 50 mM Tris(hydroxymethyl)Methylamine, 175 mM NaCl, with either 0.1%

polyethylene glycol 6000 or 2mg/ml albumin (human or bovine). Adjust to pH 8.4 with concentrated hydrochloric

acid

Human Antithrombin – Diluted to 0.125 IU/ml with above buffer

Human Thrombin – Diluted to 5 IU/ml with above buffer

Substrate – Specific for thrombin and diluted to 1.25 mM in water

Method:

Carry out two independent series of 4 dilutions of the standard and the test, within the range 0.005 to 0.05 IU/ml

using the pH 8.4 buffer. The dilutions chosen should give a linear response when the results are analysed as

absorbance against log concentrations. The assay should use a balanced order with 8 tubes for the standard and 8

tubes for the test, and be carried out at 37ºC. Volume (V) can range from 20 to 50µl depending on instrument used.

**Preliminary experiments should be carried out to determine the incubation times that are optimal for the various

stages and the reagents used.

2V Antithrombin (0.125IU/ml)

1V standard/test/buffer

Incubate for at least 1min**

0.5V Thrombin (5IU/ml)

Incubate for at least 1min**

1V substrate (1.25 mM in water)

Kinetic – obtain ∆OD/min at 405nm, no

acetic acid required Endpoint – stop with 2V 50% acetic acid after at

least 1 min** – read at 405nm

Analyse data by parallel line of

slope ratio

WHO/BS/09.2124

NIBSC Protocol for:

Anti-Xa Antithrombin Dependant Assay for Unfractionated Heparin

Materials:

Buffer - 7.5 mM Na2EDTA, 50 mM Tris(hydroxymethyl)Methylamine, 175 mM NaCl, with either 0.1%

polyethylene glycol 6000 or 2mg/ml albumin (human or bovine). Adjust to pH 8.4 with concentrated hydrochloric

acid

Human Antithrombin – Diluted to 1 IU/ml with above buffer

Bovine Factor Xa – Diluted with above buffer to a concentration that obtains an OD between 0.65 and 1.25 for a

blank sample

Substrate – Specific for Factor Xa and diluted to 1 mM in water

Method:

Carry out two independent series of 4 dilutions of the standard and the test, within the range 0.007 to 0.07 IU/ml,

using the pH 8.4 buffer. The dilutions chosen should give a linear response when the results are analysed as

absorbance against log concentrations. The assay should use a balanced order with 8 tubes for the standard and 8

tubes for the test, and be carried out at 37ºC. Volume (V) can range from 25 to 50µl depending on instrument used.

1V Antithrombin (1 IU/ml)

1V sample (or buffer)

Incubate for 2min

2V Factor Xa

Incubate for 2min

2V substrate (1 mM in water)

Kinetic – obtain ∆OD/min at

405nm, no acetic acid required Endpoint – stop with 2V 50% acetic

acid after 2 min – read at 405nm

Analyse data by parallel line

or slope ratio

WHO/BS/09.2124

Appendix 4: Study Protocol

Collaborative study on the 6th International Standard for Unfractionated Heparin (CS379)

Aims of Study

The aim of the study is to assay 6 candidate unfractionated heparin samples against the 5th International

Standard, 97/578, with a view to establish a new material as the 6th International Standard for

unfractionated heparin.

Unfractionated heparin samples provided (all samples are of porcine origin):

S - the 5th IS for Unfractionated Heparin, 97/578. Potency 2031 IU/ampoule.

T - Potency range 1900 – 2100 IU/ampoule.

V - Potency range 2400 – 2600 IU/ampoule.

W - Potency range 2000 – 2200 IU/ampoule.

X - Potency range 2000 – 2200 IU/ampoule.

Y – Potency range 1900 – 2100 IU/ampoule.

Z - Potency range 1900 – 2100 IU/ampoule.

Using the above figures as a guide for the first assay, adjust the initial dilutions of each heparin so that the

responses for the dilutions of the samples in the assays are in a similar range. Based on the results from

the first assay, if necessary, re-adjust the dilutions of the samples for the subsequent assays to be in the

same range. Note that the dilution range for different assay types (eg EP methods and anti-Xa assays)

may be different.

Storage and Reconstitution

The samples should be handled as follows:

1. Store all unopened ampoules and vials 2 – 8°C.

2. Open ampoule after ensuring all the contents are in the lower half. Reconstitute freeze-dried

ampoules at room temperature with 1.0 ml distilled water. Transfer the entire contents to a

plastic tube.

3. Keep all samples in stoppered plastic tubes at room temperature during period of assay and at

+4°C between assays carried out on different days for different methods.

Assay Methods

Each laboratory is asked to perform their chosen methods.

WHO/BS/09.2124

Design and number of Assays

A suitable design should be chosen from the schemes given below; ideally the 14 and 8 place assays are

preferred. Once a scheme has been chosen, it should be adhered to as closely as possible. In these

schemes, each coded letter represents at least 3, but preferably 4 dilutions of that material. For

identification purposes, when reporting the results, the 4 ampoules of standard should be numbered S1,

S2, S3, S4 and the test samples numbered in a similar way. All participants should carry out 4

independent assays for each method, an independent assay being defined as one with completely fresh set

of dilutions.

To allow for day to day variation, the four assays by each method should be carried out on 4 separate days.

For those laboratories performing 2 or more methods, these can be carried out on the same or different

days, using the same reconstituted solutions, but making fresh dilutions for each method.

14 place assays

Assay Order of Testing

1 S1 T1 V1 W1 X1 Y1 Z1 Z1 Y1 X1 W1 V1 T1 S1

2 S2 Z2 Y2 X2 W2 V2 T2 T2 V2 W2 X2 Y2 Z2 S2

3 S3 X3 W3 V3 Z3 T3 Y3 Y3 T3 Z3 V3 W3 X3 S3

4 S4 W4 X4 Y4 T4 Z4 V4 V4 Z4 T4 Y4 X4 W4 S4

8 place assays

Assay Run Order of Testing

1 1 S1 V1 W1 Z1 Z1 W1 V1 S1

2 S1 X1 Y1 T1 T1 Y1 X1 S1

2 1 S2 Z2 V2 W2 W2 V2 Z2 S2

2 S2 T2 X2 Y2 Y2 X2 T2 S2

3 1 S3 W3 X3 V3 V3 X3 W3 S3

2 S3 Y3 Z3 T3 T3 Z3 Y3 S3

4 1 S4 X4 V4 W4 W4 V4 X4 S4

2 S4 Z4 Y4 T4 T4 Y4 Z4 S4

WHO/BS/09.2124

6 place assays

Assay Run Order of Testing

1 1 S1 V1 Z1 Z1 V1 S1

2 S1 W1 Y1 Y1 W1 S1

3 S1 X1 T1 T1 X1 S1

2 1 S2 T2 V2 V2 T2 S2

2 S2 W2 X2 X2 W2 S2

3 S2 Z2 Y2 Y2 Z2 S2

3 1 S3 W3 T3 T3 W3 S3

2 S3 Z3 V3 V3 Z3 S3

3 S3 Y3 X3 X3 Y3 S3

4 1 S4 X4 Y4 Y4 X4 S4

2 S4 Z4 W4 W4 Z4 S4

3 S4 T4 V4 V4 T4 S4

For the 8 and 6 place assays, there should be minimal time delay between runs within an assay. Reagents

for each assay should be stored on ice, until all runs are performed.

If your laboratory cannot follow these suggested designs, please do email John Hogwood and we will

send other appropriate schemes for your laboratory.

Report of data

Raw data and estimated potencies should be recorded on the appropriate result sheets. Electronic data

sheets on EXCEL format will be forwarded to participants once the “Acknowledgement of samples

receipt form” is received by John Hogwood at NIBSC. Please email John Hogwood if you do not receive

the electronic results form ([email protected]).

WHO/BS/09.2124

Appendix 5 Summary of Results by European Pharmacopoeial Assay returned by Lab 4. Geometric

means for each candidates, as estimated by the laboratory were within the range of

geometric means obtained by other laboratories carrying out the same assay method.

Assay 1 Assay 2 Assay 3 Assay 4 Geometric

Mean GCV

T 1849 1849 1820 1837 1839 0.75%

V 1966 1977 1988 1955 1971 0.72%

W 2093 2161 2143 2092 2122 1.67%

X 2137 2161 2151 2158 2152 0.50%

Y 1895 1895 1906 1930 1906 0.87%

Z 2010 2011 2051 1963 2009 1.81%

Summary of Results by APTT Assay returned by Lab 14. Geometric means for each

candidates, as estimated by NIBSC were within the range of geometric means obtained by

other laboratories carrying out the same assay method.

Assay 1 Assay 2 Assay 3 Assay 4 Geometric

Mean GCV

T 1899 1880 1912 1880 1893 0.83%

V 1773 1786 1781 1765 1776 0.52%

W 2195 2162 2178 2169 2176 0.66%

X 2271 2263 2249 2280 2266 0.58%

Y 2026 2009 2027 2035 2024 0.54%

Z 2033 2009 2051 2049 2035 0.96%

WHO/BS/09.2124

Appendix 6: Draft Intruction for Use (IFU) for the Proposed 6th

International Standard for Unfractionated Heparin, 07/328

WHO/BS/09.2124

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