WHO/BS/2014.2245 ENGLISH ONLY

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WHO/BS/2014.2245 ENGLISH ONLY

EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION Geneva, 13 to 17 October 2014

VALUE ASSIGNMENT OF THE CANDIDATE 1^ST INTERNATIONAL STANDARD FOR ACTIVATED BLOOD COAGULATION FACTOR XI (FXIa), HUMAN, NIBSC CODE 13/100 Elaine Gray¹, John Hogwood, Helen Wilmot, Craig Thelwell, Thomas Dougall and Peter Rigsby

National Institute for Biological Standards and Control Potters Bar, Hertfordshire, EN6 3QG, UK

1Principal Investigator

NOTE:

This document has been prepared for the purpose of inviting comments and suggestions on the proposals contained therein, which will then be considered by the Expert Committee on Biological Standardization (ECBS). Comments MUST be received by 4 October 2014 and should be addressed to the World Health Organization, 1211 Geneva 27, Switzerland, attention:

Technologies, Standards and Norms (TSN). Comments may also be submitted electronically to the Responsible Officer: Dr David Wood at email: [email protected].

© World Health Organization 2014

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Summary

Seventeen laboratories from eleven different countries participated in a value assignment collaborative study for the proposed 1st International Standard for Activated Blood Coagulation Factor XI (FXIa), 13/100. Coded duplicates of the candidate together with 4 immunoglobulin preparations from 2 manufacturers were included in the study. In general the intra-laboratory variation was low indicating the laboratories were able to carry out FXIa quantitative assays with good precision. The inter-laboratory agreement was excellent for the candidate preparation with or without the inclusion of clot-based assay results; with GCV of 3.1 and 3.3% respectively.

There was no assay discrepancy between the chromogenic and clotting assays. It is

recommended that the value assignment should be based on functional chromogenic assays only.

It is therefore proposed that the candidate FXIa preparation, 13/100 be assigned as the WHO 1^st International Standard for Activated Blood Coagulation FXI (FXIa), Human, with an assigned value of 9.8 International Units (IU) per ampoule.

Introduction

The International Reference Reagent for Activated Blood Coagulation Factor XI (FXIa), Human was established by the Expert Committee on Biological Standardization (ECBS) of the World Health Organization (WHO) in October 2012 to support the measurement of FXIa in

immunoglobulin products. This Reference Reagent was assigned with an arbitrary unit of 10 u/ampoule by NIBSC and the “fit for purpose” study (1) demonstrated good improvement of intra- and inter-laboratory agreement when it was used as a standard for measurement of FXIa in IVIG products. Over 1500 ampoules have now been issued and the stock will be depleted by the end of 2014. A candidate preparation of FXIa with similar characteristics to the International Reference Reagent (IRR) has been prepared and this study serves to value assign this candidate against the IRR, thereby ensuring the continuity of the FXIa unit as defined by the International Reference Reagent. Discussions with regulators and manufacturers indicated that in addition to being used as a calibrant in the FXIa functional activity assays, the IRR has also been used as a

“standard” in the non-activated partial thromboplastin time (NAPTT) and the thrombin

generation assays (TGA). For value assignment purposes, however, only results from specific FXIa functional activity assays will be considered, as NAPTT and TGA assays are not specific for FXIa activity and these methods are not sufficiently robust to provide accurate potency estimates (Note: The results for NAPTT and TGA will be addressed in a separate report to the participants). This study also provides the opportunity to examine the performance of assay methods for detection of procoagulant activity in immunoglobulins (IgG) and therefore a selection of IgG samples were included to investigate the usefulness of a FXIa reference standard in these assays.

Candidate, NIBSC code 13/100

The bulk for the candidate was purchased. The specific activity estimated by NIBSC using a FXIa functional chromogenic activity assay relative to the International Reference Reagent for FXIa, 11/236, was approximately 11500 u/mg . The starting material was certified by the manufacturer as being negative for anti-HIV1/2, HBsAg and hepatitis C. The material was prepared by activating purified human FXI with FXIIa and subsequently purified to homogeneity by a combination of affinity chromographic methods. The single batch of material was diluted 1/3100 in 50 mM Tris, 150 mM NaCl, 5 mg/ml trehalose and 0.5% human serum albumin to give a final concentration of approximately 10 u/ml . The batches of human serum albumin used contained minimal protease activities as indicated by lack of colour development following incubation with chromogenic substrates (S2765, S2302 and S2251) for 90 minutes at 37◦C. The material was distributed in glass ampoules, filled and freeze-dried according to guidelines for

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production of international standards. The product characteristics are listed in the following table.

NIBSC Code 13/100

Presentation Sealed, siliconised glass 3 ml ampoules

Filling date 11^th April 2013

Number of Ampoules available 18925

Liquid filling weight (g) (n=698, measurements taken from all

3 pumps throughout the duration of the fill) 1.0078

CV of fill mass (%) 0.15

Homogeneity of the fill by activity: 3 ampoules selected from the start, 1st quarter, middle, 3rd quarters and end of the fill;

were assayed against the IRR 11/236 using a FXIa functional activity method(Biophen FXIa kit). 2 assays per ampoule were carried out. Effect of fill position was assessed by ANOVA of log potencies and expressed as geometric coefficient of variation (GCV).

GCV p

2.2% 0.52

Mean dry weight (g) (n=6) 0.0258 (CV 0.83%)

Mean head space oxygen (%) (n=12) 0.19 (CV 47.3%)

Residual moisture (%) (n=12) 0.150 (CV 18.1%)

Storage temperature -20°C

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

Address of present custodian NIBSC, Potters Bar, EN6 3QG, UK

Determination of Molar Concentration

The protein concentration was determined by the vendor, based on the absorbance value at 280nm and an extinction coefficient (E^1%) of 13.4 and molecular weight of 160000 Da for FXIa.

Active-site titration was carried out at NIBSC on the bulk preparation against 4

methylumbelliferyl 4-guanidinobenzoate hydrochloride hydrate (MUGB). Details of the method and results are summarised in Appendix 1. The molar concentration of 8.8 nM in the final ampoules was extrapolated from the estimation of the bulk which was found to be 27.22µM.

This information will NOT be provided on the label, but will be given in the Instruction for Use.

Participants

Twenty four laboratories agreed to participate, with 17 laboratories (2 Austria, 2 Australia, 1 Belgium, 2 France, 1 Germany, 1 Israel, 1 Spain, 1 Sweden, 1 Switzerland, 2 UK, 3 USA) returning data for Part I of the study. The participants included 3 diagnostics manufacturers, 11 therapeutic manufacturers and 3 regulatory authorities. A list of participants is given in

Appendix 2 at the end of this report. Each laboratory is referred to in this report by an arbitrarily assigned number, not necessarily representing the order of listing in the Appendix.

Samples

CODE PREPARATION

S The International Reference Reagent for Factor XIa (11/236), 10 units/ampoule - 4 ampoules supplied.

A Factor XIa preparation, NIBSC code 13/100; approx. 10 units/ampoule - 4 ampoules supplied

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B Factor XIa preparation, NIBSC code 13/100, coded duplicate of sample A – approx. 10 units/ampoule - 4 ampoules supplied

C IVIG preparation containing medium procoagulant activity, 5% protein; approx. 0.06 FXIa u/ampoule - 4 ampoules supplied.

D IVIG preparation containing high procoagulant activity, 5% protein; approx. 0.3 FXIa u/ampoule - 4 ampoules supplied.

E IVIG preparation containing low procoagulant activity, 5% protein; approx. 0.01 FXIa u/ampoule - 4 ampoules supplied.

F IVIG preparation containing high procoagulant activity, 5% protein; approx. 0.6 FXIa u/ampoule - 4 ampoules supplied.

Samples A and B were coded duplicates of the candidate standard.

Study design and assay methods

The details of the protocol are given in Appendix 3. The participants were provided with 4 sets of samples. Each laboratory was requested to determine the appropriate dilution ranges for sample S, the WHO IRR, 11/236, using their FXIa quantitation test. Once the appropriate dilutions of sample S had been determined, one independent assay was to be carried out on each of the 4 sets, preferably on 4 separate days. An example of balanced order of testing was provided.

Participants were requested to return raw data together with their own calculated estimates for all of the samples relative to sample S (the WHO IRR, 11/236) from each individual assay to

NIBSC.

Each participant was requested to perform their routine in-house functional method(s) for FXIa.

Some laboratories performed more than one method and in this case the data from each method were treated as separate sets of results and referred to as Lab 3a and Lab 3b, for example. A list of reagents, methods and instruments, together with their in-house FXIa standard (if used) by the participants is given in Appendix 4. Sixteen sets of results were returned for functional

chromogenic assays based on the conversion of FIX to FIXa by FXIa (12 used Hyphen Biomed kit, 3 used Rossix kit, 1 in house method). Two laboratories returned data for clot-based assays;

Lab 1 method was based on NAPTT using phospholipid without activator and FXI deficient plasma, while Lab 2 used one-stage clotting assay based on APTT.

Analysis of data

The potencies of all samples were calculated relative to the International Reference Reagent 11/236 (coded S) or the candidate standard (coded A) by parallel line analysis of the raw assay data at NIBSC (2). The majority of assays were analysed with a log transformation of assay response; no transformation was used for laboratories 01 (clotting), 02 (clotting), 13, 15, 19 and 22. Analysis was based on a linear section of the response range using a minimum of three dilutions for all samples. The majority of samples and assays from Lab 14 gave non-linear dose

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response curves and were not analysed further. Calculations were performed using the EDQM software CombiStats Version 5.0 (3).

Non-linearity and non-parallelism were considered in the assessment of assay validity. For chromogenic assays, samples with a high non-linearity mean square were excluded for not being linear, with cut-off values determined through visual assessment of the plotted data. A cut-off value of 0.002 was used for assays with a log transformation of assay response and 0.001 for those using no transformation. These values were chosen to allow a consistent approach to assessment of assay validity within this study and are not intended to reflect values that may be appropriate for use within all laboratories. Due to the low quantity of clotting assays, a visual assessment was used to determine linearity. Non-parallelism was assessed by calculation of the ratio of fitted slopes for the test and reference samples under consideration. The samples were concluded to be non-parallel when the slope ratio was outside of the range 0.80 – 1.25 and in these cases, no estimates are reported.

The final assay result for the candidate standard was taken as the unweighted geometric mean (GM) of the results obtained for samples A and B within each assay. Relative potency estimates from all valid assays were combined to generate an unweighted geometric mean for each

laboratory and these laboratory means were used to calculate an overall unweighted geometric mean for each sample. Variability between assays within laboratories and between laboratories has been expressed using geometric coefficients of variation (GCV = {10^s-1}×100% where s is the standard deviation of the log10 transformed estimates).

The agreement between duplicate samples within each assay was assessed by calculating the difference in log potency estimates (relative to sample S) between the duplicates, calculating the mean of the squared difference for each laboratory, taking the square root to give a root mean square (RMS) value, and expressing this as an average percentage difference.

Results and Discussions

The main aim of this study was to value assign the candidate International Standard relative to the International Reference Reagent for Activated Coagulation Factor XI, Human, NIBSC code 11/236. Only results from functional activity assays specific for FXIa were considered for value assignment since the inclusion of other semi-quantitative and non-specific assay methods such as the NAPTT or the TGA, especially when performed using normal plasma, may bias the

consensus mean potency.

The individual assay results from each lab are shown in Appendix 5. The majority of the assays were statistically valid, with invalidity arising mostly from non-linearity of dose-response curves.

Samples A and B were coded duplicates and tables 1 and 2 show potency estimates of samples A and B relative to S, the IRR. With the exception of Lab 3, Lab 12 and Lab 15, the intra-

laboratory GCV were all less than 10% indicating that the laboratories were able to carry out these assays with reasonable precision. The overall GMs for samples A and B were 9.79 and 9.80 U/ampoule respectively. The agreement between laboratories was good as indicated by the 4% inter-laboratory GCV for both samples A and B. The exclusion of clotting data did not alter the overall GM or %GCV. Laboratories 1 and 2 used clot-based methods and the potency estimates for samples A and B were not different to those obtained using chromogenic assay method. The intra-assay variability (between coded duplicate samples) is shown in Table 3. The figures represent an average percentage within-assay difference in estimates between the

duplicate samples. The majority of laboratories have average % differences of less than 10%, which represents good assay repeatability. The combined potency estimates of samples A and B,

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the coded duplicates are shown in Table 4 and also illustrated as a histogram in Figure 1. The intra-laboratory variability was low, with the majority of GCV being less than 10%. The overall GMs with or without the clotting assay results were 9.78 IU/ampoule, with an inter-laboratory GCV of 3.1% and 3.3% respectively. Although there was no difference between the clotting and chromogenic assay results, in principle, the clotting assays are not sufficiently specific for FXIa activity and therefore it is recommended to value assign the proposed IS with potency estimates obtained by functional chromogenic assays only.

Samples C, D, E and F were immunoglobulin preparations from different manufacturers, each with varying degrees of procoagulant activity. Samples C and D were also included in the 2012 collaborative study (1) for the IRR and were found to have 0.15 U/ampoule (GCV 69.6%) and 0.49 U/ampoule (GCV 54.6%) when assayed against the IRR, assuming the value of the IRR as 10 U/ampoule. The level of FXIa in sample E was found to be below the limit of quantification in the current study. The results from the chromogenic assay kits showed that FXIa could be detected, but the ranges of the responses from the standard and test curves were such that valid value quantification could not be obtained. It is possible that a standard curve covering low concentrations could give statistically valid potency for sample E, however, the results from the current study does not allow for this comparison. Tables 5 and 6 and Figures 2 and 3 present data for samples C and D respectively. The GM potency estimates of FXIa contents for samples C and D were found to be 0.07 and 0.32 U/ampoule, excluding the clotting assay results. These values were lower than the estimates obtained from the previous study, however, the inter- laboratory variation of 13.9 and 6.9% indicated an improvement in between laboratory agreement for the measurement of FXIa in these two samples. Table 7 and Figure 4 show potency estimates obtained for sample F. When the clotting assay results were excluded, the overall GM was 0.65 U/ampoule and inter-laboratory variation was low, with a GCV of 5.7%. It is interesting that the results for samples D and F, the higher procoagulant activity samples, from the two laboratories carrying out clot-based assays were different, with Lab 1 obtaining estimates close to the functional chromogenic assay estimates, while Lab 2 obtaining 7 to 8 fold higher estimates. This discrepancy was not observed for samples A, B and C. This could be explained by the differences in the principles of the clotting assays carried out by these two labs. Lab 1 used a modified NAPTT assay in combination with FXI deficient plasma while Lab 2 employed an APTT based assay. It is possible that samples D and F contained zymogen and this was detected by the APTT based assay. This shows the importance of using a specific assay for measurement of FXIa in IgG preparations.

Table 8 shows results for laboratory’s own in house controls (standards). With the exception of Lab 12, intra-laboratory variation was low indicating the laboratories are able to carry out these assays with good reproducibility.

Two functional chromogenic assay kits were used by the participants. Twelve labs used the Hyphen Biomed kit, while 3 labs used the Rossix kit. Tables 9 – 13 summarise the results obtained by these 2 kits. It is clear that there is no significant difference between the potency estimates obtained and that the values from the Rossix kit were all within the range of those obtained using the Hyphen kit.

The IRR has been used by regulators and manufacturers to develop their in-house testing

methods and also to accumulate batch data. It is therefore of paramount importance that the unit as defined by the IRR could be continued. Appendix 6 presents the individual laboratories’

overall potency estimates for each sample relative to sample A, the proposed International Standard, assuming the assigned value to be 9.8 U/ampoule. Table 14 compares the recalculated potencies for samples B, C, D and F relative to sample A with the estimates obtained against the

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IRR. The estimates were not significantly different to those obtained against the IRR indicating that there would be minimal drift in the unit of FXIa when the IS is established.

Stability studies

Accelerated degradation studies have been performed after 3 and 12 months storage at low and high temperatures. The predicted loss per year at each temperature is shown below, based on cumulative results from both time-points. The predicted percentage loss at the normal storage temperature of -20 °C is <0.001%, showing that the material is very stable at this temperature.

Continual accelerated degradation and real time monitoring will be carried out on this preparation.

Storage temperature

(°C)

Predicted loss per year (%) (relative to -150)

-150 <0.001

-70 <0.001

-20 <0.001

4 <0.001

20 0.045

37 3.567

On-bench stability studies have also been carried out and the results of the on-bench stability are as shown below. Potencies at each time-point were determined relative to a fresh ampoule of 13/100. The potency after 4 hours storage on ice overlaps well with that at 0 h, indicating the material is stable for at least 4 hours when kept on melting ice.

Time on ice

Potency % fresh ampoule (95% confidence intervals) 1 h

(n=2)

103.1%

(98.9 – 107.5) 2 h

(n=3)

103.0%

(98.1 – 108.2) 3 h

(n=1)

94.1%

(84.7 – 104.5) 4 h

(n=2)

98.6%

(91.1 – 106.7)

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Proposal and Recommendation

The proposed International Standard for Activated Coagulation Factor XI (FXIa), Human,

13/100 (samples A and B, the coded duplicates), assayed well against the International Reference Reagent for FXIa, with an overall GM of 9.8 U/ampoule, excluding the clotting assays and inter- laboratory GCV of 3.3%. In general, the intra-laboratory variability was low, indicating the participants were able to measure FXIa with precision. There was also good inter-laboratory agreement, giving confidence in the overall potency value obtained for the candidate. In terms of measurement of FXIa in IgG preparations, the inter-laboratory GCVs were much lower than those found in the previous study, demonstrating an improvement in the capability of the laboratories to carry out FXIa assays.

It is recommended that the candidate preparation, 13/100 be established as the WHO 1^st

International Standard for Activated Coagulation Factor XI (FXIa), Human, with a labelled value of 9.8 IU/ampoule.

A draft Instruction For Use (IFU) is shown in Appendix 6.

Each participant was asked to review the participants report and whether they agreed with the proposal. All participants agreed with the proposal to establish the preparation 13/100 as the 1^st International Standard for Activated Coagulation Factor XI (FXIa), Human. The experts nominated by the Factor XI and the Contact System Subcommittee of theScientific and

Standardisation Committee (SSC) of the International Society on Thrombosis and Haemostasis have also reviewed the study. The SSC has now endorsed the proposal to go forward for establishment by the ECBS.

References

1. Gray E, Wilmot H, Hogwood J and Rigsby P. Evaluation of the proposed WHO 1^st Reference Reagent for Activated Blood Coagulation Factor XI (FXIa), Human. WHO technical Report 2012.

http://apps.who.int/iris/bitstream/10665/78047/1/WHO_BS_2012.2206_eng.pdf?ua=

1

2. Finney DJ. Statistical Methods in Biological Assay. 3rd Edition. London: Charles Griffin 1978.

3. CombiStats v5.0, EDQM – Council of Europe, www.combistats.eu.

Acknowledgements

We would like to thank the participants of the collaborative study, many of whom completed the testing under tight timescales and the kind donation of IgG preparations by Octapharma and Omrix Biopharmaceuticals.

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Table 1: Potency estimates for Sample A, coded duplicate of the candidate IS, relative to S, the International Reference Reagent for FXIa

Lab Method N GM

U/ampoule

GCV

%

1 Clotting 2 9.90 .

2 Clotting 4 9.90 2.4

3 Chromogenic 2 9.34 .

4 Chromogenic 4 9.25 5.2 6 Chromogenic 4 10.37 8.7

9 Chromogenic 4 10.07 6.1 10 Chromogenic 4 9.82 5.8 11 Chromogenic 4 9.75 5.2 12 Chromogenic 5 9.28 14.5 13a Chromogenic 4 10.09 5.4 13b Chromogenic 4 9.65 2.8 15 Chromogenic 3 10.56 22.4

19 Chromogenic 4 10.22 1.8 22 Chromogenic 3 9.64 2.2 23 Chromogenic 3 9.58 3.5

Overall 17 9.79 4.0

Excl. CL 15 9.78 4.3

GM: geometric mean; GCV: geometric coefficient of variation; N: number of assays; CL:

clotting

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Table 2: Potency estimates for Sample B, coded duplicate of the candidate IS, relative to S, the International Reference Reagent for FXIa

Lab Method N GM

U/ampoule

GCV

%

1 Clotting 3 9.93 7.8

2 Clotting 4 9.68 3.3

3 Chromogenic 3 9.80 11.0

10 Chromogenic 4 9.97 3.9 11 Chromogenic 4 9.82 4.5 12 Chromogenic 5 9.45 8.2 13a Chromogenic 4 10.02 5.7 13b Chromogenic 4 9.65 3.5 15 Chromogenic 1 11.11 .

19 Chromogenic 4 9.66 6.2 22 Chromogenic 4 9.72 2.2

Overall 17 9.80 4.0

Excl. CL 15 9.80 4.3

clotting

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Table 3: Average differences of potencies between coded duplicate samples A and B within each assay for each laboratory. The majority of laboratories gave differences of less than 10% indicating good agreement of potencies for the coded duplicates

Lab Method Average % difference between A and B

1 Clotting 5.3%

2 Clotting 4.1%

3 Chromogenic 10.6%

4 Chromogenic 6.1%

6 Chromogenic 6.8%

7 Chromogenic 6.8%

9 Chromogenic 3.0%

10 Chromogenic 3.0%

11 Chromogenic 6.3%

12 Chromogenic 9.8%

13a Chromogenic 3.3%

13b Chromogenic 4.3%

15 Chromogenic .

16 Chromogenic .

19 Chromogenic 7.4%

22 Chromogenic 0.6%

23 Chromogenic 10.2%

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Table 4: Combined potency estimates for the coded duplicates, samples A and B, relative to sample S, the International Reference Reagent for FXIa. The combined potencies were the unweighted GM of the results obtained for samples A and B within each assay

Lab Method N GM

U/ampoule

GCV

%

1 Clotting 3 9.79 6.2

2 Clotting 4 9.79 2.1

13a Chromogenic 4 10.05 5.2

13b Chromogenic 4 9.65 2.0

Overall 17 9.78 3.1

Excl. CL 15 9.78 3.3

clotting

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Table 5: Potency estimates for Sample C relative to S, the International Reference Reagent for FXIa

Lab Method N GM

U/ampoule

GCV

%

1 Clotting 3 0.06 19.3

overall 16 0.07 16.0

Excl. CL 15 0.07 13.9

clotting

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Table 6: Potency estimates for Sample D relative to S, the International Reference Reagent for FXIa

Lab Method N GM

U/ampoule

GCV

%

1 Clotting 3 0.40 2.2

2 Clotting 4 3.14 2.7

Overall 17 0.37 74.8

Excl.CL 15 0.32 6.9

clotting

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Table 7: Potency estimates for Sample F relative to S, the International Reference Reagent for FXIa

Lab Method N GM

U/ampoule

GCV

%

1 Clotting 3 0.66 15.1

2 Clotting 4 4.83 2.7

Overall 17 0.73 63.3

Excl.CL 15 0.65 5.7

clotting

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Table 8: Potency estimates for in-house controls relative to S, the International Reference Reagent for FXIa

Lab Method N GM

U/ml

GCV

%

2 Clotting 4 6.39 4.5

GM: geometric mean; GCV: geometric coefficient of variation; N: number of assays

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Table 9: Comparison of Potency estimates by Hyphen and Rossix chromogenic assay kits for Sample A relative to S, the International Reference Reagent for FXIa

Method Lab N GM

U/ampoule

GCV

%

Hyphen

3 2 9.34 .

4 4 9.25 5.2

6 4 10.37 8.7

7 2 9.29 .

9 4 10.07 6.1

11 4 9.75 5.2

12 5 9.28 14.5

13b 4 9.65 2.8

15 3 10.56 22.4

16 1 9.89 .

19 4 10.22 1.8

23 3 9.58 3.5

Overall 12 9.76 4.7

Rossix

10 4 9.82 5.8

13a 4 10.09 5.4

22 3 9.64 2.2

Overall 3 9.85 2.3

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Table 10: Comparison of Potency estimates by Hyphen and Rossix chromogenic assay kits for Sample B relative to S, the International Reference Reagent for FXIa

Method Lab N GM

U/ampoule

GCV

%

Hyphen

3 3 9.80 11.0

4 4 9.69 5.6

6 3 9.71 3.7

7 2 9.12 .

9 2 9.84 .

11 4 9.82 4.5

12 5 9.45 8.2

13b 4 9.65 3.5

15 1 11.11 .

16 1 9.55 .

19 4 9.66 6.2

23 2 9.93 .

Overall 12 9.77 4.7

Rossix

10 4 9.97 3.9

13a 4 10.02 5.7

22 4 9.72 2.2

Overall 3 9.90 1.6

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Table 11: Comparison of Potency estimates by Hyphen and Rossix chromogenic assay kits for Sample C relative to S, the International Reference Reagent for FXIa

Method Lab N GM

U/ampoule

GCV

%

Hyphen

3 3 0.08 5.1

4 4 0.07 3.8

6 4 0.07 9.6

7 2 0.06 .

9 1 0.10 .

11 3 0.06 6.0

12 5 0.07 31.5

13b 4 0.07 6.5

15 2 0.08 .

16 1 0.07 .

19 4 0.08 2.7

23 2 0.08 .

Overall 12 0.07 14.0

Rossix 10 4 0.08 31.4

13a 4 0.06 5.6

Overall 2 0.07 -

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Table 12: Comparison of Potency estimates by Hyphen and Rossix chromogenic assay kits for Sample D relative to S, the International Reference Reagent for FXIa

Method Lab N GM

U/ampoule

GCV

%

Hyphen

3 4 0.34 7.1

4 4 0.31 2.4

6 3 0.33 7.1

7 1 0.31 .

9 3 0.32 7.3

11 4 0.28 10.9

12 4 0.30 8.9

13b 4 0.32 5.0

15 2 0.31 .

16 1 0.32 .

19 3 0.28 18.0

23 2 0.35 .

Overall 12 0.31 7.1

Rossix

10 3 0.34 5.0

13a 4 0.34 5.3

22 3 0.32 5.1

Overall 3 0.33 2.8

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Table 13: Comparison of Potency estimates by Hyphen and Rossix chromogenic assay kits for Sample F relative to S, the International Reference Reagent for FXIa

Method Lab N GM

U/ampoule

GCV

%

Hyphen

3 4 0.68 11.4

4 4 0.61 4.7

6 4 0.72 10.3

7 1 0.63 .

9 4 0.62 7.0

11 1 0.64 .

12 5 0.64 13.1

13b 4 0.62 6.2

15 2 0.59 .

16 1 0.69 .

19 1 0.61 .

23 3 0.68 6.9

Overall 12 0.64 6.1

Rossix

10 4 0.69 5.5

13a 4 0.65 1.8

22 4 0.65 2.1

Overall 3 0.66 3.6

Table 14: Comparison of potency estimates relative to the International Reference Reagent for FXIa or the sample A, the proposed IS (assuming potency of 9.8 U/ampoule)

Samples

All assays U/ampoule

Excl Clotting U/ampoule

Vs IRR Vs sample A Vs IRR Vs sample A

B 9.8 9.8 9.8 9.77

C 0.07 0.07 0.07 0.07

D 0.37 0.33 0.32 0.37

F 0.73 0.74 0.65 0.65

Pair t-test: p>0.5 for all samples

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Figure 1: Potency estimates from each assay for the candidate standard. Each box depicts the geometric mean potency of samples A and B in each assay.

Figure 2: Potency estimates from each assay for sample C. Results from Lab 2 were outside the range of the histogram.

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Figure 3: Potency estimates from each assay for sample D. Results from Lab 2 were outside the range of the histogram.

Figure 4: Potency estimates from each assay for sample F. Results from Lab 2 were outside the range of the histogram.

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Appendix 1: Molar concentration

Method

Active-site titration is a method for calculating the active molar concentration of a proteolytic enzyme for which a suitable titrant is available. When an active-site titrant is added to an

enzyme there is an initial burst of activity followed by a lower rate as the substrate is turned over slowly. The magnitude of this initial burst is equivalent to the molar concentration of active enzyme, which can be quantified relative to a standard curve of the released product.

Active-site titration was performed on the bulk FXIa material, prior to formulation for the fill, against the fluorimetric titrant 4-Methylumbelliferyl 4-guanidinobenzoate hydrochloride hydrate (MUGB). A stopped flow unit was used to achieve rapid mixing of FXIa with MUGB. This was coupled to a fluorimetric spectrometer to simultaneously provide an output trigger to start kinetic measurements to capture the required burst of product release from MUGB mixed with FXIa. Kinetic measurements were taken by the fluorimeter at Ex. 355 nm Em. 465 nm (5 nm slits, 0.1 s) over 200 s.

Three individual assays were performed on three separate samples. For each assay six dilutions of FXIa were made in 0.01 M HEPES (pH 7.4) with 0.15 M NaCl within an approximate range 0.1–2 µM. Each FXIa dilution was titrated against MUGB (4 µM) in replicate (n>4). For each assay a standard curve of 4-methylumbelliferone (4-MU, the fluorophore released form MUGB) was generated under identical assay conditions.

Results

The magnitude of each ‘burst’ was calculated in GraphPad Prism, based on curve fitting over 120s, and corrected for the blank reading (MUGB titrated against buffer only). For each assay the active molar concentration of FXIa was calculated relative to the 4-MU standard using a parallel line model. The results for each assay are presented in the table below, including 95%

confidence intervals (CI), with an overall concentration based on the unweighted geometric mean of the three assay results.

Assay number Active FXIa concentration µM (95% CI)

Geometric mean (95% CI)

1 26.55

(24.62 – 28.64)

27.22 (24.45 – 30.32)

2 29.64

(27.37 – 32.11)

3 26.33

(24.22 – 28.63)

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Appendix 2: List of Participants

Gerald Schrenk, Baxter AG, Austria

Iris Timmermans, Baxter Bioscience, Belgium John More, BioProducts Laboratory Ltd., UK

Steffen Kistner, Karin Fuchs & Karen Martens-Weigand, Biotest AG, Germany Innocent Bekard, CSL Behring, Australia

Patrick Schütz, CSL Behring AG, Switzerland Katherine Tull, Grifols Therapeutics Inc., USA

Ryan Dorfman, Haematologic Technologies, Inc., USA Jean Amiral, Hyphen Biomed, France

Marta José, Instituto Grifols, S.A. R&D, Spain Catherine Michalski, LFB-Biomédicaments, France Luis Figueiredo, NIBSC, UK

Martina Schwarz, Octapharma SAS, Austria Roni Mintz, Omrix, Israel

Steffen Rosén, Rossix AB, Sweden

Samuel Ling, Alison Jones, Renate Jones & Lu Liu, Therapeutic Goods Administration/OLSS, Australia

Mikhail Ovanesov, Yideng Liang & Samuel Woodle, US Food & Drug Administration (CBER), USA

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Appendix 3: Protocols for Collaborative Study

Value Assignment of the 1st International Standard for Activated Factor XI December 2013

CS504 Study Protocol

1 INTRODUCTION

In response to the urgent need of a reference preparation for activated factor XI (FXIa) to aid development of assay methods and harmonization of assay results for measurement FXIa in immunoglobulin (IgG) therapeutics, the World Health Organization established the International Reference Reagent for Activated Blood Coagulation Factor XI (FXIa), Human, in 2012. This reference reagent has proven to be very helpful, but the supply of this preparation is now running low. A collaborative study is now required to establish an international standard to ensure the availability of a reference standard and continuity of the unit defined by the International Reference Reagent.

There are two objectives to this study:

Primary: The collaborative study will value assign the functional activity of the proposed 1^st International Standard for Activated Blood Coagulation Factor XI, Human, relative to the International Reference Reagent for Activated Blood Coagulation Factor XI (FXIa), Human.

The performance of the candidate materials relative to several procoagulant IVIG samples will also be assessed. Please note that only results from functional activity methods specific for FXIa will be used for value assignment.

Secondary: This study also provides an opportunity to explore the performance of the FXIa candidates and their use in assessment of procoagulant activity by Non-activated Partial Thromboplastin Time (NAPTT) and Thrombin Generation Assay (TGA). For this purpose, a panel of IgG preparation will be included and the participants are requested to carry out either/both NAPTT and TGA on the FXIa candidates alongside the panel of IgG. The results will give an insight into the feasibility of using FXIa as a control for these procoagulant activity assays. This part of the study is optional, and additional samples have been included for this objective if the participant has agreed to take part.

Please read through this protocol before carrying out the study, if you are unclear on any aspect of the study please do get in contact (email address at the end).

2 SAMPLES FOR STUDY – PRIMARY OBJECTIVE

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CODE PREPARATION

S Proposed 1st International Reference Reagent for Factor XIa (11/236), 10 units/ampoule - 4 ampoules supplied.

A Factor XIa preparation – approx. 10 units/ampoule - 4 ampoules supplied B Factor XIa preparation – approx. 10 units/ampoule - 4 ampoules supplied C IVIG preparation containing medium procoagulant activity, 5% protein;

approx. 0.06 FXIa u/ampoule - 4 ampoules supplied.

D IVIG preparation containing high procoagulant activity, 5% protein;

E IVIG preparation containing low procoagulant activity, 5% protein; approx.

0.01 FXIa u/ampoule - 4 ampoules supplied.

F IVIG preparation containing high procoagulant activity, 5% protein;

Please also include any in-house FXIa reference reagent that you routinely use in your assay method. Note – the FXIa activity of the IVIG samples is an approximate value only

NB one set of samples have been provided for the primary objective.

SAMPLES FOR STUDY – SECONDARY OBJECTIVE

An additional set of samples, quantity as above, have been provided if you are participating in the study for the secondary objective. Due to the limit number of samples available only one set of samples can be provided even if you are carrying out both NAPTT and TGT. If available, please also include your own in-house FXIa reference reagent.

3 STORAGE AND RECONSTITUTION OF AMPOULES OF S, A, B, C, D, E and F Store all unopened ampoules at -20^oC or below. Ampoules should be allowed to warm to room temperature before reconstitution.

Directions for opening DIN ampoules

DIN ampoules have an ‘easy-open’ coloured stress point, where the narrow ampoule stem joins the wider ampoule body. Tap the ampoule gently to collect the material at the bottom (labelled) end. Ensure that the disposable ampoule safety breaker provided is pushed down on the stem of the ampoule and against the shoulder of the ampoule body. Hold the body of the ampoule in one hand and the disposable ampoule breaker covering the ampoule stem between the thumb

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and first finger of the other hand. Apply a bending force to open the ampoule at the coloured stress point, primarily using the hand holding the plastic collar.

Care should be taken to avoid cuts and projectile glass fragments that might enter the eyes, for example, by the use of suitable gloves and an eye shield. Take care that no material is lost from the ampoule and no glass falls into the ampoule. Within the ampoule is dry nitrogen gas at slightly less than atmospheric pressure. A new disposable ampoule breaker is provided with each DIN ampoule.

Reconstitute the ampoule contents by adding 1 ml of distilled water. Allow the ampoule to stand for 10 minutes at room temperature and aid reconstitution by gentle swirling. Transfer contents to a plastic tube and store on melting ice prior to the assays.

4 ASSAY DESIGN Primary Objective

The FXIa activity indicated in this protocol is an approximate value, and each laboratory should determine the appropriate dilution ranges for their FXIa quantitation test.

Once the appropriate dilutions of sample S have been determined, Assays for factor XIa should be carried out on each of the 4 sets. Please use your own in-house method. Four ampoules of each sample are provided for this. Each set should be tested on a different day (see schedule below). A balanced order of testing should be used. Please include your own in-house XIa reference, if available.

Day

1 S¹ A¹ B¹ C¹ D¹ E¹ F¹ XIa¹ XIa² F² E² D² C² B² A² S² Day

2 XIa¹ S¹ A¹ B¹ C¹ D¹ E¹ F¹ F² E² D² C² B² A² S² XIa² Day

3 F¹ XIa¹ S¹ A¹ B¹ C¹ D¹ E¹ E² D² C² B² A² S² XIa² F² Day

4 E¹ F¹ XIa¹ S¹ A¹ B¹ C¹ D¹ D² C² B² A² S² XIa² F² E²

Each letter refers to a set of three or more different dilutions (e.g. 1/10, 1/20, 1/40) and S1, S2 and A1, A2 etc. refer to separate sets of dilutions (replicates) made independently from the same ampoule. XIa refers to your own in-house reference for XIa. The range of dilutions should be chosen to lie on the most linear portion of the dose-response curve, and dilutions used should ensure that the responses (raw data points) from the standard and test preparatiuons overlap for allow for accurate potency estimation.

The assays should be completed within two hours of reconstitution of the samples. It is preferable to assay one group of samples per day.

For each sample please input dilutions, corresponding raw data and your estimated potency values against sample S into the provided excel workbook. Please also provide the expected activity of your in-house standard, if included.

Secondary Objective

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Carried out with the second set of samples

The FXIa potency estimation obtained from the primary objective can be used as a guide for dilution to use for the secondary objective. For your chosen method, TGT and/or NAPTT, we request that multiple dilutions are carried out for each sample, where feasible. Each dilution chosen for each sample will be individual and should ensure that the raw data points for each sample overlap with the data points for the standard (and preferably within a linear range). All dilutions made should be tested in replicate.

For example for S 1/400, 1/800, 1/1600, 1/3200 assuming 10 units/ampoule for C 1/2, 1/4, 1/8, 1/16 assuming 0.06 units/ampoule

(Please note this is an example and not a suggestion for dilution of S or C)

Testing by balanced order should be carried out, please use the same design as indicated in the primary objective of the study. The assays should be completed within two hours of reconstitution. It is preferable to assay one group of samples per day.

For each sample please input dilutions, corresponding raw data and your estimated potency values against sample S into the provided excel workbook. For the TGT results please fill in a separate workbook for each parameter (e.g. Lagtime, ETP, Peak Thrombin etc.). Please also provide the expected activity of your in-house standard, if included.

5 RESULTS

Please return completed excel results sheets by 28th Feb 2014, and send via email to:

[email protected]

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Appendix 4: Reagents, Methods and Instruments used by the Participants Lab Assay

Method

Machine In house

FXIa 01 Clotting

Sysmex CS2100i

Based on NAPTT with Platelet substitute and FXI deficient plasma

n/a

02 Clotting ACL-TOP using

APTT-SS HTI

03 Hyphen Plate Reader Kit

07 Hyphen Plate Reader n/a

09 Hyphen Plate Reader IVIG

10 Rossix Plate Reader Kit

11 Hyphen BSC/XP Kit

12 Hyphen Plate Reader Spiked IVIG

13a Rossix Plate Reader HTI

13b Hyphen Plate Reader HTI

14 In house Plate Reader HTI

16 Hyphen BCS/XP HTI

19 Hyphen ACL TOP n/a

22 Rossix Plate Reader Kit

23 Hyphen STAR Kit

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Appendix 5: Individual assay results- potencies relative to S, the International Reference Reagent for FXIa

Lab Sample Assay 1 Assay 2 Assay 3 Assay 4 Assay 5

1 A S nl np 9.92 9.88 .

1 B S nl 9.15 10.11 10.60 .

1 C S nl 0.05 0.07 0.07 .

1 D S nl 0.40 0.41 0.40 .

1 F S nl 0.57 0.74 0.70 .

2 A 10.19 9.98 9.76 9.67 .

2 B 9.82 9.62 9.28 10.01 .

2 C Nl nl nl nl .

2 D 3.10 3.05 3.15 3.25 .

2 F 4.70 4.98 4.89 4.76 .

3 A 9.63 nl np 9.06 .

3 B 11.01 nl 8.99 9.51 .

3 C 0.08 np 0.08 0.07 .

3 D 0.37 0.33 0.36 0.31 .

3 F 0.76 0.65 0.71 0.59 .

4 A 9.04 9.72 9.54 8.71 .

4 B 9.17 10.45 9.66 9.55 .

4 C 0.07 0.07 0.07 0.07 .

4 D 0.30 0.32 0.31 0.31 .

4 F 0.62 0.59 0.59 0.65 .

6 A 9.29 10.97 10.15 11.15 .

6 B 9.31 nl 9.82 9.99 .

6 C 0.06 0.07 0.06 0.08 .

6 D 0.31 0.33 0.36 nl .

6 F 0.62 0.74 0.73 0.78 .

7 A 8.99 np np 9.59 .

7 B 9.40 np np 8.84 .

7 C Nr 0.05 . 0.07 .

7 D Nr . . 0.31 .

7 F . nl nl 0.63 .

9 A 9.70 9.68 9.99 10.97 .

9 B 9.60 10.09 nl nl .

9 C 0.10 nl nl nl .

9 D 0.30 nl 0.35 0.31 .

9 F 0.66 0.61 0.57 0.66 .

10 A 9.14 9.74 9.94 10.49 .

10 B 9.69 9.71 9.98 10.52 .

10 C 0.06 0.11 0.06 0.07 .

10 D Nl 0.33 0.34 0.36 .

10 F 0.64 0.69 0.73 0.70 .

11 A 9.50 10.11 9.20 10.23 .

11 B 10.42 9.50 9.51 9.89 .

11 C 0.06 np 0.06 0.07 .

11 D 0.24 0.28 0.30 0.29 .

11 F Nl nl 0.64 np .

12 A 8.97 10.07 10.40 7.44 9.84

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Lab Sample Assay 1 Assay 2 Assay 3 Assay 4 Assay 5

12 B 9.56 9.37 10.72 8.74 8.99

12 C 0.05 0.07 0.10 0.07 0.06

12 D . 0.31 0.34 0.28 0.29

12 F 0.59 0.65 0.67 0.77 0.55

13a A 10.61 10.29 9.38 10.12 .

13a B 10.86 9.78 9.58 9.90 .

13a C 0.07 0.07 0.06 0.06 .

13a D 0.36 0.34 0.32 0.33 .

13a F 0.66 0.65 0.63 0.65 .

13b A 9.55 9.71 9.35 9.99 .

13b B 10.15 9.60 9.42 9.44 .

13b C 0.07 0.07 0.06 0.07 .

13b D 0.34 0.33 0.30 0.32 .

13b F 0.58 0.67 0.60 0.62 .

15 A 12.41 8.41 11.27 S nl .

15 B 11.11 nl nl S nl .

15 C 0.08 0.07 nl S nl .

15 D 0.34 0.28 nl S nl .

15 F 0.66 0.53 nl S nl .

16 A 9.89 S nl nl S nl .

16 B 9.55 S nl nl S nl .

16 C 0.07 S nl nl S nl .

16 D Nl S nl 0.32 S nl .

16 F Nl S nl 0.69 S nl .

19 A 10.45 10.19 10.21 10.02 .

19 B 10.09 10.27 9.20 9.16 .

19 C 0.08 0.08 0.08 0.08 .

19 D Np 0.32 0.29 0.23 .

19 F Np nl 0.61 nl .

22 A 9.89 9.53 9.50 nl .

22 B 9.97 9.58 9.52 9.84 .

22 C 0.06 0.06 nl 0.06 .

22 D 0.34 0.32 . 0.31 .

22 F 0.66 0.64 0.63 0.66 .

23 A 9.52 np 9.93 9.28 .

23 B 9.28 nl nl 10.63 .

23 C Np 0.09 nl 0.08 .

23 D 0.35 nl nl 0.34 .

23 F 0.72 np 0.68 0.63 .

nl: non-linear; S nl: sample S non-linear: np: non-parallel; nr: not in range of standard

Lab 14 - the majority of samples and assays from this lab gave non-linear dose response curves and were not analysed further.

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Appendix 6: Potency estimates relative to Sample A, the proposed International Standard, assuming an assigned value of 9.8 U/ampoule

Sample B

Lab Method N GM GCV

U/ampoule %

1 Clotting 3 10.37 3.3

2 Clotting 4 9.58 3.9

13b Chromogenic 4 9.80 5

Overall 17 9.80 4.5

Excl.CL 15 9.77 4.5

clotting

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Sample C

Lab Method N GM GCV

U/ampoule %

1 Clotting 2 0.07 -

10 Chromogenic 4 0.08 32.2 11 Chromogenic 3 0.06 2.9 12 Chromogenic 5 0.07 31.7 13a Chromogenic 4 0.06 2.9 13b Chromogenic 4 0.07 6.1

Overall 15 0.07 18.7

Excl. CL 14 0.07 19.5

clotting

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Sample D

Lab Method N GM GCV

U/ampoule %

1 Clotting 3 0.42 6.8

2 Clotting 4 3.14 4.8

Overall 16 0.37 78.0

Excl.CL 14 0.32 8.4

clotting

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Sample F

Lab Method N GM GCV

U/ampoule %

1 Clotting 3 0.69 5.5

2 Clotting 4 4.80 3.9

Overall 16 0.74 65.5

Excl.CL 14 0.65 6.9

clotting

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Appendix 6: Draft IFU

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