Final report on the evaluation of the WHO reference reagent for activated blood coagulation factor XI (FXIa), human

WHO/BS/2013.2230 Addendum 2 ENGLISH ONLY EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION Geneva, 21 to 25 October 2013

Final report on the evaluation of the WHO Reference Reagent for Activated Blood Coagulation Factor XI (FXIa), Human Helen Wilmot1,3, John Hogwood1, Peter Rigsby2 and Elaine Gray1 Haemostasis Section, 2Biostatistics Section, National Institute for Biological Standards and Control Potters Bar, Hertfordshire, EN6 3QG, UK 3 Principal Investigator 1

© World Health Organization 2013 All rights reserved. Publications of the World Health Organization are available on the WHO web site (www.who.int) or can be purchased from WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel: +41 22 791 3264; fax: +41 22 791 4857; e-mail: bookorders@who.int). Requests for permission to reproduce or translate WHO publications – whether for sale or for noncommercial distribution – should be addressed to WHO Press through the WHO web site (http://www.who.int/about/ licensing/copyright_form/en/index.html). The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement. The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. All reasonable precautions have been taken by the World Health Organization to verify the information contained in this publication. However, the published material is being distributed without warranty of any kind, either expressed or implied. The responsibility for the interpretation and use of the material lies with the reader. In no event shall the World Health Organization be liable for damages arising from its use. The named authors alone are responsible for the views expressed in this publication.

.

WHO/BS/2013.2230 Addendum 2 Page 2

Summary Twelve laboratories from nine different countries participated in a “fit for purpose” collaborative study on the Reference Reagent for Activated Blood Coagulation Factor XI (FXIa), 11/236. The study had two parts; Part A being a fit for purpose study to examine whether the material could serve as a reference for FXIa in IVIG and Part B examining the effect of different IVIG matrices on the performance of the material in the assays. Overall in Part A, intra-laboratory variability was quite high, however, most laboratories were able to quantify FXIa in IVIG samples, including those from different manufacturers. A coded duplicate of the reference material was included in this part of the study and it was reassuring to note that although the intra-lab variability as expressed by GCV was ranged from 2.15% to 17.23%, the inter-lab variability was low (GCV 5.55%) for this sample. In Part B, participants were asked to spike the reference material into their own IVIG samples at 3 different dilutions. The recovery of the spiked FXIa was then measured. The inter- and intra-laboratory variation of labs using FXIa-specific assays was low (around 10%) and these labs were able to recover close to the expected amount of FXIa in the samples. Overall the results support the Reference Reagent for FXIa as a reference reagent for different IVIG preparations and very well as a reagent to create IVIG matrix-specific references within each laboratory. It also serves to unite the FXIa units used by different laboratories, as previously there was no common unit for measurement of FXIa. The FXIa reference material was assigned as the WHO Reference Reagent for FXIa, with an arbitrary unitage of 10 units per ampoule in October 2012. This report provides the full results from the fit for purpose study carried out in 2012, which were not available in time for the ECBS report submission deadline in 2012.

Introduction Between 2008 and 2010, a cluster of thrombotic events was associated with the administration of intravenous immunoglobulins (IVIGs). Following investigation by the European Official Medicines Control Laboratories (OMCLs), European Medicines Agency (EMA), the United States Food and Drug Administration (US FDA) and the manufacturer, the thrombotic cause was identified as factor XIa, though other procoagulant components were not completely ruled out. Subsequently, IVIG products from one manufacturer were suspended. IVIGs from other manufacturers were also investigated, some of which were found to contain similar procoagulant activity. The European Pharmacopoeia monograph for Human Normal Immunoglobulin for Intravenous Administration was revised with effect from January 2012 and now requires step(s) in the production method that have been shown to remove thrombosis generating agents and that the product does not exhibit thrombogenic (procoagulant) activity. The European regulators requested the manufacturers submit batch data by July 2012 that supports the removal or absence of procoagulant activity from their products. An international collaborative study carried out by NIBSC in 2011 indicated that there is an urgent need to standardize assay methods for procoagulant activity in IVIG products. It was recognised that although FXIa is one of the major procoagulant components, it would be important to have orthogonal methods to detect thrombogenicity. The assay methods being considered are Non-Activated Partial Thromboplastin Time (NAPTT), Thrombin Generation Test (TGT) and FXIa assays. Currently, there are no common qualitative or quantitative reference materials or system suitability controls available to develop assay methods and harmonise the assay results for these tests. It was agreed by the stakeholders (OMCLs, regulatory bodies, WHO Collaborating Centres (Paul Ehrlich Institut (PEI), Center for Biologics Evaluation and Research (CBER), NIBSC) and

WHO/BS/2013.2230 Addendum 2 Page 3 manufacturers) that in order to ensure the safety of IVIG products, internationally harmonised reference materials should be developed for these tests. As FXIa is the major procoagulant component, a proposal has been made to produce an international standard for FXIa. However to produce a new well characterised international standard will take 2 to 3 years and therefore an interim material is required to cover this period. A proposal was made to produce an Reference Reagent (RR) for Blood Coagulation Factor XIa, Human. It is envisaged that this RR will be replaced by an International Standard for FXIa in 2 to 3 years. This interim reference reagent is assigned with an arbitrary unit of 10 u/ampoule by NIBSC and this study serves as a “fit for purpose” study to assess the suitability of the Reference Reagent as a standard for measurement of FXIa in IVIG products. A preliminary report1 with partial analysis of the collaborative study was submitted to ECBS in October 2012 and the ECBS endorsed the establishment of this candidate as the Reference Reagent for Activated Blood Coagulation Factor XI (FXIa), Human with the proviso that a report with full analysis of all the results and stability data should be presented to the ECBS in October 2013. This report covers the analysis of data submitted by all the participants, together with accelerated degradation and on–bench stability results.

Participants Twelve laboratories from 9 different countries (1 Austria, 1 Belgium, 1 Canada, 1 France, 1 Germany, 1 Spain, 1 Sweden, 2 UK, 3 USA) agreed to participate in the study. The participants included 3 diagnostics manufacturers, 5 therapeutic manufacturers and 4 regulatory authorities. A list of participants is given in Appendix 1 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.

The WHO Reference Reagent for Factor XIa, NIBSC code 11/236 The bulk for the RR was purchased and the certificate of analysis from the vendor indicated that it had a specific activity of 606 U/mg, based on the activity in factor XI clotting assays, where 1 unit equals FXI activity in 1 ml of normal plasma. 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 at NIBSC in 50 mM Tris, 150 mM NaCl, 5 mg/ml trehalose and 0.5% human serum albumin. The material was distributed in glass ampoules, filled and freeze-dried according to guidelines for production of international standards. The product characteristics are listed in the following table.

WHO/BS/2013.2230 Addendum 2 Page 4 NIBSC Code Presentation Filling date Number of Ampoules available Liquid filling weight (g) (n=587, measurements taken from all 3 pumps throughout the duration of the fill) CV of fill mass (%) Homogeneity of the fill by activity: 3 ampoules selected from the beginning of the fill and the end of the fill and were assayed against an in-house FXIa standard using an in-house method (adapted Biophen FIXa kit). 2 assays per ampoule were carried out. Effect of fill position was assessed by ANOVA of log potencies. Mean dry weight (g) (n=6) Mean head space oxygen (%) (n=12) Residual moisture (%) (n=11) Storage temperature Address of processing facility Address of present custodian 11/236 Sealed, glass 5 ml DIN ampoules 24th May 2012 2775 1.0082 0.3069 GCV p

FXIa

0.701

0.574

0.0260 (CV 0.95%) 0.53 (CV 37.13%) 0.189 (CV 16.06%) -20°C NIBSC, Potters Bar, EN6 3QG, UK NIBSC, Potters Bar, EN6 3QG, UK

Study design This was a two part study. PART A: A “fit for purpose” study for the WHO reference reagent for FXIa (11/236) assessing the performance in FXIa quantitative assays and the ability to serve as a reference reagent for FXIa in IVIG samples with various levels of FXIa activity. PART B: An optional study for the participants with an aim to investigate the effect of different IVIG matrices on the validity of the assays. In-house IVIG preparations were required for this part. Two groups of participants were recruited. The first group of participants was requested to carry out both Part A and Part B of the study, while a second group of participants carried out Part B only. Participants were requested to carry out four assays for FXIa using fresh ampoules of all the samples provided in each assay. Within each assay, participants were requested to assay at least three dilutions of each of the samples in replicate, according to balanced assay designs (study protocols shown in Appendix 2). Participants were requested to return raw data together with calculated estimates for all of the samples relative to sample S (the IRR, 11/236) from each individual assay.

Samples Part A: S: WHO Reference Reagent for Factor XIa (11/236) – 10 units/ampoule - 6 ampoules supplied. A: Factor XIa preparation- 10 units/ampoule – 4 ampoules supplied B: IVIG preparation containing high procoagulant activity, 5% protein (10/224) - 4 ampoules supplied.

WHO/BS/2013.2230 Addendum 2 Page 5 C: IVIG preparation containing moderate procoagulant activity, 5% protein (10/222) - 4 ampoules supplied. D: IVIG preparation containing low procoagulant activity, 5% protein (10/220) - 4 ampoules supplied. E: IVIG preparation containing moderate procoagulant activity, 5% protein (10/282) - 4 ampoules supplied.

Part B: S: WHO Reference Reagent for Factor XIa (11/236) – 10 units/ampoule - 6 ampoules supplied.

Assay methods 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 Lab3b, for example. A list of reagents, methods and instruments, together with their in-house FXIa standard and IVIGs used by the participants is given in Appendix 3. For Part A, Lab 1, 2, 3a and 4 carried out functional activity assays that were based on the conversion of FIX to FIXa. Lab 6a added a FXI/FXIa immune-capture step before detection of FXIa by chromogenic substrate. These assays are referred to in the subsequent Results and Discussion section as “specific tests”. Labs 3b and 5 carried out chromogenic substrate assay methods that could be influenced by other proteases and these are referred to as “other tests”. For part B, 4 laboratories used FXIa-specific chromogenic kits, which are referred to as “specific tests”. One lab (10) carried out a FXI clotting assay and Lab 12a used a fluorogenic substrate assay. Finally, two laboratories carried out ELISAs for FXIa.

Statistical analysis An independent statistical analysis of raw data was performed at NIBSC. Potency estimates of the test samples in the study, relative to the RR, were calculated by parallel-line analysis2 of log transformed assay response against log concentration, independently for each test sample included in each assay. Assay validity was assessed by analysis of variance and any deviations from linearity and parallelism were considered significant at the 1% level (p<0.01). Where significant deviations from the model appeared to result from underestimation of residual error, linearity was assessed by visual inspection of the plotted data and non-parallelism was assessed using deviations from linearity as an alternative residual error. Any assays rejected for deviations from linearity or parallelism are indicated in the tables of results. Results from all valid assays were combined to generate unweighted geometric mean potencies for each laboratory and these laboratory means were used to calculate overall unweighted geometric mean potencies. Variability between assays and laboratories has been expressed using geometric coefficients of variation (GCV = {10s-1}×100% where s is the standard deviation of the log10 transformed potencies). Laboratory 2 performed each assay using two microtitre plates, with all samples present on both plates. The data from each plate were therefore analysed separately, and the results from the two plates combined to give an overall potency estimate for each assay. Data from this laboratory were found not to lie in the linear portion of the dose-response curve, therefore the data from the highest dilution was excluded and the data were assessed for parallelism using two dilutions only.

WHO/BS/2013.2230 Addendum 2 Page 6 Laboratory 6a returned single data points for each dilution, therefore the assays were assessed by using deviations from linearity as the estimate of residual error. Laboratory 12a returned data from single dilutions only for all samples other than S, therefore the results could not be analysed by parallel line bioassay. The results presented are therefore the laboratory’s own calculated data. Data from part B, the spiking experiments, were evaluated by plotting the observed log potencies against their expected values and also assessing the differences between observed and expected values.

Results and discussion Part A The main aim of this study was to evaluate the performance of the RR for FXIa as a reference material for measurement of FXIa in IVIG products. This is the first reference material for FXIa. The collaborative study carried out in 2011/2012 showed the dynamic ranges of the assays and the amount of FXIa detected in the IVIG products. Based on this information, the RR has been assigned an arbitrary unitage of 10 u/ampoule. This unitage has no relationship with the International Unit of Factor XI assigned to the 1st International Standard for Blood Coagulation Factor XI, Plasma, 04/102. As this is quantitative measurement, it was important that the participants carried out quantitative methods for this study. The individual assay results from each lab are shown in Appendix 4a. With the exception of labs 3b, 5 and 6a, all laboratories returned results obtained using functional activity assays that were based on the activation of FIX, with FXa generation as the final measurement readout. Lab 6a did not use an assay based on FXa generation, but employed an antibody capture step (anti-FXI Ab) to make the assay specific, followed by use of a chromogenic substrate (S2288). Lab 3b used a fluorogenic substrate that directly interacts with FXIa. However, this substrate is not specific for FXIa and results can be influenced by the presence of kallikrein and other proteases. Similarly, lab 5 used a chromogenic substrate, S2366, which is also not specific for FXIa and can be influenced by other proteases. Tables 1 to 4 show the summarised data for samples A, B, C and E. Sample D was a low procoagulant activity IVIG sample and none of the laboratories reported quantifiable levels of FXIa in this sample. Each table shows the data from the specific assays (Labs 1, 2, 3a, 4 and 6a), together with the data from the other assays (Labs 3b and 5) and finally the combined data. Sample A (Table 1) was a coded duplicate of sample S, the RR, and the results therefore provide a good indication of the precision and accuracy within labs. As the RR has an arbitrary unitage of 10 u/ampoule, the potency estimates for sample A should be close to this assigned value. Table 1 and Figure 1 show clearly that the range of potency estimates for sample A calculated relative to sample S was around 10 u/amp, with a geometric mean of 10.03 u/amp and a GCV less than 5%. Therefore, overall agreement was good. However, the intra-lab variability could be improved upon, with only one of the labs performing specific tests having a GCV of less than 10% (Lab 4; 2.15%) and the range of potency estimates from 8.01 to 12.94 u/amp in individual assays. Samples B and E were high procoagulant activity IVIG samples from different manufacturers. Most laboratories were able to quantify FXIa in both samples (Tables 2 and 4 and Figures 2 and 4) and generated statistically valid assays, suggesting that sample S, the RR, would be a suitable calibrant for different IVIG preparations. For sample B, all the laboratories carrying out specific tests for FXIa were able to quantify FXIa, though the ranges of overall geometric means was quite wide (0.32 to 1.31 u/amp) and the %GCV was high overall. Apart from Lab 4, with a GCV

WHO/BS/2013.2230 Addendum 2 Page 7 of 1.8%, the %GCVs from within each laboratory were high (15.24 to 39.69), however it is encouraging that labs using similar methods (1, 2, 3a and 4) reported similar results (between 0.32 and 0.85 u/amp). For sample E, Labs 2 and 6a were not able to quantify FXIa. For Lab 6a the responses for sample E were outside the range of the standard curve. For Lab 2 the results were not parallel to the standard and this could be as a result of the doses for the standard and sample not lying in the most linear part of the response. The labs that could quantify FXIa were in relatively close agreement with each other, giving an overall value of 0.14 u/amp. Assays carried out by Labs 3b and 5 on samples B and E were not specific for FXIa and in general the results were found to be higher than those obtained using specific tests. Other proteases present in the IVIG samples could have contributed to these overall values. Samples C and D were produced from the same IVIG product as sample B. None of the labs were able to detect FXIa in sample D, which was a low procoagulant activity sample. Sample C had intermediate activity and only labs 1, 3a and 4 were able to quantify the FXIa content (Table 3 and Fig. 3). Lab 2 did show a dose response for sample C however the results were not parallel to the standard. The reason for this is unknown but it is unlikely that this was due to matrix effect as sample B, C and D were all produced from the same product. The interlab %GCV for labs 1, 3a and 4 was very high at 165% due to the results from Lab 4 being around 3 fold higher than for Labs 1 and 3a. Overall, the intra-laboratory variability was quite high for the IVIG samples, with only one laboratory consistently having a %GCV of <5. This could be partly due to the inexperience of the laboratories in carrying out these assays. However, it is encouraging that most laboratories were able to quantify FXIa in the high procoagulant activity samples and since these were from different manufacturers it does suggest that the FXIa RR can serve as a reference material for a range of IVIG preparations.

Part B Part B of the study was designed to assess how well the RR would perform as a reference in different IVIG preparations. For this part of the study, participants were asked to spike sample S into their own IVIG preparation at 1/50, 1/100 and 1/200 dilutions. The recovery of the FXIa from the spiked samples against the purified reference gives an indication of how the reference may be affected by the different IVIG matrices. Labs 7, 8, 9, 10, 11 and 12 participated in this part of the study. Lab 10 used the FXI clotting assay and spiked with FXIa dilutions of 1/5, 1/10 and 1/20. The individual assay results from each laboratory are shown in Appendix 4b. Labs 1, 2, 3a and 3b from part A also participated, but were asked to spike at FXIa at a single dilution of 1/100. Lab 6a also participated, spiking at a dilution of 1/10. On the whole, the %GCV was good, with most labs having intra-laboratory variability of less than 10%. Table 5 shows the summarised data from all labs for sample X (1/50 dilution of FXIa into IVG), Y (1/100 dilution) and Z (1/200 dilution). Although the inter-lab variation was high (up to 147%), the variation was reduced to around 10% when only FXIa specific assays were used in the calculations. Also when FXIa specific assays were used, the overall geometric mean for recovery of the spiked FXIa was close to that expected for each dilution. Figures 5 to 10 show the data from Labs 7 to 12a, with observed log potencies plotted against their expected values (a), and also the difference (observed minus expected) against the expected values (b). These figures can be used to assess how the assay results vary depending on the dilution used and also how the recovery of the spiked FXIa compares to that expected. Overall, the laboratories’ results did not vary much according to dilution (figures 5-10 (a)) and the difference between expected and observed was close to zero (Figures 5-10 (b)). Regression

WHO/BS/2013.2230 Addendum 2 Page 8 analysis of the data (from figures (a) in each case) was significant for laboratories 10 and 12a only (p<0.001 in both cases), suggesting that the recovery of activity in the spiked samples was not as expected for these assays. The results from Labs 9 and 10 (Figs. 7 and 8) indicate that there was a trend towards more variation in the potency estimates as the dilution factor increased. This could be due to the way in which the dilutions were prepared or due to the dynamic range of the assays. Since Lab 9 used the same assay kit as Labs 8 and 11, in this case it is probably due to the preparation of the dilutions. Lab 10 used the FXI clotting assay, spiking IVIG at 1/5, 1/10 and 1/20 dilutions. For this lab the assay variability appeared to increase with higher dilutions but it is interesting to note that the recovery was always less than expected. Lab 12a used a non-specific assay for FXIa, based on cleavage of chromogenic substrate SN13a. The data shown for Lab 12a is the laboratory’s own calculated data. Here, the assay variability did not appear to be affected by dilution, however there was an inhibitory effect on activity at lower dilutions (Figure 10 (b)) because the difference between observed and expected potency was closest to zero at the 1/200 dilutions. The inhibitory effect could either be a result of the chromogenic substrate binding competitively to a non-active substance, or a result of a component of the IVIG preparation inhibiting the FXIa activity at lower dilutions. Some inhibition of activity was also seen in data from Lab 6a (Appendix 4a, Table 7), who spiked FXIa at a 1/10 dilution into IVIG. In this case only around 75% recovery was observed. Several different IVIG preparations were used in this part of the study. The protein content and excipient for each preparation are detailed in the table in Appendix 3. The protein content varied from 5 to 20% and the excipients used included glycine, maltose, sorbitol and L-proline. The fact that all labs using specific FXIa assays had good recovery of FXIa when spiked at 1/50, 1/100 or 1/200 shows that the matrices of different IVIG preparations have minimal influence over the activity of FXIa in these assays and therefore this reference reagent will work well as a reference material when spiked into various different IVIG preparations. This suggests that using the reference material in this way provides an excellent reference material for a particular IVIG product. Two laboratories carried out ELISAs for FXI/FXIa. The data are presented in Tables 6 and 7 and represents that laboratory’s own calculated data. In the case of Lab 6b, the values are calculated relative to the laboratory’s own in-house standard and thus cannot be directly compared to the activity data from Lab 6a presented in Appendix 3. However, it was interesting to note that samples C and E appeared to have similar antigen values despite having quite different activities (Tables 3 and 4) and that samples B, C and D had decreasing amounts of antigen, which aligns well with the different overall potencies reported (Tables 2 and 3; no activity reported for sample D). Lab 12b used S as the standard in their assay and also included S as a duplicate sample (Table 8). Comparing the ELISA data to the data from the functional assay (Appendix 4b; Table 6), the overall antigen values agree with the overall potency estimates, with the exception of X (IVIG spiked with 1/50 FXIa), where the antigen value was twice that of the potency estimate. However, as discussed previously, the functional assay appeared to show inhibition, so that probably explains the observed differences.

Supplementary data provided by Lab 3c Laboratory 3 carried out the thrombin generation test on the samples, with the value estimated taken against their in-house standard for FXIa. The results are tabulated in Appendix 5. The data reported is the peak thrombin (nM) that each sample generated in congenital factor XI deficient plasma. The returned results have a GCV range from 19.8% to 42.6%. Re-analysis of

WHO/BS/2013.2230 Addendum 2 Page 9 samples against S using parallel line analysis reduced the GCV range to 13.0 to 28.7%. An internal IVIG sample included did not give measurable peak thrombin, but when spiked with 1/100 sample S a peak thrombin level was observed which gave a high GCV 89.3% which was not improved when recalculated against S, GCV 141.2%.

Conclusion Overall sample S, the WHO RR, performed well as a reference reagent for FXIa quantification in different preparations of IVIG in different laboratories and the excellent recovery in the spiking experiments also indicates the suitability of this reference reagent for measurement of FXIa present in IVIG products. The reference material also serves to resolve the difference in the unitage of FXIa used throughout different labs, as currently units vary between mass units and activity units. The reference material (11/236) was established as the WHO Reference Reagent for Activated Coagulation Factor XI, Human, in October 2012. Over 1000 ampoules of this RR have been issued and feedback from some of the Official Medicine control Laboratories (OMCLs) and manufacturers for IVIG products indicated that this RR is helpful in the harmonization of results from procoagulant activity assays generated within and between laboratories. The data from this study and the usage rate of this RR support the need for an international standard for FXIa.

Stability studies On-bench and accelerated degradation studies have been carried out. The results of the on-bench stability are shown below, with potencies representing 2 assays at each time-point and determined relative to a fresh ampoule of 11/236 at each time. The potency after 3 hours storage on ice overlaps well with that at 0 h, indicating the material is stable for at least 3 hours when kept on ice. Time on ice 0h 1h 2h 3h Potency u/ml – combined (95% confidence intervals) 9.77 (9.40-10.15) 9.94 (9.51-10.39) 9.40 (9.22-9.59) 9.80 (9.08-10.59)

Accelerated degradation studies have been performed after 3 and 9 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. Degradation studies are on-going.

WHO/BS/2013.2230 Addendum 2 Page 10 Storage temperature (°C) -150 -70 -20 4 20 37 Predicted loss per year (%) (relative to -150) <0.001 <0.001 0.001 0.067 1.089 14.302

A statement on the stability of this material has been inserted into the instructions for use (see appendix 6).

Participant responses All participants were sent the report for comment and all agreed with the proposal to establish 11/236 as the Reference Reagent for FXIa. Two participants suggested that a different unitage could be used (one suggesting 1 unit/ampoule, the other 100 unit/ampoule), however neither objected to 10 units per ampoule. NIBSC explained that at the start of the study the FXIa content of various IVIG products was unknown, hence 10 units was selected to give a wide enough scope to cover a range of eventualities. It was also important to distinguish the FXIa unit from that of the FXI plasma unit to avoid confusion. One participant also asked for comment on the variability of the results for the reference reagent, to which NIBSC responded that the coded duplicates (samples S and A) showed that variability was consistent with other collaborative studies and that the homogeneity of the batch was good.

Acknowledgments The authors would like to thank the participants of the collaborative study, many of whom completed the testing under tight timescales.

References 1. Elaine Gray, Helen Wilmot, John Hogwood and Peter Rigsby. Evaluation of the proposed WHO 1st Reference Reagent for Activated Blood Coagulation Factor XI (FXIa), Human. WHO/BS/2012.2206 2. Finney DJ. Statistical Methods in Biological Assay. 3rd Edition. London: Charles Griffin 1978.

WHO/BS/2013.2230 Addendum 2 Page 11 Table 1: Overall results for Sample A, a coded duplicate of Sample S Sample A GM 95% CL Specific Lab 1 10.25 8.71- 12.05 Tests Lab 2 9.38 7.28-12.09 Lab 3a 10.38 8.90- 12.10 Lab 4 9.62 9.30- 9.95 Lab 6a 10.67 8.48-13.41 Overall GM 10.05 9.40-10.75 (u/amp) Range (u/amp) 8.01-12.94 Other Tests Lab 3b 9.82 9.57- 10.08 Lab 5 10.16 10.55-10.81 Overall GM 9.82 NA (u/amp) Range (u/amp) 9.56-10.51 All Tests Overall GM 10.03 9.62-10.46 (u/amp) Range (u/amp) 8.01-12.94 %GCV 10.71 17.23 10.12 2.15 15.45 5.55 2.48 3.98 NA

4.64

GM: geometric mean; 95% CL: 95% confidence limits; %GCV: geometric coefficient of variation; NA: not applicable

Table 2: Overall results for Sample B, a high procoagulant activity IVIG Sample B GM 95% CL Specific Lab 1 0.37 Tests Lab 2 0.32 0.26-0.40 Lab 3a 0.53 0.38-0.76 Lab 4 0.85 0.82-0.89 Lab 6a 1.31 0.96-1.79 Overall GM 0.59 0.28-1.22 (u/amp) Range (u/amp) 0.27-1.66 Other Tests Lab 3b 1.26 0.97 - 1.62 Lab 5 Overall GM NA NA (u/amp) Range (u/amp) 1.16-1.60 All Tests Overall GM 0.67 0.35-1.26 (u/amp) Range (u/amp) 0.27-1.66

%GCV 15.24 39.69 1.80 21.52 79.66 17.44 NA

83.98

GM: geometric mean; 95% CL: 95% confidence limits; %GCV: geometric coefficient of variation; NA: not applicable

WHO/BS/2013.2230 Addendum 2 Page 12 Table 3: Overall results for Sample C, an intermediate procoagulant activity IVIG Sample C GM 95% CL %GCV Specific Lab 1 0.02 0.004-0.100 88.57 Tests Lab 2 NP Lab 3a 0.05 0.04 - 0.07 31.68 Lab 4 0.14 0.13-0.15 3.77 Lab 6a LOQ Overall GM 0.052 0.004-0.583 164.72 (u/amp) Range (u/amp) 0.01-0.14 Other Tests Lab 3b ODR NA NA Lab 5 LOQ NA NA Overall GM NA NA NA (u/amp) Range (u/amp) NA All Tests Overall GM 0.052 0.29-1.19 164.72 (u/amp) Range (u/amp) 0.01-0.14 GM: geometric mean; 95% CL: 95% confidence limits; %GCV: geometric coefficient of variation; NP: Not parallel; LOQ: Below limit of quantification; ODR: outside detection range; NA: not applicable Table 4: Overall results for Sample E, a high procoagulant activity IVIG Sample E GM 95% CL Specific Lab 1 0.08 0.057- 0.1018 Tests Lab 2 NP Lab 3a 0.14 0.11 - 0.18 Lab 4 0.23 0.23 - 0.24 Lab 6a LOQ Overall GM 0.137 0.07-0.51 (u/amp) Range (u/amp) 0.07-0.24 Other Tests Lab 3b 1.17 0.97-1.41 Lab 5 4.30 3.87-4.81 Overall GM NA NA (u/amp) Range (u/amp) 0.99-4.72 All Tests Overall GM 0.419 0.05-3.22 (u/amp) Range (u/amp) 0.07-4.72

%GCV 15.62 16.569 2.49 69.61 12.49 7.05 NA

416.35

GM: geometric mean; 95% CL: 95% confidence limits; %GCV: geometric coefficient of variation; NP: Not parallel; LOQ: Below limit of quantification; NA: not applicable

WHO/BS/2013.2230 Addendum 2 Page 13

Figure 1: Potency estimates from each laboratory for Sample A. The geometric mean from each laboratory is indicated by the horizontal line.

Figure 2: Potency estimates from each laboratory for Sample B. The geometric mean from each laboratory is indicated by the horizontal line.

WHO/BS/2013.2230 Addendum 2 Page 14

Figure 3: Potency estimates from each laboratory for Sample C. The geometric mean from each laboratory is indicated by the horizontal line.

Figure 4: Potency estimates from each laboratory for Sample E. The geometric mean from each laboratory is indicated by the horizontal line.

WHO/BS/2013.2230 Addendum 2 Page 15 Table 5: Summary of results from FXIa spiked in IVIG Assay results in u/ml IVIG + 1/100 FXIa (Y) 0.100 0.11 0.12 0.13 0.099 0.098 0.097 0.103 0.075 0.102 17.92 0.089-0.117 0.107 11.95

Expected recovery Lab 1 Lab 2 Lab 3a Lab 7 Lab 8 Lab 9 Lab 11 Lab 12a* GM (overall) %GCV (overall) 95% CL (overall) GM (specific assays) %GCV (specific assays) 95% CL (specific 0.187-0.220 0.096-0.119 0.044-0.057 assays) * Non-specific assay. GM: geometric mean; %GCV: geometric coefficient of variation; 95% CL: 95% confidence limits Lab 10 used dilutions of 1/5, 1/10 and 1/20 and therefore the data is not shown above. Lab 3b took part (1/100 dilution only) but the results were outside the detection range and are therefore not shown.

IVIG + 1/50 FXIa (X) 0.200 0.200 0.198 0.219 0.197 0.027 0.135 146.92 0.044-0.417 0.203 5.12

IVIG + 1/200 FXIa (Z) 0.050 0.050 0.053 0.046 0.055 0.051 0.050 6.98 0.046-0.055 0.050 8.1

WHO/BS/2013.2230 Addendum 2 Page 16 (a)

Lab 7 0

log u/ml observed

-0.4 y = 0.9975x - 0.0029 R² = 0.9974

-0.8

-1.2 -1.2 -0.8 -0.4 log u/ml expected 0

-1.6

-1.6

(b)

Lab 7 0.5 0.4 0.3 0.2 0.1 0 -0.1 0 -0.2 -0.3 -0.4 -0.5 obs-exp (log u/ml)

-1.4

-1.2

-1

-0.8

-0.6

-0.4

-0.2

log u/ml expected

Figure 5: Plots of (a) expected versus observed log u/ml and (b) difference versus expected log u/ml for IVIG samples spiked with 0.2, 0.1 and 0.05 u/ml FXIa (Lab 7)

WHO/BS/2013.2230 Addendum 2 Page 17 (a)

Lab 8 0 -0.2 log u/ml observed -0.4 y = 0.9388x - 0.0538 R² = 0.988 -0.6 -0.8 -1 -1.2 -1.4 -1.2 -1 -0.8 -0.6 -0.4 -1.4 -0.2 0

log u/ml expected

(b)

Lab 8 0.5 0.4 0.3 0.2 obs-exp 0.1 0 -1.2 -1 -0.8 -0.6 -0.4 -0.2 -0.1 0 -0.2 -0.3 -0.4 log u/ml expected -0.5

-1.4

Figure 6: Plots of (a) expected versus observed log u/ml and (b) difference versus expected log u/ml for IVIG samples spiked with 0.2, 0.1 and 0.05 u/ml FXIa (Lab 8)

WHO/BS/2013.2230 Addendum 2 Page 18 (a)

Lab 9 0 -0.2 log u/ml observed -0.4 y = 1.1201x + 0.1188 R² = 0.9712 -0.6 -0.8 -1 -1.2

-1.4 -1.6 -1.4 -1.2 -1 -0.8 -0.6 -0.4 -1.6 -0.2 0

log u/ml expected

(b)

Lab 9 0.5 0.4 0.3 0.2 obs-exp 0.1

0 -1.2 -1 -0.8 -0.6 -0.4 -0.2 -0.1 0 -0.2 -0.3 -0.4 log u/ml expected -0.5

-1.4

Figure 7: Plots of (a) expected versus observed log u/ml and (b) difference versus expected log u/ml for IVIG samples spiked with 0.2, 0.1 and 0.05 u/ml FXIa (Lab 9)

WHO/BS/2013.2230 Addendum 2 Page 19 (a)

Lab 10 0.4 log u/ml observed y = 1.1054x - 0.0785 R² = 0.9816 0.2 0 -0.2 -0.4 -0.6

-0.4

-0.2

0 log u/ml expected

0.2

0.4

(b)

Lab 10 0.5 0.3 obs-exp 0.1 -0.3 -0.2 -0.1 -0.1 0 -0.3 -0.5 log u/ml expected 0.1 0.2 0.3 0.4

-0.4

Figure 8: Plots of (a) expected versus observed log u/ml and (b) difference versus expected log u/ml for IVIG samples spiked with 0.2, 0.1 and 0.05 u/ml FXIa. Please note lab 10 carried out FXI clotting assay with spiked FXIa at 1/5, 1/10 and 1/20 (Lab 10)

WHO/BS/2013.2230 Addendum 2 Page 20 (a)

Lab 11 0 -0.2 log u/ml observed -0.4 y = 0.9158x - 0.066 R² = 0.9699 -0.6 -0.8 -1 -1.2 -1.4 -1.4 -1.2 -1 -0.8 -0.6 -0.4 -0.2 0 log u/ml expected

(b)

Lab 11 0.5 0.3 obs-exp

0.1 -1.2 -1 -0.8 -0.6 -0.4 -0.2 -0.1 0 -0.3 log u/ml expected -0.5

-1.4

Figure 9: Plots of (a) expected versus observed log u/ml and (b) difference versus expected log u/ml for IVIG samples spiked with 0.2, 0.1 and 0.05 u/ml FXIa (Lab 11)

WHO/BS/2013.2230 Addendum 2 Page 21 (a)

Lab 12 0 -0.2 log u/ml observed -0.4 -0.6 -0.8 -1 -1.2 -1.4 -1.6 -1.4 -1.2 -1 -0.8 -0.6 -0.4 -1.6 -0.2 0

y = 0.4719x - 0.8008 R² = 0.9672

log u/ml expected

(b)

Lab 12 0.5 0.4 0.3 0.2 0.1 0 -0.1 0 -0.2 -0.3 -0.4 -0.5

obs-exp

-1.4

-1.2

-1

-0.8

-0.6

-0.4

-0.2

Log u/ml expected

Figure 10: Plots of (a) expected versus observed log u/ml and (b) difference versus expected log u/ml for IVIG samples spiked with 0.2, 0.1 and 0.05 u/ml FXIa. Data represents the laboratory’s own calculated data (Lab 12a)

WHO/BS/2013.2230 Addendum 2 Page 22 Table 6: Lab 6b ELISA data (relative to in-house FXIa standard) FXI total antigen (ng/ml) Sample mean S.D. S 2011.2 141.5 A 2119.1 108.0 B 1804.1 51.0 C 647.2 35.7 D 523.6 71.3 E 694.9 74.8 IVIG 405.8 30.1 S.D.: standard deviation Table 7: Lab 12b ELISA data (relative to S) u/amp Sample Assay 1 Assay 2 Assay 3 Assay 4 GM %GCV 95% CL W 0.02 0.04 0.02 0.05 0.029 0.014-0.063 60.55 X 0.164 0.218 0.096 0.174 0.156 0.089-0.271 41.54 Y 0.078 0.110 0.055 0.051 0.070 0.039-0.122 42.37 Z 0.045 0.075 0.035 0.077 0.054 0.029-0.101 47.55 IHIXa 0.35 0.37 0.31 0.35 0.344 0.305-0.387 7.75 S 11.68 12.03 9.01 10.00 10.61 8.55-13.16 14.53 GM: geometric mean; 95% CL: 95% confidence limits; %GCV: geometric coefficient of variation; IHXIa: in-house FXIa preparation (different preparation to that used in the functional assay in Appendix 4).

WHO/BS/2013.2230 Addendum 2 Page 23

Appendix 1: Participants Michael Etscheid and Nathalie Beer, Paul Ehrlich Institute, Lange, Germany Tenzin W. Lhakhang, Richard Jenny and Ryan Dorfman, Haematologic Technologies, Inc., Vermont, USA Berta Pons and Marta José, Instituto Grifols SA, Barcelona, Spain Yideng Liang, Samuel Woodle and Mikhail Ovanesov, CBER/FDA, Bethesda, USA Helen Wilmot, NIBSC, Potters Bar, UK Steffen Rosén, Pia Bryngelhed and Per Rosén, Rossix, Molndal, Sweden Rebecca Scott, Tara Dolan, Myfanwy Owen, Martyn Paddick and John More, Bio Products Laboratory, Elstree, UK Geoffrey Pot, Iris Timmermans and Peter Turecek, Baxter Bioscience, Lessines, Belguim Dagmar Krause, Martina Schwarz, Gerda Wiry, Andrea Buchacher, Octapharma Pharmazeutika Produktions GesmbH, Vienna, Austria Russell Pronyk and Derek Toth, Cangene Corporation, Winnipeg, Canada Glenn Stevenson, Melanie Williams and Pete Vandeberg, Grifols Therapeutics Inc., Raleigh, USA Valérie Lievre and Laurent Fleury, Agence nationale de sécurité du médicament et des produits de santé, Saint-Denis cedex, France

WHO/BS/2013.2230 Addendum 2 Page 24

Appendix 2: Protocols for Collaborative Study Collaborative Study on the proposed 1st WHO Reference Reagent for Factor XIa. CS487 Study Protocol A 1 INTRODUCTION

Following the reports of thromboembolic events associated with the clinical use of IVIG, investigations indicated that the procoagulant component could be FXIa. Currently a reference material is not available to aid development of assay methods and to improve intra- and interlaboratory agreement on the measurement of FXIa. A reference standard for FXIa is urgently needed to ensure harmonisation of measurement of FXIa in IVIG products. NIBSC has produced a FXIa reference reagent as an interim measure prior to the development of an International Standard. This interim reference reagent will be assigned with an arbitrary unit by NIBSC and this study serves as a “fit for purpose” study to assess the suitability of the proposed Reference Reagent as a standard for measurement of FXIa in IVIG products. There are two parts to the study: PART A: A “fit for purpose” study for the proposed WHO reference reagent for XIa (11/236) assessing the performance in FXIa quantitative assays and the ability to serve as a reference reagent for FXIa in IVIG samples with various levels of FXIa activity. PART B: An optional study to investigate the effect of different IVIG matrices on the validity of the assays. For this part, in-house IVIG preparations must be available for use. Part B is optional; however we would like to include as many participants as possible to take part. Part B (if chosen) must be carried out at the same time (within the same assays) as Part A. 2 SAMPLES FOR ASSAY – PART A

CODE

PREPARATION

S Proposed 1st Reference Reagent for Factor XIa (11/236) – 10 units/ampoule - 6 ampoules supplied. Two extra ampoules are provided for pre-optimisation of your established method(s) to establish the working dilutions necessary for optimal performance. A Factor XIa preparation- 10 units/ampoule – 4 ampoules supplied

B IVIG preparation containing high procoagulant activity, 5% protein (10/224) - 4 ampoules supplied. C IVIG preparation containing moderate procoagulant activity, 5% protein (10/222) - 4 ampoules supplied.

WHO/BS/2013.2230 Addendum 2 Page 25 D IVIG preparation containing low procoagulant activity, 5% protein (10/220) - 4 ampoules supplied. E IVIG preparation containing moderate procoagulant activity, 5% protein (10/282) - 4 ampoules supplied. IHXIa Participants’ own in-house FXIa standard, if used routinely SAMPLES FOR ASSAY – PART B Your chosen IVIG sample (fresh sample each day) Your chosen IVIG sample spiked at 1/100 with sample S (prepared fresh each day) For Part B, please include an IVIG sample (same batch in each assay), both spiked and unspiked with a 1/100 dilution of sample S before dilution for the assay. For example, if the assay requires 1/10, 1/20 and 1/40 working dilutions, please spike the undiluted sample 1/100 with sample S (11/236), before performing the 1/10, 1/20 and 1/40 dilutions. Please use the same ampoule of S as the standard in your assay. 3 STORAGE AND RECONSTITUTION OF AMPOULES OF S, A, B, C, D AND E

Store all unopened ampoules at -20oC or below. Ampoules should be allowed to warm to room temperature before reconstitution. Directions for opening DIN ampoules DIN ampoules have an ‘easy-open’ coloured stress point, where the narrow ampoule stem joins the wider ampoule body. Tap the ampoule gently to collect the material at the bottom (labelled) end. Ensure that the disposable ampoule safety breaker provided is pushed down on the stem of the ampoule and against the shoulder of the ampoule body. Hold the body of the ampoule in one hand and the disposable ampoule breaker covering the ampoule stem between the thumb 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 at 4 oC prior to the assays. 4 ASSAY DESIGN

PART A Two extra ampoules of S are provided; please use these to determine the most appropriate dilutions for sample S for use in your in-house assay method before beginning the study.

WHO/BS/2013.2230 Addendum 2 Page 26 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, ampoule set 1 Day 2, ampoule set 2 Day 3, ampoule set 3 Day 4, ampoule set 4 S1 IHXI a1 E1 D1 A1 S1 IHXI a1 E1 B1 A1 S1 IHXIa 1

C 1

D 1

E1 D 1

IHXIa 1

IHXIa 2

E2 D 2

D 2

C 2

B2 A2 S2 IHXIa 2

A2 S2 IHXIa 2

S2 IHXIa 2

B 1

C 1

E1 D1 C1

E2 D2 C2

C 2

B 2

A 1

B 1

C 1

C 2

B 2

A 2

E2 D2

S1

A 1

B 1

B 2

A 2

S2

E2

Each letter refers to a set of three 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. IHXIa 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 relationship. The same range of dilutions should be used for all three materials (S, A, B, C, D, E). The assays should be completed within two hours of reconstitution. It is preferable for the whole study to be carried out over four days. PART B – to be carried out at the same time as Part A For Part B of the collaborative study, please assay your chosen IVIG product (a fresh sample per day) within the same assays as above. The samples should be assayed both with and without XIa (sample S) spiked into the sample at a 1/100 dilution. This spiking should be carried out on undiluted material and then the sample diluted for assay as per your normal method. Please use the same ampoule of S as the standard in your assay. The testing schedule is shown on the following page.

5

RESULTS

Raw data (e.g. absorbance) should be recorded on the results sheet. Please return your raw data and calculated potency estimates by 13th July 2012 to: helen.wilmot@nibsc.hpa.org.uk

WHO/BS/2013.2230 Addendum 2 Page 27 Testing schedule if taking part in both parts A and B: Day 1, Ampoule set 1 Day 2, Ampoule set 2 Day 3, Ampoule set 3 Day 4, Ampoule set 4 S1 IVIG+ XIa1 IVIG1 IHXIa 1

A1 S1 IVIG+ XIa1 IVIG1

B1 A1 S1 IVIG+ XIa1

C1 B1 A1 S1

D1 C1 B1 A1

E1 D1 C1 B1

IHXIa1 E1 D1 C1

IVIG1 IHXIa 1

IVIG+ XIa1 IVIG1 IHXIa1 E1

IVIG+ XIa2 IVIG2 IHXIa2 E2

IVIG2 IHXIa 2

IHXIa 2

E2 D2 C2 B2

D2 C2 B2 A2

C2 B2 A2 S2

B2 A2 S2 IVIG+ XIa2

A2 S2 IVIG+ XIa2 IVIG2

S2 IVIG+ XIa2 IVIG2 IHXIa2

E2 D2 C2

E1 D1

E2 D2

Each letter refers to a set of three 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. IHXIa refers to your own in-house reference for XIa; IVIG refers to your in-house IVIG preparation and IVIG+XIa refers to your in-house IVIG preparation spiked 1/100 with sample S (XIa). The range of dilutions should be chosen to lie on the most linear portion of the dose-response relationship.

WHO/BS/2013.2230 Addendum 2 Page 28 Collaborative Study on the proposed 1st WHO Reference Reagent for Factor XIa. CS487 Study Protocol (B) 1 INTRODUCTION

Following the reports of thromboembolic events associated with the clinical use of IVIG, investigations indicated that the procoagulant component could be FXIa. Currently a reference material is not available to aid development of assay methods and to improve intraand inter-laboratory agreement on the measurement of FXIa. A reference standard for FXIa is urgently needed to ensure harmonisation of measurement of FXIa in IVIG products. NIBSC has produced a FXIa reference reagent as an interim measure prior to the development of an International Standard. This interim reference reagent will be assigned with an arbitrary unit by NIBSC and this study serves as a “fit for purpose” study to assess the suitability of the proposed Reference Reagent as a standard for measurement of FXIa in IVIG products. 2. AIM OF STUDY

To investigate the effect of different IVIG matrices on the validity of quantitative FXIa assays. For this study, in-house IVIG preparations must be available for use. 3 SAMPLES FOR ASSAY

Please use the same batch of IVIG product throughout the whole study Your chosen IVIG sample (fresh sample each day) Your same chosen IVIG sample spiked at 1/50, 1/100 and 1/200 with sample S (prepared fresh each day) Your in-house FXIa standard Please assay your chosen IVIG product (a fresh sample per day). The samples should be assayed both with and without XIa (sample S) spiked into the sample at a 1/50, 1/100 and 1/200 dilution. This spiking should be carried out on undiluted material and then the sample diluted for assay as per your normal method. For example, if the assay requires 1/10, 1/20 and 1/40 working dilutions, please spike the undiluted sample 1/50, 1/100 or 1/200 with sample S (11/236), before performing the 1/10, 1/20 and 1/40 dilutions. Please use the same ampoule of S for spiking as the standard in your assay. CODE PREPARATION

S (11/236) Proposed 1st WHO Reference Reagent for Factor XIa (11/236) – 10 units/ampoule - 6 ampoules supplied. Two extra ampoules are provided for preoptimisation of your established method(s) to establish the working dilutions necessary for optimal performance.

WHO/BS/2013.2230 Addendum 2 Page 29 W X Y Z Your chosen IVIG product Spike sample S at 1 in 50 dilution to your chosen IVIG product Spike sample S at 1 in 100 dilution to your chosen IVIG product. Spike sample S at 1 in 200 dilution to your chosen IVIG product.

IHXIa Participants’ own in-house FXIa standard, if used routinely 4 STORAGE AND RECONSTITUTION OF AMPOULES OF S

Store all unopened ampoules at -20oC or below. Ampoules should be allowed to warm to room temperature before reconstitution. Directions for opening DIN ampoules DIN ampoules have an ‘easy-open’ coloured stress point, where the narrow ampoule stem joins the wider ampoule body. Tap the ampoule gently to collect the material at the bottom (labelled) end. Ensure that the disposable ampoule safety breaker provided is pushed down on the stem of the ampoule and against the shoulder of the ampoule body. Hold the body of the ampoule in one hand and the disposable ampoule breaker covering the ampoule stem between the thumb 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 at 4 oC prior to the assays. 5 ASSAY DESIGN

Two extra ampoules of S are provided; please use these to determine the most appropriate dilutions for sample S for use in your in-house assay method before beginning the study. 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.

WHO/BS/2013.2230 Addendum 2 Page 30 Day 1, ampoule set 1 Day 2, ampoule set 2 Day 3, ampoule set 3 Day 4, ampoule set 4 S1 IHXI a1 Z1 Y1 W1 S1 IHXI a1 Z1 X1 W1 S1 IHXI a1 Y1 X1 W1 S1 Z1 Y1 X1 W1 IHXI a1 Z1 Y1 X1 IHXI a2 Z2 Y2 W1 Z2 Y2 X2 X1 Y2 W1 W2 S2 X2 X1 S2 IHXI a2 W2 S2 IHXI a2 Z2 S2 IHXI a2 Z2 Y2

Each letter refers to a set of three different dilutions (e.g. 1/10, 1/20, 1/40) and S1, S2 and W1, W2 etc. refer to separate sets of dilutions (replicates) made independently from the same ampoule. IHXIa 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 relationship. The same range of dilutions should be used for all samples. The assays should be completed within two hours of reconstitution. It is preferable for the whole study to be carried out over four days. 6 RESULTS

Raw data (e.g. absorbance) should be recorded on the results sheet. Please return your raw data and calculated potency estimates by 20th July 2012 to: helen.wilmot@nibsc.hpa.org.uk

WHO/BS/2013.2230 Addendum 2 Page 31

Appendix 3: Reagents, Methods and Instruments used by the Participants Lab number 1 2 Method Biophen FXIa kit In-house method based on addition of recombinant FIX to Biophen FIXa kit Biophen FXIa kit In-house method based on mixing samples 50%/50% with fluorogenic substrate SN-13a in a flat-bottom half-area microplate In-house method based on addition of plasma derived FIX to Biophen FIXa kit In-house method using S2366 In-house method using Ab capture followed by S2288 FXI ELISA (affinity Biologicals) Rossix Rox Factor XIa Biophen FXIa kit Biophen FXIa kit In-house Factor XI assay Biophen FXIa kit ANSN-based Fluorogenic substrate #SN13a AssayMax Human FXI ELISA kit, AssayPro Machine Plate reader MTP-reader temperature controlled BioTek Synergy H4 Hybrid Plate Reader In-house XIa reference standard CAL from Biophen kit 5% IVIG Protein content and excipient of IVIG 10%; Glycine 5%; Maltose 20%; L-proline, polysorbate 80

3a

Purified FXIa (HTI)

3b

BioTek Synergy H4 Hybrid Plate Reader

Purified FXIa (HTI)

20%; L-proline, polysorbate 80

4

ACL TOP 500 Plate reader Versamax microplate reader Tecan Sunrise microplate reader Tecan Sunrise Thermomax Plate reader Plate reader SpectraMAX M5 Plate Reader ACL TOP 700 BMG Fluostar Optima Spectramax M2e Microplate Spectrophotometer Spectramax M2e Microplate Spectrophotometer

Purified FXIa (ERL)

Part A participant

5 6a 6b 7 8 9 10 11 12a

Purified FXIa (Kordia) Purified FXIa (Calbiochem) Purified FXIa (Calbiochem) In-house purified FXIa calibrator CAL from Biophen kit CAL from Biophen kit HemosIL Calibration Plasma Purified FXIa (ERL) Purified FXIa (HTI) Human FXI from AssayMax ELISA kit

Part A participant 5%; Glycine 5%; Glycine 5%; Maltose 10%; Sorbitol 10%; Glycine 10%; Glycine, NaCl, Poly 80 5%; Sorbitol 5%; Maltose, Polysorbate 80 5%; Maltose, Polysorbate 80

12b

WHO/BS/2013.2230 Addendum 2 Page 32

Appendix 4a: Assay results from each laboratory, Part A Table 1: Lab 1 - Potency estimates and intra-laboratory GCV for test samples relative to sample S, the RR for FXIa u/ml Sample %GCV Assay 1 Assay 2 Assay 3 Assay 4 GM 95% CL A 11.71 9.15 10.27 10.04 10.25 8.71 - 12.05 10.71 B NP NP 0.37 0.38 0.37 C 0.01 NP 0.03 0.03 0.02 0.004 - 0.100 88.57 D LOQ LOQ LOQ LOQ E NP 0.07 0.09 0.09 0.08 0.057 - 0.102 15.62 IHXIa 0.04 NP 0.04 0.04 0.04 IVIG LOD LOD LOD LOD IVIG+XI 0.10 0.11 0.11 0.11 0.11 0.10 - 0.12 4.88 a GM: geometric mean; 95% CL: 95% confidence limits; %GCV: geometric coefficient of variation; NP: Not parallel; LOQ: Below limit of quantification; LOD: Below limit of detection; IHXIa: in-house FXIa preparation; IVIG+XIa: IVIG spiked with 1/100 of sample S Table 2: Lab 2 - Potency estimates and intra-laboratory GCV for test samples relative to sample S, the RR for FXIa u/ml Assay Sample %GCV Assay 1 Assay 2 Assay 4 GM 95% CL 3 A 8.59 11.50 8.01 9.79 9.38 7.28 – 12.09 17.23 B 0.27 0.31 0.33 0.38 0.32 0.26 - 0.40 15.24 C NP NP NP NP D LOQ LOQ LOQ LOQ E NP NP NP NP IHXIa 0.29 0.35 0.25 0.32 0.30 0.24 - 0.38 15.50 IVIG LOQ LOQ LOQ LOQ IVIG+XI 0.09 0.11 0.14 0.16 0.12 0.08 - 0.18 29.10 a GM: geometric mean; 95% CL: 95% confidence limits; %GCV: geometric coefficient of variation; NP: Not parallel; LOQ: Below limit of quantification; IHXIa: in-house FXIa preparation; IVIG+XIa: IVIG spiked with 1/100 of sample S

WHO/BS/2013.2230 Addendum 2 Page 33 Table 3: Lab 3a - Potency estimates and intra-laboratory GCV for test samples relative to sample S, the RR for FXIa Sample A B C D E IVIG IVIG+XIa Assay 1 11.47 0.75 0.06 LOQ 0.18 LOQ 0.26 Assay 2 10.33 0.57 0.04 LOQ 0.13 LOQ 0.17 Assay 3 10.74 0.59 0.05 LOQ 0.13 LOQ 0.09 Assay 4 VR 0.28 VR LOQ VR LOQ 0.28 Assay 5 NP NP NL LOQ NL LOQ NP u/ml Assay 6 NL NP 0.09 LOQ NL LOQ NP Assay 7 9.12 0.59 0.05 LOQ 0.14 LOQ 0.08 Assay 8 NP 0.56 0.05 LOQ NL LOQ 0.06 GM 10.38 0.53 0.05 LOQ 0.14 LOQ 0.13 95% CL 8.90 - 12.10 0.38 - 0.76 0.04 - 0.07 0.11 - 0.18 0.07 - 0.26 %GCV 10.12 39.69 31.68 16.569 91.42

GM: geometric mean; 95% CL: 95% confidence limits; %GCV: geometric coefficient of variation; NP: Not parallel; NL: not linear; VR: highly variable results; LOQ: Below limit of quantification; IHXIa: in-house FXIa preparation; IVIG+XIa: IVIG spiked with 1/100 of sample S Table 4: Lab 3b - Potency estimates and intra-laboratory GCV for test samples relative to sample S, the RR for FXIa Sample A B C D E IVIG IVIG+XIa Assay 1 NT NT NT NT NT NT NT Assay 2 NT NT NT NT NT NT NT Assay 3 9.78 1.60 ODR ODR 1.28 ODR ODR Assay 4 9.65 1.16 ODR ODR 1.18 ODR ODR Assay 5 9.72 ODR ODR ODR NL ODR ODR u/ml Assay 6 9.56 1.16 ODR ODR 0.99 ODR ODR Assay 7 10.17 1.16 ODR ODR NL ODR ODR Assay 8 10.07 ODR ODR ODR 1.26 ODR ODR GM 9.82 1.26 1.17 95% CL 9.57 - 10.08 0.97 - 1.62 0.97 - 1.41 %GCV 2.48 17.44 12.49 -

NT: not tested; GM: geometric mean; 95% CL: 95% confidence limits; %GCV: geometric coefficient of variation; NL: not linear; ODR: Outside detection range; IHXIa: in-house FXIa preparation; IVIG+XIa: IVIG spiked with 1/100 of sample S Table 5: Lab 4 - Potency estimates and intra-laboratory GCV for test samples relative to sample S, the RR for FXIa u/ml Sample Assay 1 Assay 2 Assay 3 Assay 4 %GCV GM 95% CL A 9.34 9.82 9.69 9.64 9.62 9.30 - 9.95 2.15 B 0.85 0.84 0.87 0.85 0.82 - 0.89 1.80 C 0.13 0.14 0.14 0.14 0.14 0.13 - 0.15 3.77 D LOD LOD LOD LOD E 0.23 0.24 0.23 0.24 0.23 0.23 - 0.24 2.49 727.80 IHXIa NT 775.32 806.25 843.14 807.76 4.29 896.51 GM: geometric mean; 95% CL: 95% confidence limits; %GCV: geometric coefficient of variation; LOQ: Below limit of quantification; NT: not tested; IHXIa: in-house FXIa preparation

WHO/BS/2013.2230 Addendum 2 Page 34 Table 6: Lab 5 - Potency estimates and intra-laboratory GCV for test samples relative to sample S, the RR for FXIa u/ml Assay Sample %GCV Assay 2 Assay 3 Assay 4 GM 95% CL 1 A 10.51 10.34 10.21 9.61 10.16 10.55-10.81 3.98 B NP NP NP NP C LOQ LOQ LOQ LOQ D LOQ LOQ LOQ LOQ E 4.16 4.72 4.01 4.22 4.30 3.87-4.81 7.05 IHXIa 6.92 7.04 6.87 6.78 6.90 6.73-7.08 1.58 GM: geometric mean; 95% CL: 95% confidence limits; %GCV: geometric coefficient of variation; LOQ: Below limit of quantification; NT: not tested; IHXIa: in-house FXIa preparation

Table 7: Lab 6a - Potency estimates and intra-laboratory GCV for test samples relative to sample S, the RR for FXIa u/ml Sample %GCV Assay 1 Assay 2 Assay 3 Assay 4 GM 95% CL A 9.18 10.70 12.94 10.18 10.67 8.48-13.41 15.45 B 1.03 1.31 1.66 1.32 1.31 0.96-1.79 21.52 C LOQ LOQ LOQ LOQ D LOQ LOQ LOQ LOQ E LOQ LOQ LOQ LOQ IHXIa 3.25 3.80 4.25 3.82 3.82 3.16-4.49 11.68 IVIG LOQ LOQ LOQ LOQ IVIG+XI 0.58 0.80 0.89 0.75 0.75 0.55-1.00 20.42 a (1/10) Note – assessed by deviations from linearity as only one data point from each dilution GM: geometric mean; 95% CL: 95% confidence limits; %GCV: geometric coefficient of variation; LOQ: Below limit of quantification; NT: not tested; IHXIa: in-house FXIa preparation

WHO/BS/2013.2230 Addendum 2 Page 35

Appendix 4b: Assay results from each laboratory, Part B Table 1: Lab 7 - Potency estimates and intra-laboratory GCV for test samples relative to sample S, the RR for FXIa u/ml Sample Assay 1 Assay 2 Assay 3 Assay 4 GM %GCV 95% CL W LOQ LOQ LOQ LOQ 0.192 X 0.193 0.203 0.202 0.203 0.200 2.48 0.208 0.093 Y 0.094 0.099 0.102 0.102 0.099 3.93 0.105 0.047 Z 0.048 0.051 0.052 0.05 0.050 3.48 0.053 0.063 IHIXa 0.064 0.071 0.069 0.067 0.068 4.53 0.072 GM: geometric mean; 95% CL: 95% confidence limits; %GCV: geometric coefficient of variation; LOQ: Below limit of quantification; IHXIa: in-house FXIa preparation Table 2: Lab 8 - Potency estimates and intra-laboratory GCV for test samples relative to sample S, the RR for FXIa u/ml Sample Assay 1 Assay 2 Assay 3 Assay 4 GM %GCV 95% CL W LOQ LOQ LOQ LOQ 0.184 X 0.209 0.197 0.187 0.200 0.198 4.70 0.213 0.087 Y 0.102 0.099 0.089 0.105 0.098 7.48 0.110 0.048 Z 0.052 0.057 0.050 0.057 0.053 6.85 0.059 0.032 IHIXa 0.036 0.037 0.033 0.039 0.036 7.23 0.040 GM: geometric mean; 95% CL: 95% confidence limits; %GCV: geometric coefficient of variation; LOQ: Below limit of quantification; IHXIa: in-house FXIa preparation Table 3: Lab 9 - Potency estimates and intra-laboratory GCV for test samples relative to sample S, the RR for FXIa u/ml Sample Assay 1 Assay 2 Assay 3 Assay 4 GM %GCV 95% CL W LOQ LOQ LOQ LOQ X 0.223 0.241 0.201 0.213 0.219 0.194-0.247 7.96 Y 0.092 0.103 0.084 0.114 0.097 0.078-0.120 14.22 Z 0.038 0.050 0.045 0.054 0.046 0.036-0.059 16.43 IHIXa 0.041 0.037 0.036 0.039 0.038 0.034-0.041 5.93 GM: geometric mean; 95% CL: 95% confidence limits; %GCV: geometric coefficient of variation; LOQ: Below limit of quantification; IHXIa: in-house FXIa preparation

WHO/BS/2013.2230 Addendum 2 Page 36 Table 4: Lab 10 - Potency estimates and intra-laboratory GCV for test samples relative to sample S, the RR for FXIa u/ml Sample Assay 1 Assay 2 Assay 3 Assay 4 GM %GCV 95% CL IVIG LOQ LOQ LOQ LOQ + 1/5 1.783 1.818 1.748 1.691 1.759 1.674-1.848 3.14 FXIa + 1/10 0.894 0.764 0.830 0.880 0.840 0.750-0.941 7.37 FXIa + 1/20 0.384 0.319 0.404 0.457 0.387 0.305-0.491 16.10 FXIa 21.62 20.302IHIXa 20.991 22.583 22.138 20.833 4.04 3 23.029 GM: geometric mean; 95% CL: 95% confidence limits; %GCV: geometric coefficient of variation; LOQ: Below limit of quantification; IHXIa: in-house FXIa preparation Table 5: Lab 11 - Potency estimates and intra-laboratory GCV for test samples relative to sample S, the RR for FXIa u/ml Sample Assay 1 Assay 2 Assay 3 Assay 4 %GCV GM 95% CL W LOQ LOQ LOQ LOQ X 0.173 0.224 0.177 0.222 0.197 0.158-0.246 15.07 Y 0.100 0.108 0.100 0.106 0.103 0.097-0.110 4.06 Z 0.049 0.063 0.058 0.053 0.055 0.046-0.066 11.52 6114.7 7977.1 5501.0 7917.3 5042.22IHIXa 6789.104 20.56 4 1 6 5 9142.30 GM: geometric mean; 95% CL: 95% confidence limits; %GCV: geometric coefficient of variation; LOQ: Below limit of quantification; IHXIa: in-house FXIa preparation Table 6: Lab 12 - Potency estimates and intra-laboratory GCV for test samples relative to sample S, the RR for FXIa - (Laboratory reported results) u/ml Sample Assay 1 Assay 2 Assay 3 Assay 4 GM %GCV 95% CL W 0.028 0.026 0.026 0.030 0.027 0.024-0.030 7.12 X 0.077 0.075 0.074 0.076 0.075 0.073-0.077 1.72 Y 0.053 0.050 0.049 0.053 0.051 0.048-0.054 4.12 Z 0.040 0.038 0.036 0.043 0.039 0.034-0.044 7.86 455.0 388.33IHIXa 450.67 474.85 398.22 503.13 10.48 5 533.23 S 10.54 11.10 10.60 11.44 10.91 10.26-11.61 3.98 GM: geometric mean; 95% CL: 95% confidence limits; %GCV: geometric coefficient of variation; IHXIa: in-house FXIa preparation. S was included as a duplicate test sample in these assays.

WHO/BS/2013.2230 Addendum 2 Page 37

Appendix 5: Thrombin Generations Assay results from laboratory 3c. Table 1: Lab 3c Peak Thrombin (nM) and intra-laboratory GCV from the samples Thrombin Generation Results values as reported Assay Assay Assay Assay Assay Assay Assay Assay Sample GM 95% CL 1 2 3 4 5 6 7 8 9045 A 7020 16412 11704 10196 9495 12606 12208 11576 11113 13652 B C D E S IVIG IVIG +XIa ODR 99.6 56.8 ODR 8491 45.2 111 541 40.1 ODR 100 632 49.8 ODR 144 380 32.0 ODR 72.1 12628 ODR 67.0 475 44.1 ODR 109 11602 ODR 16.3 589 47.3 ODR 119 14562 ODR 76.9 498 37.6 ODR 116 11473 ODR 52.8 396 34.2 ODR 72.0 12676 ODR 55.1 494 45.1 102 12363 63.5 413 – 589 33.5 – 60.7 79.9 - 129 10628 14381 37.2 - 108

%GCV 27.9 21.1 42.6 29.7 19.8 89.3

15055 13762 ODR 136 ODR 71.5

ODR = Outside detection range (as reported) Table 2: Lab 3c Peak thrombin values and intra-laboratory GCV for test samples taken relative to sample S, the RR for FXIa, calculated by NIBSC Assay Assay Assay Assay Assay Assay Assay Assay Sample GM 95% CL 1 2 3 4 5 6 7 8 8.74 – A 10.59 11.43 8.45 8.34 8.49 8.32 9.84 9.28 9.28 10.28 B C D E IVIG IVIG +XIa 4.71 0.362 ODR 1.02 ODR 3.19 3.73 0.275 ODR 0.752 ODR 0.93 4.51 0.364 ODR 1.08 ODR 0.533 3.04 0.25 ODR 0.567 ODR 0.531 4.19 0.395 ODR 1.02 ODR 0.135 4.02 0.317 ODR 0.81 ODR 0.488 4.34 0.328 ODR 0.952 ODR 0.447 3.26 0.281 ODR 0.592 ODR 0.454 3.93 0.317 0.827 0.57 3.45 – 4.48 0.278 – 0.363 0.67 – 1.021 0.273 – 1.191

%GCV 13.0 16.9 17.3 28.7 141.2

ODR = Outside detection range NIBSC reanalysis by parallel line using log transformation, values given in table 2 are for valid results with standard 'v' test; validity acceptance criteria is p < 0.05

WHO/BS/2013.2230 Addendum 2 Page 38

Appendix 6: Instructions for use

WHO/BS/2013.2230 Addendum 2 Page 39