WHO/BS/2015.2261 ENGLISH ONLY
EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION Geneva, 12 to 16 October 2015
COLLABORATIVE STUDY REPORT ON BLOOD COAGULATION FACTOR IX A. Value assignments:
1. Addition of Factor IX antigen value to The 4th International Standard for Blood Coagulation Factors II, VII, IX, X, Plasma, 09/172
2. The WHO 5th International Standard for Blood Coagulation Factor IX, Concentrate and The Ph Eur BRP for Human Coagulation Factor IX, Concentrate, Batch 3
B. Investigation of the suitability of the 4th International Standard for Blood Coagulation Factor IX, Concentrate as a potency standard for purified full length recombinant FIX
1Elaine Gray, John Hogwood, Thomas Dougall* and Peter Rigsby*
Haemostasis Section and *Biostatistics Section National Institute for Biological Standards and Control
Potters Bar, Hertfordshire, UK.
Eriko Terao
Department of Biological Standardisation, OMCL network and HealthCare (DBO) European Directorate for the Quality of Medicines and HealthCare (EDQM)
Council of Europe, Strasbourg, France
1Principal Investigator
NOTE:
This document has been prepared for the purpose of inviting comments and suggestions on the proposals contained therein, which will then be considered by the Expert Committee on
Biological Standardization (ECBS). Comments MUST be received by 14 September 2015 and should be addressed to the World Health Organization, 1211 Geneva 27, Switzerland, attention:
Technologies, Standards and Norms (TSN). Comments may also be submitted electronically to the Responsible Officer: Dr M Nübling at email: [email protected]
© World Health Organization 2015
All rights reserved. Publications of the World Health Organization are available on the WHO web site (www.who.int) or can be purchased from WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax:
+41 22 791 4857; e-mail: [email protected]).
Requests for permission to reproduce or translate WHO publications – whether for sale or for noncommercial distribution – should be addressed to WHO Press through the WHO web site: (http://www.who.int/about/licensing/copyright_form/en/index.html).
The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement.
The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters.
All reasonable precautions have been taken by the World Health Organization to verify the information contained in this publication.
However, the published material is being distributed without warranty of any kind, either expressed or implied. The responsibility for the interpretation and use of the material lies with the reader. In no event shall the World Health Organization be liable for damages arising from its use. The named authors alone are responsible for the views expressed in this publication.
Summary
There are three main aims for this collaborative study:
1. to add a FIX antigen value to the 4th International Standard for Blood Coagulation Factors II, VII, IX and X, Plasma, Human (09/172);
2. to value assign a World Health Organisation (WHO) replacement International Standard (IS) for Blood Coagulation Factor IX, Concentrate, Human and a replacement Pharmacopoeial (Ph Eur) Biological Reference Preparation (BRP) for Blood Coagulation FIX, and
3.To investigate the suitability of the 4th International Standard for Blood Coagulation Factor IX, Concentrate as a potency standard for purified full length recombinant FIX.
Assignment of antigen value to the 4th International Standard for Blood Coagulation Factors II, VII, IX and X, Plasma, Human (09/172) relative to local normal plasma pool was carried out by 15 laboratories returning 17 sets of data in total for analysis. Only 5 sets of data gave intra-
laboratory GCVs greater than 10%. There was good inter-laboratory agreement, GCV was 7.9%.
It was clear that local pools or the 4th IS for FIX Plasma could not be used as an antigen
measurement standard for recombinant products since some reagent kits gave much lower results than other kits and this is reflected in the high inter-laboratory variation (GCVs A: 26.6%; D;
31.5%). It is recommended that a FIX antigen value of 0.90 IU/ampoule be added to the label of the 4th IS for International Standard for Blood coagulation Factors II, VII, IX, X, Plasma.
Fifty laboratories from 18 countries took part for the value assignment of the plasma derived 5th IS for FIX, Concentrate and Ph Eur BRP for FIX Concentrate, batch 3 relative to the 4th IS for FIX Concentrate, with 55 sets of clotting assay and 15 sets of chromogenic assay results returned for analysis. The intra-laboratory variability was reasonably low, with the majority of geometric coefficient of variation (GCV) below 10%. Lower inter-laboratory agreement was obtained for sample B, 14/148 than for sample C, 14/162. Although there were no assay discrepancies
between clotting and chromogenic assays for either samples, a significantly lower (~6%) potency was obtained for sample C with clotting assays when buffer rather than FIX deficient plasma was used as pre-diluent. It is recommended that sample B, 14/148 be established as the 5th
International Standard for Blood Coagulation Factor IX, Concentrate and the Ph Eur BRP for Blood coagulation Factor IX Batch 3, with an assigned value of 10.5 IU/ampoule.
The study also investigated the comparability of the plasma derived concentrate standard with the full length recombinant FIX products and considered the feasibility of establishing an
International Standard for Recombinant FIX. There are currently 3 full length recombinant FIX
products licenced and these were all represented in this collaborative study. Data from 49 laboratories (55 sets of clotting, 15 sets of chromogenic results) for two candidate recombinant samples, A and D were received with additional results for another full length recombinant test sample (Purple) returned by 6 laboratories. The intra-laboratory variability when samples A, D and Purple were assayed against the 4th IS for FIX Concentrate was acceptably low, majority being less than 10%. In terms of inter-laboratory agreement, the clotting assay variability was markedly high. For recombinant sample A, the potencies ranged 7.7 - 12.4 IU/ampoule (up to 62% discrepancy), with overall inter-laboratory GCV of 11.6% and for sample D, the potencies ranged 6.6 – 12.4 IU/ampoule (up to 88% discrepancy), with overall inter-laboratory GCV of 13.4%. There were no major differences in the inter-laboratory GCVs for the chromogenic assays. The inter-laboratory GCVs for overall potency estimates (including both clotting and chromogenic assays) relative to the 4th IS were high for both recombinant samples A (15.5%) and D (16.8%); however, the GCV was reduced to 5.7% for Sample A when it was reanalysed using sample D as the putative standard. In addition, reanalysis of clotting and chromogenic data for sample A relative to sample D also markedly reduced the clotting and chromogenic discrepancy observed when sample A was assayed against the 4th IS for FIX Concentrate
(clotting/chromogenic ratios: vs 4th IS – 1.25 (p <0.001) vs D – 0.98 (p=0.282). Although the full length recombinant FIX could be assayed against the plasma derived 4th IS Concentrate and provided statistically valid results, there was high assay discrepancies amongst the clotting assays using different APTT reagents and there was also significant clotting and chromogenic assay discrepancies. The data from the present study indicated that a recombinant standard for recombinant products will minimise assay discrepancies and improve inter-laboratory agreement.
Except for one laboratory who disagreed with a proposal of establishing an International Standard for Recombinant FIX on the grounds that this may cause a shift in the amount of proteins in the final products, all other participants agreed with the proposal. The data have also been evaluated by 8 experts nominated by the Scientific and Standardisation Committee (SSC) of the
International Society for Thrombosis and Haemsotasis (ISTH). Seven SSC experts agreed with the proposal, with 2 experts strongly support the establishment of an International Standard for Recombinant FIX based on the reduction in assay discrepancies. One SSC Expert strongly opposed and indicated that the use of multiple International Standards for the same coagulation factor should be avoided whenever possible. Because of these 2 objections and after discussion with the ISTH/WHO Liaison Group, it is unlikely that SSC will endorse such a proposal and therefore the ECBS will not be requested to consider the establishment of an International Standard for Recombinant FIX. The results and analysis for this investigation presented in this report is for information only.
Proposals for establishment:
Sample P, NIBSC code 09/172: FIX antigen value: 0.90 IU/ampoule
WHO 4th International Standard for Blood Coagulation Factors II, VII, IX, X, Plasma
Sample B, NIBSC code 14/148: Functional activity - 10.5 IU/ampoule
WHO 5th International Standard for Blood Coagulation Factor IX, Concentrate
Introduction
The 4th International Standard (IS) for Blood Coagulation Factor IX, Concentrate, Human (07/182) was established by the Expert Committee on Biological Standardisation (ECBS) of the World Health Organisation (WHO) in October 20081,2. Part of this batch of material was also established as the European Pharmacopoeia Biological Reference Preparation (BRP) Batch 2 and the US FDA reference standard for Blood Coagulation Factor IX Concentrate. The stock level of the WHO 4th IS and the Ph Eur BRP reference standards are now near depletion and replacement standards are required. The aim of the study is to assay factor IX concentrate candidate
preparations against the 4th International Standard, 07/182, with a view to establish a new material as the 5th International Standard for Blood Coagulation Factor IX, Concentrate, and EP Human Coagulation Factor IX Concentrate Batch 3. In addition, as there are now three
recombinant FIX products licensed, this study also included these 3 recombinant preparations (2 of which could be candidate International Standard for Recombinant FIX) with a view to assess the need and possible establishment of an International Standard for recombinant FIX.
This study also serves to value assign a factor IX antigen value to the 4th International Standard for Blood Coagulation Factors II, VII, IX and X, Human, Plasma, 09/172 and provides an opportunity to assess the factor IX unitage as defined by the concentrate and plasma standards.
The replacement of 4th IS for FIX, Concentrate project and the antigen value assignment to the 4th IS for FII, VII, IX and X, plasma were endorsed by the WHO Expert Committee on Biological Standardisation in October 2012 and October 2014 respectively.
Participants
Fifty laboratories agreed to participate, with 49 laboratories (6 Austria, 1 Australia, 4 Canada, 1 China, 1 Croatia, 2 Denmark, 5 France, 6 Germany, 1 Italy, 1 Japan, 1 Korea, 3 Netherlands, 1 Portugal, 2 Spain, 1 Sweden, 1 Switzerland, 6 UK, 6 USA) returning data for the study. The participants included 7 diagnostic reagents manufacturers, 19 therapeutic manufacturers, 14 regulatory authorities and 9 clinical laboratories. A list of participants is given in Appendix I at the end of this report. Each laboratory is referred to in this report by an arbitrarily assigned number, not necessarily representing the order of listing in the Appendix.
Samples
Coded samples included in the study were:
S – the 4th I.S. Factor IX, Concentrate, 07/182, potency 7.9 IU/ampoule
P – the 4th I.S. Blood Coagulation Factor II, VII, IX, X, Plasma, 09/172, FIX potency 0.86 IU/ampoule
A – candidate sample, 07/142, recombinant FIX, nominal potency 8 – 10 IU/ampoule B – candidate sample, 14/148, plasma derived FIX, nominal potency 10 – 12 IU/ampoule C – candidate sample, 14/162, plasma derived FIX, nominal potency 8 – 10 IU/ampoule D – candidate sample, 14/180, recombinant FIX, nominal potency 8 – 10 IU/ampoule
An additional frozen FIX concentrate sample, coded Pr (Purple) was also sent and assayed by 6 consenting participants.
The plasma pools for both the plasma derived candidates, the final products, the excipient human albumin and the two International Standards have been tested negative for HBsAg, anti-HCV, anti-HIV 1/2, and HCV RNA by PCR. All the candidates were prepared and freeze-dried according to recommendations for the preparation, characterization and establishment of
international and other biological reference materials3. The product characteristics of each of the candidates are summarised in Table 1. In addition, clotting times from activated coagulation factors test (Non-activated partial Thromboplastin times, NAPTT), carried out in accordance with Ph Eur method (2.6.22) and levels of activated factor IXa in the candidates are presented in Tables 13 and 14 respectively.
With the exception of the local normal plasma pools, all samples were provided by NIBSC and the participants were requested to reconstitute the samples according to protocol provided (Appendix II).
Study design and assay methods
Details of the assay design were as stated in the protocol which is attached as Appendix II.
Briefly, each participant was requested to carry out 4 independent assays on 4 sets of samples and to follow one of the suggested balanced assay designs as described in the study protocol.
Each participant was requested to perform their routine in-house method for FIX activity.
Multiple result sets returned by a participating laboratory were treated as results from an independent laboratory and were given a separate lab code, e.g. Lab 2a, Lab 2b.
Statistical Analysis
Relative potencies of all samples in all assays were calculated by parallel line analysis with a log transformation of assay response, using a minimum of three dilutions on a linear section of the dose-response curve. Calculations were performed using the EDQM software CombiStats Version 5.04. Non-linearity and non-parallelism were considered in the assessment of assay validity. All dose-response lines showing no significant non-linearity (p>0.01) were accepted for further analysis. All instances of significant non-linearity (p<0.01) were assessed visually and those showing clear departures from linearity were excluded from further analysis. Non- parallelism was assessed by calculation of the ratio of fitted slopes for the test and reference
samples under consideration. The samples were concluded to be non-parallel when the slope ratio was outside of the range 0.80 – 1.25 and no estimates are reported.
Relative potency estimates from all valid assays were combined to generate an unweighted geometric mean (GM) for each laboratory and these laboratory means were used to calculate overall unweighted geometric means for each sample. Variability between assays within laboratories and between laboratories has been expressed using geometric coefficients of variation (GCV = {10s-1}×100% where s is the standard deviation of the log10 transformed estimates). Comparisons between assay methods were made by two-tailed t-test of log transformed laboratory mean estimates (paired or unpaired as appropriate for the comparison being made).
The relative contents of the accelerated thermal degradation samples were used to fit an
Arrhenius equation relating degradation rate to absolute temperature assuming first-order decay5 and hence predict the degradation rates when stored at -20°C.
Results and Discussions
FIX Antigen value assignment to 4
thIS for FII, VII, IX, X, Plasma relative to local normal plasma pools
The Candidate, 4
thIS for FII, VII, IX, X, Plasma, 09/172
The candidate 09/172, coded sample P in the study was established by the ECBS/WHO in 2010 as the 4th IS for FII, VII, IX, X, Plasma, with functional potencies assigned for these 4 factors. It was produced from a pool of 85 donations of platelet poor normal plasma (The Welsh Blood Service), each collected in CPD-adenine and buffered with 0.05 M HEPES. The final product was filled and freeze-dried according to guidelines for production of international biological standards (WHO TRS, 20063). Each individual plasma donation has been tested and found negative for anti-HIV 1/2, HBsAg and anti-hepatitis C. The product characteristics are shown in the following table:
NIBSC Code 09/172
Presentation Sealed, glass 5 ml DIN ampoules
Number of Ampoules available 16,000
Liquid filling weight (g) Mean=1.1078; Range=1.1000 – 1.1140
CV of fill mass (%) 0.20 (n=849)
Mean dry weight (g, n = 5) 0.1034
Mean head space oxygen (%) 0.14 (n =12)
Residual moisture (%) 0.225 (n = 12)
Storage temperature -20°C
Address of processing facility NIBSC, Potters Bar, EN6 3QG, UK Address of present custodian NIBSC, Potters Bar, EN6 3QG, UK
Assay methods
The FIX antigen value assignment was relative to the local normal pooled plasmas. Participants were requested to collect fresh plasma on two separate days to prepare two normal plasma pools.
It was requested that each fresh pool was tested in the study on the day of collection and that a sample of the same pool should be frozen for use on a second day. The normal pool was to be used as the standard in the antigen assays. With the exception of Lab 28 and Lab 40, the laboratories were not able to prepare fresh plasma pools and used in-house frozen pools or commercial normal plasma pools; the number of donors across all pools was > 1800.
In total, 17 sets of antigen results (66 assays in total) from 15 labs were returned. Five
commercial ELISA kits were used (Table 2C). Laboratory 2 returned three sets of assays coded 2a, 2b and 2c. All laboratories performed four independent assays except Lab 48 which was only able to perform 2 assays.
This study also offered the opportunity to assess whether a plasma antigen standard would be an appropriate comparator for therapeutic concentrates as purified plasma derived and recombinant FIX concentrates (samples A, B, C and D) were included in this study.
Assay validity
The majority of antigen assays gave valid estimates of relative potency. A slightly higher
proportion of non-linear and non-parallel exclusions (both 2-4%) was observed, but this partially reflects the use of a log transformation of assay response which was consistently applied in all laboratories and this may not reflect the best transformation choice in a small number of cases.
Individual cases of exclusions due to non-linearity or non-parallelism are indicated in Appendix III, Table S3.
Results
The antigen value was assessed relative to the local normal plasma pools (L). Individual assay results are shown in Appendix III, Table S3. Individual laboratory mean and overall potency estimates for antigen are presented in Tables 8 and 9A.
Antigen relative to local normal plasma pools
o The intra-laboratory variation (%GCV) ranged 1.8 – 27.1%, with 5 laboratories having %GCV greater than 10% (Table 8, 9A).
o The inter-laboratory variability was 7.9% (Table 8, 9A) and there was no obvious assay kit bias or any detectable outliers.
o The overall FIX antigen value for the 4th IS for FII, VII, IX, X, plasma against local pool was 0.90 u/ml (Table 8, 9A).
Antigen values for samples S, A, B, C, D relative to local normal pooled plasma, L and sample P, assuming antigen value of 0.90 u/ml
o When samples S, B and C, the plasma derived samples were assayed against L, the local normal pooled plasma, there was good laboratory agreement of antigen values for sample S, B and C; the inter-laboratory %GCVs were 9.2%, 9.8% and
8.6% respectively (Table 9A) and these were slightly reduced when these samples were assayed against P, the plasma IS, with an assumed value of 0.90 u/ml.
o The inter-laboratory %GCVs for A and D, the recombinant samples, were extremely high (28.9% and 30.7% for samples A and D) and they were not reduced when the samples were reanalysed against sample P (25.2% for A and 30.2% for D). The ranges of potencies against P for samples A and D were found to be 3.8 – 9.4 u/ml and 3.1 – 8.1 u/ml respectively.
Discussion
Antigen measurements are carried out to assess phenotypes of haemophilia B, expression of antigen in gene therapy and characterisation of therapeutic products. Since this is the first time a FIX antigen value is assigned to an International Standard, the calibration is relative to local normal plasma pool. Ideally, fresh normal plasma pool with large numbers of donors is used for the comparison. However, for logistical reasons, it is difficult for laboratories to collect and prepare fresh plasma to be used for assays on the same day. NIBSC have carried out in-house studies to show that there was no significant difference in the antigen values of fresh plasmas and their corresponding frozen/thawed samples (data not shown), the study therefore allowed the use of fresh frozen plasmas as calibrators. With the exception of 2 laboratories (labs 28 and 40), all laboratories used frozen pools (Table 2C) and although the total number of donors was not known, it was >1800. Five different commercial reagents/kits were used: Asserachrom (8), Visulize (5) and Zymutest (2), AssayPro (1) and Cedarlane (1) by 17 laboratories. The following summarises the essential points for consideration:
Antigen value of sample P relative to local pooled plasma, L
o Intra-laboratory variability: 5 laboratories gave GCVs greater than 10% and only one laboratory obtained GCV of 27% (Tables 8A, 9A). This indicates the kits and the laboratories were able to measure antigen in plasma with reasonable precision.
o Inter-laboratory agreement: There was no significant difference in values by different assay kits and outliers were not detected. Considering that the calibration was against local pools, good agreement of antigen values was obtained; GCV was 7.9% (Tables 8, 9A). This may be partly aided by the sufficiently large number of donors in each pool to offset the differences in individual donors. The overall value for sample P was 0.90 u/ampoule.
Antigen values of samples S, A, B, C and D relative to local normal pool plasma, L or sample P, assuming assigned value of 0.90 u/ampoule
Low intra-laboratory variability was observed with the majority of GCVs less than 10% for all 4 samples (Table 9A) when assayed against L. The GCVs were
slightly reduced when these samples were assayed against P. (Table 9B)
The inter-laboratory GCVs for samples B and C, the plasma derived preparations was reasonable when assayed against the local pool and were only marginally reduced when assayed against P (Tables 9A, 9B).
The inter-laboratory GCVs for A and D, the recombinant samples, were extremely high, being close to 30% when assayed against L (Table 9A) and were not reduced when the samples were reanalysed against sample P (A: 26.6%; D 31.5%). The
ranges of potencies against P for samples A and D were found to be 3.8 – 9.4 U/ml and 3.1 – 8.1 U/ml respectively. This indicates that antigen measurement of recombinant products, including FIX expressed by gene therapy vectors, against normal plasma, using different antigen kits or antibody pairs may give
substantially different results and product specific standards may be required for accurate measurement of antigen in recombinant FIX products.
Long term and on-bench stability of the 4
thIS for FII, VII, IX and X Plasma
The 4th IS for FII, VII, IX, X, Plasma, 09/172 was established in 2010. Accelerated degradation study for FIX antigen at 6 month time point showed no loss of activity. Stability data on other analytes (functional activities of FII, VII, IX and X) showed that this reference standard is highly stable. As with all International Standards, the proposed International Standards will also be under real time stability monitoring throughout the life time of the standard.
Assessment of on-bench stability was carried out at NIBSC by storage of the reconstituted sample on melting ice. Samples were reconstituted at 0 h, 2 h, and 3 h and stored on melting ice. After storage of the first sample for 4 hours on melting ice, all samples were assayed relative to freshly
reconstituted ampoules of 09/172. The results indicated that FIX antigen would be stable for up to 4 hours after reconstitution when stored on melting ice.
Time point Potency % fresh ampoule (95% confidence intervals)
2 h 96.1 (93.3 -100.0)
3 h 101.0 (98.1 – 103.8)
4 h 98.1 (95.2 -101.0)
Conclusions
Intra- and inter-laboratory variability were low when P, the 4th IS for FIX Plasma was assayed against local normal pooled plasma (L).
Sample P improved antigen estimates agreement for samples S, B and C, the plasma derived concentrates when used as a putative standard.
Both local pooled plasma (L) and the 4th IS for plasma FIX (P) gave highly variable results for samples A and D, the recombinant products.
It is recommended that an antigen value of 0.90 IU/ampoule be included in the labelling of the 4th International Standard for FII, VII, IX and X, Plasma.
However, it should be noted that the use of this International Standard for measurement of antigen in recombinant/modified products, post-infusion plasma from patients who have been treated with recombinant/modified products or gene therapy products is not recommended. If this standard is used for these purposes, it needs to be qualified and validated by the end-user with their own reagents and methods. The draft Instruction for Use for this proposed IS is illustrated in Appendix IV.
Value assignment of the 5
thIS for IX, Concentrate and
Ph Eur BRP for FIX, Batch 3relative to the 4
thIS for FIX, Concentrate, 07/182 and comparability of full length recombinant FIX with plasma derived concentrate IS
The Candidates, Samples B (14/148) and C (14/162)
The characteristics of the candidates are as described in the Sample Section.
Recombinant FIX, Samples A (07/142), D (14/180) and Purple (Pr)
The characteristics of the candidates are as described in the Sample Section.
Assay methods
Each participant was requested to perform their routine in-house method for FIX activity.
Multiple result sets returned by a participating laboratory were treated as results from an independent laboratory and were given a separate lab code, e.g. Lab 2a, Lab 2b.
FIX functional activity assays
Clotting: The details of the instruments and reagents used by the participants are listed in Table 2A. With the exception of 3 laboratories that returned more than one set of results (Lab 2 – 5 sets;
Lab 8 – 4 sets; Lab 28 – 3 sets with 1 assay for two sets and 2 assays for the third set), all other laboratories returned one set of results. In total 55 sets of clotting assays were analysed. Of the clotting assays, 35 sets of results were obtained using FIX deficient plasma as pre-diluent for the concentrate preparations (samples A, B, C and D), as stated in the European Pharmacopoeia monograph for assay of human coagulation factor IX (01/2008:20711). Twenty sets of results were from assays using buffer for pre-dilution (Table 2A). Sample P, the plasma IS did not require pre-dilution as its activity is less than 1 IU/ml.
Chromogenic: In total, 15 sets of chromogenic assay results from 13 labs were returned. Two commercial kits, Biophen Factor IX (Hyphen Biomed) and Rox Factor IX (Rossix AB) were employed (Table 2B). The participants carried out the assays as described by the instruction given by the manufacturers of the kit and pre-dilutions were carried out using kit buffers recommended.
Purple sample
In total, 10 sets of clotting results were returned from 5 labs (1 lab returned 5 sets), 4 sets of chromogenic results from 3 labs (1 lab returned 2 sets) and 2 sets of antigen assays from 1 lab.
Assay data returned
Clotting assays
Fifty-five sets of results from clotting assays (213 assays in total) were returned. Laboratory 2 performed five sets of assays which are coded 2a, 2b, 2c, 2d and 2e. Laboratory 8 performed four sets of assay assigned codes 8a, 8b, 8c and 8d. Laboratory 31 performed 2 sets of assays, coded 31a and 31b. All laboratories performed four independent assays, apart from laboratory 33 which performed 5 assays (extra assay performed using samples supplied for antigen testing).
Laboratory 28 performed four assays, but with three different APTT agents and results were 28a, 28b and 28c. Sample P was not tested by laboratory 42.
Chromogenic assays
Thirteen laboratories performed chromogenic assays (59 assays in total). Laboratory 2 and 12 both performed two sets of assays, coded 2a & 2b and 12a & 12b respectively. All laboratories performed four independent assays, except laboratory 48 which was only able to perform 3 assays.
Assay validity
The majority of clotting and chromogenic assays gave valid estimates of relative potency when assessed as described in the analysis section above. Samples omitted for showing a non-linear dose-response accounted for around 1% of cases. Reference-test pairs concluded to be non- parallel accounted for only 1-2% of cases. A high number of slope ratios were in the range 0.90 – 1.11 indicating that more stringent criteria for parallelism could be applied to these assays.
Individual cases of exclusions due to non-linearity or non-parallelism are indicated in Appendix III. In general, relative to the plasma derived 4th International Standard for FIX Concentrate, statistically valid assays by all method types were obtained for all the test samples, including recombinant preparations.
Results
Samples A, B, C and D relative to the 4th IS for FIX Concentrate
The potencies of samples A, B, C and D have been calculated relative to sample S, the 4th IS for FIX Concentrate. Individual assay results are shown in Appendix III. Laboratory mean and overall potency estimates for clotting and chromogenic assays are presented in Tables 3A and 3B respectively and are also illustrated as histograms in Figures 1 – 4.
Clotting assays
o Of the 55 laboratories, <10 laboratories had an intra-laboratory GCV >10% and over 60% of laboratories had intra-laboratory GCV <5% for all 4 samples.
o Inter-laboratory variability for the clotting assays was 4.8% and 8.4% for the plasma derived samples, B and C and higher at 11.6% and 13.4% for the recombinant samples, A and D. Figure 5 shows that there was no obvious effect with regards to APTT reagents on the potencies obtained, but wider ranges of estimates were observed for the recombinant samples A and D as confirmed by the higher inter- laboratory variation for these two samples.
o With the exception of sample C, there was no effect of the diluents used for pre- dilution of the samples on the overall potency estimates by clotting assay (Table 4).
For sample C, there was a 6% (including all results) - 8% (excluding Lab 15) decrease in potency when the samples were diluted in buffer rather than in FIX deficient
plasma.
o Tables 5A and 5B show the overall potency estimates by clotting methods were 9.8, 10.5, 8.7 and 9.4 IU/ml for samples A, B, C and D. Results from Lab 15 were identified as outliers for samples B, C and D. Mean potency estimates excluding results from Lab 15 were marginally increased for samples A (9.9 IU/ml) and C (8.8 IU/ml), with a reduction in %GCV for samples B, C and D.
Chromogenic assays
o Intra-lab variability for chromogenic assays was higher than for clotting assays, but GCVs in the majority of cases were still <10%.
o Inter-laboratory variability for the chromogenic assays was 9.3% and 8.6% for the plasma derived samples, B and C, and only slightly higher at 10.2% and 10.4% for the recombinant samples, A and D.
o The overall potencies by chromogenic methods were found to be 7.8, 10.6, 9.1 and 7.3 IU/ml for samples A, B, C and D respectively. Results from Lab 21 were identified as outliers for samples B and C. Potency estimates excluding results from Lab 21 were marginally reduced for samples A, B and C, with a marked reduction in %GCV also for samples B and C (Tables 5A and 5B).
o The results from the 2 chromogenic assay kits used in the study showed no significant differences in the potencies obtained for plasma derived samples B and C, although the Hyphen kit gave approximately 10% lower potencies for the recombinant samples A and D.
Comparison of clotting and chromogenic assays (Table 5A and 5B)
o It is clear that there were no clotting and chromogenic assay discrepancies for the plasma derived samples B and C, including or excluding the outliers.
o For recombinant samples A and D, significantly higher potencies (approximately 30%) were obtained by clotting assays.
Overall potency estimates by clotting and chromogenic assays
o Table 7 presents the overall geometric mean potencies obtained by both clotting and chromogenic assays for samples A, B, C and D, including and excluding Lab 15 for clotting assay and Lab 21 for chromogenic assay. The GM estimates were 9.3, 10.5, 8.8, 8.9 IU/ml for samples A, B, C and D when all the assays were included. With the exception of sample A, which gave a potency estimate of 9.4 IU/ml, the GM potency did not change when labs 15 (clotting) and 21 (chromogenic) were excluded. The inter-laboratory variation was markedly lower for samples B and C (5.9% and 8.6%
respectively) than for samples A (15.4%) and D (17.1%). The %GCV for samples A and D were not changed by the exclusion of labs 15 and 21, but they were reduced by approximately 2% for samples B and C.
Sample A relative to Sample S, the 4th IS for FIX Concentrate and sample D, the putative recombinant standard
Results for sample A were reanalysed relative to sample D with an assumed assigned value of 9.4 IU/ml based on clotting assays (Dcl) only or 8.9 IU/ml based on overall potency estimates
(Dcl+ch).
Table 10A shows comparison of potencies for sample A relative to sample S, the 4th IS for FIX Concentrate and sample D, the putative recombinant standard. For clotting assays, similar ranges of intra-laboratory variability as expressed by %GCVs were obtained when A was assayed against S or D, with 5 labs giving >10% GCV. For
chromogenic assays, the majority of the labs gave intra-laboratory GCVs of <10% (Table 10B).
There was no significant difference between the potency estimates by clotting assays obtained against sample S and sample Dcl (Table 10A; p = 0.985), but there was significant difference when compared with results against sample Dcl+ch (Table 10A;
p=0.003). The inter-laboratory variability as expressed by GCVs was markedly reduced for both cases from 11.6% against sample S to 6.1% against samples Dcl or Dcl+ch.
There was a significant difference between the potency estimates by chromogenic assays obtained against sample S, sample Dcl or sample Dcl+ch (Table 10B, p<0.001); however, the inter-laboratory variation was reduced from 10.2% against sample S to 3.6% against both samples D.
Clotting and chromogenic assays discrepancy (clotting and chromogenic ratio= 1.25, p
<0.001) was apparent when sample A was assayed against sample S. There was no clotting and chromogenic discrepancy when sample A was assayed against either sample Dcl or Dcl+ch (clotting and chromogenic ratio of 0.98; p = 0.282).
Sample Purple relative to Sample S, the 4th IS for FIX Concentrate and using sample A and sample D as putative recombinant standards
In total, 14 sets of results, 10 clotting and 4 chromogenic assays, were returned for analysis.
Results for sample Pr were analysed relative to sample S and then recalculated against samples A (Acl) and D (Dcl) using assigned values (based on clotting assays only, 9.8 IU/ml for Acl and 9.4 IU/ml for Dcl)) and against samples A (Acl+ch) and D (Dcl+ch) using assigned values (based on overall potency estimates, 9.4 IU/ml for Acl and 8.9 IU/ml for Dcl))
Similar ranges of intra-laboratory GCVs were obtained for both clotting and chromogenic assays when sample Pr was assayed against samples S, A or D (Tables 11A, 11B). Only 1 lab gave GCV >10% for chromogenic assay when Purple was assayed against A; for clotting assays, 2 labs gave GCVs just over 10% when assayed against sample A and 3 labs gave GCVs over 10% when assayed against sample D (Tables 11A, 11B).
Some significant clotting and chromogenic assay discrepancies were observed (Table 11C: vs S, p < 0.001; vs Acl, p = 0.038; vs Dcl, p = 0.02). However, the clotting and chromogenic ratio was reduced from 1.26 when sample S was used as the standard to 0.90 and 0.88 when against samples A and D.
The overall potency was around 4.8% higher against Acl and was higher, at 6.0%, when assayed against Dcl (Table 11A). Good agreement of overall estimates was obtained when Pr was assayed against samples Dcl+ch and Acl+ch and it was only 1.2% higher than against sample S (Table 11B). The inter-laboratory variability as expressed by GCVs were reduced from 14.6% against sample S to 9.5% against A and 10.7% against sample D.
Sample P, the 4th IS for FII, VII, IX and X Plasma relative to the 4th IS for FIX Concentrate The FIX potency of sample P was calculated relative to sample S, the 4th IS for FIX concentrate.
Individual assay results are shown in Appendix III. Individual laboratory mean and overall potency estimates for clotting and chromogenic assays are presented in Table 8 and are also shown in Figure 6.
Clotting and chromogenic assays against 4th IS for FIX Concentrate
o The intra-laboratory variation (%GCV) ranges were 0.7 – 14.9% for clotting assays and narrower, 1.6 – 7.9%, for the chromogenic assays. Only 4 out of 54 laboratories had %GCV >10% and 25 laboratories had %GCVs <5% for the clotting assays (Table 8).
o The inter-laboratory variability was high, at 16.3% for the clotting assays and lower, at 8.6% for the chromogenic assays (Tables 8, 5A, 5B).
o Overall potency estimates for clotting and chromogenic assays were 0.91 and 0.81 IU/ml and remained the same when Lab 15 for clotting and Lab 21 for
chromogenic were excluded. (Tables 5A, 5B).
o A significant assay discrepancy was observed with estimates by clotting methods (12% higher than chromogenic assay results) yielding a clotting to chromogenic ratio of 1.11 (Table 5B).
o The overall potency by both clotting and chromogenic assay was 0.88 IU/ml and this equates to 2.3% difference to the labelled potency of 0.86 IU/ml. However this difference is not statistically significant (unpaired t-test p=0.195). The inter- laboratory GCV, including both clotting and chromogenic assay results was 15.4%
(Table 7)
Long term stability of the candidates and samples
Accelerated degradation studies have been initiated for all four concentrate preparations. Table 12 shows the predicted loss of clotting activity for samples A, B, C and D after being stored at various elevated temperatures (-70, –20, +4, +20, +37 and +45°C). All samples showed low predicted loss of activity at storage temperature of -20 °C. However, it should be noted that these data are preliminary and further results are required to fully evaluate the long term stability of these samples.
Discussion
The most important prerequisite for a potency reference standard is improvement of laboratory agreement and this is evidenced by its ability to improve agreement of potency estimates within a method and between method types. This study generated data from 55 laboratories, using
different methods to support the choice of candidates and value assignment of these candidates as potency standards for FIX. There were two main aims to this part of the study:
a. to assign the blood coagulation factor IX functional activity values to the replacement WHO International Standard for Blood Coagulation Factor IX, Concentrate and Ph
Eur. BRP for Human Blood Coagulation Factor IX, relative the 4th IS for FIX, Concentrate
b. To assess the comparability of the plasma derived IS for FIX concentrate with recombinant FIX preparations and calibrate an IS for recombinant FIX relative to the 4th IS for FIX, Concentrate
Overall, the majority of the assays were statistically valid even when the recombinant samples (A, D, Purple) were compared with the plasma derived International Standard for FIX concentrate, thus supporting potency labelling of recombinant FIX products in IU. The majority of the intra- laboratory GCVs were under 10% and a high proportion were less than 5% for all assay method types, thus indicating the laboratories were able to perform the assays with good reproducibility.
Results from only 2 laboratories were identified as outliers, Lab 15 for clotting assays and Lab 21 for chromogenic assays, and these were excluded in the value assignment of the proposed
International Standards and Ph Eur BRP.
Value assignment to the 5th IS for FIX, Concentrate and the Ph Eur BRP for FIX, Batch 3 The current (4th IS) and the previous International Standards were value assigned using 1-stage clotting assays only. In the 2008 collaborative study that value assigned the 4th IS, the Ph Eur BRP Batch 2 and the FDA/CBER standard, two chromogenic kits were used, however, since there was only one set of data for each assay kit returned for analysis, it was decided that assignment of the three standards should exclude results from chromogenic assays. Since the 2008 study, the 2 commercial chromogenic assay kits have become more widely used, especially in the development of new generation products and in the estimation of low levels of FIX in haemophilic patients. In the present study, 15 laboratories returned data for chromogenic assays and since discussions with manufacturers and regulators indicate that chromogenic assays are used as part of the characterisation package for FIX products, value assignment for the 5th IS for FIX, Concentrate also considered results from chromogenic assays as well as those obtained using 1-stage clotting assays. For the Ph Eur BRP, it is clear that the calibration should consider results only from the Monograph method and this is the 1-stage clotting assay, using FIX
deficient plasma as diluent. There are 2 plasma derived candidates, samples B and C, both could be considered as the replacement IS and Ph Eur BRP. The following summarises the essential points for consideration:
o Intra-laboratory variability: Both samples gave similar ranges of GCVs for all assay methods when assayed against sample S, the 4th IS for FIX, Concentrate (Tables 3A, 3B) o Effect of pre-diluent: For sample B, the potency estimates were 10.5 IU/ml, using either
FIX deficient plasma or buffer as pre-diluent. However, for sample C, the estimates were 9.0 IU/ml when FIX deficient plasma was used as pre-diluent and 8.5 IU/ml, 6% lower, when buffer was used (Table 4). The inter-laboratory agreement was better for sample B (GCVs – FIX deficient plasma: 3.4%, buffer: 4.6%) than sample C (GCVs –FIX deficient plasma: 4.8%, buffer: 7.8%) regardless of diluent, and the agreement was improved when FIX deficient plasma was used, variation as expressed by GCVs was lower. Both samples were prepared from the same FIX therapeutic product and the major difference between
samples B and C is the formulation. It is possible that sample B which has a similar formulation as the current IS, and different formulation of sample C caused this discrepant result. This interesting effect will be further investigated by NIBSC.
o Effect of APTT reagents: There was no obvious bias with the 15 APTT reagents
employed in this study on the potency estimates obtained using different APTT reagents for either samples B and C (Figure 5).
o Effect of Chromogenic assay kits: For samples B and C, when assayed against the 4th IS, Concentrate , there was no significant difference in potency estimates obtained using either kits; the Hyphen to Rossix potency ratios were 0.98 and 0.99 for samples B and C respectively. The Inter-laboratory GCVs were less than 8% for both samples and both kits (Table 6).
o Stability: Preliminary accelerated degradation studies on both samples B and C predicted no loss of activity when stored at -20°C.
o Number of ampoules available: For harmonisation purpose, it would be ideal for the IS and BRP to be the same batch of material, providing there is sufficient stock to support this. There are approximately 20,000 ampoules of sample B and 24000 ampoules of sample C. Although more ampoules of sample C are available, there should be sufficient ampoules of sample B to cover both reference standards.
In summary, given that sample B gave agreement of potencies by all assay method types and that sample C gave discrepant results in clotting assays using FIX deficient plasma or buffer as diluents, it is proposed that sample B be the 5th IS for FIX, Concentrate and the Ph Eur BRP Batch 3. The assignment of the 5th IS is based on results from 1-stage clotting with both FIX deficient plasma and buffer as diluents and chromogenic assays, while the value for the BRP is assigned with value from 1-stage clotting assays using only FIX deficient plasma as diluent. For both standards, the unitage proposed is 10.5 IU/ampoule.
Assessment of on-bench stability of sample B was carried out at NIBSC by storage of the reconstituted sample on melting ice. The potency at 1 h, 2 h, 3 h and 4 h were estimated relative to freshly reconstituted 14/148 at each time-point. Two assays were carried. No significant difference was observed between the potency values for the time points. This indicates that the material would be stable for up to 4 hours storage on melting ice.
Time point Potency % fresh ampoule (95% confidence intervals)
1 h 100.0 (96.5 – 104.4)
2 h 101.7 (98.2 - 105.3)
3 h 100.0 (94.7 - 105.3)
4 h 98.2 (93.8 – 103.5)
Rationale for route of value assignment to an International Standard for Recombinant FIX Results from a previous international collaborative study (NIBSC Phase II study 2013) indicated that although the 3 recombinant FIX products could be assayed validly against the 4th IS for FIX Concentrate, there were substantial potency discrepancies within 1-stage clotting assays and between clotting and chromogenic methods. Agreement of potencies was obtained when the 3
recombinant products were assayed against a recombinant reference preparation. The current study aimed to investigate these discrepancies further and confirm the need for a recombinant reference standard to harmonise assay methods and improve agreement of potency estimates within and between laboratories. The present study included 2 recombinant products, samples A and D. Additionally, a third recombinant product, coded Purple, was also sent to 6 laboratories so that the 3 recombinant products could be compared.
The current practice for potency labelling of recombinant FIX products is by one-stage clotting assays against the 4th IS for FIX Concentrate or a reference standard that is traceable to the 4th IS.
The impact of value assignment using a recombinant putative standard labelled with overall potency by both clotting and chromogenic assay results or labelled with estimate by clotting assays only was evaluated. For sample A, in addition to analysis of data exploring the effect of using sample D as a putative standard with value assigned from all assays (Dcl+ch), with the overall potency estimate, 8.9 IU/ml against sample S, the results were also reanalysed using the clotting assay GM for sample D (Dcl) at 9.4 IU/ml.
Potency estimates for sample A relative to sample S, the 4th IS for FIX Concentrate and sample D:
o The intra-laboratory variation was similar when sample A was assayed against sample S or sample Dcl or sample Dcl+ch (Table 10A, 10B)
o The inter-laboratory variability for both clotting and chromogenic assays were reduced by approximately two-fold (Table 10C).
o There was significant clotting and chromogenic assay discrepancy when sample A was assayed against sample S, with chromogenic assays giving 25% lower
potency than the clotting assays (p<0.001) This discrepancy was eliminated when sample A was reanalysed against either sample Dcl or Dcl+ch (clotting and chromogenic ratio 0.98, p = 0.282).
o Potency estimates
The clotting potency estimates for sample A against samples S and Dcl were the same, giving 9.8 IU/ml, but approximately 5% lower, at 9.3 IU/ml when assayed against Dcl+ch (Table 10C).
The estimates by chromogenic assays against S were over 20% lower than those obtained against Dcl or Dcl+ch.
It is clear that there is improvement in agreement of potency by both clotting and chromogenic assays when sample A was assayed against sample D (compare Figures 1 and 7). While the overall potency estimate generated against Dcl+ch was not significantly different to values from assays against S (Table 10C, p =0.956), potency obtained against Dcl was significantly different (Table 10C, p = 0.003).
o This indicates that if sample D is a standard for sample A, it will reduce both inter- laboratory variability and improve agreement of potencies by both clotting and chromogenic assays.
o Taking into account that the current recombinant products are labelled against the 4th IS for FIX concentrate using clotting assays, the clotting potency of sample A relative to sample S, the 4th IS for FIX concentrate was compared with the overall potency estimates obtained against Dcl and Dcl+ch. Table 10C shows that there was good agreement of potency against samples S and Dcl (A vs S clotting: 9.8 IU/ml; A vs Dcl overall: 9.8 IU/ml; p =0.779). However there was significant differences in potencies when compared with the overall value obtained with Dcl+ch (A vs S clotting: 9.8 IU/ml; A vs Dcl+ch overall: 9.3 IU/ml; p =0.003).
Therefore if sample D is to be the standard for recombinant products, value
assigned using clotting assays results only, this will ensure the continuity of the IU and there should be minimal shift in potency labelling of recombinant products.
The recombinant standard will also help to reduce inter-laboratory variability and harmonise clotting and chromogenic assay results.
Potency estimates for sample Purple relative to sample S, the 4th IS for FIX Concentrate, sample A and sample D:
o The intra-laboratory variation was similarly low when sample Purple was assayed against samples S, A, or D (Table 11A, 11B), indicating that the laboratories were able to assay the recombinant products with precision and reproducibility.
o While the inter-laboratory GCVs were slightly reduced for the clotting assay when Purple was assayed against A or D than against S (Tables 11C, 11D; GCVs vs S:
8.3%; vs A: 7.1%; vs D: 8.0%;), the GCVs for the chromogenic assays were not reduced (Tables 11C, 11D; GCVs vs S: 10.1%; vs A: 10.6%; vs D: 10.8%;).
o Although some clotting and chromogenic discrepancies were found to be
statistically significant, the clotting to chromogenic ratio was reduced from 1.26 when assayed against S to 0.90 against A and 0.88 against D, (Tables 11C, 11D).
o Although there was no significant difference to the overall potency estimates against the different standards, using results from all methods (Tables 11A. 11B), the overall potency agreement was improved when Purple was assayed against samples A or D as exemplified by the approximately 5% reduction in the inter- laboratory variability as expressed by GCVs. (Tables 11A, 11B, 11C, 11D).
o These results indicate that if sample A or D was used as a standard for sample Purple, it will minimise clotting and chromogenic discrepancies and improve potency agreement by reduction of overall inter-laboratory variability
The reanalysis of data for samples A, D and Purple confirms finding from the previous study that assaying recombinant product against a recombinant standard helps to reduce assay discrepancies and increase potency agreement between laboratories.
Conclusions
The 5th IS for Blood Coagulation Factor IX, Concentrate and Ph Eur BRP batch 3
The high inter-laboratory %GCV (Table 7, 15.4%) obtained when the 4th IS for FIX Plasma was assayed against the 4th IS for FIX Concentrate support the rationale of a concentrate potency reference standard for plasma derived FIX products.
Both candidates B, C gave similarly low within laboratory variability.
There was no clotting and chromogenic discrepancy for either candidate.
Preliminary accelerated degradation studies showed that both candidates are predicted to be equally stable.
Candidate B gave slightly lower inter-laboratory variability
Different pre-diluent gave discrepant clotting assay results for candidate C.
Since candidate B gave agreement of potencies by different methods and pre-diluents, it is recommended that candidate B, 14/148 be the 5th International Standard for Blood Coagulation Factor IX, Concentrate and EP BRP for Human Coagulation Factor IX Concentrate, Batch 3.
Both reference standards are value assigned relative to the 4th International Standard for Blood Coagulation Factor IX, Concentrate, with the 5th International Standard for Blood Coagulation FIX, Concentrate potency labelled based on all functional activity assays and the Ph Eur BRP labelled based on one-stage clotting assays using FIX deficient plasma as pre-diluent. In both cases, the value is 10.5 IU/ampoule. The draft Instruction for Use for this proposed IS is illustrated in Appendix IV.
Should an International Standard for Recombinant FIX be established?
It is clear that statistically valid assays can be obtained when the current recombinant FIX products are assayed against the plasma derived IS for FIX Concentrate and that one of these products has been potency labelled against the IS for the last decade. Unlike FVIII, there is global agreement that 1-stage clotting assays is the assay of choice for potency labelling and therefore clotting and chromogenic assay discrepancies have not so far been an issue. Nonetheless, chromogenic assay kits for FIX are now available and these kits will suit the clinical need for assays with higher sensitivity for low level of FIX. The results from this study showed that there are significant chromogenic and clotting assay discrepancies when the recombinant samples were assayed against the current IS. In addition, the availability of new recombinant and modified products has brought attention to assay discrepancies within the 1-stage clotting assays. The package insert from one of the most recently licensed recombinant full-length FIX indicates that the FIX potency results for this product can be affected by the type of APTT reagent and
reference standard used in the assay and that differences of up to 40% have been observed. The current study, using a range of APTT reagents routinely used by manufacturers, regulators and clinical laboratories indicated that up to >60 % and >80% differences in potencies could be seen when recombinant samples A and D were assayed by clotting assays against the plasma derived IS. The APTT reagent discrepancy for sample A was reduced to ~20% and was reflected by the decrease of inter-laboratory GCV from 11.6 to 6.4% when sample D was used as the reference
standard. In addition when sample D was used as the putative standard for samples A or Purple, apart from lowering the overall inter-laboratory variability, the clotting and chromogenic
discrepancies was eliminated or reduced to an acceptable level.
Although having a standard for recombinant products in addition to the plasma derived concentrate IS may cause some initial confusion, precedent has been set for a number of
Biotherapeutics (Prolactin: WHO 3rd International Standard for Prolactin, Human, NIBSC code:
84/500 and WHO 1st International Reference Reagent for Prolactin, Human, recombinant. NIBSC code: 97/714; Follicle Stimulating Hormone (FSH): WHO 2nd International Standard for Follicle- Stimulating Hormone, human, recombinant, for bioassay, NIBSC code: 08/282 and WHO 5th International Standard for Follicle Stimulating Hormone, Luteinizing Hormone human, urinary for bioassay, NIBSC code:10/286) which have co-existing International Standards for
plasma/human derived and recombinant proteins and both of these International Standards serve well to support potency labelling and clinical measurement in patient samples of the different forms of the same therapeutic product types. The results from the study also support the
continuity of the FIX IU when transferring to the recombinant standard. This is illustrated by the same clotting potency of A, 9.8 IU/ml against S and sample D (when sample D is assigned using the clotting potency value; Tables 10C). The replacement strategy for such a standard should also be considered carefully and it is proposed that successive replacement recombinant standards should follow the same route as other International Standards i.e. value assignment relative to the previous recombinant standard but study should include current plasma derived concentrate standard so that any drift of IUs would be monitored.
While the establishment of an IS for recombinant full length FIX based on the data from the current study will serve well against the current licensed products, there is a possibility that more recombinant FIX products will be on market and that the future recombinant FIX products may not compare well with this recombinant IS. The NIBSC Phase II study has already identified that the modified recombinant products (e.g. pegylated-FIX) do not compare well against full length recombinant products and recombinant full length FIX should not be used as potency standard for these modified products. This study showed that the 3 recombinant full length products compared better against each other than against the plasma derived IS and the recombinant IS will promote better comparability of potencies amongst these products. The likelihood that another
recombinant full length FIX will behave substantially different to these 3 products is low, but not impossible and should be investigated should another recombinant full length FIX becomes available. There is also another advantage of the availability of an IS for recombinant FIX.
There have been numerous discussions amongst regulators, manufacturers and clinical laboratories on assay discrepancies related to clinical monitoring. The sensitivity and the
reproducibility of chromogenic assays are being considered alongside the conventional one-stage clotting assay and is supported by the US National Hemophilia Foundation who urged the US FDA to accelerate the approval of chromogenic assay kit and clinical laboratories to establish the chromogenic assays. The clinical laboratories will require support to improve intra- and inter- laboratory agreement and minimise assay discrepancies whenever they change reagents,
instruments and operators. Since product specific standards are not yet feasible, the availability
of a global recombinant International Standard will go a long way to aid the development and continual validation of assay methods for measurement of FIX activity following replacement therapy with recombinant full length FIX. The following table summarises the pros and cons of establishing an International Standard for Recombinant FIX and on balance, having a global standard for recombinant FIX would be useful to the community as the disadvantages could be minimised by careful and considered risk management:
Pros Cons
Improve intra-laboratory agreement, less reliant on calibration against the plasma derived IS and stability of manufacturers’ own in-house standards
Minimize the substantial assay discrepancies within and between clotting and chromogenic assays
Improve inter-laboratory agreement, giving more
confidence in label potencies and interchangeability of products if and when required
Useful independent resource for clinical labs to develop assays for measurement of recombinant FIX in patient samples
Too many standards, can be confusing to end-users
Future recombinant full length FIX may not assay so well against recombinant standard
Complex considerations for route of value assignment
Replacement strategy require careful consideration
Possible discontinuity of for labelling of licensed recombinant products
In consideration of which candidate should go forward as the 1st International Standard for Recombinant FIX, both candidates have similar physical characteristics in terms of coefficient of variation, residual moisture and oxygen headspace and passed all the recommended specifications for a WHO international biological reference standards. They gave similar intra- and inter-
laboratory variability. Candidate A, 07/142, was ampouled in 2007 with fewer ampoules
available than candidate D, 14/180. In addition, although all the candidates including samples A and D passed the activated coagulation factors test (all test clotting times >150s; Table 13), the amount of FIXa estimated in sample A was 5 times higher than that obtained for sample D (Table 14). High levels of FIXa in a FIX reference standard may have some influence on the potency estimates of test preparations. So it is recommended that candidate D be the 1st International Standard for Recombinant FIX. In terms of value assignment, since the Ph Eur monograph method for labelling is the one-stage clotting assay and globally the current licensed products are labelled by one-stage clotting assay, to ensure good continuity of the IU, it is recommended that the 1st IS for recombinant FIX be value assigned with overall clotting potency of 9.4 IU/ampoule, against the 4th IS for FIX concentrate. The established standard can be used as a reference
standard for measurement of recombinant FIX activity by either 1-stage clotting or chromogenic assays.
Based on the above rationale, it was proposed to the participants that Sample D, 14/180 be established as the WHO 1st International Standard for Recombinant Human Blood Coagulation Factor IX, with the assigned value for functional activity of 9.4 IU/ampoule.
Participants Comments and Responses
Thirty out of the 49 participating laboratories returned comments on the study. Most of the comments were associated with typos and queries related to analysis of results for individual assays. Following investigation of the revised data, the statisticians concluded that there will not be any major impact on the overall potency estimates or the recommendations for establishment of the standards and the tables have been updated for the ECBS report. All responded participants agreed with the proposal of the value assignment of the 5th International Standard for Blood Coagulation Factor IX, Concentrate and the addition of the FIX antigen value to the 4th International standard for Factors II, VII, IX and X, Plasma.
For the proposal of an International Standard for Recombinant FIX, with the exception of one lab (see comment A), who did not agree with the proposal, all others agree with the proposed establishment. There were 2 other significant comments (comments B and C).
Comment A
“At this time, we would recommend not supporting the proposal to establish an
International Standard (IS) for recombinant Factor IX (FIX) because this will complicate the meaning of the International Unit (IU) as it has been understood for decades by users of FIX products. Although we know that the IU is dependent on many factors, having one IU for all FIX products provides a reference with which one can gauge the effect of the variables on the resultant potency values. If we agree to having an IS for recombinant FIX concentrates, manufacturers of currently licensed recombinant FIX products would, in practice, need to recalibrate their in-house recombinant product-specific standards, which may result in adjustment of FIX protein content in the final container. As a result, we would have to re-assign the potency of all currently licensed recombinant FIX
products (both traditional and long-acting analogues), which would most likely
necessitate changes in dosing regimens. In addition, we can expect that these two FIX IS will be replaced at different times and experience different drifts, which will further exacerbate the differences between the two standards.
On the other hand, we know that plasma-derived and recombinant FIX preparations do behave differently as we have seen in laboratory studies comparing different APTT reagents. It may be worthwhile to investigate the possibility of harmonizing potency assignment and product description in the labelling of different recombinant and long- acting FIX products. To this end, the availability of a recombinant FIX reference reagent would be helpful in such studies. Thus, we can support establishing the proposed
candidate as a WHO Reference Reagent for recombinant FIX concentrates.”
Response from NIBSC
We like to make clear that a recombinant FIX International Standard or International Reference Reagent should not be used for assays of long-acting products or any other modified products and this will be explicit in the Instruction for Use to the end-users.
There is a possibility that a recombinant FIX International Standard may cause a shift in labelling of the current licensed (or product in development) recombinant FIX products. The current study data (Table 10A) showed that the overall potency estimate for sample A (a recombinant product) against the 4th International
Standard (sample S) was the same as the value against sample D (another
recombinant product). However, due to the variability of the APTT reagents, there will be discrepancies within individual laboratories. So results on recombinant products against the proposed International Standard from manufacturers will be needed to assess the impact of the recombinant standard on the potency labelling of their products. Even if there is a slight shift of the unit, this one-time transfer will have the long-lasting advantage that a recombinant standard will diminish the APTT reagents and the clotting and chromogenic discrepancies.
Comment B
“1. The mere fact that only “the OS method” is prescribed by regulatory authorities as the method to be used for release testing, and the results of which are hence proposed for Sample D assignment, should not be taken as an argument to prevent introduction of CS methods for release testing. We also want to state that there is no support for any tentative view that the OS results for Sample D are more true than the CS results. The ratio of close to 1.3 for OS/CS (close to 1.2 if only including the seven Rox Factor IX results) when A and D were value assigned vs the 4th IS may encourage follow-up work to investigate why the difference appears.
2. The data from the study clearly show a significantly lower inter-lab GCV for CS methods when Sample A was value assigned vs Sample D, no matter whether Dcl or Dcl+ch was used for D. Such results strongly indicate that CS methods may well improve consistency in FIX potency assignments in manufacturers´QC labs. Again, it would we quite unfortunate if regulatory authorities will not consider CS methods to be allowed for potency assignments by referring to the Sample D value assignment.
3. The proposed value and use of Sample D (14/180) will not solve the problems with OS methods on analysis of long-life rFIX concentrates. The data from CS methods,
although so far limited, clearly indicate that CS methods may be a better alternative.”
Response from NIBSC
The value assignment of the proposed standard based on clotting assays only is to
minimize the risk of a shift in potency labelling of current licensed products which were licensed on potency labelling by the clotting assay. The recombinant standard should reduce clotting and chromogenic assay discrepancy and allow the development of the chromogenic assay not only as an alternative for potency labelling but also for clinical monitoring.
