WHO/BS/2012.2207 ENGLISH ONLY
EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION Geneva, 15 to 19 October 2012
COLLABORATIVE STUDY FOR VALUE ASSIGNMENT OF THE 3rd INTERNATIONAL STANDARD FOR LOW MOLECULAR WEIGHT HEPARIN Elaine Gray1,3, John Hogwood1, and Peter Rigsby2 1
Haemostasis Section, 2Biostatistics, 3Principal Investigator National Institute for Biological Standards and Control Potters Bar, Hertfordshire, EN6 3QG, UK
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 01 October 2012 and should be submitted electronically to the Responsible Officer: Dr Ana Padilla at email: padillaa@who.int, with a copy to Dr David Wood at email: woodd@who.int.
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WHO/BS/2012.2207 Page 2 Summary Twenty-two laboratories from 13 different countries participated in a collaborative study to value assign the 3rd International Standard for Low Molecular Weight Heparin against the 2nd International Standard for Low Molecular Weight Heparin. Two candidates, sample A (NIBSC code 11/174) and sample B (NIBSC code 11/176) were included in the study The intra-laboratory variability was low for both candidates. Over 80% of the laboratories obtained potency estimates that had intra-laboratory geometric coefficients of variation (GCV) of less than 5% when the test samples were assayed against the 2nd International Standard for both anti-Xa and anti-IIa activities, indicating that the participants performed these assays reproducibly and with high precision. For both anti-Xa and anti-IIa activities, inter-laboratory variability was lower for sample B, 11/176 than for sample A, 11/174. The GCVs for anti-Xa and anti-IIa assays for sample B were 3.1% and 3.2% respectively, compared with 6.1% and 8.2% for the anti-Xa and anti-IIa assays for sample A. Taking into consideration the lower inter-laboratory variation for sample B, we propose to recommend sample B, 11/176, be the 3rd International Standard for Low Molecular Weight Heparin. It is therefore recommended that the proposed 3rd International Standard for Low Molecular Weight Heparin (11/176) be assigned with potencies for anti-Xa and anti-IIa activity, relative to the 2nd International Standard for Low Molecular Weight Heparin (01/608): Activity Anti-Xa Anti-IIa Proposed 3rd IS IU/ampoule 1068 342
Introduction There are at least 8 licensed low molecular weight heparin products and a number of biosimilars available worldwide. The 1st International Standard for Low Molecular Weight heparin established in 1987 successfully harmonised potency labelling of clinical low molecular weight products. The 2nd International Standard (IS) for Low Molecular Weight Heparin (01/608) was established by the Expert Committee on Biological Standardisation (ECBS) of the World Health Organisation (WHO) in October 2003. Because of low stock level, it is now necessary to replace this IS and this study served to evaluate two candidates with a view to value assign a replacement IS against the 2nd IS. Two prior studies were carried out before the two candidates were chosen for this study. The licensed products are diverse in their biological activity profiles with anti-Xa and anti-IIa ratios ranging from 1.5 to 10. In addition, there are now a new generation of ultra-low low molecular weight heparins with anti-Xa to anti-IIa ratio greater than 80 in phase II and III clinical trials. These pilot studies were required to ensure the best comparators were selected as candidates. The first study was a NIBSC in-house to investigate how well the new generation ultra-low low molecular weight heparin products compare with the current international standard (IS). The results indicated that the current IS can serve as a standard for these products. The second study included 7 licensed low molecular weight heparins and the nine participants of this study were NIBSC, manufacturers of the low molecular weight heparins and pharmacopoeial laboratories. Based on how well they compare with the current IS and other products in anti-Xa and anti-IIa
WHO/BS/2012.2207 Page 3 assays, two preparations were chosen to go forward in the present study as candidates for the replacement of the 2nd IS for Low Molecular Weight Heparin.
Participants Twenty-three laboratories agreed to take part in the study, with 22 participants from 13 different countries (2 Austria, 1 Australia, 1 China, 1 Denmark, 4 France, 3 Germany, 1 India, 1 Italy, 2 Japan, 1 Sweden, 1 Switzerland, 1 UK, 3 USA) returning data in time for the statistical analysis. The participants included 8 heparin manufacturers, 3 clinical laboratories, 2 Pharmacopoeias, 4 assay kit manufacturers and 5 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. Candidates Two candidates, NIBSC codes 11/174 and 11/176 were included in the study. The bulk materials were clinical grade APIs. Approximately 10mg/ml was dissolved in sterile water and distributed in glass ampoules, filled and freeze-dried according to guidelines for production of international standards1 Samples The protocol of the study and the following coded samples were sent to each participant: Sthe 2nd International Standard (IS) for Low Molecular Weight Heparin, 01/608 Assigned potency anti-Xa: 1097 IU/ampoule; anti-IIa 326 IU/ampoule. NIBSC code 11/174 Potency range anti-Xa: 925 – 975; anti-IIa 220 – 260 IU/ampoule NIBSC code 11/176 Potency range – anti-Xa: 1000 – 1050; anti-IIa 325 – 375 IU/ampoule
A-
B.
Assay Methods Each participant was requested to perform their routine method(s) for anti-Xa and anti-IIa activities. Some laboratories performed more than one method and the data from each method were treated as separate sets of results e.g. Lab 17a and Lab 17b. A list of methods used by the participants is given in Appendix 3. For both anti-Xa and anti-IIa assays, 5 laboratories carried out the United States Pharmacopoeial (USP) monograph methods and seven laboratories used the European Pharmacopoeial (EP) assays for low molecular mass heparin. One laboratory used the Japanese Pharmacopoeial method. Seven laboratories returned data from their in-house methods. All these methods employed purified antithrombin and proteases (FXa and thrombin). Three laboratories used commercial anti-Xa assay kits that employ plasma as a source of antithrombin, while one laboratory used a commercial kit that involved purified antithrombin. One laboratory used HEPTEST, an anti-Xa clot based assay. Lab 17 also returned data obtained using the Prothrombinase Induced Clotting Time (PiCT) and the Activated Partial Thromboplastin Time (APTT). The laboratory calculated the potencies of the candidates against the anti-Xa value of sample S, the 2nd IS. Since the APTT is based on thrombin inhibition and it is unclear whether
WHO/BS/2012.2207 Page 4 FXa or thrombin inhibition predominates in the PICT assay, the results from these methods were not included in the analysis for the overall potency estimates of samples A and B. Study Design Participants were requested to carry out four assays for each activity using fresh ampoules of samples S, A, and B in each assay. Laboratories were requested to assay anti-Xa and anti-IIa activities on the same ampoules. Within each assay, participants were requested to assay three dilutions (preferably four dilutions) of each of the samples S, A, and B in replicate, according to balanced assay designs. Raw assay data were returned together with calculated estimates for samples A (11/174) and B (11/176) relative to sample S (2nd IS, 01/608) from each individual assay.
Statistical Analysis An independent statistical analysis of raw data was performed at NIBSC. Potency estimates relative to the 2nd International Standard (01/608), were calculated by parallel-line analysis2 of assay response (absorbance or clotting time) against log concentration, independently for each test sample included in each assay. For the majority of laboratories untransformed assay responses were used in analysis, with log transformed responses being used in a small number of laboratories in order to obtain a linear dose-response relationship. 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). Grubbs’ Test2 was applied to the log transformed laboratory mean potencies in order to detect any significant outliers. Comparisons between methods have been made by unpaired t-tests of log transformed laboratory mean potencies. In general, the laboratories’ reported potency estimates were close to those calculated centrally by NIBSC (data not shown). Results Assay Data Twenty two participants returned a total of 188 assays which comprised 100 anti-Xa assays, and 80 anti-IIa assays. Lab 17 returned 8 assays from APTT and PiCT methods and these were not centrally analysed by NIBSC. The individual assay results calculated by NIBSC, together with the geometric mean potencies (GM) and the intra-laboratory variation expressed as GCV are presented in Table 1a, 1b, 2a and 2b. Assay Validity All assays were valid as defined by criteria set out in the Statistical Analysis Section of this report.
WHO/BS/2012.2207 Page 5 Sample A, 11/174 Intra- and inter- laboratory variability Estimates of intra-laboratory variability (between assays) for both anti-Xa and anti-IIa activities in sample A are given as geometric coefficients of variation (GCV) for potency estimates relative to sample S (Tables 1a and 2a). These exceeded 5% in 5 and 4 laboratories for anti-Xa and anti-IIa assays respectively, with 80% of cases giving GCV lower than 5%. Within each assay type, as shown by the range of intra-laboratory GCVs, there were no obvious differences between the reproducibility of the different tests methods used by the participants. Inter-laboratory variability was low for both anti-Xa and anti-IIa assays, being 6.1 and 8.2%, respectively, showing reasonably good agreement between laboratories. Potency estimates The overall potency estimates for sample A (11/174) were calculated relative to the assigned values for the 2nd IS. Laboratory mean potency estimates and overall potency estimates, together with 95% confidence limits, for anti-Xa and anti-IIa activities are shown in Table 1a and Table 2a. The potency estimates relative to the 2nd IS from individual assays are also presented in histogram form in Figures 1a and 2a. The histograms illustrate good agreement between laboratories for sample A relative to the 2nd IS for activities; with no outliers detected. The overall geometric mean anti-Xa and anti-IIa potency estimates were 1030 and 275 IU/ampoule respectively, with an anti-Xa to antiIIa ratio of 3.75 (Table 3). Sample B, 11/176 Intra- and inter- laboratory variability Estimates of intra-laboratory variability for both anti-Xa and anti-IIa activities in sample B are given as geometric coefficients of variation (GCV) for potency estimates relative to sample S (Table 1b and 2b). These exceeded 5% in only 6 and 3 laboratories for anti-Xa and anti-IIa assays respectively, with 80% of cases giving a GCV lower than 5%. Inter-laboratory variability was exceptionally low at 3.1 and 3.2%, respectively, for anti-Xa and anti-IIa assays, showing good agreement between laboratories. Potency estimates The overall potency estimates and 95% confidence limits for test sample B calculated relative to the assigned values for the 2nd IS are shown in Tables 1b and 2b, together with laboratory mean potency estimates. The potency estimates relative to the 2nd IS from individual assays are also presented in histogram form in Figures 2a and 2b. The histograms illustrate good agreement between laboratories for sample B relative to the 2nd IS for both activities; with no detectable outliers. The overall geometric mean anti-Xa and anti-IIa potency estimates were 1068 and 342 IU/ampoule respectively, with the anti-Xa to anti-IIa ratio being 3.12 (Table 3). Discussion The primary aim of this study was to value assign a replacement International Standard for Low Molecular Weight Heparin. For both anti-Xa and anti-IIa activities, over 80% of the participants carried out methods that employed purified antithrombin, factor Xa and thrombin. The majority of the intra-laboratory GCVs were below 5%, indicating that these assays are reasonably robust and reproducible within each laboratory. Approximately 60% of the laboratories carried out pharmacopoeial methods.
WHO/BS/2012.2207 Page 6 There were no significant differences in the anti-Xa and anti-IIa potencies obtained using either the USP or EP monograph methods for both samples(Tables 4a and 4b). The inter-laboratory agreement as expressed using GCVs for both pharmacopoeial methods was also similar. The overall geometric mean potencies by each pharmacopoeial method for sample B, 11/176, were closer to the overall geometric mean potencies than those obtained for sample A (Tables 4a and 4b). No outliers were detected for either anti-Xa or anti-IIa assays for both samples A and B. The results of PiCT and APTT assays returned by Lab 17 were not included in the calculation of the overall geometric mean anti-Xa potencies of samples A and B as it was unclear whether these data should be included into the anti-Xa group (as performed by the participant). Better agreement between laboratories was obtained for sample B than for sample A. The interlaboratory variability for sample A, 11/174, expressed as GCVs was 6.1% and 8.2% for anti-Xa and anti-IIa activity respectively. For sample B, 11/176, the inter-laboratory GCVs was 3.1% and 3.2% for anti-Xa and anti-IIa activity (Tables 1a, 1b, 2a and 2b). Table 6 shows the physical characteristics of sample B, in terms of the variability of the fill, residual moisture content and head oxygen space and homogeneity by functional activities were acceptable. In addition, preliminary accelerated degradation study on ampoules of sample B that have been stored at elevated temperature for 5 months showed that there is no apparent loss of anti-Xa and anti-IIa activities when stored at 20°C (Table 7a). Table 7b shows that sample B is relatively stable following reconstitution and no loss of activity was detected 5 hours upon reconstitution when stored at ambient temperature. Since sample B, 11/176, gave lower inter-laboratory variation, closer potency estimates to the overall values by EP and USP methods and reasonable stability, it will serve well as a replacement for the current 2nd IS. Proposal to Participants Overall, sample B, 11/176, gave better inter-laboratory agreement for both anti-Xa and anti-IIa activity and the overall geometric mean potencies of sample B were in good agreement with the mean estimates by both the EP and USP monograph methods. It is therefore recommended that sample B, 11/176, be the replacement international standard for low molecular weight heparin, with value assigned using all laboratory mean estimates for both anti-Xa and anti-IIa activities. Responses from participants and the experts nominated by the SSC/ISTH Control of Anticoagulation Sub-Committee All participants and SSC nominated experts who have sent in responses agreed with the proposals. There were no comments in relation to the analysis or interpretation of the data in the study. Proposal and Recommendation to the ECBS Sample B, 11/176, be the 3rd International Standard for Low Molecular Weight Heparin, 11/176, relative to the 2nd IS Activity Anti-Xa Anti-IIa Proposed 3rd IS IU/ampoule 1068 342
A draft of “The Instruction for Use” for the proposed Standard, 11/176 is illustrated in Appendix 4.
WHO/BS/2012.2207 Page 7 References (1) Finney DJ. Statistical Method in Biological Assay. 3rd Edition. London: Charles Griffin 1978. (2) Grubbs F. Procedures for Detecting Outlying Observations in Samples. Technometrics, 1969; 11: 1-21. Acknowledgements We would like to thank the participants of the study, and the support of the Control of Anticoagulation Subcommittee of the SSC/ISTH and the following manufacturers who donated material for the two pilot and definitive WHO studies: – Sanofi – Pfizer – Leo – Opocrin – Rovi – GSK
WHO/BS/2012.2207 Page 8 Table 1a: Table 1a: Potency estimates, intra- and inter-laboratory GCV for Anti-Xa activity in sample A relative to sample S, the 2nd IS for Low Molecular Weight Heparin Estimates (IU/ampoule) Assay 1 Assay 2 Assay 3 Assay 4 GM GCV 972 963 970 974 970 0.5% 987 991 993 986 989 0.3% 976 941 973 964 963 1.7% 1056 1019 1014 1005 1023 2.2% 1040 1006 1041 1032 1030 1.6% 973 1021 1008 1094 1023 5.0% 1180 1166 1202 1134 1170 2.5% 1059 1064 1071 1036 1057 1.5% 983 985 983 1019 993 1.8% 1073 1063 1127 1044 1076 3.3% 956 948 962 944 952 0.9% 1024 968 970 987 987 2.6% 1026 1003 972 975 994 2.6% 1021 1024 960 1092 1023 5.4% 1074 1154 996 1149 1091 7.2% 1154 1151 1197 1131 1158 2.4% 981 1025 978 1033 1004 2.9% 1083 1046 1078 1079 1072 1.6% 1009 955 1009 976 987 2.7% 909 928 991 926 938 3.8% 1042 1049 1219 1088 1097 7.5% 1110 1063 1149 1186 1126 4.8% 990 956 1034 1121 1023 7.1% 1072 1046 1058 1033 1052 1.6% 859 999 1090 1022 989 10.6% 1030 Overall GM IU/ampoule 1005 - 1055 95% CL 6.1% Between Lab GCV GM: geometric mean; GCV: geometric coefficient of variation; CL: confidence limits. Purified AT: assays using purified antithrombin; Plasma AT; assays using plasma as source of antithrombin Lab 1 2 4 5 7 8 9a 9b 10 11 13 14 16 17a 17b 18 19 20 21 22 23 3 6 12 17c Method Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT plasma AT plasma AT plasma AT HEPTEST
WHO/BS/2012.2207 Page 9 Table 1b: Potency estimates, intra- and inter-laboratory GCV for Anti-Xa activity in sample B relative to sample S, the 2nd IS for Low Molecular Weight Heparin Estimates (IU/ampoule) Assay 1 Assay 2 Assay 3 Assay 4 GM GCV 1013 1011 1005 1017 1012 0.5% 1076 1085 1124 1061 1086 2.5% 1062 1085 1080 1077 1076 0.9% 1036 1069 1058 1047 1052 1.3% 1059 1073 1060 1073 1066 0.7% 1105 1165 1137 1135 1135 2.2% 1007 1059 1076 1087 1057 3.5% 1051 1094 1066 1057 1067 1.8% 1019 1073 1085 1098 1068 3.3% 1036 1042 1096 1103 1069 3.4% 968 990 1059 1077 1022 5.3% 1098 1022 1075 1052 1061 3.1% 1047 1036 1015 1017 1029 1.5% 1029 1047 1033 1086 1048 2.5% 1131 1092 984 1073 1068 6.1% 1035 1035 1098 1085 1063 3.2% 1069 1101 1022 997 1047 4.6% 1091 1064 1095 1096 1087 1.4% 1061 1060 1061 1009 1048 2.5% 1012 1164 1017 1084 1068 6.8% 1065 1062 1268 1106 1122 8.7% 1009 1040 1014 1041 1026 1.7% 1065 988 1134 1297 1115 12.2% 1095 1072 1069 1032 1067 2.5% 1048 1208 1165 1157 1143 6.3% 1068 Overall GM IU/ampoule 1054 - 1081 95% CL 3.1% Between Lab GCV GM: geometric mean; GCV: geometric coefficient of variation; CL: confidence limits. Purified AT: assays using purified antithrombin; Plasma AT; assays using plasma as source of antithrombin Lab 1 2 4 5 7 8 9a 9b 10 11 13 14 16 17a 17b 18 19 20 21 22 23 3 6 12 17c Method Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT plasma AT plasma AT plasma AT HEPTEST
WHO/BS/2012.2207 Page 10 Table 2a: Potency estimates, intra- and inter-laboratory GCV for Anti-IIa activity in sample A relative to sample S, the 2nd IS for Low Molecular Weight Heparin Estimates (IU/ampoule) Assay 1 Assay 2 Assay 3 Assay 4 GM 249 249 257 236 248 271 278 280 285 278 254 244 241 252 248 279 250 266 275 267 272 266 268 273 270 287 271 279 290 281 303 361 300 288 312 280 279 276 266 275 293 288 297 292 293 268 268 274 269 270 251 256 244 250 250 288 287 283 295 288 312 278 271 284 286 313 314 288 320 308 246 260 285 304 273 288 288 291 292 290 263 262 253 274 263 241 218 230 229 229 268 271 279 283 275 309 308 308 311 309 275 Overall GM IU/ampoule 265 - 285 95% CL 8.2% Between Lab GCV
Lab 1 2 4 5 7 8 9a 10 11 13 14 16 17 18 19 20 21 22 23 9b
Method Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT JP Clot
GCV 3.5% 2.1% 2.6% 5.1% 1.2% 3.1% 10.6% 2.5% 1.2% 1.2% 1.9% 1.7% 6.3% 4.8% 9.9% 0.8% 3.3% 4.1% 2.6% 0.5%
GM: geometric mean; GCV: geometric coefficient of variation; CL: confidence limits. Purified AT: assays using purified antithrombin; JP Clot: Japanese Pharmacopoeia clot-based assays
WHO/BS/2012.2207 Page 11 Table 2b: Potency estimates, intra- and inter-laboratory GCV for Anti-IIa activity in sample B relative to sample S, the 2nd IS for Low Molecular Weight Heparin Estimates (IU/ampoule) Assay 1 Assay 2 Assay 3 Assay 4 GM 347 351 361 368 356 330 328 340 345 336 354 349 354 354 353 333 349 314 342 334 327 329 326 333 329 347 354 350 367 354 349 348 334 333 341 344 344 352 345 346 362 335 334 339 342 331 328 334 338 333 341 332 332 335 335 319 329 321 326 324 355 319 331 319 331 340 352 312 340 336 359 358 346 334 349 352 346 343 347 347 348 340 336 356 345 372 346 355 353 356 316 313 352 339 330 353 359 367 359 359 342 Overall GM IU/ampoule 337 - 347 95% CL 3.2% Between Lab GCV
Lab 1 2 4 5 7 8 9a 10 11 13 14 16 17 18 19 20 21 22 23 9b
Method Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT Purified AT JP Clot
GCV 2.7% 2.4% 0.6% 4.7% 0.9% 2.4% 2.6% 1.2% 3.9% 1.4% 1.3% 1.5% 5.3% 5.3% 3.4% 1.0% 2.5% 3.2% 5.8% 1.6%
GM: geometric mean; GCV: geometric coefficient of variation; CL: confidence limits. Purified AT: assays using purified antithrombin; JP Clot: Japanese Pharmacopoeia clot-based assays
WHO/BS/2012.2207 Page 12 Table 3: Summary of Potency estimates and anti-Xa to anti-IIa ratios for samples A and B Sample A, 11/174 1030 275 3.75 Sample B, 11/176 1068 342 3.12
Anti-Xa activity IU/ampoule Anti-IIa activity IU/ampoule Anti-Xa:anti-IIa ratio
Table 4a: Comparison of potency estimates from USP and EP methods for sample A relative to the 2nd IS EP Methods IU/ampoule % from the GCV overall mean potency USP GCV IU/ampoule 995 (n = 5) 264 (n = 5) 3.1% 6.1% % from the overall mean potency 3.4 4.0
T-Test (p) >0.05 >0.05
Anti-Xa 982 (n = 7) 3.0% 4.7 Anti-IIa 261 (n = 7) 6.9% 5.1 GCV: geometric coefficient of variation;
Table 4b: Comparison of potency estimates from USP and EP methods for sample B relative to the 2nd IS % from the overall Methods mean IU/ampoule potency Anti-Xa 1055 (n = 7) 1.9% 1.2 Anti-IIa 341 (n = 7) 2.6% 0.3 GCV: geometric coefficient of variation; GCV EP USP GCV IU/ampoule 1071 (n = 5) 347 (n = 5) 4.2% 3.3% % from the overall mean potency 0.3 1.6
T-Test (p) >0.05 >0.05
Table 5a. Sample A, 11/174: Lab 17, Laboratory reported results for PiCT and APTT Assay 1 978.2 952.8 Assay 2 968.1 973.6 Assay 3 930.8 1034.3 Assay 3 894.4 941.2 Mean 942.9 975.5
PiCT APTT
Table 5b. Sample B, 11/176: Lab 17, Laboratory reported results for PiCT and APTT Assay 1 1192.4 1204.2 Assay 2 1201.4 1322.3 Assay 3 1140.1 1347.0 Assay 3 999.9 1101.0 Mean 1133.5 1243.6
PiCT APTT
WHO/BS/2012.2207 Page 13 Table 6: Product characteristics for sample B, 11/176 NIBSC Code Presentation Filling date Number of Ampoules available Liquid filling weight (g) (n=879, 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, after every 5000 ampoules and at the end of the fill were assayed against the 2nd IS using EP assays. 2 assays per ampoule were carried out. Effect of fill position assessed by ANOVA of log potencies. Mean dry weight (g) (n=25) Mean head space oxygen (%) (n=24) Residual moisture (%) (n=3) Storage temperature Address of processing facility Address of present custodian 11/176 Sealed, glass 3 ml DIN ampoules 22th Sept 2011 24,000 1.0068 (Range 1.0045 - 1.0090) 0.097 GCV Anti-Xa Anti-IIa 2.62% 0.85% P 0.814 0.974
0.00922 (CV 1.39%) 0.47 (CV 33.56%) 3.41 (CV 24.23%) -20°C NIBSC, Potters Bar, EN6 3QG, UK NIBSC, Potters Bar, EN6 3QG, UK
WHO/BS/2012.2207 Page 14 Table 7a: Accelerated Degradation Study on sample B, 11/176 Anti-Xa activity Confidence Anti-IIa activity Confidence IU/ampoule limits (95 %) IU/ampoule limits (95 %) (relative to -150°C) (relative to -150°C) 1.055 1.000-1.114 0.995 0.926-1.070 -70°C 1.093 1.035-1.153 1.019 0.948-1.100 -20°C 4°C 1.048 0.993-1.106 1.002 0.932-1.077 1.018 0.965-1.075 0.950 0.884-1.022 20°C 0.946 0.896-0.998 0.928 0.863-0.997 37°C 0.979 0.928-1.033 0.903 0.840-0.971 45°C 0.947 0.897-0.999 0.824 0.766-0.886 56°C An accelerated degradation study of 11/176 was carried out at NIBSC, employing anti-Xa and antiIIa assays with purified reagents. Table indicates the residual potency for ampoules stored at elevated temperatures for 5 months relative to ampoules stored at -150°C. Data from one timepoint (5 months) indicated that the material is stable. No significant loss of activity is observed after storage up to 20°C after 5 months compared to -150°C. Continual real time degradation studies of the –20°C against ampoules stored at –150°C and further accelerated degradation studies at elevated temperatures will be carried out to monitor the stability of the replacement standard. Table 7b: On-bench stability of sample B, 11/176 Anti-Xa activity Confidence Anti-IIa activity Confidence (IU/ampoule) limits (95 %) (IU/ampoule) limits (95 %) 1110 1079-1143 341 329-353 0h 1113 1083-1144 358 339-377 5h Assessment of on-bench stability was carried out at NIBSC by storage of the reconstituted sample at room temperature (22°C). The anti-Xa and anti-IIa activities at 0 h, and 5 h were estimated relative to freshly reconstituted 2nd IS LMWH 01/608 at each time-point. Two assays were carried out for each method. No significant difference is observed between the potency values for the two time points. This shows that the material is stable after 5 hours storage at room temperature of 22°C. Time Storage temperature
WHO/BS/2012.2207 Page 15 Figure 1a: Individual assay potency estimates for anti-Xa activity in sample A (11/146), relative to sample S, the 2nd IS. The number in the square denotes the laboratory code.
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WHO/BS/2012.2207 Page 16 Figure 2a: Individual assay potency estimates for anti-Xa activity in sample B (11/147), relative to sample S, the 2nd IS. The number in the square denotes the laboratory code.
30
GM: 1068IU/amp; GCV: 3.1%;
25
20
15 12 5 3 16 1 17a 22 18 19 17a 16 16 11 3 3 14 1 17c 22 18 17a 16 10 11 5 1
10
5 17b 13 6
0 900
13
21 19 1 9a 3
14 9b 2 21 13 12 9b 7 5 23 21 20 9a 5 23 21 19 9b 7 6 4
Number of estimates
22 18 17b 17a 13 23 9a 20 11 7 10 4 14 20 10 18 9a 11 19 4 12 17b 10 9b 20 7 14 4 12 2 8 2
2
8 17c 8 17c 22 6 17b 8
17c
23
6
1000
1100 Potency (IU/ampoule)
1200
1300
Figure 2b: Individual assay estimates for anti-IIa activity in sample B (11/147), relative to sample S, the 2nd IS. The number in the square denotes the laboratory code.
30
25
GM: 342 IU/amp; GCV: 3.2%;
20
15
10
5 18 5 23
0 200 250 300
17 16 17 23 16
19 16 9a 7 17 14 13 11 9a 7 16 14 13 11 7 2 13 7 5 2
23 18 14 13 11 5 21 20 2 21 10 18 14 10
22 20 10 4 2 23 19 10 8 4 22 20 18 9a 8 5 4 1 9b 21 20 9a 8 4 1
Number of estimates
9b 21 22 1 9b 19 19 17 11
8 1 9b 22
350
400
Potency (IU/ampoule)
WHO/BS/2012.2207 Page 17 Appendix 1: List of Participants Patrick Shaklee, BioCascade Incorporated, USA Susanne Alban, Christian-Albrechts-University of Kiel, Germany Stephaine Berrier and Caroline Bisson, Diagnostica Stago, France Guy Rautmann and Gwenaelle Cozic, European Directorate for the Quality of Medicines, France C.S. Venkatesan, Gland Pharma Ltd, India Patrick Rousseau, GlaxoSmithKline, France Bjørn Fehrmann and Trine Larsen, LEO Pharma A/S, Denmark Jeanine Walenga and Debra Hoppensteadt, Loyola University Chicago, USA Fan Huihong and Li Jing, National Institute for Food and Drug Control, P.R. China Takuo Suzuki, Akiko Ishii and Nana Kawasaki, National Institute of Health Sciences, Japan John Hogwood, National Institute for Biological Standards and Control, UK Hinrich Nagel, Nordmark Arzneimittel GmbH & Co KG, Germany Bruna Parma, Opocrin SpA, Italy Anders Svensson, Pfizer Health AB, Sweden Yukari Nakagawa, Pharmaceutical and Medical Device Regulatory Science Society of Japan, Japan Andreas Weiler and Manuel Seebacher, Sandoz GmbH, Austria Céline Martinez, Sanofi-Aventis, France Wolfgang Klein, Siemens Healthcare Diagnostics Products GmbH, Germany Roger Meier, Swissmedic, Switzerland Sabine Geiter and Matrina Leiter, Technoclone GmbH, Austria Allison Jones and Chong Loh, Therapeutics Goods Administration, Australia Michael Ambrose and Jeanne Fringer, US Pharmacopia, USA
WHO/BS/2012.2207 Page 18 Appendix 2: Protocol of the study Collaborative study to establish the 3rd International Standard for Low Molecular Weight Heparin Aims of Study The aim of this study is to assay two candidate low molecular weight heparin materials against the 2nd International Standard, 01/608, with the view to establish a new material as the 3rd International Standard for Low Molecular Weight Heparin
Low molecular weight heparin samples provided: Sthe 2nd IS for Low Molecular Weight Heparin, 01/608 Potency anti-Xa: 1097 IU/ampoule; anti-IIa 326 IU/ampoule. Potency range – anti-Xa: 925 – 975; anti-IIa 220 – 260 IU/ampoule Potency range – anti-Xa: 1000 – 1050; anti-IIa 325 – 375 IU/ampoule
AB-
Storage and Reconstitution The samples should be handled as follows: 1. Store all unopened ampoules -20°C or below 2. Ampoules should be allowed to warm to room temperature before reconstitution. 3. Please ensure that all material is at the bottom of the ampoule before opening. 4. 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. 5. 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. 6. 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°C prior to the assays.
Assay Methods Please perform in-house routine anti-Xa and anti-IIa methods for low molecular weight heparin, and provide details of the method in the results sheet provide..
WHO/BS/2012.2207 Page 19 Design and number of Assays Four sets of ampoules are provided. Assays for anti-IIa and anti-Xa should be carried out on each of the 4 sets. Each set should be tested on a different day (see schedule below). A balanced order of testing should be used.
Day 1, ampoule set 1 Day 2, ampoule set 2 Day 3, ampoule set 3 Day 4, ampoule set 4
S1 A1 B1 S1
A1 S1 A1 B1
B1 B1 S1 A1
B2 B2 S2 A2
A2 S2 A2 B2
S2 A2 B2 S2
Each letter refers to a set of three (preferably four) different dilutions (e.g. 1/10, 1/20, 1/40) and A1, A2 and S1, S2 etc. refer to separate sets of dilutions (replicates) made independently from the same ampoule. The range of dilutions should be chosen to lie on the linear portion of the dose-response curve. Report of data Raw data and estimated potencies should be recorded on in the forwarded excel sheets, sent to participants with this protocol once the “Acknowledgement of samples receipt form” has been returned. Results should be returned by Thursday 15th March 2012, results returned after this date may not be included in the analysis. Please contact john.hogwood@nibsc.hpa.org.uk if this will cause any issues, or you require any further information about the study.
WHO/BS/2012.2207 Page 20 Appendix 3: Methods used by the Participants Lab 01 02 03 Anti-Xa USP EP Commercial Kit, plasma as source of antithrombin USP Anti-IIa USP EP NT Other methods
04 05 06
USP
EP EP NT Commercial Kit, plasma as source of antithrombin 07 USP USP 08 USP USP 09 a. In-house a. In-house Clot based, possibly b. JP APTT 10 USP USP 11 In-house In-house 12 Commercial Kit, plasma as source of NT antithrombin 13 EP EP 14 EP EP 15 Results not returned Results not returned 16 In-house In-house In-house APTT and PICT, but 17 a. Commercial used anti-Xa potency Kit, purified of the standard to antithrombin calculate the estimates b. In-house of the test samples c. HEPTEST 18 USP USP 19 EP EP 20 In-house In-house 21 EP EP 22 EP EP 23 In-house In-house USP: United States Pharmacopoeia; EP: European Pharmacopoeia; JP: Japanese Pharmacopoeia; NT: not tested
WHO/BS/2012.2207 Page 21 Appendix 4: Draft Instruction for Use (IFU) for the Proposed 3rd International Standard for Low Molecular Weight Heparin 11/176
WHO/BS/2012.2207 Page 22