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Evaluation of Fourth International Standard for Whole Cell Pertussis Vaccine
Biologicals 38 (2010) 644e651
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Evaluation of Fourth International Standard for Whole Cell Pertussis Vaccine R. Gaines Das, P. Newland, C. Asokanathan, A. Douglas-Bardsley, M. Corbel, D. Xing* Division of Bacteriology, National Institute for Biological Standards and Control, Health Protection Agency, Potters Bar, Hertfordshire EN6 3QG, UK
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 June 2010 Accepted 23 July 2010
Whole cell pertussis vaccine is still widely used in many countries. An International Standard is needed for its potency control. The Third International Standard for Pertussis Vaccine was prepared about 40 years ago and its replacement was recommended by the Expert Committee for Biological Standardisation (ECBS) of the WHO. Material in ampoules coded 94/532 was prepared as a candidate replacement and has been evaluated in international collaborative studies which consisted of two parts. The first part, to assess the suitability of the candidate standard by comparing it with the Second International Standard for Pertussis Vaccine (IS2) involved 14 laboratories in 11 countries. The second part to compare the candidate standard with the Third International Standard for Pertussis Vaccine (IS3) involved 16 laboratories in 14 countries. Since 1995 various other studies have included the international standards and the results of these are also considered in assessing likely continuity of the IU for potency of whole cell pertussis vaccine. The preparation in ampoules coded 94/532 was adopted by the WHO ECBS in October 2006 as the 4th International Standard for whole cell pertussis vaccine and assigned an activity of 40 IU per ampoule on the basis of the studies reported here. Ó 2010 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.
Keywords: Whole cell pertussis vaccine International standard Potency
1. Introduction Although whole cell pertussis vaccine has been largely replaced by acellular pertussis vaccines in North America, Western Europe and some Asian countries, whole cell pertussis vaccine is still widely used in many countries and is likely to continue in use for a considerable time due to the fact that most whole cell pertussis vaccines are safe, effective, and cheaper to produce than acellular pertussis vaccines. Therefore an International Standard for whole cell pertussis vaccine is needed for potency control test (Kendrick test) [2]. The Third International Standard (IS3, ampoule code 66/ 303) was established in 1998 [1]. This material was originally prepared from the same bulk material as the Second International Standard (IS2, ampoule code 66/302), and included in a collaborative study with it [3]. Its stability was confirmed by a subsequent study before its establishment as IS3 in 1998 [4]. The candidate standard (ampoule code 94/532) was prepared in 1994 from a bulk material kindly donated by CSL, Australia. A collaborative study to compare this candidate with IS2 was organized by Dr Gert Albert Hansen, Statens Seruminstitut (SSI), Copenhagen. This study was carried out by 14 laboratories in 1995e1996, together with some preliminary stability testing. In 1997, the remaining ampoules of this material were transferred to
* Corresponding author. Tel.: þ44 (0) 1707 641 433; fax: þ44 (0) 1707 641 054. E-mail address: [email protected] (D. Xing).
National Institute for Biological Standards and Control (NIBSC), UK, together with the data for this study. At that time there were some questions about the stability of IS2 [5], and the full statistical analysis and report of this study were not completed pending clarification of this issue. Several subsequent studies have included IS2 and have not confirmed the suggested instability [4]. The IS3 was prepared about 40 years ago and the stock was running low. Therefore there is a need for replacement. The Expert Committee for Biological Standardisation (ECBS) suggested that the stability of the candidate replacement standard (94/532) should be assessed and the candidate standard should be compared with the IS3. The ECBS also suggested that this collaborative study should include regional reference standard(s) if possible [6]. This publication reports the results of two international collaborative studies and summarizes other related information. The first international study was launched in 1995 with the aim of comparing the candidate standard 94/532 with the Second International Standard for Pertussis Vaccine and was carried out by 14 laboratories in 11 countries. Throughout this report, this part is denoted O1. The second study, denoted N2 was launched in 2005. In this study the candidate 94/532 was compared with the Third International Standard for Pertussis Vaccine by 16 laboratories in 14 countries. The primary aim of each study was to assess the suitability of the candidate standard 94/532 to serve as an International Standard for whole cell Pertussis Vaccine and, subject to its suitability and stability, to assign to it a unitage which as far as possible maintains
1045-1056/$36.00 Ó 2010 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.biologicals.2010.07.006
R.G. Das et al. / Biologicals 38 (2010) 644e651
continuity of the international unit assigned to pertussis vaccine. Additionally, study N2 was to include the Pan American Health Organisation (PAHO) regional standard. It was further anticipated that information from N2 would provide information regarding the relationship among the various preparations studied. 2. Participants A total of 16 laboratories from 14 countries participated in N2 (Annex 1) and 14 laboratories from 11 countries participated in O1 (Annex 2). Throughout this report, each laboratory in study N2 has been assigned a code number from 1 to 16, and each laboratory in study O1 has been assigned a code number from 1 to 14 (except that the numbers 9 and 13 are not used, code numbers 11.1 and 11.2 were assigned to two different laboratories, and code 7.1 has been used to indicate a modification of the assay method). These numbers are not related to the order of listing of the participants in the Annexes which are listed alphabetically by country. Participants in study N2 were sent copies of this report for comment and confirmation of their data. It was not feasible to contact participants in study O1, and data have been taken as reported at the time of that study. 3. Materials 3.1. Materials included in the N2 study
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3.1.3. Whole cell pertussis vaccine preparation U.S. Lot no. 11 (ampoule coded PV93 e 1, denoted US11 throughout this report) Freezeedried preparation, established in 1994. The potency of this preparation was defined to be 90 IU/ampoule based on a collaborative study comparing it with the IS2 and U.S. Master Standard pertussis vaccine Lot 4 [7]. 3.2. Materials included in the O1 study 3.2.1. Whole cell pertussis vaccine preparation 66/302 (denoted IS2 throughout this report) IS2 was established in 1980 [8] with assigned potency of 46 IU/ ampoule. It was produced at NIH, Bethesda, USA and was freeze dried at NIBSC in 1966 and distributed by SSI. Remaining stock (<200 ampoules) was shipped back to NIBSC in 1997. 3.2.2. Whole cell pertussis vaccine preparation (94/532): (denoted CIS4 throughout this report) As described in 3.1.2 for study N2 above. 3.2.3. Whole cell pertussis vaccine preparation CPV (PERV): (denoted IS1 throughout this report) This preparation was the discontinued (1980) 1st IS for pertussis vaccine which had been established in 1957. This preparation had an assigned unitage of 35 IU/ampoule and had been calibrated against the US Pertussis Vaccine Master Lot 4 in 1956 [9]. It was used as a control pertussis vaccine in this study.
3.1.1. Whole cell pertussis vaccine preparation 66/303 (denoted IS3 in this report) IS3 (a freezeedried preparation of whole cell pertussis vaccine) was established in 1998 [1]. This material was originally prepared from the same bulk material as the IS2, and included in a collaborative study with it [3]. The stability of this material was confirmed by subsequent study before its establishment in 1998 [4]. This standard has been assigned an activity of 46 IU/ampoule.
4. Assay method
3.1.2. Whole cell pertussis vaccine preparation 94/532: (denoted CIS4 through out this report) Six litres of killed Bordetella pertussis suspension was generously supplied by CSL Australia in 1994 (Lot 0588196) and was stated by the manufacturer to have a density of 150 International Opacity Units in terms of the International Opacity Standard. One ml volumes of the material (in phosphate buffered saline (pH 6.8e7.4) with 8% dextran and 5% glucose and maintained at þ4 C) were filled into glass ampoules and then freeze dried on a five day cycle (starting shelf temperature of 40 C) followed by secondary desiccation for a further six days over phosphorus pentoxide under vacuum and the ampoules finally back filled with high purity nitrogen before sealing. The precision of fill was determined by weighing 75 ampoules taken at 2e3 min time intervals and the results showed a mean of 1.018 gs with a CV of 0.26%. The sealed ampoules were integrity tested by immersion in water under vacuum with added dye. No failure was detected. Moisture content determinations were carried out by Karl Fischer titration, showed a mean of 1.69% of dry weight with a CV of 0.78%. The ampoules were coded 94/532 and the entire batch was sent to the International Laboratory for Biological Standards (SSI) in Copenhagen in 1994. A collaborative study to compare this candidate preparation with the Second International Standard (ampoule code 66/302) was organized by SSI, Copenhagen and carried out in 1995e1996, together with some preliminary stability testing. The remaining ampoules of this material were transferred back to NIBSC in 1997.
5.1. N2 study
The Active Mouse Protection Test [2] was used by all laboratories in both studies. Assays were carried out using intracerebral challenge of mice from local sources with the strain of B. pertussis customarily in use in the individual laboratory. 5. Study design
Participants were asked to perform two independent assays each to include CIS4, IS3, US11 and their IHR (if available) using their own routine procedures, reagents and analytical methods. Results were reported back to NIBSC on standardised forms for analysis. 5.2. O1 study Each participant received ampoules of CIS4, IS2 and the IS1. The ampoules of CIS4 had been separately numerically coded. Participants were requested to perform at least four potency assays. As an option, participants were encouraged to do two additional potency assays (a total of six assays). Half of the assays included two independent ampoule(s) of CIS4 as individual samples and the standard vaccine IS2 and the remaining assays included one ampoule of CIS4, the positive control IS1 and standard vaccine IS2. The national or inhouse reference was also included in these assays (if available). Standardised forms and a methods questionnaire were also included for the participants to return their results to SSI for analysis. 6. Statistical analysis For the results reported here, all data were analysed at NIBSC, with the preliminary analysis for study O1 carried out at SSI used for confirmation that those data had been correctly entered and interpreted.
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All data were entered and preliminary analysis was carried out considering each assay as a multiple parallel-line assay. Overall consistency with the parallel-line assay model was assessed using probit transformed responses with the assumed ‘natural’ response limits of 0% and 100% responses. However, the asymptotic limits of the responses for each preparation in each assay were also fitted. Data from assays with preparations showing response limits significantly different from 0% or 100% and assays which showed statistically significant deviations from the parallel-line model (p < 0.05) were considered in detail, and in some cases anomalous responses or preparations were omitted before further analysis. For a small number of preparations the largest response was less than 50% or the smallest response was greater than 50%. In no case was the largest response less than 40% or the smallest response greater than 62%, and these preparations have not been omitted from analysis. The estimates of relative potency reported are based on analysis of the contributed assays as multiple parallel-line assays using probit transformed responses and assuming the ‘natural’ response limits of 0% and 100% for numbers responding. Assays included did not show statistically significant deviations from parallelism. The slope based on all preparations included in the assay was therefore considered to be more reliable than the slopes which would have been obtained for pair-wise comparisons only. Nevertheless, slopes were separately examined for any consistent differences across all assays which might have indicated slight non-parallelism which was not detected as statistically significant in the individual assays. After deletion of anomalous data as detailed in results, any assays for which the deviations from either linearity or parallelism are significant (p < 0.05) and for which the total deviations from the model are also significant (p < 0.05) are taken to be invalid, and estimates from these assays are not included in the combined mean estimates. Estimates of the number of organisms giving an LD50 have been obtained by fitting (using SASÒ PROC PROBIT)[10] a function relating the probit response to the logarithm of the dose assuming the ‘natural’ response limits of 0% and 100%. Estimates of potency have been assessed for homogeneity using a chi-square test for homogeneity. Estimates have been combined as geometric means. As noted with the combined estimate, these are unweighted geometric means or, for some groups of estimates which do not show significant deviations from homogeneity, weighted geometric means. In the case of weighted geometric means, the 95% limits for the mean estimate are based on the total weights of the estimates combined. In the case of unweighted geometric means, the 95% limits are based on the variance of the logarithms of the estimates combined. 7. Results 7.1. Preliminary consideration of data A total of 31 assays from 16 laboratories were contributed to study N2, and a total of 70 assays from 14 laboratories were contributed to study O1. Preliminary analysis of the data showed that for the majority of preparations the fitted response limits did not deviate significantly from the ‘natural’ limits of 0% and 100% and that the majority of assays did not deviate significantly from the linear parallel-line model. However, there were a few laboratories in which there were a number of preparations with fitted limits significantly different from the ‘natural’ limits and these tended also to be laboratories in which several assays showed significant deviations from the linear parallel-line model. Most notable were laboratories 10 and 14 in study N2 and laboratories 1, 8 and 12 in study O1, with occasional
anomalous assays in other laboratories. In most of these assays, the natural response rate was significantly larger than 0%; that is, small numbers of mice survived at low doses of vaccine, and the doseeresponse line became non-linear as the dose of vaccine decreased. The available data suggest that the ‘true’ response limit is likely to differ between different assays as well as between different laboratories. However, these data are insufficient for reliable estimation of the response limit. Therefore, for these assays low doses of vaccine giving small responses which are not consistently increasing have been omitted from analysis. There were also a limited number of cases in which one preparation in an assay gave anomalous responses which were not consistent with the responses of that preparation in other assays in the same laboratory, and data from these preparations have also been omitted from the final analysis. Data omitted from analysis are of doses giving responses apparently at the maximum (or minimum) of the response range, and for which responses showed no further increase (or decrease) when compared with smaller (or larger) doses of the same preparations. Thus, the smallest doses of vaccine were omitted from most preparations in most assays of laboratories 10 and 14 in study N2 and of laboratories 1 and 12 in study O1, and also from most preparations in assays 1.1 and 2.1 in laboratory 7 and assay 4 in laboratory 8 in study O1. The largest doses of vaccine were omitted from most preparations in assay 6 of laboratory 4 and assay 3 of laboratory 12 in study O1. In addition to these deletions of doses giving responses apparently at the limits of the response range, five anomalies were noted and omitted from the total of 463 preparations, namely for two preparations (US11 and IHR, all doses omitted) in assay 2 of laboratory 13 in study N2, for two preparations (CIS4 and IHR, one dose from each omitted) in assay 3 of laboratory 6 and one preparation (IS1, one dose omitted) in assay 1 of laboratory 8 in study O1. After omission of these non-monotonic or anomalous responses, all assays showed a satisfactory fit to the parallel-line model, with the probability of deviations from the model greater than 0.05 for all except two assays. These were assays 3 from laboratory 1 and 2 from laboratory 12, both in study O1. 7.2. Comparison of doseeresponse lines Analysis of the slopes of the log dose-probit response lines for each preparation in each assay did not indicate any significant differences among the preparations compared, based on the values of the chi-square test for deviations from the parallel-line model (data not shown). Slopes were additionally assessed for any consistent differences across all assays in the study. Comparison of the slopes within the same assay for duplicate ampoules of CIS4 in study O1 showed no significant difference between them (probability of paired t-test w 0.7). Similar comparisons of slopes for CIS4 and IS2 in study O1 and for CIS4 and IS3 in study N2 showed no consistent differences between these pairs (probability of paired ttest > 0.3 in each case). Comparison of slopes for either IS3 or CIS4 and US11 in study N2 did not show any consistent differences. In contrast, comparison of slopes for IS1 with either CIS4 or IS2 in study O1 suggested that there may be a tendency for the slopes of the doseeresponse lines for IS1 to be less than those for CIS4 or IS2 (probability of paired t-test < 0.05 for each comparison). 7.3. Comparison of duplicate ampoules of CIS4 In one assay of study N2, and in 26 assays in study O1, independent ampoules of CIS4 were directly compared in terms of potency with one another. These 27 individual estimates were homogeneous (p chi-square test w 0.5) and gave a weighted
R.G. Das et al. / Biologicals 38 (2010) 644e651
geometric mean of 1.08 with 95% limits 0.95e1.23. This observed value is in good agreement with the expected value of 1 for identical ampoules. The geometric mean weight over these assays was 39. This provides an estimate of the within assay variance and corresponds to 95% limits for an individual estimate of potency from 48% to 209% of the estimated value. The unweighted geometric mean of these estimates was 1.02 with corresponding weight 21 (determined as the reciprocal of the variance of log10 of the potency estimates) which corresponds to 95% limits for an individual estimate of potency from 37% to 273% of the estimated value. Thus the directly observed variance (pooled over assays and laboratories) is somewhat larger than the average estimated within assay variance, but these differences are not significant (assuming the ratio of these variances follows an F distribution, p > 0.05). 7.4. Calibration of CIS4 Estimates of the potency and their associated weights for CIS4 in terms of IS3 (study N2) and in terms of IS2 (study O1) are shown in Tables 1and 2 respectively. Distribution of estimates is shown in Fig. 1. The mean estimates for each study separately, and for both studies combined under the assumption that the IU of the IS2 and IS3 are equivalent to one another are also given in Table 3. It is noted that the geometric mean weight of assay weights for individual estimates for study O1 is approximately 40 while that for study N2 is approximately 30. For study O1 all individual estimates of potency, and similarly the laboratory geometric mean estimates of potency are homogeneous. For study N2 all individual estimates of potency are heterogeneous. Most laboratories contributing data to this study carried out two assays, with several laboratories contributing data from a single assay. Thus the laboratory geometric mean estimates of potency for CIS4 from study N2 are also heterogeneous. Three estimates contributed excessively to the chi-square for heterogeneity, namely assays 1 from each of laboratories 2, 7 and 8. Omission of these three of the 29 estimates (10% of estimates) from study N2 gave a variance of log potency estimates for the remaining 26 estimates which was only 44% of the variance of the 29 estimates, and the 26 estimates of potency did not deviate significantly from homogeneity. Mean estimates using this homogeneous group of estimates are also shown. In view of the data from O1, it is likely that if the laboratories from which the ‘heterogeneous’ estimates came had carried out up to six assays each, the laboratory geometric means from N2 would not be
647
Table 2 Estimated potency of candidate standard CIS4 and of the proposed regional standard US11 expressed as IU of the Second International Standard (study O1) per ampoule. Weight is the reciprocal of the log10 of the estimated potency. Geometric means marked * are unweighted means with weight determined as the reciprocal of the variance of the estimates combined. The symbol ‘u’ denotes that an estimate is not available. Laboratory
Total no. of assay
CIS4
Weight
US11
Weight
1 2 3 4 5 6 7 7.1 8 10 11.1 11.2 12 14
4 6 4 6 4 6 4 4 4 4 6 5 6 6
33.16 44.63 41.62 37.83 38.18 29.13 41.09 44.01 33.31 49.22 34.59 56.28 45.07 25.82
130 356 117 468 373 156 133 214 178 294 184 104 270 170
u 87.61* 83.30* u u u u u u u u u u u
u 67 31 u u u u u u u u u u u
heterogeneous. That is, to some extent, the apparent heterogeneity may be an artefact of the limited data. It is suggested that the combined estimate which is most representative of these assays is the weighted geometric mean of estimates from both studies N2 and O1 (excluding the three estimates from study N2 which contributed excessively to heterogeneity), namely 40 IU per ampoule with 95% limits 37e43 IU per ampoule (Table 3). This estimate is consistent with the combined estimate for each study separately, and is also consistent with the unweighted geometric mean for study N2 including all estimates. This estimate maintains overall continuity of unitage with both IS2 and IS3. Continuity is also broadly maintained for the individual laboratories. Laboratory geometric means from study O1, with a minimum of four assays from each laboratory, ranged from 26 IU (95% limits 18e36 IU) to 56 IU (95% limits 36e88 IU) per ampoule and were not heterogeneous. Laboratory geometric means from study N2 ranged from 9 IU per ampoule (95% limits 3e30 based on a single assay) to 78 IU per ampoule (95% limits 45e136 IU based on two assays), and were heterogeneous. This heterogeneity may reflect both the greater variability of the individual assays and the
Table 1 Geometric means (GM) estimated potency of candidate standard CIS4 and of the proposed regional standard US11 expressed as IU of the Third International Standard (study N2). Weight is the reciprocal of the log10 of the estimated potency. The symbol ‘u’ denotes that an estimate is not available. Laboratory
Total no. of assay
CIS4
Weight
US11
Weight
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
2 2 1 2 2 2 2 1 2 2 1 2 2 3 3 2
42.07 14.92 42.24 49.02 47.27 50.32 15.36 9.08 65.36 78.27 34.20 36.70 22.70 65.96 34.14 35.21
51 59 49 66 51 29 70 14 82 69 82 58 91 63 73 105
90.81 29.54 u 113.30 107.62 81.66 31.02 47.52 68.07 127.26 64.62 58.05 86.67 109.04 63.38 56.29
51 59 u 55 51 29 69 15 83 69 78 25 27 63 56 104
Fig. 1. Histogram showing the distribution of estimates of potency for CIS4 from each assay in each laboratory. Each square represents one estimate and the label in the square identifies the study as N2 (first character of label is N) or study O1 (first character of label is O) and the remaining label identifies the laboratory number. Estimates from study O1 are also identified by shading.
648
R.G. Das et al. / Biologicals 38 (2010) 644e651
Table 3 Estimated potency of candidate standard CIS4 and of the proposed regional standard US11 expressed as IU of the Third International Standard (study N2) or of the Second International Standard (study O1) per ampoule. Weight is the reciprocal of the log10 of the estimated potency. Geometric means marked * are unweighted means with weight determined as the reciprocal of the variance of the estimates combined. CIS4 Study N2 GM All Individual Estimates 95% Interval GM Laboratory GMs 95% Interval Study O1 GM All individual Estimates 95% Interval GM Laboratory GMs 95% Interval
39.26 36.23e42.55 39.25 36.27e42.46
Studies O1 and N2 GM All individual Estimates 95% Interval GM Laboratory GMs 95% Interval
37.89* 34.50e41.61 36.70* 31.00e43.43
35.95* 28.91e44.70 34.96* 25.50e47.94
Weight 469 242
3154 3302
2381 780
US11
Weight
71.53* 57.64e88.77 69.56* 54.39e88.96
482
39.70 36.99e42.60 39.70 36.99e42.60
4081
85.86* 51.98e141.80 86.22 54.14e137.92
108
75.25* 61.82e91.60 71.25* 57.38e88.49
565
4228
82.81* 69.62e98.49 78.93 67.57e92.21
Study
Laboratory
n
IS2
Weight
CIS4
Weight
O1
01.0 02.0 03.0 04.0 05.0 06.0 07.0 07.1 08.0 10.0 11.1 11.2 12.0 14.0
2 4 4 3 2 6 2 2 4 4 3 3 3 3
39.93 27.02a 22.17a 28.58 29.56 28.76 26.17 21.14 44.20 37.15 48.22 31.33 30.00 48.08
133 80 34 192 126 157 59 86 172 248 80 61 145 82
34.11 23.60 21.19 26.03 28.56 18.11 23.24 27.76 32.21 39.10 37.07 44.10 41.60 31.00
129 205 116 195 126 157 59 86 175 266 80 61 145 81
1861
29.46 26.54e32.70 29.58* 25.38e34.49
403
98
508
Omit three assays from study N2, assays 1 from laboratories 2, 7 and 8 Study N2 GM All Individual Estimates 41.20 926 78.03 749 95% Interval 35.52e47.80 66.14e92.05 GM Laboratory GMs 41.20 926 78.03 749 95% Interval 35.51e47.81 66.13e92.06 Studies O1 and N2 GM All individual Estimates 95% Interval GM Laboratory GMs 95% Interval
Table 4 Estimates of the potency (GM) of IS2 and CIS4 expressed as IU of IS1. Weight denotes the reciprocal of the variance of log10 of the potency estimate.
731 847
limited number of assays in each laboratory in study N2. Although individual estimates from study O1 were generally more precise than those from study N2, individual assay estimates covered a similar range. Individual estimates in study O1 ranged from 17 IU per ampoule to 139 IU per ampoule and those in study N2 ranged from 9 IU per ampoule to 80 IU per ampoule. Direct comparisons of US11 with the candidate standard CIS4 can be obtained from Tables 1e3. These estimates do not deviate significantly from homogeneity within laboratories, within either study, or when considered over both studies. The weighted geometric mean of all estimates for US11 based on assumed ampoule content for CIS4 of 40 IU is 74.5 IU per ampoule (95% limits 65.5e84.9 IU). This estimate is consistent with the estimate for US11 in terms of IS2 and IS3, and indicates that overall continuity of unitage of US11 will be maintained if CIS4 is assigned an ampoule content of 40 IU. 7.5. Comparisons of the First International Standard for pertussis Vaccine with the Second International Standard IS2 and with the candidate standard CIS4 At the time of study O1 there was a remaining stock of the First International Standard, and ampoules of the preparation were included in some assays. Estimates of IS2 and CIS4 in terms of this preparation, denoted IS1 are given in Table 4. The geometric mean estimate for IS2 in terms of IS1 is 33 IU per ampoule which is about 70% of the assigned potency for IS2 of 46 IU per ampoule. Similarly, the geometric mean estimate for CIS4 in terms of IS1 is 30 IU per ampoule which is about 75% of the estimate for CIS4 in terms of either IS2 or IS3. 7.6. Stability study After the candidate standard was transferred to NIBSC, in June 2003, a total of 298 ampoules of that material were stored at
GM, individual estimates 95% limits GM, laboratory GMs 95% limits a
32.13 28.94e35.68 33.09 29.61e36.98
1657
1881 1052
Unweighted combination of heterogeneous estimates.
elevated temperatures to provide information about thermal degradation (18 at 56 C, 30 at 45 C, 30 at 37 C, 30 at 20 C, 30 at þ4 C). Ampoules of CIS4 (94/532) which had been stored at elevated temperatures for up to 2.5 years were assessed in several mouse protection assays in one laboratory. The potency of the vaccine in ampoules stored at 37 C for one year relative to that for ampoules stored continuously at 20 C was 1.18 (95% limits 0.64 to 2.18). The relative potency of samples stored at 4 C for 19.5 months and of samples stored at 20 C for 19.5 months or 32.4 months also did not differ significantly from 1 (estimates of relative potency 1.05, 1.01 and 1.05 respectively). Since none of these samples show loss of activity, no degradation rate can be reliably predicted. Additional information about stability can be inferred from the comparisons of the candidate standard with other established reference preparations in 1995 and 2005. These comparisons do not indicate any differential changes in relative activity, and thus are consistent with stability of these preparations. These results indicate that the candidate standard is sufficiently stable to serve as an international standard. Experience, with reconstituted material of similar type to the candidate standard suggests that such material may not be stable after reconstitution and it is recommended that freshly reconstituted material should be used for each assay. 8. Discussion This report summarizes the currently available information about international standards for whole cell pertussis vaccine and brings together data from two international studies to evaluate the candidate international standard. The results from the two studies together with results from previous studies are given in Table 5 for comparison. The two studies described in this report provide a consistent calibration of 40 IU per ampoule for CIS4 in terms of IS2 and IS3, and no apparent differences in doseeresponse curves were detected. The calibration of CIS4 in terms of IS1 obtained in study O1 is about 25% smaller than its calibration in terms of IS2/IS3. However, differences in slope between CIS4 and IS1 were noted in study O1 and were also observed between IS2 and IS1 (data not shown). This may reflect differences in the responses of these different preparations in some assays. Differences between IS1 and the more recent international standards and CIS4 may also give rise to the heterogeneity observed in some previous comparisons, e.g. IS2 and IS1 in the study [3], IS3 and IS1 in another study [4].
R.G. Das et al. / Biologicals 38 (2010) 644e651 Table 5 Comparison of results from various studies. Preparation Ref. used
Results (IU/ amp.)
Study
1st IS
US NIH, Lot 4
34.7 IU
US Master Lot 4
33.9 (29e40)
O. Maaloe, WHO/BS/338, 1956 Seagroatt et al., 1981
1st IS U.S. Master Lot 4 U.S. Master Lot 4
Seagroatt et al, 1981 Seagroatt et al, 1981 Jansen et al, 1996
1st IS
45.9 (36e59)a 38.9 (29e52)a 41.4 (35.1e48.3)b 33.1 (29.6e37.0)e 36 (14e90)a
3rd IS
1st IS 2nd IS 1st IS
46.3 (40e53) 46.0 (41e53) 41 (17e98)a
2nd IS
56.1 (46e68)
Seagroatt et al, 1981 Seagroatt et al, 1981 Xing et al., WHO/BS/98, 1880, 1998 Xing et al., 2001
US Lot 11
2nd IS US Lot 4
91.1 (80e103) 79.8 (68.9e92.4) 86.2 (54.1e138) 69.56 (54.4e89.0)c 78.0 (66.1e92.1)d 71.3 (57.4e88.5)c 78.9 (67.6e92.2)d 76.5 (61.6e94.9) 73.5 (62.5e86.4) 74.6 (65.5e84.9)
2nd IS
1st IS
2nd IS 3rd IS
2nd & 3rd IS (combined)
CIS4 CIS4 CIS4 CIS4
1st IS 2nd IS 3rd IS
2nd & 3rd IS (combined)
29.6 (25.4e34.5)e 39.3 (36.3e42.5) 35.0 (25.5e47.9)c 41.2 (35.5e47.8)d 36.8 (31.1e43.5)a 39.7 (37.0e42.5)d
O1995 Xing et al., WHO/BS/98, 1880, 1998
Jansen et al, 1996 Jansen et al, 1996 O1995 N2005
649
activity of these preparations. The consistency of comparisons of US11 with the various international standards over this period of time also does not suggest differential changes in activity among these preparations. These data are consistent with stability of the preparations compared. Limited data from thermally accelerated degradation studies indicate that CIS4 is likely to be stable at the usual storage temperature of 20 C. Thus CIS4 appears to be sufficiently stable to serve as an international standard. Data from calibration of standards using the active mouse protection assay (Kendrick test) over the period of these studies (from 1980 to 2005) suggest that over this time the assay has performed consistently and reliably. Estimates based on limited assays will show variation, and there is some heterogeneity of estimates possibly reflecting differences between preparations which are detected by some assays. The reported methods and challenge organisms show great diversity and some of these reported factors may be interrelated (data not shown). This study is not designed to explore these issues. As previously noted [3], in spite of variations, results considered over a number of laboratories are generally concordant. This was also the finding of a recent proficiency study of this method [12]. Comments on this report were obtained from 12 of 16 laboratories participating in study N2 and all of these participants agreed with the proposed unitage for CIS4. 9. Conclusions and recommendations
O1995 & N2005
O1995 N2005 O1995 & N2005 O1955
On the basis of the results of the studies reported here and previous studies indicating stability and continuity of unitage among International Standards for Pertussis Vaccine, the ECBS of WHO established the material in ampoules coded 94/532 (CIS4) as the Fourth International Standard for Whole Cell Pertussis Vaccine and that it be assigned an activity of 40 IU per ampoule on the basis of its calibration in terms of the Second and Third International Standards for Pertussis Vaccine (Whole Cell) [13].
O1955
Acknowledgements
N2005
The contribution of the participants to studies O1 and N2 is gratefully acknowledged. The organization and preliminary consideration of O1 by Dr. Gert A. Hansen and staff at SSI is also gratefully acknowledged. We thank WHO for supporting the studies.
O1995 & N2005
Annex 1. Participant list (N2 study)
a
Unweighted combination of heterogeneous estimates. US4 and IS2 did not give comparable potency estimates in some laboratories. c Unweighted geometric mean of heterogeneous laboratory geometric means (all data). d Weighted geometric mean of homogeneous laboratory geometric means after omitting 3 assays as described in text. e Consistent difference in slopes of CIS4 or IS2 compared with IS1, as discussed in text. b
US11 was included in the majority of laboratories in study N2 and has also been included in a number of previous studies. The various estimates from these studies are shown in Table 5. The comparison of US11 with IS2/IS3 in the two studies reported here gives a value about 10% smaller than that assigned to US11 in terms of IS2 by a previous study. However, these results are consistent with the comparison of US11 with US4 in a previous study and of US11 with CIS4 in the recent studies. Questions had been raised regarding the stability of IS2. Previous studies have confirmed the apparent stability of IS2 [5,11] and of IS3 [4]. Comparison of CIS4 with IS2 and after a period of 10 years with IS3 are consistent and thus do not suggest differential changes in
Dr Rajiv Jayasena QC Compliance CSL Ltd 45 Poplar Rd Parkville Victoria 3052 Australia Mr Frederic Mortiaux GlaxoSmithKline Biologicals Rue de l’Institut 89 B e 1330 Rixensart Belgium Dr. Humberto Pinheiro de Araujo Ministerio da Saude do Brasil Fundacao Oswaldo Cruz FIOCRUZ Instituto Nacional de Controle de Qualidade em Saude INCQS Av. Brasil 4365, Manguinhos, Rio de Janeiro CEP 21045 900 Brazil
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Dr Zhang Shumin Tiantan Xili No 2 National Institute for the Control of Pharmaceutical & Biological Products (NICPBP) Temple of Heaven Beijing 100050 China Dr Patrice Chagnaud and Ms El-zaouk Annie Bacterial Vaccines Control Unit and Bioactivity and Radio-analysis Control Unit Agence Francaise de Securite Sanitaire des Produits de Sante (AFSSAPS) 321, avenue Jean Jaurès F e 69007 Lyon France Dr Christophe Cochet Sanofi Pasteur 1541 Av. Marcel Merieux 69289 Marcy l’Etoile France Dr A.K. Tahlan Joint Director, Central Drugs Laboratory Central Research Institute Division of Biological Products Kasauli 1732504 Himachal Pradesh India Dr Suresh S. Jadhav and Dr S. Gairola Serum Institute of India Ltd 212/2 Hadapsar Pune 411 028 Maharashtra State India Dr Syahrial Tahir and Dr H. Moch.Ma’roef National Quality Control Lab of Drug and Food (NQCLDF) Jalan Percetaken Negara No 23 Jakarta Pusat 10560 Indonesia Dr Yoshinobu Horiuchi Department of Bacterial and Blood Products National Institute of Infectious Diseases (NIID) 4-7-1, Gakuen, Musashimurayama-shi Tokyo 208-0011 Japan Mrs Angélica López Sotelo and Ms Adriana Santiago Echauri Laboratorios de Biologicos y Reactivos de Mexico, S.A. de C.V. Departmento de Control Biologico Mariano Escobedo No. 20 Col Popotla CP 11 400 Mexico, D.F. Dr Lonneke Levels Netherlands Vaccine Institute (NVI) Antonie van Leewenhoeklaan 9-11 Postbus 457 3720 MA Bilthoven The Netherlands Dr Anna Gzyl Dept of Sera and Vaccines Evaluation National Institute of Hygiene (NIH) National Centre for Disease Control and Prevention 24 Chocimska Street 00791 Warsaw Poland
Mr Apichai Supasansatorn and Ms Teeranart Jivapaisarnpong Division of Biological Products Department of Medical Sciences Tivanon Rd Ministry of Public Health Nonthaburi 11000 Thailand Dr D. Xing and Dr M.J. Corbel Division of Bacteriology National Institute for Biological Standards and Control Blanche Lane South Mimms Potters Bar Herts EN6 3QG UK Dr Ma. Teresa Ibarz and Dr Maria Luz Pombo Division Control Nacional de Productos Biologicos Instituto Nacional de Higiene “Rafael Rangel” Ciudad Universitaria UCV, Detras del Hospital Clinico Universitario de Caracas Los Chaguaramos Caracas 1051, Venezuela.
Annex 2. Participant list (O1 study) Dr K. Healy CSL Ltd 45 Poplar Road Parkville Victoria 3052 Australia Dr E. Rommel SmithKline Beecham Biologicals S.A. Rue de l’Institut 89 B e 1330 Rixensart Belgium Dr Hou Qiming Division of Bacterial Vaccines National Institute for the Control of Pharmaceutical & Biological Products Temple of Heaven Beijing 100050 China Dr L. Peyron Pasteur Mérieux Val de Reuil France Dr L. Peyron Pasteur Mérieux 1541 Ave. Marcel e Mérieux F-69280 Marcy l’Etoile France Dr K. Enssle Chiron Behring GmbH & Co Emil e von e Behring e Str, 76 D e 35041 Marburg Germany Dr Z. Nagy HUMAN Serum Production and Medicine Manufacturing Co Ltd 2100 Godollo
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Tancsics Mihaly H-2100 Hungary Dr Yoshikazu Tada BIKEN The Research Foundation for Microbial Diseases of Osaka University Kanonji Institute Kanonji City Kagawa 768 Japan Dr H. van de Donk RIVM Antonie van Leeuwenhoeklaan 9 NL-3720 BA Bilthoven The Netherlands Dr S.J. Varallyay Swiss Serum and Vaccine Institute Rehhagstrasse 79 CH-3001 Berne Switzerland Dr K. Readhead and Dr M.J. Corbel, Division of Bacteriology National Institute for Biological Standards and Control Blanche Lane, South Mimms Potters Bar EN6 3QG, UK Dr Bruce Meade Laboratory of Pertussis Center for Biologics Evaluation and Research Food and Drug Administration 1401 Rockville Pike Bethesda
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MD 20852 USA Dr Bonnie Brock American Cyanamid Co. Lederle e Praxis Biologicals Division 401 N. Middletown Rd Pearl River NY 10965 USA References [1] WHO Technical Report Series 897, WHO ECBS 49th Report, 2000 (Collab study: WHO BS/98 1880). [2] Kendrick PL, Eldering G, Dixon MK, Misner J. Mouse protection test in the study of pertussis vaccines: a comparative series using intracerebral route of challenge. American Journal of Public Health 1947;37:803e10. [3] Seagroatt V, Sheffield F. A collaborative assay of the proposed second international standard for pertussis vaccine and of the proposed first British standard for pertussis vaccine. Journal of Biological Standardization 1981;9:351e65. [4] Xing D, Gaines Das R, Newland, Corbel M. Third international standard for pertussis vaccine: International confirmation study of activity of British standard for pertussis vaccine, coded 66/303. Biologicals 2001;29:133e6. [5] Dobbelaer R. The stability of the second International Standard for Pertussis Vaccine e results of an inquiry. WHO BS/89.1617, Geneva; 1989 . [6] WHO Technical Report Series 932, WHO ECBS 55th Report; 2005. [7] Jansen DL, Meade BD. Preparation and standardisation of US standard pertussis vaccine lot 11. Biologicals 1996;24:363e70. [8] WHO Technical report series 658; 1981. p. 18 [9] WHO Technical Report Series 127, 1957. [10] SAS Institute, Inc. SAS/STAT user’s guide, version 6. 4th ed. Cary, NC: SAS, Institute, Inc; 1989. [11] Redhead K, Gaines Das R. A collaborative assay of the proposed third British reference preparation for pertussis vaccine and the relative potencies of the second international standard and the second British reference preparation for pertussis vaccine. Biologicals 1991;19:107e11. [12] Xing D, Gaines Das R, O’Neill T, Corbel M, Dellepiane N, Milstien and participants J. Laboratory testing of whole cell pertussis vaccine: a WHO proficiency study using the Kendrick test. Vaccine 2002;20:342e51. [13] WHO Technical Report, WHO ECBS 57th Report, 2006 (Collab study: WHO/BS/ 06.2036), in press.
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Biologicals journal homepage: www.elsevier.com/locate/biologicals
Evaluation of Fourth International Standard for Whole Cell Pertussis Vaccine R. Gaines Das, P. Newland, C. Asokanathan, A. Douglas-Bardsley, M. Corbel, D. Xing* Division of Bacteriology, National Institute for Biological Standards and Control, Health Protection Agency, Potters Bar, Hertfordshire EN6 3QG, UK
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 June 2010 Accepted 23 July 2010
Whole cell pertussis vaccine is still widely used in many countries. An International Standard is needed for its potency control. The Third International Standard for Pertussis Vaccine was prepared about 40 years ago and its replacement was recommended by the Expert Committee for Biological Standardisation (ECBS) of the WHO. Material in ampoules coded 94/532 was prepared as a candidate replacement and has been evaluated in international collaborative studies which consisted of two parts. The first part, to assess the suitability of the candidate standard by comparing it with the Second International Standard for Pertussis Vaccine (IS2) involved 14 laboratories in 11 countries. The second part to compare the candidate standard with the Third International Standard for Pertussis Vaccine (IS3) involved 16 laboratories in 14 countries. Since 1995 various other studies have included the international standards and the results of these are also considered in assessing likely continuity of the IU for potency of whole cell pertussis vaccine. The preparation in ampoules coded 94/532 was adopted by the WHO ECBS in October 2006 as the 4th International Standard for whole cell pertussis vaccine and assigned an activity of 40 IU per ampoule on the basis of the studies reported here. Ó 2010 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.
Keywords: Whole cell pertussis vaccine International standard Potency
1. Introduction Although whole cell pertussis vaccine has been largely replaced by acellular pertussis vaccines in North America, Western Europe and some Asian countries, whole cell pertussis vaccine is still widely used in many countries and is likely to continue in use for a considerable time due to the fact that most whole cell pertussis vaccines are safe, effective, and cheaper to produce than acellular pertussis vaccines. Therefore an International Standard for whole cell pertussis vaccine is needed for potency control test (Kendrick test) [2]. The Third International Standard (IS3, ampoule code 66/ 303) was established in 1998 [1]. This material was originally prepared from the same bulk material as the Second International Standard (IS2, ampoule code 66/302), and included in a collaborative study with it [3]. Its stability was confirmed by a subsequent study before its establishment as IS3 in 1998 [4]. The candidate standard (ampoule code 94/532) was prepared in 1994 from a bulk material kindly donated by CSL, Australia. A collaborative study to compare this candidate with IS2 was organized by Dr Gert Albert Hansen, Statens Seruminstitut (SSI), Copenhagen. This study was carried out by 14 laboratories in 1995e1996, together with some preliminary stability testing. In 1997, the remaining ampoules of this material were transferred to
* Corresponding author. Tel.: þ44 (0) 1707 641 433; fax: þ44 (0) 1707 641 054. E-mail address: [email protected] (D. Xing).
National Institute for Biological Standards and Control (NIBSC), UK, together with the data for this study. At that time there were some questions about the stability of IS2 [5], and the full statistical analysis and report of this study were not completed pending clarification of this issue. Several subsequent studies have included IS2 and have not confirmed the suggested instability [4]. The IS3 was prepared about 40 years ago and the stock was running low. Therefore there is a need for replacement. The Expert Committee for Biological Standardisation (ECBS) suggested that the stability of the candidate replacement standard (94/532) should be assessed and the candidate standard should be compared with the IS3. The ECBS also suggested that this collaborative study should include regional reference standard(s) if possible [6]. This publication reports the results of two international collaborative studies and summarizes other related information. The first international study was launched in 1995 with the aim of comparing the candidate standard 94/532 with the Second International Standard for Pertussis Vaccine and was carried out by 14 laboratories in 11 countries. Throughout this report, this part is denoted O1. The second study, denoted N2 was launched in 2005. In this study the candidate 94/532 was compared with the Third International Standard for Pertussis Vaccine by 16 laboratories in 14 countries. The primary aim of each study was to assess the suitability of the candidate standard 94/532 to serve as an International Standard for whole cell Pertussis Vaccine and, subject to its suitability and stability, to assign to it a unitage which as far as possible maintains
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R.G. Das et al. / Biologicals 38 (2010) 644e651
continuity of the international unit assigned to pertussis vaccine. Additionally, study N2 was to include the Pan American Health Organisation (PAHO) regional standard. It was further anticipated that information from N2 would provide information regarding the relationship among the various preparations studied. 2. Participants A total of 16 laboratories from 14 countries participated in N2 (Annex 1) and 14 laboratories from 11 countries participated in O1 (Annex 2). Throughout this report, each laboratory in study N2 has been assigned a code number from 1 to 16, and each laboratory in study O1 has been assigned a code number from 1 to 14 (except that the numbers 9 and 13 are not used, code numbers 11.1 and 11.2 were assigned to two different laboratories, and code 7.1 has been used to indicate a modification of the assay method). These numbers are not related to the order of listing of the participants in the Annexes which are listed alphabetically by country. Participants in study N2 were sent copies of this report for comment and confirmation of their data. It was not feasible to contact participants in study O1, and data have been taken as reported at the time of that study. 3. Materials 3.1. Materials included in the N2 study
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3.1.3. Whole cell pertussis vaccine preparation U.S. Lot no. 11 (ampoule coded PV93 e 1, denoted US11 throughout this report) Freezeedried preparation, established in 1994. The potency of this preparation was defined to be 90 IU/ampoule based on a collaborative study comparing it with the IS2 and U.S. Master Standard pertussis vaccine Lot 4 [7]. 3.2. Materials included in the O1 study 3.2.1. Whole cell pertussis vaccine preparation 66/302 (denoted IS2 throughout this report) IS2 was established in 1980 [8] with assigned potency of 46 IU/ ampoule. It was produced at NIH, Bethesda, USA and was freeze dried at NIBSC in 1966 and distributed by SSI. Remaining stock (<200 ampoules) was shipped back to NIBSC in 1997. 3.2.2. Whole cell pertussis vaccine preparation (94/532): (denoted CIS4 throughout this report) As described in 3.1.2 for study N2 above. 3.2.3. Whole cell pertussis vaccine preparation CPV (PERV): (denoted IS1 throughout this report) This preparation was the discontinued (1980) 1st IS for pertussis vaccine which had been established in 1957. This preparation had an assigned unitage of 35 IU/ampoule and had been calibrated against the US Pertussis Vaccine Master Lot 4 in 1956 [9]. It was used as a control pertussis vaccine in this study.
3.1.1. Whole cell pertussis vaccine preparation 66/303 (denoted IS3 in this report) IS3 (a freezeedried preparation of whole cell pertussis vaccine) was established in 1998 [1]. This material was originally prepared from the same bulk material as the IS2, and included in a collaborative study with it [3]. The stability of this material was confirmed by subsequent study before its establishment in 1998 [4]. This standard has been assigned an activity of 46 IU/ampoule.
4. Assay method
3.1.2. Whole cell pertussis vaccine preparation 94/532: (denoted CIS4 through out this report) Six litres of killed Bordetella pertussis suspension was generously supplied by CSL Australia in 1994 (Lot 0588196) and was stated by the manufacturer to have a density of 150 International Opacity Units in terms of the International Opacity Standard. One ml volumes of the material (in phosphate buffered saline (pH 6.8e7.4) with 8% dextran and 5% glucose and maintained at þ4 C) were filled into glass ampoules and then freeze dried on a five day cycle (starting shelf temperature of 40 C) followed by secondary desiccation for a further six days over phosphorus pentoxide under vacuum and the ampoules finally back filled with high purity nitrogen before sealing. The precision of fill was determined by weighing 75 ampoules taken at 2e3 min time intervals and the results showed a mean of 1.018 gs with a CV of 0.26%. The sealed ampoules were integrity tested by immersion in water under vacuum with added dye. No failure was detected. Moisture content determinations were carried out by Karl Fischer titration, showed a mean of 1.69% of dry weight with a CV of 0.78%. The ampoules were coded 94/532 and the entire batch was sent to the International Laboratory for Biological Standards (SSI) in Copenhagen in 1994. A collaborative study to compare this candidate preparation with the Second International Standard (ampoule code 66/302) was organized by SSI, Copenhagen and carried out in 1995e1996, together with some preliminary stability testing. The remaining ampoules of this material were transferred back to NIBSC in 1997.
5.1. N2 study
The Active Mouse Protection Test [2] was used by all laboratories in both studies. Assays were carried out using intracerebral challenge of mice from local sources with the strain of B. pertussis customarily in use in the individual laboratory. 5. Study design
Participants were asked to perform two independent assays each to include CIS4, IS3, US11 and their IHR (if available) using their own routine procedures, reagents and analytical methods. Results were reported back to NIBSC on standardised forms for analysis. 5.2. O1 study Each participant received ampoules of CIS4, IS2 and the IS1. The ampoules of CIS4 had been separately numerically coded. Participants were requested to perform at least four potency assays. As an option, participants were encouraged to do two additional potency assays (a total of six assays). Half of the assays included two independent ampoule(s) of CIS4 as individual samples and the standard vaccine IS2 and the remaining assays included one ampoule of CIS4, the positive control IS1 and standard vaccine IS2. The national or inhouse reference was also included in these assays (if available). Standardised forms and a methods questionnaire were also included for the participants to return their results to SSI for analysis. 6. Statistical analysis For the results reported here, all data were analysed at NIBSC, with the preliminary analysis for study O1 carried out at SSI used for confirmation that those data had been correctly entered and interpreted.
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All data were entered and preliminary analysis was carried out considering each assay as a multiple parallel-line assay. Overall consistency with the parallel-line assay model was assessed using probit transformed responses with the assumed ‘natural’ response limits of 0% and 100% responses. However, the asymptotic limits of the responses for each preparation in each assay were also fitted. Data from assays with preparations showing response limits significantly different from 0% or 100% and assays which showed statistically significant deviations from the parallel-line model (p < 0.05) were considered in detail, and in some cases anomalous responses or preparations were omitted before further analysis. For a small number of preparations the largest response was less than 50% or the smallest response was greater than 50%. In no case was the largest response less than 40% or the smallest response greater than 62%, and these preparations have not been omitted from analysis. The estimates of relative potency reported are based on analysis of the contributed assays as multiple parallel-line assays using probit transformed responses and assuming the ‘natural’ response limits of 0% and 100% for numbers responding. Assays included did not show statistically significant deviations from parallelism. The slope based on all preparations included in the assay was therefore considered to be more reliable than the slopes which would have been obtained for pair-wise comparisons only. Nevertheless, slopes were separately examined for any consistent differences across all assays which might have indicated slight non-parallelism which was not detected as statistically significant in the individual assays. After deletion of anomalous data as detailed in results, any assays for which the deviations from either linearity or parallelism are significant (p < 0.05) and for which the total deviations from the model are also significant (p < 0.05) are taken to be invalid, and estimates from these assays are not included in the combined mean estimates. Estimates of the number of organisms giving an LD50 have been obtained by fitting (using SASÒ PROC PROBIT)[10] a function relating the probit response to the logarithm of the dose assuming the ‘natural’ response limits of 0% and 100%. Estimates of potency have been assessed for homogeneity using a chi-square test for homogeneity. Estimates have been combined as geometric means. As noted with the combined estimate, these are unweighted geometric means or, for some groups of estimates which do not show significant deviations from homogeneity, weighted geometric means. In the case of weighted geometric means, the 95% limits for the mean estimate are based on the total weights of the estimates combined. In the case of unweighted geometric means, the 95% limits are based on the variance of the logarithms of the estimates combined. 7. Results 7.1. Preliminary consideration of data A total of 31 assays from 16 laboratories were contributed to study N2, and a total of 70 assays from 14 laboratories were contributed to study O1. Preliminary analysis of the data showed that for the majority of preparations the fitted response limits did not deviate significantly from the ‘natural’ limits of 0% and 100% and that the majority of assays did not deviate significantly from the linear parallel-line model. However, there were a few laboratories in which there were a number of preparations with fitted limits significantly different from the ‘natural’ limits and these tended also to be laboratories in which several assays showed significant deviations from the linear parallel-line model. Most notable were laboratories 10 and 14 in study N2 and laboratories 1, 8 and 12 in study O1, with occasional
anomalous assays in other laboratories. In most of these assays, the natural response rate was significantly larger than 0%; that is, small numbers of mice survived at low doses of vaccine, and the doseeresponse line became non-linear as the dose of vaccine decreased. The available data suggest that the ‘true’ response limit is likely to differ between different assays as well as between different laboratories. However, these data are insufficient for reliable estimation of the response limit. Therefore, for these assays low doses of vaccine giving small responses which are not consistently increasing have been omitted from analysis. There were also a limited number of cases in which one preparation in an assay gave anomalous responses which were not consistent with the responses of that preparation in other assays in the same laboratory, and data from these preparations have also been omitted from the final analysis. Data omitted from analysis are of doses giving responses apparently at the maximum (or minimum) of the response range, and for which responses showed no further increase (or decrease) when compared with smaller (or larger) doses of the same preparations. Thus, the smallest doses of vaccine were omitted from most preparations in most assays of laboratories 10 and 14 in study N2 and of laboratories 1 and 12 in study O1, and also from most preparations in assays 1.1 and 2.1 in laboratory 7 and assay 4 in laboratory 8 in study O1. The largest doses of vaccine were omitted from most preparations in assay 6 of laboratory 4 and assay 3 of laboratory 12 in study O1. In addition to these deletions of doses giving responses apparently at the limits of the response range, five anomalies were noted and omitted from the total of 463 preparations, namely for two preparations (US11 and IHR, all doses omitted) in assay 2 of laboratory 13 in study N2, for two preparations (CIS4 and IHR, one dose from each omitted) in assay 3 of laboratory 6 and one preparation (IS1, one dose omitted) in assay 1 of laboratory 8 in study O1. After omission of these non-monotonic or anomalous responses, all assays showed a satisfactory fit to the parallel-line model, with the probability of deviations from the model greater than 0.05 for all except two assays. These were assays 3 from laboratory 1 and 2 from laboratory 12, both in study O1. 7.2. Comparison of doseeresponse lines Analysis of the slopes of the log dose-probit response lines for each preparation in each assay did not indicate any significant differences among the preparations compared, based on the values of the chi-square test for deviations from the parallel-line model (data not shown). Slopes were additionally assessed for any consistent differences across all assays in the study. Comparison of the slopes within the same assay for duplicate ampoules of CIS4 in study O1 showed no significant difference between them (probability of paired t-test w 0.7). Similar comparisons of slopes for CIS4 and IS2 in study O1 and for CIS4 and IS3 in study N2 showed no consistent differences between these pairs (probability of paired ttest > 0.3 in each case). Comparison of slopes for either IS3 or CIS4 and US11 in study N2 did not show any consistent differences. In contrast, comparison of slopes for IS1 with either CIS4 or IS2 in study O1 suggested that there may be a tendency for the slopes of the doseeresponse lines for IS1 to be less than those for CIS4 or IS2 (probability of paired t-test < 0.05 for each comparison). 7.3. Comparison of duplicate ampoules of CIS4 In one assay of study N2, and in 26 assays in study O1, independent ampoules of CIS4 were directly compared in terms of potency with one another. These 27 individual estimates were homogeneous (p chi-square test w 0.5) and gave a weighted
R.G. Das et al. / Biologicals 38 (2010) 644e651
geometric mean of 1.08 with 95% limits 0.95e1.23. This observed value is in good agreement with the expected value of 1 for identical ampoules. The geometric mean weight over these assays was 39. This provides an estimate of the within assay variance and corresponds to 95% limits for an individual estimate of potency from 48% to 209% of the estimated value. The unweighted geometric mean of these estimates was 1.02 with corresponding weight 21 (determined as the reciprocal of the variance of log10 of the potency estimates) which corresponds to 95% limits for an individual estimate of potency from 37% to 273% of the estimated value. Thus the directly observed variance (pooled over assays and laboratories) is somewhat larger than the average estimated within assay variance, but these differences are not significant (assuming the ratio of these variances follows an F distribution, p > 0.05). 7.4. Calibration of CIS4 Estimates of the potency and their associated weights for CIS4 in terms of IS3 (study N2) and in terms of IS2 (study O1) are shown in Tables 1and 2 respectively. Distribution of estimates is shown in Fig. 1. The mean estimates for each study separately, and for both studies combined under the assumption that the IU of the IS2 and IS3 are equivalent to one another are also given in Table 3. It is noted that the geometric mean weight of assay weights for individual estimates for study O1 is approximately 40 while that for study N2 is approximately 30. For study O1 all individual estimates of potency, and similarly the laboratory geometric mean estimates of potency are homogeneous. For study N2 all individual estimates of potency are heterogeneous. Most laboratories contributing data to this study carried out two assays, with several laboratories contributing data from a single assay. Thus the laboratory geometric mean estimates of potency for CIS4 from study N2 are also heterogeneous. Three estimates contributed excessively to the chi-square for heterogeneity, namely assays 1 from each of laboratories 2, 7 and 8. Omission of these three of the 29 estimates (10% of estimates) from study N2 gave a variance of log potency estimates for the remaining 26 estimates which was only 44% of the variance of the 29 estimates, and the 26 estimates of potency did not deviate significantly from homogeneity. Mean estimates using this homogeneous group of estimates are also shown. In view of the data from O1, it is likely that if the laboratories from which the ‘heterogeneous’ estimates came had carried out up to six assays each, the laboratory geometric means from N2 would not be
647
Table 2 Estimated potency of candidate standard CIS4 and of the proposed regional standard US11 expressed as IU of the Second International Standard (study O1) per ampoule. Weight is the reciprocal of the log10 of the estimated potency. Geometric means marked * are unweighted means with weight determined as the reciprocal of the variance of the estimates combined. The symbol ‘u’ denotes that an estimate is not available. Laboratory
Total no. of assay
CIS4
Weight
US11
Weight
1 2 3 4 5 6 7 7.1 8 10 11.1 11.2 12 14
4 6 4 6 4 6 4 4 4 4 6 5 6 6
33.16 44.63 41.62 37.83 38.18 29.13 41.09 44.01 33.31 49.22 34.59 56.28 45.07 25.82
130 356 117 468 373 156 133 214 178 294 184 104 270 170
u 87.61* 83.30* u u u u u u u u u u u
u 67 31 u u u u u u u u u u u
heterogeneous. That is, to some extent, the apparent heterogeneity may be an artefact of the limited data. It is suggested that the combined estimate which is most representative of these assays is the weighted geometric mean of estimates from both studies N2 and O1 (excluding the three estimates from study N2 which contributed excessively to heterogeneity), namely 40 IU per ampoule with 95% limits 37e43 IU per ampoule (Table 3). This estimate is consistent with the combined estimate for each study separately, and is also consistent with the unweighted geometric mean for study N2 including all estimates. This estimate maintains overall continuity of unitage with both IS2 and IS3. Continuity is also broadly maintained for the individual laboratories. Laboratory geometric means from study O1, with a minimum of four assays from each laboratory, ranged from 26 IU (95% limits 18e36 IU) to 56 IU (95% limits 36e88 IU) per ampoule and were not heterogeneous. Laboratory geometric means from study N2 ranged from 9 IU per ampoule (95% limits 3e30 based on a single assay) to 78 IU per ampoule (95% limits 45e136 IU based on two assays), and were heterogeneous. This heterogeneity may reflect both the greater variability of the individual assays and the
Table 1 Geometric means (GM) estimated potency of candidate standard CIS4 and of the proposed regional standard US11 expressed as IU of the Third International Standard (study N2). Weight is the reciprocal of the log10 of the estimated potency. The symbol ‘u’ denotes that an estimate is not available. Laboratory
Total no. of assay
CIS4
Weight
US11
Weight
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
2 2 1 2 2 2 2 1 2 2 1 2 2 3 3 2
42.07 14.92 42.24 49.02 47.27 50.32 15.36 9.08 65.36 78.27 34.20 36.70 22.70 65.96 34.14 35.21
51 59 49 66 51 29 70 14 82 69 82 58 91 63 73 105
90.81 29.54 u 113.30 107.62 81.66 31.02 47.52 68.07 127.26 64.62 58.05 86.67 109.04 63.38 56.29
51 59 u 55 51 29 69 15 83 69 78 25 27 63 56 104
Fig. 1. Histogram showing the distribution of estimates of potency for CIS4 from each assay in each laboratory. Each square represents one estimate and the label in the square identifies the study as N2 (first character of label is N) or study O1 (first character of label is O) and the remaining label identifies the laboratory number. Estimates from study O1 are also identified by shading.
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Table 3 Estimated potency of candidate standard CIS4 and of the proposed regional standard US11 expressed as IU of the Third International Standard (study N2) or of the Second International Standard (study O1) per ampoule. Weight is the reciprocal of the log10 of the estimated potency. Geometric means marked * are unweighted means with weight determined as the reciprocal of the variance of the estimates combined. CIS4 Study N2 GM All Individual Estimates 95% Interval GM Laboratory GMs 95% Interval Study O1 GM All individual Estimates 95% Interval GM Laboratory GMs 95% Interval
39.26 36.23e42.55 39.25 36.27e42.46
Studies O1 and N2 GM All individual Estimates 95% Interval GM Laboratory GMs 95% Interval
37.89* 34.50e41.61 36.70* 31.00e43.43
35.95* 28.91e44.70 34.96* 25.50e47.94
Weight 469 242
3154 3302
2381 780
US11
Weight
71.53* 57.64e88.77 69.56* 54.39e88.96
482
39.70 36.99e42.60 39.70 36.99e42.60
4081
85.86* 51.98e141.80 86.22 54.14e137.92
108
75.25* 61.82e91.60 71.25* 57.38e88.49
565
4228
82.81* 69.62e98.49 78.93 67.57e92.21
Study
Laboratory
n
IS2
Weight
CIS4
Weight
O1
01.0 02.0 03.0 04.0 05.0 06.0 07.0 07.1 08.0 10.0 11.1 11.2 12.0 14.0
2 4 4 3 2 6 2 2 4 4 3 3 3 3
39.93 27.02a 22.17a 28.58 29.56 28.76 26.17 21.14 44.20 37.15 48.22 31.33 30.00 48.08
133 80 34 192 126 157 59 86 172 248 80 61 145 82
34.11 23.60 21.19 26.03 28.56 18.11 23.24 27.76 32.21 39.10 37.07 44.10 41.60 31.00
129 205 116 195 126 157 59 86 175 266 80 61 145 81
1861
29.46 26.54e32.70 29.58* 25.38e34.49
403
98
508
Omit three assays from study N2, assays 1 from laboratories 2, 7 and 8 Study N2 GM All Individual Estimates 41.20 926 78.03 749 95% Interval 35.52e47.80 66.14e92.05 GM Laboratory GMs 41.20 926 78.03 749 95% Interval 35.51e47.81 66.13e92.06 Studies O1 and N2 GM All individual Estimates 95% Interval GM Laboratory GMs 95% Interval
Table 4 Estimates of the potency (GM) of IS2 and CIS4 expressed as IU of IS1. Weight denotes the reciprocal of the variance of log10 of the potency estimate.
731 847
limited number of assays in each laboratory in study N2. Although individual estimates from study O1 were generally more precise than those from study N2, individual assay estimates covered a similar range. Individual estimates in study O1 ranged from 17 IU per ampoule to 139 IU per ampoule and those in study N2 ranged from 9 IU per ampoule to 80 IU per ampoule. Direct comparisons of US11 with the candidate standard CIS4 can be obtained from Tables 1e3. These estimates do not deviate significantly from homogeneity within laboratories, within either study, or when considered over both studies. The weighted geometric mean of all estimates for US11 based on assumed ampoule content for CIS4 of 40 IU is 74.5 IU per ampoule (95% limits 65.5e84.9 IU). This estimate is consistent with the estimate for US11 in terms of IS2 and IS3, and indicates that overall continuity of unitage of US11 will be maintained if CIS4 is assigned an ampoule content of 40 IU. 7.5. Comparisons of the First International Standard for pertussis Vaccine with the Second International Standard IS2 and with the candidate standard CIS4 At the time of study O1 there was a remaining stock of the First International Standard, and ampoules of the preparation were included in some assays. Estimates of IS2 and CIS4 in terms of this preparation, denoted IS1 are given in Table 4. The geometric mean estimate for IS2 in terms of IS1 is 33 IU per ampoule which is about 70% of the assigned potency for IS2 of 46 IU per ampoule. Similarly, the geometric mean estimate for CIS4 in terms of IS1 is 30 IU per ampoule which is about 75% of the estimate for CIS4 in terms of either IS2 or IS3. 7.6. Stability study After the candidate standard was transferred to NIBSC, in June 2003, a total of 298 ampoules of that material were stored at
GM, individual estimates 95% limits GM, laboratory GMs 95% limits a
32.13 28.94e35.68 33.09 29.61e36.98
1657
1881 1052
Unweighted combination of heterogeneous estimates.
elevated temperatures to provide information about thermal degradation (18 at 56 C, 30 at 45 C, 30 at 37 C, 30 at 20 C, 30 at þ4 C). Ampoules of CIS4 (94/532) which had been stored at elevated temperatures for up to 2.5 years were assessed in several mouse protection assays in one laboratory. The potency of the vaccine in ampoules stored at 37 C for one year relative to that for ampoules stored continuously at 20 C was 1.18 (95% limits 0.64 to 2.18). The relative potency of samples stored at 4 C for 19.5 months and of samples stored at 20 C for 19.5 months or 32.4 months also did not differ significantly from 1 (estimates of relative potency 1.05, 1.01 and 1.05 respectively). Since none of these samples show loss of activity, no degradation rate can be reliably predicted. Additional information about stability can be inferred from the comparisons of the candidate standard with other established reference preparations in 1995 and 2005. These comparisons do not indicate any differential changes in relative activity, and thus are consistent with stability of these preparations. These results indicate that the candidate standard is sufficiently stable to serve as an international standard. Experience, with reconstituted material of similar type to the candidate standard suggests that such material may not be stable after reconstitution and it is recommended that freshly reconstituted material should be used for each assay. 8. Discussion This report summarizes the currently available information about international standards for whole cell pertussis vaccine and brings together data from two international studies to evaluate the candidate international standard. The results from the two studies together with results from previous studies are given in Table 5 for comparison. The two studies described in this report provide a consistent calibration of 40 IU per ampoule for CIS4 in terms of IS2 and IS3, and no apparent differences in doseeresponse curves were detected. The calibration of CIS4 in terms of IS1 obtained in study O1 is about 25% smaller than its calibration in terms of IS2/IS3. However, differences in slope between CIS4 and IS1 were noted in study O1 and were also observed between IS2 and IS1 (data not shown). This may reflect differences in the responses of these different preparations in some assays. Differences between IS1 and the more recent international standards and CIS4 may also give rise to the heterogeneity observed in some previous comparisons, e.g. IS2 and IS1 in the study [3], IS3 and IS1 in another study [4].
R.G. Das et al. / Biologicals 38 (2010) 644e651 Table 5 Comparison of results from various studies. Preparation Ref. used
Results (IU/ amp.)
Study
1st IS
US NIH, Lot 4
34.7 IU
US Master Lot 4
33.9 (29e40)
O. Maaloe, WHO/BS/338, 1956 Seagroatt et al., 1981
1st IS U.S. Master Lot 4 U.S. Master Lot 4
Seagroatt et al, 1981 Seagroatt et al, 1981 Jansen et al, 1996
1st IS
45.9 (36e59)a 38.9 (29e52)a 41.4 (35.1e48.3)b 33.1 (29.6e37.0)e 36 (14e90)a
3rd IS
1st IS 2nd IS 1st IS
46.3 (40e53) 46.0 (41e53) 41 (17e98)a
2nd IS
56.1 (46e68)
Seagroatt et al, 1981 Seagroatt et al, 1981 Xing et al., WHO/BS/98, 1880, 1998 Xing et al., 2001
US Lot 11
2nd IS US Lot 4
91.1 (80e103) 79.8 (68.9e92.4) 86.2 (54.1e138) 69.56 (54.4e89.0)c 78.0 (66.1e92.1)d 71.3 (57.4e88.5)c 78.9 (67.6e92.2)d 76.5 (61.6e94.9) 73.5 (62.5e86.4) 74.6 (65.5e84.9)
2nd IS
1st IS
2nd IS 3rd IS
2nd & 3rd IS (combined)
CIS4 CIS4 CIS4 CIS4
1st IS 2nd IS 3rd IS
2nd & 3rd IS (combined)
29.6 (25.4e34.5)e 39.3 (36.3e42.5) 35.0 (25.5e47.9)c 41.2 (35.5e47.8)d 36.8 (31.1e43.5)a 39.7 (37.0e42.5)d
O1995 Xing et al., WHO/BS/98, 1880, 1998
Jansen et al, 1996 Jansen et al, 1996 O1995 N2005
649
activity of these preparations. The consistency of comparisons of US11 with the various international standards over this period of time also does not suggest differential changes in activity among these preparations. These data are consistent with stability of the preparations compared. Limited data from thermally accelerated degradation studies indicate that CIS4 is likely to be stable at the usual storage temperature of 20 C. Thus CIS4 appears to be sufficiently stable to serve as an international standard. Data from calibration of standards using the active mouse protection assay (Kendrick test) over the period of these studies (from 1980 to 2005) suggest that over this time the assay has performed consistently and reliably. Estimates based on limited assays will show variation, and there is some heterogeneity of estimates possibly reflecting differences between preparations which are detected by some assays. The reported methods and challenge organisms show great diversity and some of these reported factors may be interrelated (data not shown). This study is not designed to explore these issues. As previously noted [3], in spite of variations, results considered over a number of laboratories are generally concordant. This was also the finding of a recent proficiency study of this method [12]. Comments on this report were obtained from 12 of 16 laboratories participating in study N2 and all of these participants agreed with the proposed unitage for CIS4. 9. Conclusions and recommendations
O1995 & N2005
O1995 N2005 O1995 & N2005 O1955
On the basis of the results of the studies reported here and previous studies indicating stability and continuity of unitage among International Standards for Pertussis Vaccine, the ECBS of WHO established the material in ampoules coded 94/532 (CIS4) as the Fourth International Standard for Whole Cell Pertussis Vaccine and that it be assigned an activity of 40 IU per ampoule on the basis of its calibration in terms of the Second and Third International Standards for Pertussis Vaccine (Whole Cell) [13].
O1955
Acknowledgements
N2005
The contribution of the participants to studies O1 and N2 is gratefully acknowledged. The organization and preliminary consideration of O1 by Dr. Gert A. Hansen and staff at SSI is also gratefully acknowledged. We thank WHO for supporting the studies.
O1995 & N2005
Annex 1. Participant list (N2 study)
a
Unweighted combination of heterogeneous estimates. US4 and IS2 did not give comparable potency estimates in some laboratories. c Unweighted geometric mean of heterogeneous laboratory geometric means (all data). d Weighted geometric mean of homogeneous laboratory geometric means after omitting 3 assays as described in text. e Consistent difference in slopes of CIS4 or IS2 compared with IS1, as discussed in text. b
US11 was included in the majority of laboratories in study N2 and has also been included in a number of previous studies. The various estimates from these studies are shown in Table 5. The comparison of US11 with IS2/IS3 in the two studies reported here gives a value about 10% smaller than that assigned to US11 in terms of IS2 by a previous study. However, these results are consistent with the comparison of US11 with US4 in a previous study and of US11 with CIS4 in the recent studies. Questions had been raised regarding the stability of IS2. Previous studies have confirmed the apparent stability of IS2 [5,11] and of IS3 [4]. Comparison of CIS4 with IS2 and after a period of 10 years with IS3 are consistent and thus do not suggest differential changes in
Dr Rajiv Jayasena QC Compliance CSL Ltd 45 Poplar Rd Parkville Victoria 3052 Australia Mr Frederic Mortiaux GlaxoSmithKline Biologicals Rue de l’Institut 89 B e 1330 Rixensart Belgium Dr. Humberto Pinheiro de Araujo Ministerio da Saude do Brasil Fundacao Oswaldo Cruz FIOCRUZ Instituto Nacional de Controle de Qualidade em Saude INCQS Av. Brasil 4365, Manguinhos, Rio de Janeiro CEP 21045 900 Brazil
650
R.G. Das et al. / Biologicals 38 (2010) 644e651
Dr Zhang Shumin Tiantan Xili No 2 National Institute for the Control of Pharmaceutical & Biological Products (NICPBP) Temple of Heaven Beijing 100050 China Dr Patrice Chagnaud and Ms El-zaouk Annie Bacterial Vaccines Control Unit and Bioactivity and Radio-analysis Control Unit Agence Francaise de Securite Sanitaire des Produits de Sante (AFSSAPS) 321, avenue Jean Jaurès F e 69007 Lyon France Dr Christophe Cochet Sanofi Pasteur 1541 Av. Marcel Merieux 69289 Marcy l’Etoile France Dr A.K. Tahlan Joint Director, Central Drugs Laboratory Central Research Institute Division of Biological Products Kasauli 1732504 Himachal Pradesh India Dr Suresh S. Jadhav and Dr S. Gairola Serum Institute of India Ltd 212/2 Hadapsar Pune 411 028 Maharashtra State India Dr Syahrial Tahir and Dr H. Moch.Ma’roef National Quality Control Lab of Drug and Food (NQCLDF) Jalan Percetaken Negara No 23 Jakarta Pusat 10560 Indonesia Dr Yoshinobu Horiuchi Department of Bacterial and Blood Products National Institute of Infectious Diseases (NIID) 4-7-1, Gakuen, Musashimurayama-shi Tokyo 208-0011 Japan Mrs Angélica López Sotelo and Ms Adriana Santiago Echauri Laboratorios de Biologicos y Reactivos de Mexico, S.A. de C.V. Departmento de Control Biologico Mariano Escobedo No. 20 Col Popotla CP 11 400 Mexico, D.F. Dr Lonneke Levels Netherlands Vaccine Institute (NVI) Antonie van Leewenhoeklaan 9-11 Postbus 457 3720 MA Bilthoven The Netherlands Dr Anna Gzyl Dept of Sera and Vaccines Evaluation National Institute of Hygiene (NIH) National Centre for Disease Control and Prevention 24 Chocimska Street 00791 Warsaw Poland
Mr Apichai Supasansatorn and Ms Teeranart Jivapaisarnpong Division of Biological Products Department of Medical Sciences Tivanon Rd Ministry of Public Health Nonthaburi 11000 Thailand Dr D. Xing and Dr M.J. Corbel Division of Bacteriology National Institute for Biological Standards and Control Blanche Lane South Mimms Potters Bar Herts EN6 3QG UK Dr Ma. Teresa Ibarz and Dr Maria Luz Pombo Division Control Nacional de Productos Biologicos Instituto Nacional de Higiene “Rafael Rangel” Ciudad Universitaria UCV, Detras del Hospital Clinico Universitario de Caracas Los Chaguaramos Caracas 1051, Venezuela.
Annex 2. Participant list (O1 study) Dr K. Healy CSL Ltd 45 Poplar Road Parkville Victoria 3052 Australia Dr E. Rommel SmithKline Beecham Biologicals S.A. Rue de l’Institut 89 B e 1330 Rixensart Belgium Dr Hou Qiming Division of Bacterial Vaccines National Institute for the Control of Pharmaceutical & Biological Products Temple of Heaven Beijing 100050 China Dr L. Peyron Pasteur Mérieux Val de Reuil France Dr L. Peyron Pasteur Mérieux 1541 Ave. Marcel e Mérieux F-69280 Marcy l’Etoile France Dr K. Enssle Chiron Behring GmbH & Co Emil e von e Behring e Str, 76 D e 35041 Marburg Germany Dr Z. Nagy HUMAN Serum Production and Medicine Manufacturing Co Ltd 2100 Godollo
R.G. Das et al. / Biologicals 38 (2010) 644e651
Tancsics Mihaly H-2100 Hungary Dr Yoshikazu Tada BIKEN The Research Foundation for Microbial Diseases of Osaka University Kanonji Institute Kanonji City Kagawa 768 Japan Dr H. van de Donk RIVM Antonie van Leeuwenhoeklaan 9 NL-3720 BA Bilthoven The Netherlands Dr S.J. Varallyay Swiss Serum and Vaccine Institute Rehhagstrasse 79 CH-3001 Berne Switzerland Dr K. Readhead and Dr M.J. Corbel, Division of Bacteriology National Institute for Biological Standards and Control Blanche Lane, South Mimms Potters Bar EN6 3QG, UK Dr Bruce Meade Laboratory of Pertussis Center for Biologics Evaluation and Research Food and Drug Administration 1401 Rockville Pike Bethesda
651
MD 20852 USA Dr Bonnie Brock American Cyanamid Co. Lederle e Praxis Biologicals Division 401 N. Middletown Rd Pearl River NY 10965 USA References [1] WHO Technical Report Series 897, WHO ECBS 49th Report, 2000 (Collab study: WHO BS/98 1880). [2] Kendrick PL, Eldering G, Dixon MK, Misner J. Mouse protection test in the study of pertussis vaccines: a comparative series using intracerebral route of challenge. American Journal of Public Health 1947;37:803e10. [3] Seagroatt V, Sheffield F. A collaborative assay of the proposed second international standard for pertussis vaccine and of the proposed first British standard for pertussis vaccine. Journal of Biological Standardization 1981;9:351e65. [4] Xing D, Gaines Das R, Newland, Corbel M. Third international standard for pertussis vaccine: International confirmation study of activity of British standard for pertussis vaccine, coded 66/303. Biologicals 2001;29:133e6. [5] Dobbelaer R. The stability of the second International Standard for Pertussis Vaccine e results of an inquiry. WHO BS/89.1617, Geneva; 1989 . [6] WHO Technical Report Series 932, WHO ECBS 55th Report; 2005. [7] Jansen DL, Meade BD. Preparation and standardisation of US standard pertussis vaccine lot 11. Biologicals 1996;24:363e70. [8] WHO Technical report series 658; 1981. p. 18 [9] WHO Technical Report Series 127, 1957. [10] SAS Institute, Inc. SAS/STAT user’s guide, version 6. 4th ed. Cary, NC: SAS, Institute, Inc; 1989. [11] Redhead K, Gaines Das R. A collaborative assay of the proposed third British reference preparation for pertussis vaccine and the relative potencies of the second international standard and the second British reference preparation for pertussis vaccine. Biologicals 1991;19:107e11. [12] Xing D, Gaines Das R, O’Neill T, Corbel M, Dellepiane N, Milstien and participants J. Laboratory testing of whole cell pertussis vaccine: a WHO proficiency study using the Kendrick test. Vaccine 2002;20:342e51. [13] WHO Technical Report, WHO ECBS 57th Report, 2006 (Collab study: WHO/BS/ 06.2036), in press.