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Collaborative study for the establishment of a European Phamacopoeia biological reference preparation for Bordetella pertussis mouse antiserum for serological potency testing of acellular pertussis vaccines
BIOLOGICALS Biologicals 31 (2003) 25–38 www.elsevier.com/locate/biologicals
Collaborative study for the establishment of a European Phamacopoeia biological reference preparation for Bordetella pertussis mouse antiserum for serological potency testing of acellular pertussis vaccines Bertrand Poirier a*, Nicole Bornstein a, Murielle Andre a, Denis Marmonier a, Monique Pares b, Gerard Vanhooren c, Guy Rautmann d, Marie-Emmanuelle Behr-Gross d, Roland Dobbelaer c, Florence Fuchs a a
b
Agence Franc¸aise de Se´curite´ Sanitaire des Produits de Sante´, Site de Lyon, 321 avenue Jean Jaure`s, 69007 Lyon, France Agence Franc¸aise de Se´curite´ Sanitaire des Produits de Sante´, Site de Montpellier, 635 rue de la Garenne, 34740 Vendargues, France c Institut Scientifique de la Sante´ Publique – Louis Pasteur 14, rue Juliette Wytsman B-1050 Bruxelles, Belgique d Council of Europe, European Directorate for the Quality of Medicines, Division IV B.P. 907 F-67029 Strasbourg Cedex 1, France Received 30 May 2001; revised 13 August 2001; accepted 20 August 2002
Abstract A collaborative study was organised by the European Directorate For the Quality of Medicines (EDQM) to assess the suitability of a candidate mouse antiserum as a European Pharmacopoeia Biological reference preparation (BRP) for acellular pertussis vaccine potency testing. The candidate antiserum was obtained by immunising mice with a five-component acellular pertussis vaccine: pertussis toxin (PT), filamentous haemagglutinin (FHA), pertactin (PRN) and Fimbrial 2/Fimbrial 3 (Fim 2&3). The study has been divided into two separate phases. Phase I was a pre-qualification study including three laboratories. This phase was aimed at pre-qualifying the candidate BRP (cBRP) and at documenting the impact of differences in the antibody detection methodology enzyme linked immunosorbent assay (ELISA) procedures on results of pertussis antisera calibration versus the currently used standard US standard pertussis antiserum (mouse) Lot 1 (SPAM-1) (United States Food and Drug Administration (USFDA) reference serum) and the cBRP. As no significant difference between the antibody titres determined by using the different ELISA methodologies was found, a large-scale study enrolling 13 laboratories (Phase II) was carried out, each participant performing its in-house methodology. Its aim was to calibrate the cBRP (in terms of the SPAM-1 reference) and to demonstrate its equivalence or superiority to internal references. The study showed that there was no difference in positive sera titres expressed relative to their corresponding internal reference (homologous situation) or the proposed standard (heterologous situation) reference. The cBRP can, therefore, reliably act as replacement for the in-house reference preparations. Further analysis of the outcome of this study enabled to assign to the cBRP a potency of 39, 138, 34 and 56 ELISA unit per millilitre, respectively, to its anti-PT, anti-FHA, anti-PRN and anti-Fim 2&3 antibody contents. The cBRP has been adopted by the European Pharmacopoeia Commission at its June 2000 session as Bordetella pertussis mouse anti-serum Ph Eur. BRP batch 1. 2003 The International Association for Biologicals. Published by Elsevier Science Ltd. All rights reserved. Keywords: Collaborative study; Statistics; Acellular pertussis vaccine; Enzyme linked immunosorbent assay; European Pharmacopoeia biological reference preparation; Potency assay
1. Introduction * Corresponding author. Tel.: +33-4-72-76-0610; fax: +33-4-72-76-0615 E-mail address: [email protected] (B. Poirier).
Following the development of a variety of acellular pertussis vaccines, different laboratory tests were introduced for monitoring safety, potency, physico-
1045-1056/03/$30.00 2003 The International Association for Biologicals. Published by Elsevier Science Ltd. All rights reserved. doi:1 0 . 1 0 1 6 / S 1 0 4 5 - 1 0 5 6 ( 0 2 ) 0 0 0 7 5 - 1
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B. Poirier et al. / Biologicals 31 (2003) 25–38
chemical and immunological characteristics of new vaccine lots, comparatively to an homologous reference vaccine clinically evaluated [1,2]. No entirely satisfactory method is available for potency monitoring. Several in vivo protection model assays are under study, including intracerebral, intranasal and aerosol challenge models [3,4], but mouse immunogenicity assays remain useful for checking consistency of production from batch to batch even if they do not necessarily correlate with protection in humans [2,5]. The European Pharmacopoeia monographs [6] and WHO guidelines [7] describe this test for potency assay on final lots of acellular pertussis vaccines currently being marketed. The mouse immunogenicity assay is based on the measurement of the specific antibody responses to the different antigenic components present in a test vaccine. Serum antibody levels are measured by enzyme linked immunosorbent assay (ELISA) and the immunogenicity is compared with that of a reference vaccine. Antibody responses to any given dose of an acellular pertussis component have been found to vary considerably between individual mice, and the ELISA part of the potency test is itself subject to some variability. Therefore, in order to allow a valid quantitation of antibody levels, a calibrated reference antiserum has to be included in every ELISA assay (this procedure allows the selection of serum dilutions within the quantitation limits of an ELISA assay and to express results in ELISA units (ELU) that are consistent between assays [8]). Some manufacturers of acellular pertussis vaccines as well as some control authorities, e.g. United States Food and Drug Administration (USFDA) and Japanese National Institute (JNIH) have established such reference antisera. Recently, an International reference reagent for acellular pertussis antiserum was calibrated and established against the existing reference antisera [9,10]. In Europe, various in-house reference antisera, usually calibrated against the USFDA reference antiserum (SPAM-1) are used by manufacturers and official medicines control laboratories (OMCLs). Replacement of different in-house references by an European Pharmacopoeia biological reference preparation (BRP) for pertussis antiserum in routine release assays by both manufacturers and OMCLs should improve inter-laboratory comparability of results within Europe. In order to produce a candidate BRP (cBRP), a large batch of Bordetella pertussis antiserum obtained by immunising CD1 mice with a five component acellular pertussis vaccine similar in composition to that used for generating the US standard pertussis antiserum (mouse) Lot 1; SPAM-1 (SPAM-1) has been aliquoted and freeze-dried at the European Directorate for the Quality of Medicines (EDQM). A preliminary characterisation study by Western blot and ELISA analysis showed that
the candidate serum contained quantifiable amounts of specific antibodies directed against the five pertussis antigens present in the vaccine. The preparation, still in the liquid state, was included in the WHO study previously mentioned. A pre-qualification phase, referred to as Phase I (expert report, private communication), has been run between June and October 1999. This study was performed in order to pre-qualify the cBRP and to document the impact of differences in ELISA methodologies on the estimation of the antibody content of pertussis antisera. It consisted of a complete crossover study between five different methodologies and antisera (internal references and control sera) generated by different acellular vaccines in the market. Following successful completion of Phase I, an international collaborative study, referred to as Phase II, was run between November 1999 and April 2000. Phase II was aimed at calibrating the B. pertussis mouse antiserum Ph. Eur. cBRP in terms of anti-pertussis toxin (PT), anti-filamentous haemagglutinin (FHA), anti-pertactin (PRN) and anti-Fimbriae type 2&3 (Fim 2&3) antibody titres expressed in ELISA unit per millilitre relative to the US FDA reference serum (SPAM-1) on a sufficiently large database.
2. Materials 2.1. Reference sera SPAM-1 (Center for Biologics Evaluation and Research (CBER), FDA, Bethesda, MA, USA) and International reference reagent anti-pertussis serum (IRR (1999) coded 97/642; NIBSC, London, UK) are lyophilised preparations, which have an assigned unitage after reconstitution with 0.5 ml distilled water of 170 ELU/ml anti-PT, 540 ELU/ml anti-FHA, 150 ELU/ml anti-PRN and 200 ELU/ml anti-Fim 2&3 for SPAM-1 and 34 ELU/ml anti-PT, 286 ELU/ml antiFHA, 60 ELU/ml anti-PRN, 64 ELU/ml anti-Fim 2&3 for IRR. Production of the cBRP has been previously described (expert report, private communication). The immunisation and blood collection were performed by the French Health Products Safety Agency (AFSSAPS) following a similar immunisation schedule as for SPAM-1 production by the CBER. Briefly, a five component acellular pertussis vaccine (Pasteur Merieux Connaught, Canada) was used to inoculate 1200 mice of the CD-1 strain. The vaccine, provided as combined diphtheria–tetanus–pertussis formulations adsorbed to alum, was reported to contain 10 µg/0.5 ml of glutaraldehyde-inactivated PT, 5 µg/0.5 ml of FHA, 3 µg/ml of PRN, 5 µg/0.5 ml of Fim 2&3, 15 Lf tetanus toxoid and 15 Lf diphtheria toxoid. Animals were bled 12 days after a second booster immunisation. Mini pools of serum were prepared and subsequently characterised
B. Poirier et al. / Biologicals 31 (2003) 25–38
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Fig. 1. Influence of the method in relation to the component tested. FHA antigen (A), PT antigen (B), PRN antigen (C). Mean of titres between the three participating laboratories.
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B. Poirier et al. / Biologicals 31 (2003) 25–38
by Western blot analysis for the presence of antibodies against each of the five pertussis antigens. All sera were then pooled in a large batch that was aliquoted in 0.5 ml fractions prior to freeze-drying. Finally, a lyophilised serum batch of 4000 ampoules was produced at the EDQM. 2.2. Participants Thirteen laboratories routinely using ELISA assays for measuring mouse anti-FHA, anti-PT, anti-PRN and/or anti-Fim 2&3 antibodies were invited to participate in the study. These included vaccine manufacturers and National control laboratories. A list of participants is given at the end of the report (Appendix A). They are referred to by using an arbitrary assigned number, not related to the order of listing at the end of this report. All the 13 laboratories contributed to Phase II, but only three of them were involved in Phase I. 2.3. Study design and methods Phase I: This study consisted of a complete crossover study between different methodologies and antisera (in-house reference sera and control sera) generated with different vaccines. The different coating antigens, reagents and ELISA procedures were provided by four manufacturers and an official control laboratory. The three laboratories involved received all in-house references and control sera as well as all reagents and standard operating procedures necessary to perform assays by means of five different ELISA methodology groups referred to as A–E (Method A: PT, FHA; Method B: PT, FHA, PRN; Method C: PT, FHA, PRN; Method D: PT; Method E: PT, FHA, PRN). All methodologies used an indirect ELISA: passive adsorption of the antigens onto microtiter plates followed by the addition of the serum samples and then detection of antigen-specific antibodies by enzyme-conjugated antimouse antibodies and substrate. For each methodology, each participant was requested to test all in-house reference and control sera, as well as reference sera previously cited (SPAM-1, IRR, cBRP), and to assay eight consecutive dilutions of each serum in three independent ELISA assays performed in different weeks. Participants were asked to return all the absorbance raw data from microtitration plates to permit an independent statistical analysis. For feasibility reasons (availability of reagents), some of them did not strictly follow the provided procedures. The Fim 2&3 were not analysed due to lack of corresponding coating antigens and reagents. Phase II: Each participating laboratory was requested to test the three reference sera (SPAM-1, IRR, cBRP) as per the ELISA method established in its laboratory, to include its control and internal reference sera, and to
Fig. 2. Frequency distribution of cBRP antiserum titres for: anti-FHA (A), anti-PT (B), anti-PRN (C), anti-Fim 2&3 (D). Numbers in boxes are laboratory code numbers.
B. Poirier et al. / Biologicals 31 (2003) 25–38
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calculate results according to its standard procedures. Participants were requested to perform three independent assays. Each laboratory was requested to use the same plate layout and to assay eight consecutive dilutions of each serum. Only values derived from valid assays, as determined by the individual laboratory, were to be reported. Laboratories were asked to provide details about the methodology and reagents used in their assays so that the effect of these factors could be assessed. Participants were also asked to return calculated results using their in-house mathematical models, as well as the raw data for each assay to permit recalculation using a common method. The number of laboratories contributing data for each antigen reflects the assays run in each of the laboratories. All 13 laboratories submitted data for PT, but not all laboratories submitted data for the other antigens: only 12 carried out anti-FHA assays, 11 carried out anti-PRN assays and 4 carried out anti-Fim 2&3 assays. To calculate sample titres against the reference serum, four laboratories (numbers 2, 3, 6, 13) used a fourParameter logistic fit. Other calculation methods used included a parallel line model with log dose transformation (laboratories 9, 11, 12), parallel line model with log dose/log OD transformation (laboratories 1, 5, 8), and ‘reference line calculation’ (laboratories 4 and 10). Laboratory 7 did not report its calculation methodology. 2.4. Statistical analysis The raw data returned by each laboratory were used in the statistical analysis performed by the AFSSAPS. Unit calculations were performed with the , version 2.0 software [11]. ELISA unitage for each sample was assigned relative to the reference sera placed on each microtiter plate (SPAM-1, IRR, cBRP). A four-parameter dose response curve was generated for the reference serum by plotting the absorbance as a function of the logarithmically transformed antibody concentration data in ELISA unit per millilitre. The equation fit was (aⳮd)/[1⫹(x/c)b]⫹d, where a and d represent the OD values of the asymptotes, c the logarithmic value of the concentration for which the response value will be centred between the two asymptotes (the inflection point) and b the slope of the linear part of the curve (central part of the curve). The quantitative range was identified as the absorbance range between 10 and 80% of the asymptotic absorbances. A serum was considered non-quantifiable when ODs were below or above the quantification limits. In order to obtain a normal distribution of the antibody unitage data, a logarithmic transformation was applied. The statistical study required analysis of variance (ANOVA) and usual statistical tests. The geometric coefficient of variation (gCV), defined as 100(10s1),
Fig. 3. Segregation of titres and variability by laboratory: anti-FHA titres (A), anti-PT titres (B), anti-PRN titres (C), anti-Fim 2&3 titres (D).
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B. Poirier et al. / Biologicals 31 (2003) 25–38
where s represents the standard deviation on log10 scale, was calculated. Comparisons between laboratories were evaluated by ANOVA and Newman–Keuls test [12]. The ANOVA decomposes the variability into contributions from various factors. The Newman–Keuls multiple comparison procedure discriminates among the means. With the latter method, there is a 5.0% risk of calling one or more pairs significantly different when their actual difference equals 0.
3. Results 3.1. Impact of the ELISA method factor In phase I, the impact of the method factor was analysed by comparing titration results obtained by the different methodologies for all tested sera (internal controls and standards, cBRP, IRR) versus reference serum SPAM-1. For all the tested sera, titres and variability according to the method were homogenous as it is shown by Fig. 1. The maximum titre difference between methods did not exceed 0.5 log10. Only one method (method E) raised problems of quantification for PT and PRN antigens (expert report, private communication). This is probably due to the choice of the conjugate, the working dilutions and the incubation time. The other results obtained in Phase I (expert report, private communication) as serum calibration and positive control discrepancies when titred in homologous and heterologous situations will not be detailed since they were confirmed by the Phase II with more consistent data due to a greater number of participants. 3.2. Calibration of the cBRP In phase II, the cBRP antiserum was calibrated versus SPAM-1 antiserum. Overall analysis of the data generated by all participating laboratories showed the following (Fig. 2): • Anti-FHA antibody titration: Laboratory 10 gave statistically different results compared to the other laboratories. When the results of Laboratory 10 were excluded from the analysis, no significant difference in the results was observed (P⫽0.726). The maximal difference observed between any two laboratories’ mean titres did not exceed 0.4 log10. • Anti-PT antibody titration: All laboratories gave statistically different results, but none can be shown in evidence as really different (P⫽0.0004). The maximal difference observed between any two laboratories’ mean titres did not exceed 0.4 log10. • Anti-PRN antibody titration: There is no statistically significant difference between laboratories (P⫽0.362). The maximal difference observed
between any two laboratories’ mean titres did not exceed 0.3 log10. • Anti-Fim 2&3 antibody titration: No difference between laboratories reached the statistical significance (P⫽0.721). The maximal difference observed between any two laboratories’ mean titres did not exceed 0.2 log10. Concerning the estimation of the variation of cBRP titrations versus SPAM-1 reference antiserum for all laboratories, the overall gCV is around 36, 28, 40 and 31%, for FHA, PT, PRN and Fim 2&3 components, respectively. Segregation of variability by laboratory (Fig. 3) indicates that the gCVs for each component are roughly equivalent and relatively low. It should be noted, however, that laboratories 4 (for FHA, PRN and Fim 2&3) and 7 (for FHA and PT) had gCV higher than 50%. Assays from Laboratory 10 showed relatively high variability for all components, but below 50%. A combination of all the individual results enabled the assignment of antibody titres to the five components of acellular pertussis vaccines. After reconstitution of the lyophilisate with 500 µl of distilled water, the unitage of cBRP antiserum is assigned as follows: FHA: 138 ELU/ml PT: 39 ELU/ml PRN: 34 ELU/ml Fim 2&3: 56 ELU/ml 3.3. Behaviour of internal controls and internal references The relative antibody contents of internal references versus SPAM-1 reference obtained in this study showed differences with those previously established by some laboratories (Table 1). Antibody contents of internal controls were expressed relative to the corresponding in-house reference serum (referred to as homologous situation) or relative to the cBRP antiserum (referred to as heterologous situation). It was shown (Fig. 4) that significant differences exist between titres obtained in homologous or heterologous situations. This may be explained by the differences between the unitages of ‘in-house’ antisera estimated in this study versus SPAM-1 and the titres established by some manufacturers; these differences are roughly equivalent (Table 1). 3.4. Comparison of results from the WHO [13] and EDQM collaborative studies The International reference reagent and the candidate European Biological reference preparation were both included in the WHO and EDQM collaborative studies.
Table 1 Origin of differences between the homologous and heterologous situations Laboratory
Antigen FHA Assigned/ihr
1
2
4
5
6
7
Calculated/cBRP
2600 3.41 0.77 75 1.88 0.73
15466 4.19
Titres (ELU/ml) Titres (log10) Difference (assigned–calculated)b Titres (ELU/ml) Titres (log10) Difference (ihr–cBRP)c
713 2.85 0.05 2896 3.46 0.10
631 2.80
Titres (ELU/ml) Titres (log10) Difference (assigned–calculated)b Titres (ELU/ml) Titres (log10) Difference (ihr–cBRP)c
713 2.85 0.00 3080 3.49 0.09
Titres (ELU/ml) Titres (log10) Difference (assigned–calculated)b Titres (ELU/ml) Titres (log10) Difference (ihr–cBRP)c
540 2.73 0.03 531 2.72 0.01
Titres (ELU/ml) Titres (log10) Difference (assigned–calculated)b Titres (ELU/ml) Titres (log10) Difference (ihr–cBRP)c
–a –a –a –a –a –a
Titres (ELU/ml) Titres (log10) Difference (assigned–calculated)b Titres (ELU/ml) Titres (log10) Difference (ihr–cBRP)c
713 2.85 0.13 3414 3.53 0.10
Titres (ELU/ml) Titres (log10) Difference (assigned–calculated)b Titres (ELU/ml) Titres (log10) Difference (ihr–cBRP)c
*d *d *d *d *d *d
400 2.60
2307 3.36 717 2.86 2517 3.40 500 2.70 548 2.74
Assigned/ihr
Calculated/cBRP
1400 3.15 0.46 134 2.13 0.41
4028 3.61
403 2.61 0.05 1056 3.02 0.02
447 2.65
403 2.61 0.03 1202 3.08 0.02 170 2.23 0.07 *d *d *d
–a –a
1.832 0.26
345 2.54
1009 3.00 427 2.63 1136 3.06 144 2.16 *d *d *d *d
*d –a –a 526 2.72 2725 3.44 1349 3.13 1919 3.28
90 1.95 0.24
52 1.72
403 2.61 0.07 1335 3.13 0.01
349 2.54
*d *d *d *d *d *d
1306 3.12 124 2.09 366 2.56
Assigned/ihr
Fim 2&3 Calculated/cBRP
690 2.84 0.03 47 1.67 0.09
645 2.81
109 2.04 0.11 115 2.06 0.09
139 2.14
109 2.04 0.03 118 2.07 0.01
118 2.07
150 2.18 0.17 160 2.20 *d
102 2.01
–a –a –a –a –a –a 109 2.04 0.07 119 2.07 0.07 *d *d *d *d *d *d
38 1.58
142 2.15
114 2.06
*d *d –a –a –a –a 94 1.97 138 2.14 445 2.65 262 2.42
Assigned/ihr a
Calculated/cBRP
– –a –a –a –a –a
–a –a
–a –a –a –a –a –a
–a –a
–a –a –a –a –a –a
–a –a
200 2.30 0.05 227 2.36 0.01
179 2.25
–a –a
–a –a
–a –a
220 2.34
–a –a –a –a –a –a
–a –a
–a –a –a –a –a –a
–a –a
*d *d *d *d *d *d
268 2.43
–a –a
–a –a
187 2.27
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Titres (ELU/ml) Titres (log10) Difference (assigned–calculated)b Titres (ELU/ml) Titres (log10) Difference (ihr–cBRP)c
PRN
B. Poirier et al. / Biologicals 31 (2003) 25–38
3
PT
(continued)
Laboratory
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Table 1
Antigen FHA Assigned/ihr
8
PT Calculated/cBRP
286 2.46 0.02 188 2.27 0.09
Titres (ELU/ml) Titres (log10) Difference (assigned–calculated)b Titres (ELU/ml) Titres (log10) Difference (ihr–cBRP)c
1598 3.20 0.07 2876 3.46 0.06
1894 3.28
10 Titres (ELU/ml) Titres (log10) Difference (assigned–calculated)b Titres (ELU/ml) Titres (log10) Difference (ihr–cBRP)c
2600 3.41 0.36 44 1.64 0.59
5878 3.77
11 Titres (ELU/ml) Titres (log10) Difference (assigned–calculated)b Titres (ELU/ml) Titres (log10) Difference (ihr–cBRP)c
3000 3.48 0.08 498 2.70 0.04
3584 3.55
12 Titres (ELU/ml) Titres (log10) Difference (assigned–calculated)b Titres (ELU/ml) Titres (log10) Difference (ihr–cBRP)c
111220 5.05 1.34 119308 5.08 1.61
5044 3.70
13 Titres (ELU/ml) Titres (log10) Difference (assigned–calculated)b Titres (ELU/ml) Titres (log10) Difference (ihr–cBRP)c
675 2.83 0.03 1206 3.08 0.05
9
a b c d
275 2.44 154 2.19
3320 3.52
172 2.24
548 2.74
2900 3.46 718 2.86 1081 3.03
34 1.53 0.12 20 1.30 0.10 90 1.95 0.05 *d *d *d
Calculated/cBRP 44 1.65 25 1.40 101 2.00 117 2.07
1400 3.15 0.81 93 1.97 0.73
9045 3.96
2700 3.43 0.57 1246 3.10 0.56
723 2.86
20724 4.32 1.73 23251 4.37 1.48 369 2.57 0.04 414 2.62 0.10
494 2.69
343 2.53 384 2.58 775 2.89 409 2.61 329 2.52
Assigned/ihr
Fim 2&3 Calculated/cBRP
60 1.78 0.06 5 0.69 0.07
52 1.72
52 1.72 0.07 397 2.60 *d
44 1.64
6 0.75
*d *d
690 2.84 0.23 20 1.30 0.34
409 2.61
–a –a –a –a –a –a
–a –a
4901 3.69 1.23 6360 3.80 1.24
286 2.46
144 2.16 0.06 91 1.96 0.04
9 0.96
–a –a
368 2.57 126 2.10 83 1.92
Assigned/ihr 64 1.81 0.05 210 2.32 0.04 74 1.87 0.30 88 1.95 0.31
Calculated/cBRP 72 1.86 232 2.37 147 2.17 180 2.26
–a –a –a –a –a –a
–a –a
–a –a –a –a –a –a
–a –a
–a –a –a –a –a –a
–a –a
–a –a –a –a –a –a
–a –a
–a –a
–a –a
–a –a
–a –a
Not performed. Difference between internal reference serum value assigned by the manufacturer and the titre calculated in the collaborative study versus SPAM-1 reference serum expressed in log10. Difference between titres of the internal control in the homologous and heterologous situations expressed in log10. Data not given or not quantifiable.
B. Poirier et al. / Biologicals 31 (2003) 25–38
Titres (ELU/ml) Titres (log10) Difference (assigned–calculated)b Titres (ELU/ml) Titres (log10) Difference (ihr–cBRP)c
Assigned/ihr
PRN
B. Poirier et al. / Biologicals 31 (2003) 25–38
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Table 2 Comparison of results between the WHO and EDQM collaborative studies for IRR or cBRP (titration versus SPAM-1) EDQM collaborative study
WHO collaborative study
Antibody
log10
ELU/ml
log10
ELU/ml
IRR
Anti-FHA Anti-PT Anti-PRN Anti-Fim 2&3
2.35 1.47 1.66 1.66
223 29 46 45
2.46 1.53 1.78 1.81
286 34 60 64
cBRP
Anti-FHA Anti-PT Anti-PRN Anti-Fim 2&3
2.14 1.59 1.53 1.75
138 39 34 56
2.25 1.68 1.56 1.83
178 48 36 68
IRR antiserum titration versus SPAM-1 antiserum (Table 2): Results obtained in both studies are similar. cBRP antiserum titration versus SPAM-1 antiserum (Table 2): The cBRP titres obtained in this phase II study are close to those obtained in the WHO collaborative study. 3.5. Comparison between laboratories’ and AFSSAPS calculations of unitage relative to SPAM-1 The results obtained by each participant did not differ substantially from those obtained by AFSSAPS with the exception of Laboratory 4, which obtained lower results with its own calculation method (Table 3).
4. Discussion
Fig. 4. Behaviour of internal controls in homologous and heterologous situations (meanSEM): anti-FHA titres (A), anti-PT titres (B), anti-PRN titres (C) anti-Fim 2&3 titres (D). +, Titre of internal control versus ‘internal reference’ serum (homologous situation); +, Titre of internal control versus cBRP serum (heterologous situation).
Phase I of this study has shown that the cBRP for B. pertussis mouse antiserum contains significant levels of specific antibodies to the B. pertussis antigens PT, FHA, PRN and Fim 2&3 present in acellular pertussis vaccines. This study allowed to conclude that all methodologies tested seemed to be equivalent regarding their ability to calibrate the cBRP, IRR and in-house references relative to SPAM-1. In general, the variability (repeatability and reproducibility) observed was shown to be satisfactory and in the same range as the variability observed for ELISAs used in routine potency control of acellular pertussis vaccines (data not shown). No significant bias or particular advantage related to methodology could be identified during phase I. Therefore, it was decided to run the enlarged phase (international collaborative study), referred to as Phase II, allowing participants to use their own in-house methods. As regards the relative homogeneity of results obtained by different methodologies, the conclusion of Phase I was confirmed in Phase II, thus, justifying the use of
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Table 3 Individual assay results (calibration against SPAM-1 reference) Assay
Serum potency expressed in ELU/ml cBRP calculated
cBRP Lab. IRR calculated
IRR Lab. In-house calculated
In-house stated
1
FHA FHA FHA PT PT PT PRN PRN PRN
1 2 3 1 2 3 1 2 3
138 154 150 33 39 38 44 * 43
144 152 157 34 39 38 36 34 38
226 238 245 23 22 22 57 * 53
219 231 235 26 25 24 56 52 53
14 167 15 459 16 892 4420 3975 3721 618 * 673
13 885 15 776 15 445 4234 3878 3447 628 764 715
2
FHA FHA FHA FHA PT PT PT PT PRN PRN PRN PRN
1 2 3 4 1 2 3 4 1 2 3 4
140 135 170 161 43 39 49 44 33 29 37 41
138 138 171 160 42 38 48 43 33 29 37 41
200 225 278 269 35 37 45 42 52 43 60 60
199 224 276 269 34 36 44 41 52 43 60 59
481 841 569 687 356 583 434 444 128 115 177 143
3
FHA FHA FHA PT PT PT PRN PRN PRN
1 2 3 1 2 3 1 2 3
150 181 176 44 42 40 35 38 41
150 181 176 44 42 40 35 38 41
295 290 296 38 40 47 55 55 53
295 290 296 38 40 47 55 55 53
4
FHA FHA FHA PT PT PT PRN PRN PRN FIM 2/3 FIM 2/3 FIM 2/3
1 2 3 1 2 3 1 2 3 1 2 3
206 68 95 * 46 55 80 21 16 81 36 46
192 65 78 31 30 25 31 11 8 46 26 19
303 145 175 * 45 57 78 45 37 57 31 34
306 147 174 22 43 41 54 28 34 42 34 16
Internal control calculated
Internal control Lab.
49
518 385 420 326 347 270 30 33 41
477 836 568 681 344 565 425 433 127 113 174 142
2307 2290 2985 2796 1047 1131 1323 1206 125 136 175 157
2294 2272 2970 2781 1010 1094 1297 1173 125 133 175 156
768 662 725 380 437 469 100 126 131
768 662 725 379 436 468 100 126 131
3196 2931 2994 1182 1196 1426 114 127 138
3196 2930 2994 1180 1194 1424 114 127 138
1022 302 406
1058 302 362 130 133 113 203 73 59 380 145 126
1060 279 385 * 208 169 209 85 68 339 145 168
1200 284 353 202 165 146 199 80 65 346 145 147
* 153 136 196 86 63 359 129 124
516 387 434 354 388 312 47 *
B. Poirier et al. / Biologicals 31 (2003) 25–38
Laboratory Antigen
Table 3
(continued)
Laboratory Antigen
Assay
Serum potency expressed in ELU/ml cBRP calculated
cBRP Lab. IRR calculated
IRR Lab. In-house calculated
PT PT PT
1 2 3
43 45 34
41 45 37
34 46 26
36 47 29
6
FHA FHA FHA PT PT PT PRN PRN PRN
1 2 3 1 2 3 1 2 3
124 146 119 34 35 34 23 24 28
124 146 119 34 35 34 23 24 28
200 234 195 23 26 24 36 38 37
200 234 195 23 26 24 36 38 37
7
FHA FHA FHA PT PT PT PRN PRN PRN FIM 2/3 FIM 2/3 FIM 2/3
1 2 3 1 2 3 1 2 3 1 2 3
88 171 245 32 39 73 29 * 44 50 55 101
* * * * * * * * * * * *
51 167 268 13 20 41 16 * 58 25 42 59
* * * * * * * * * * * *
8
FHA FHA FHA PT PT PT PRN PRN PRN FIM 2/3 FIM 2/3 FIM 2/3
1 2 3 1 2 3 1 2 3 1 2 3
156 126 147 41 42 36 26 28 25 67 50 50
149 117 143 39 40 37 26 27 25 68 49 54
237 280 281 29 34 40 29 53 47 59 60 62
237 277 285 27 33 39 27 51 48 50 59 64
1.28 1.24 1.24 433 634 531 331 389 331 88 90 104 235 1953 5350 82 93 249 197 * 1006 150 179 719 308 226 299 57 39 39 52 52 52 83 66 68
1.19 1.22 1.28
Internal control calculated
Internal control Lab.
64 58 60
63 59 63
433 634 531 331 389 331 88 90 104
2285 2902 2517 1166 1178 1160 92 94 115
2285 2902 2517 1166 1178 1160 92 94 115
* * * * * * * * * * * *
840 2160 5844 357 252 934 199 * 209 80 572 231
* * * * * * * * * * * *
300 222 287 56 39 38 54 50 53 86 61 67
208 138 190 32 22 25 4 4 5 281 197 217
202 117 177 33 22 24 5 9 4 289 178 252
B. Poirier et al. / Biologicals 31 (2003) 25–38
5
In-house stated
35
(continued)
Laboratory Antigen
9
36
Table 3
Assay
Serum potency expressed in ELU/ml cBRP Lab. IRR calculated
IRR Lab. In-house calculated
In-house stated
Internal control calculated
Internal control Lab.
129 144 142 * 34 * * * 28 50 49 56
* * * * * * * * * * * *
232 * 208 * 35 * * * 38 44 47 43
* * * * * * * * * * * *
* * * * * * * * * * * *
3115 * 3456 * 120 * * * 388 183 178 157
* * * * * * * * * * * *
131 * 91 9 * 9 45 33 32
136 88 104 9 10 9 49 31 32
* 12215 299 294 777
4470 5378 5382 7082 6077 10684 305 307 743
4022 3356 3412 793 698 683
FHA FHA FHA PT PT PT PRN PRN PRN FIM 2/3 FIM 2/3 FIM 2/3
1 2 3 1 2 3 1 2 3 1 2 3
FHA FHA FHA PT PT PT PRN PRN PRN
1 2 3 1 2 3 1 2 3
* 23 68 36 53
57 84 73 33 30 25 59 28 43
11
FHA FHA FHA PT PT PT
1 2 3 1 2 3
139 147 148 37 36 39
137 143 151 36 36 40
255 278 233 22 20 20
245 260 238 22 20 20
12
FHA FHA FHA PT PT PT PRN PRN PRN
1 2 3 1 2 3 1 2 3
* 170 * 24 24 25 * 30 *
156 173 163 26 24 25 30 30 36
* 283 * 64 * * * 45 47
309 272 264 63 51 62 54 45 52
10
52 * 78 34
1961 * 1830 * 101 * * * 44 146 152 142 5281 * 6542 6698
* 5044 * 467 324 375 * 321 255
74
456 14 12 14
96 128 97 577 421 428 10 11 12
3947 3280 3450 782 706 689
649 530 544 330 322 346
645 532 552 366 332 360
5979 5203 4904 709 555 559 312 280 247
* 5016 * 523 377 407 * 332 295
6844 5083 3930 746 543 555 269 329 291
* 95 591 *
B. Poirier et al. / Biologicals 31 (2003) 25–38
cBRP calculated
Table 3
(continued)
Laboratory Antigen
13
FHA FHA FHA PT PT PT PRN PRN PRN
Assay
1 2 3 1 2 3 1 2 3
Serum potency expressed in ELU/ml cBRP calculated
cBRP Lab. IRR calculated
IRR Lab. In-house calculated
In-house stated
Internal control calculated
Internal control Lab.
161 163 163 54 51 54 32 32 36
161 163 163 57 52 57 32 32 36
309 322 295 53 49 48 55 54 60
733 740 687 392 414 437 121 124 132
1299 1275 1256 460 441 477 74 84 83
1299 1275 1256 481 438 517 74 84 83
309 322 295 51 49 46 55 54 60
729 740 687 382 419 429 121 124 132
Calculated, AFSSAPS calculations; Lab, Laboratory calculations; *, Data not given or not quantifiable. B. Poirier et al. / Biologicals 31 (2003) 25–38 37
38
B. Poirier et al. / Biologicals 31 (2003) 25–38
in-house methods rather than that of a single standardised methodology for each pertussis component tested. In phase II, results (i.e. unitages of the sera expressed in reference to SPAM-1) calculated by AFSSAPS using a standard calculation methodology were similar to those reported by the participants. Results were overall comparable and exhibited a homogeneous variability among the laboratories. In order to assess that the proposed European standard (cBRP) could be used instead of the internal references, the positive control titres calculated relative to their corresponding internal reference (homologous situation) or the proposed European standard (heterologous situation) were compared. The most remarkable discrepancy observed between both situations was up to 1.7 log10 (Fig. 4) and seemed to be linked to the preliminary calibration of internal references. Indeed, it appeared in each case that unitages obtained when testing internal references versus SPAM-1 were different from those established in the procedures (Table 1). These results emphasise the need for a common reference [14]. The analysis of the outcome of this study has shown that the cBRP is suitable to be used as a reference antiserum. Therefore, the cBRP has been adopted by the Ph. Eur. commission during the 107th Session in June 2000 as B. pertussis mouse antiserum Ph. Eur. BRP batch 1 (EDQM product reference B1142000) with the following unitages (after reconstitution of the lyophilisate with 500 µl of distilled water): • • • •
Anti-PT: 39 ELU/ml Anti-FHA: 138 ELU/ml Anti-PRN: 34 ELU/ml Anti-Fim 2&3: 56 ELU/ml
Acknowledgements The organisers express their sincere thanks to all the participants for their contributions to this study. The study was run by the EDQM under the aegis of the Biological Standardisation Programme supported by the Council of Europe and the European Commission (project number BSP031). Appendix A. Participants R. Dobbelaer, G. Vanhooren, Institut Scientifique de la Sante´ Publique – Louis Pasteur, Brussels (Belgium)* M. Ducheˆne, E. Remy, Glaxo SmithKline Beecham Biologicals S.A., Rixensart (Belgium) G. Calver, N. Shakerchi, W. Wilson, Therapeutic Products Programme, Health Canada, Ottawa (Canada) B. Willumsen, Statens Serum Institut, Copenhagen (Denmark)
F. Fuchs, N. Bornstein, Agence Franc¸aise de Se´curite´ Sanitaire des Produits de Sante´, Lyon (France)* R. Bandet, A. Sabouraud, Aventis Pasteur, Marcy l’Etoile (France) G. Rautmann, B. Wild, C. Raphalen, EDQM, Council of Europe, Strasbourg, (France)* A. Zott, Paul Ehrlich Institut, Langen (Germany) P. Mastrantonio, Instituto Superiore di Sanita`, Roma (Italy) M. Duccio, N. Hug, Chiron – S.p.A., Siena (Italy) P. Newland, D. Xing, National Institute for Biological Standards and Control, Potters Bar (UK) J. Arciniega, B. Meade, Food and Drug Administration, Bethesda (USA) R. Gupta, Wyeth–Lederle Vaccines and Pediatrics, Pearl River (USA) *Laboratories involved in both Phase I and Phase II collaborative studies References [1] Corbel MJ, Xing DKL. The current status of acellular pertussis vaccines. J Med Microbiol 1997;46:817–8. [2] Corbel MJ, Xing DKL, Bolgiano B, Hockley D. Approaches to the control of acellular pertussis vaccines. Biologicals 1999; 27:133–41. [3] Corbel MJ, Xing DKL, Kreftenberg JG. Informal consultation with manufacturers and WHO Ad Hoc Working Group on mouse protection models for acellular pertussis vaccines. Biologicals 1999;27:189–93. [4] Xing D, Das R. International collaborative study: mouse protective models for acellular pertussis vaccines, September 2000 (unpublished report). [5] Mastrantonio P, Cerquetti M, Cardines R, Lande R, Ausiello CM, Cassone A. Immunogenicity issues in the quality control of the new acellular pertussis vaccines. Biologicals 1999;27:119–21. [6] European Pharmacopoeia. 4th ed. Pertussis vaccine (acellular, component, adsorbed), monograph 1356; Pertussis vaccine (acellular, co-purified, adsorbed), monograph 1595, 2002. [7] WHO guidelines for the production and control of the acellular pertussis component of monovalent or combined vaccines. WHO Tech Rep Ser 1998;878:57–76. [8] Andre´ M, Poirier B, Bornstein N, Marmonier D, El Zaouk A, Fuchs F. Key points for the development of mouse immunogenicity test as potency assay for acellular pertussis vaccines. Biologicals 2000;28(4):217–25. [9] BS/99.1901, WHO, 20 avenue Appia CH-1211 Geneva 27 Switzerland. [10] Wood DJ, Padilla A, Griffith E. WHO expert committee on Biological Standardization: highlights of the 50th meeting, October 1999. Biologicals 2000;28:199–206. [11] . Version 2.0. User’s manual, 103p. [12] . Manugistics. Version 3. User manual. [13] Gaines Das R, Xing D, Rigsby P, Newland P, Corbel M. International collaborative study: evaluation of proposed international reference reagent of pertussis antiserum (mouse). Biologicals 2001;29(2):137–48. [14] Fuchs F, Dobbelaer R. Collaborative study for the establishment of a European Pharmacopoeia biological reference preparation for Bordetella pertussis mouse antiserum for serological potency testing of acellular pertussis vaccines. Pharmeuropa Bio 2000; 2:25–42.
Collaborative study for the establishment of a European Phamacopoeia biological reference preparation for Bordetella pertussis mouse antiserum for serological potency testing of acellular pertussis vaccines Bertrand Poirier a*, Nicole Bornstein a, Murielle Andre a, Denis Marmonier a, Monique Pares b, Gerard Vanhooren c, Guy Rautmann d, Marie-Emmanuelle Behr-Gross d, Roland Dobbelaer c, Florence Fuchs a a
b
Agence Franc¸aise de Se´curite´ Sanitaire des Produits de Sante´, Site de Lyon, 321 avenue Jean Jaure`s, 69007 Lyon, France Agence Franc¸aise de Se´curite´ Sanitaire des Produits de Sante´, Site de Montpellier, 635 rue de la Garenne, 34740 Vendargues, France c Institut Scientifique de la Sante´ Publique – Louis Pasteur 14, rue Juliette Wytsman B-1050 Bruxelles, Belgique d Council of Europe, European Directorate for the Quality of Medicines, Division IV B.P. 907 F-67029 Strasbourg Cedex 1, France Received 30 May 2001; revised 13 August 2001; accepted 20 August 2002
Abstract A collaborative study was organised by the European Directorate For the Quality of Medicines (EDQM) to assess the suitability of a candidate mouse antiserum as a European Pharmacopoeia Biological reference preparation (BRP) for acellular pertussis vaccine potency testing. The candidate antiserum was obtained by immunising mice with a five-component acellular pertussis vaccine: pertussis toxin (PT), filamentous haemagglutinin (FHA), pertactin (PRN) and Fimbrial 2/Fimbrial 3 (Fim 2&3). The study has been divided into two separate phases. Phase I was a pre-qualification study including three laboratories. This phase was aimed at pre-qualifying the candidate BRP (cBRP) and at documenting the impact of differences in the antibody detection methodology enzyme linked immunosorbent assay (ELISA) procedures on results of pertussis antisera calibration versus the currently used standard US standard pertussis antiserum (mouse) Lot 1 (SPAM-1) (United States Food and Drug Administration (USFDA) reference serum) and the cBRP. As no significant difference between the antibody titres determined by using the different ELISA methodologies was found, a large-scale study enrolling 13 laboratories (Phase II) was carried out, each participant performing its in-house methodology. Its aim was to calibrate the cBRP (in terms of the SPAM-1 reference) and to demonstrate its equivalence or superiority to internal references. The study showed that there was no difference in positive sera titres expressed relative to their corresponding internal reference (homologous situation) or the proposed standard (heterologous situation) reference. The cBRP can, therefore, reliably act as replacement for the in-house reference preparations. Further analysis of the outcome of this study enabled to assign to the cBRP a potency of 39, 138, 34 and 56 ELISA unit per millilitre, respectively, to its anti-PT, anti-FHA, anti-PRN and anti-Fim 2&3 antibody contents. The cBRP has been adopted by the European Pharmacopoeia Commission at its June 2000 session as Bordetella pertussis mouse anti-serum Ph Eur. BRP batch 1. 2003 The International Association for Biologicals. Published by Elsevier Science Ltd. All rights reserved. Keywords: Collaborative study; Statistics; Acellular pertussis vaccine; Enzyme linked immunosorbent assay; European Pharmacopoeia biological reference preparation; Potency assay
1. Introduction * Corresponding author. Tel.: +33-4-72-76-0610; fax: +33-4-72-76-0615 E-mail address: [email protected] (B. Poirier).
Following the development of a variety of acellular pertussis vaccines, different laboratory tests were introduced for monitoring safety, potency, physico-
1045-1056/03/$30.00 2003 The International Association for Biologicals. Published by Elsevier Science Ltd. All rights reserved. doi:1 0 . 1 0 1 6 / S 1 0 4 5 - 1 0 5 6 ( 0 2 ) 0 0 0 7 5 - 1
26
B. Poirier et al. / Biologicals 31 (2003) 25–38
chemical and immunological characteristics of new vaccine lots, comparatively to an homologous reference vaccine clinically evaluated [1,2]. No entirely satisfactory method is available for potency monitoring. Several in vivo protection model assays are under study, including intracerebral, intranasal and aerosol challenge models [3,4], but mouse immunogenicity assays remain useful for checking consistency of production from batch to batch even if they do not necessarily correlate with protection in humans [2,5]. The European Pharmacopoeia monographs [6] and WHO guidelines [7] describe this test for potency assay on final lots of acellular pertussis vaccines currently being marketed. The mouse immunogenicity assay is based on the measurement of the specific antibody responses to the different antigenic components present in a test vaccine. Serum antibody levels are measured by enzyme linked immunosorbent assay (ELISA) and the immunogenicity is compared with that of a reference vaccine. Antibody responses to any given dose of an acellular pertussis component have been found to vary considerably between individual mice, and the ELISA part of the potency test is itself subject to some variability. Therefore, in order to allow a valid quantitation of antibody levels, a calibrated reference antiserum has to be included in every ELISA assay (this procedure allows the selection of serum dilutions within the quantitation limits of an ELISA assay and to express results in ELISA units (ELU) that are consistent between assays [8]). Some manufacturers of acellular pertussis vaccines as well as some control authorities, e.g. United States Food and Drug Administration (USFDA) and Japanese National Institute (JNIH) have established such reference antisera. Recently, an International reference reagent for acellular pertussis antiserum was calibrated and established against the existing reference antisera [9,10]. In Europe, various in-house reference antisera, usually calibrated against the USFDA reference antiserum (SPAM-1) are used by manufacturers and official medicines control laboratories (OMCLs). Replacement of different in-house references by an European Pharmacopoeia biological reference preparation (BRP) for pertussis antiserum in routine release assays by both manufacturers and OMCLs should improve inter-laboratory comparability of results within Europe. In order to produce a candidate BRP (cBRP), a large batch of Bordetella pertussis antiserum obtained by immunising CD1 mice with a five component acellular pertussis vaccine similar in composition to that used for generating the US standard pertussis antiserum (mouse) Lot 1; SPAM-1 (SPAM-1) has been aliquoted and freeze-dried at the European Directorate for the Quality of Medicines (EDQM). A preliminary characterisation study by Western blot and ELISA analysis showed that
the candidate serum contained quantifiable amounts of specific antibodies directed against the five pertussis antigens present in the vaccine. The preparation, still in the liquid state, was included in the WHO study previously mentioned. A pre-qualification phase, referred to as Phase I (expert report, private communication), has been run between June and October 1999. This study was performed in order to pre-qualify the cBRP and to document the impact of differences in ELISA methodologies on the estimation of the antibody content of pertussis antisera. It consisted of a complete crossover study between five different methodologies and antisera (internal references and control sera) generated by different acellular vaccines in the market. Following successful completion of Phase I, an international collaborative study, referred to as Phase II, was run between November 1999 and April 2000. Phase II was aimed at calibrating the B. pertussis mouse antiserum Ph. Eur. cBRP in terms of anti-pertussis toxin (PT), anti-filamentous haemagglutinin (FHA), anti-pertactin (PRN) and anti-Fimbriae type 2&3 (Fim 2&3) antibody titres expressed in ELISA unit per millilitre relative to the US FDA reference serum (SPAM-1) on a sufficiently large database.
2. Materials 2.1. Reference sera SPAM-1 (Center for Biologics Evaluation and Research (CBER), FDA, Bethesda, MA, USA) and International reference reagent anti-pertussis serum (IRR (1999) coded 97/642; NIBSC, London, UK) are lyophilised preparations, which have an assigned unitage after reconstitution with 0.5 ml distilled water of 170 ELU/ml anti-PT, 540 ELU/ml anti-FHA, 150 ELU/ml anti-PRN and 200 ELU/ml anti-Fim 2&3 for SPAM-1 and 34 ELU/ml anti-PT, 286 ELU/ml antiFHA, 60 ELU/ml anti-PRN, 64 ELU/ml anti-Fim 2&3 for IRR. Production of the cBRP has been previously described (expert report, private communication). The immunisation and blood collection were performed by the French Health Products Safety Agency (AFSSAPS) following a similar immunisation schedule as for SPAM-1 production by the CBER. Briefly, a five component acellular pertussis vaccine (Pasteur Merieux Connaught, Canada) was used to inoculate 1200 mice of the CD-1 strain. The vaccine, provided as combined diphtheria–tetanus–pertussis formulations adsorbed to alum, was reported to contain 10 µg/0.5 ml of glutaraldehyde-inactivated PT, 5 µg/0.5 ml of FHA, 3 µg/ml of PRN, 5 µg/0.5 ml of Fim 2&3, 15 Lf tetanus toxoid and 15 Lf diphtheria toxoid. Animals were bled 12 days after a second booster immunisation. Mini pools of serum were prepared and subsequently characterised
B. Poirier et al. / Biologicals 31 (2003) 25–38
27
Fig. 1. Influence of the method in relation to the component tested. FHA antigen (A), PT antigen (B), PRN antigen (C). Mean of titres between the three participating laboratories.
28
B. Poirier et al. / Biologicals 31 (2003) 25–38
by Western blot analysis for the presence of antibodies against each of the five pertussis antigens. All sera were then pooled in a large batch that was aliquoted in 0.5 ml fractions prior to freeze-drying. Finally, a lyophilised serum batch of 4000 ampoules was produced at the EDQM. 2.2. Participants Thirteen laboratories routinely using ELISA assays for measuring mouse anti-FHA, anti-PT, anti-PRN and/or anti-Fim 2&3 antibodies were invited to participate in the study. These included vaccine manufacturers and National control laboratories. A list of participants is given at the end of the report (Appendix A). They are referred to by using an arbitrary assigned number, not related to the order of listing at the end of this report. All the 13 laboratories contributed to Phase II, but only three of them were involved in Phase I. 2.3. Study design and methods Phase I: This study consisted of a complete crossover study between different methodologies and antisera (in-house reference sera and control sera) generated with different vaccines. The different coating antigens, reagents and ELISA procedures were provided by four manufacturers and an official control laboratory. The three laboratories involved received all in-house references and control sera as well as all reagents and standard operating procedures necessary to perform assays by means of five different ELISA methodology groups referred to as A–E (Method A: PT, FHA; Method B: PT, FHA, PRN; Method C: PT, FHA, PRN; Method D: PT; Method E: PT, FHA, PRN). All methodologies used an indirect ELISA: passive adsorption of the antigens onto microtiter plates followed by the addition of the serum samples and then detection of antigen-specific antibodies by enzyme-conjugated antimouse antibodies and substrate. For each methodology, each participant was requested to test all in-house reference and control sera, as well as reference sera previously cited (SPAM-1, IRR, cBRP), and to assay eight consecutive dilutions of each serum in three independent ELISA assays performed in different weeks. Participants were asked to return all the absorbance raw data from microtitration plates to permit an independent statistical analysis. For feasibility reasons (availability of reagents), some of them did not strictly follow the provided procedures. The Fim 2&3 were not analysed due to lack of corresponding coating antigens and reagents. Phase II: Each participating laboratory was requested to test the three reference sera (SPAM-1, IRR, cBRP) as per the ELISA method established in its laboratory, to include its control and internal reference sera, and to
Fig. 2. Frequency distribution of cBRP antiserum titres for: anti-FHA (A), anti-PT (B), anti-PRN (C), anti-Fim 2&3 (D). Numbers in boxes are laboratory code numbers.
B. Poirier et al. / Biologicals 31 (2003) 25–38
29
calculate results according to its standard procedures. Participants were requested to perform three independent assays. Each laboratory was requested to use the same plate layout and to assay eight consecutive dilutions of each serum. Only values derived from valid assays, as determined by the individual laboratory, were to be reported. Laboratories were asked to provide details about the methodology and reagents used in their assays so that the effect of these factors could be assessed. Participants were also asked to return calculated results using their in-house mathematical models, as well as the raw data for each assay to permit recalculation using a common method. The number of laboratories contributing data for each antigen reflects the assays run in each of the laboratories. All 13 laboratories submitted data for PT, but not all laboratories submitted data for the other antigens: only 12 carried out anti-FHA assays, 11 carried out anti-PRN assays and 4 carried out anti-Fim 2&3 assays. To calculate sample titres against the reference serum, four laboratories (numbers 2, 3, 6, 13) used a fourParameter logistic fit. Other calculation methods used included a parallel line model with log dose transformation (laboratories 9, 11, 12), parallel line model with log dose/log OD transformation (laboratories 1, 5, 8), and ‘reference line calculation’ (laboratories 4 and 10). Laboratory 7 did not report its calculation methodology. 2.4. Statistical analysis The raw data returned by each laboratory were used in the statistical analysis performed by the AFSSAPS. Unit calculations were performed with the , version 2.0 software [11]. ELISA unitage for each sample was assigned relative to the reference sera placed on each microtiter plate (SPAM-1, IRR, cBRP). A four-parameter dose response curve was generated for the reference serum by plotting the absorbance as a function of the logarithmically transformed antibody concentration data in ELISA unit per millilitre. The equation fit was (aⳮd)/[1⫹(x/c)b]⫹d, where a and d represent the OD values of the asymptotes, c the logarithmic value of the concentration for which the response value will be centred between the two asymptotes (the inflection point) and b the slope of the linear part of the curve (central part of the curve). The quantitative range was identified as the absorbance range between 10 and 80% of the asymptotic absorbances. A serum was considered non-quantifiable when ODs were below or above the quantification limits. In order to obtain a normal distribution of the antibody unitage data, a logarithmic transformation was applied. The statistical study required analysis of variance (ANOVA) and usual statistical tests. The geometric coefficient of variation (gCV), defined as 100(10s1),
Fig. 3. Segregation of titres and variability by laboratory: anti-FHA titres (A), anti-PT titres (B), anti-PRN titres (C), anti-Fim 2&3 titres (D).
30
B. Poirier et al. / Biologicals 31 (2003) 25–38
where s represents the standard deviation on log10 scale, was calculated. Comparisons between laboratories were evaluated by ANOVA and Newman–Keuls test [12]. The ANOVA decomposes the variability into contributions from various factors. The Newman–Keuls multiple comparison procedure discriminates among the means. With the latter method, there is a 5.0% risk of calling one or more pairs significantly different when their actual difference equals 0.
3. Results 3.1. Impact of the ELISA method factor In phase I, the impact of the method factor was analysed by comparing titration results obtained by the different methodologies for all tested sera (internal controls and standards, cBRP, IRR) versus reference serum SPAM-1. For all the tested sera, titres and variability according to the method were homogenous as it is shown by Fig. 1. The maximum titre difference between methods did not exceed 0.5 log10. Only one method (method E) raised problems of quantification for PT and PRN antigens (expert report, private communication). This is probably due to the choice of the conjugate, the working dilutions and the incubation time. The other results obtained in Phase I (expert report, private communication) as serum calibration and positive control discrepancies when titred in homologous and heterologous situations will not be detailed since they were confirmed by the Phase II with more consistent data due to a greater number of participants. 3.2. Calibration of the cBRP In phase II, the cBRP antiserum was calibrated versus SPAM-1 antiserum. Overall analysis of the data generated by all participating laboratories showed the following (Fig. 2): • Anti-FHA antibody titration: Laboratory 10 gave statistically different results compared to the other laboratories. When the results of Laboratory 10 were excluded from the analysis, no significant difference in the results was observed (P⫽0.726). The maximal difference observed between any two laboratories’ mean titres did not exceed 0.4 log10. • Anti-PT antibody titration: All laboratories gave statistically different results, but none can be shown in evidence as really different (P⫽0.0004). The maximal difference observed between any two laboratories’ mean titres did not exceed 0.4 log10. • Anti-PRN antibody titration: There is no statistically significant difference between laboratories (P⫽0.362). The maximal difference observed
between any two laboratories’ mean titres did not exceed 0.3 log10. • Anti-Fim 2&3 antibody titration: No difference between laboratories reached the statistical significance (P⫽0.721). The maximal difference observed between any two laboratories’ mean titres did not exceed 0.2 log10. Concerning the estimation of the variation of cBRP titrations versus SPAM-1 reference antiserum for all laboratories, the overall gCV is around 36, 28, 40 and 31%, for FHA, PT, PRN and Fim 2&3 components, respectively. Segregation of variability by laboratory (Fig. 3) indicates that the gCVs for each component are roughly equivalent and relatively low. It should be noted, however, that laboratories 4 (for FHA, PRN and Fim 2&3) and 7 (for FHA and PT) had gCV higher than 50%. Assays from Laboratory 10 showed relatively high variability for all components, but below 50%. A combination of all the individual results enabled the assignment of antibody titres to the five components of acellular pertussis vaccines. After reconstitution of the lyophilisate with 500 µl of distilled water, the unitage of cBRP antiserum is assigned as follows: FHA: 138 ELU/ml PT: 39 ELU/ml PRN: 34 ELU/ml Fim 2&3: 56 ELU/ml 3.3. Behaviour of internal controls and internal references The relative antibody contents of internal references versus SPAM-1 reference obtained in this study showed differences with those previously established by some laboratories (Table 1). Antibody contents of internal controls were expressed relative to the corresponding in-house reference serum (referred to as homologous situation) or relative to the cBRP antiserum (referred to as heterologous situation). It was shown (Fig. 4) that significant differences exist between titres obtained in homologous or heterologous situations. This may be explained by the differences between the unitages of ‘in-house’ antisera estimated in this study versus SPAM-1 and the titres established by some manufacturers; these differences are roughly equivalent (Table 1). 3.4. Comparison of results from the WHO [13] and EDQM collaborative studies The International reference reagent and the candidate European Biological reference preparation were both included in the WHO and EDQM collaborative studies.
Table 1 Origin of differences between the homologous and heterologous situations Laboratory
Antigen FHA Assigned/ihr
1
2
4
5
6
7
Calculated/cBRP
2600 3.41 0.77 75 1.88 0.73
15466 4.19
Titres (ELU/ml) Titres (log10) Difference (assigned–calculated)b Titres (ELU/ml) Titres (log10) Difference (ihr–cBRP)c
713 2.85 0.05 2896 3.46 0.10
631 2.80
Titres (ELU/ml) Titres (log10) Difference (assigned–calculated)b Titres (ELU/ml) Titres (log10) Difference (ihr–cBRP)c
713 2.85 0.00 3080 3.49 0.09
Titres (ELU/ml) Titres (log10) Difference (assigned–calculated)b Titres (ELU/ml) Titres (log10) Difference (ihr–cBRP)c
540 2.73 0.03 531 2.72 0.01
Titres (ELU/ml) Titres (log10) Difference (assigned–calculated)b Titres (ELU/ml) Titres (log10) Difference (ihr–cBRP)c
–a –a –a –a –a –a
Titres (ELU/ml) Titres (log10) Difference (assigned–calculated)b Titres (ELU/ml) Titres (log10) Difference (ihr–cBRP)c
713 2.85 0.13 3414 3.53 0.10
Titres (ELU/ml) Titres (log10) Difference (assigned–calculated)b Titres (ELU/ml) Titres (log10) Difference (ihr–cBRP)c
*d *d *d *d *d *d
400 2.60
2307 3.36 717 2.86 2517 3.40 500 2.70 548 2.74
Assigned/ihr
Calculated/cBRP
1400 3.15 0.46 134 2.13 0.41
4028 3.61
403 2.61 0.05 1056 3.02 0.02
447 2.65
403 2.61 0.03 1202 3.08 0.02 170 2.23 0.07 *d *d *d
–a –a
1.832 0.26
345 2.54
1009 3.00 427 2.63 1136 3.06 144 2.16 *d *d *d *d
*d –a –a 526 2.72 2725 3.44 1349 3.13 1919 3.28
90 1.95 0.24
52 1.72
403 2.61 0.07 1335 3.13 0.01
349 2.54
*d *d *d *d *d *d
1306 3.12 124 2.09 366 2.56
Assigned/ihr
Fim 2&3 Calculated/cBRP
690 2.84 0.03 47 1.67 0.09
645 2.81
109 2.04 0.11 115 2.06 0.09
139 2.14
109 2.04 0.03 118 2.07 0.01
118 2.07
150 2.18 0.17 160 2.20 *d
102 2.01
–a –a –a –a –a –a 109 2.04 0.07 119 2.07 0.07 *d *d *d *d *d *d
38 1.58
142 2.15
114 2.06
*d *d –a –a –a –a 94 1.97 138 2.14 445 2.65 262 2.42
Assigned/ihr a
Calculated/cBRP
– –a –a –a –a –a
–a –a
–a –a –a –a –a –a
–a –a
–a –a –a –a –a –a
–a –a
200 2.30 0.05 227 2.36 0.01
179 2.25
–a –a
–a –a
–a –a
220 2.34
–a –a –a –a –a –a
–a –a
–a –a –a –a –a –a
–a –a
*d *d *d *d *d *d
268 2.43
–a –a
–a –a
187 2.27
31
Titres (ELU/ml) Titres (log10) Difference (assigned–calculated)b Titres (ELU/ml) Titres (log10) Difference (ihr–cBRP)c
PRN
B. Poirier et al. / Biologicals 31 (2003) 25–38
3
PT
(continued)
Laboratory
32
Table 1
Antigen FHA Assigned/ihr
8
PT Calculated/cBRP
286 2.46 0.02 188 2.27 0.09
Titres (ELU/ml) Titres (log10) Difference (assigned–calculated)b Titres (ELU/ml) Titres (log10) Difference (ihr–cBRP)c
1598 3.20 0.07 2876 3.46 0.06
1894 3.28
10 Titres (ELU/ml) Titres (log10) Difference (assigned–calculated)b Titres (ELU/ml) Titres (log10) Difference (ihr–cBRP)c
2600 3.41 0.36 44 1.64 0.59
5878 3.77
11 Titres (ELU/ml) Titres (log10) Difference (assigned–calculated)b Titres (ELU/ml) Titres (log10) Difference (ihr–cBRP)c
3000 3.48 0.08 498 2.70 0.04
3584 3.55
12 Titres (ELU/ml) Titres (log10) Difference (assigned–calculated)b Titres (ELU/ml) Titres (log10) Difference (ihr–cBRP)c
111220 5.05 1.34 119308 5.08 1.61
5044 3.70
13 Titres (ELU/ml) Titres (log10) Difference (assigned–calculated)b Titres (ELU/ml) Titres (log10) Difference (ihr–cBRP)c
675 2.83 0.03 1206 3.08 0.05
9
a b c d
275 2.44 154 2.19
3320 3.52
172 2.24
548 2.74
2900 3.46 718 2.86 1081 3.03
34 1.53 0.12 20 1.30 0.10 90 1.95 0.05 *d *d *d
Calculated/cBRP 44 1.65 25 1.40 101 2.00 117 2.07
1400 3.15 0.81 93 1.97 0.73
9045 3.96
2700 3.43 0.57 1246 3.10 0.56
723 2.86
20724 4.32 1.73 23251 4.37 1.48 369 2.57 0.04 414 2.62 0.10
494 2.69
343 2.53 384 2.58 775 2.89 409 2.61 329 2.52
Assigned/ihr
Fim 2&3 Calculated/cBRP
60 1.78 0.06 5 0.69 0.07
52 1.72
52 1.72 0.07 397 2.60 *d
44 1.64
6 0.75
*d *d
690 2.84 0.23 20 1.30 0.34
409 2.61
–a –a –a –a –a –a
–a –a
4901 3.69 1.23 6360 3.80 1.24
286 2.46
144 2.16 0.06 91 1.96 0.04
9 0.96
–a –a
368 2.57 126 2.10 83 1.92
Assigned/ihr 64 1.81 0.05 210 2.32 0.04 74 1.87 0.30 88 1.95 0.31
Calculated/cBRP 72 1.86 232 2.37 147 2.17 180 2.26
–a –a –a –a –a –a
–a –a
–a –a –a –a –a –a
–a –a
–a –a –a –a –a –a
–a –a
–a –a –a –a –a –a
–a –a
–a –a
–a –a
–a –a
–a –a
Not performed. Difference between internal reference serum value assigned by the manufacturer and the titre calculated in the collaborative study versus SPAM-1 reference serum expressed in log10. Difference between titres of the internal control in the homologous and heterologous situations expressed in log10. Data not given or not quantifiable.
B. Poirier et al. / Biologicals 31 (2003) 25–38
Titres (ELU/ml) Titres (log10) Difference (assigned–calculated)b Titres (ELU/ml) Titres (log10) Difference (ihr–cBRP)c
Assigned/ihr
PRN
B. Poirier et al. / Biologicals 31 (2003) 25–38
33
Table 2 Comparison of results between the WHO and EDQM collaborative studies for IRR or cBRP (titration versus SPAM-1) EDQM collaborative study
WHO collaborative study
Antibody
log10
ELU/ml
log10
ELU/ml
IRR
Anti-FHA Anti-PT Anti-PRN Anti-Fim 2&3
2.35 1.47 1.66 1.66
223 29 46 45
2.46 1.53 1.78 1.81
286 34 60 64
cBRP
Anti-FHA Anti-PT Anti-PRN Anti-Fim 2&3
2.14 1.59 1.53 1.75
138 39 34 56
2.25 1.68 1.56 1.83
178 48 36 68
IRR antiserum titration versus SPAM-1 antiserum (Table 2): Results obtained in both studies are similar. cBRP antiserum titration versus SPAM-1 antiserum (Table 2): The cBRP titres obtained in this phase II study are close to those obtained in the WHO collaborative study. 3.5. Comparison between laboratories’ and AFSSAPS calculations of unitage relative to SPAM-1 The results obtained by each participant did not differ substantially from those obtained by AFSSAPS with the exception of Laboratory 4, which obtained lower results with its own calculation method (Table 3).
4. Discussion
Fig. 4. Behaviour of internal controls in homologous and heterologous situations (meanSEM): anti-FHA titres (A), anti-PT titres (B), anti-PRN titres (C) anti-Fim 2&3 titres (D). +, Titre of internal control versus ‘internal reference’ serum (homologous situation); +, Titre of internal control versus cBRP serum (heterologous situation).
Phase I of this study has shown that the cBRP for B. pertussis mouse antiserum contains significant levels of specific antibodies to the B. pertussis antigens PT, FHA, PRN and Fim 2&3 present in acellular pertussis vaccines. This study allowed to conclude that all methodologies tested seemed to be equivalent regarding their ability to calibrate the cBRP, IRR and in-house references relative to SPAM-1. In general, the variability (repeatability and reproducibility) observed was shown to be satisfactory and in the same range as the variability observed for ELISAs used in routine potency control of acellular pertussis vaccines (data not shown). No significant bias or particular advantage related to methodology could be identified during phase I. Therefore, it was decided to run the enlarged phase (international collaborative study), referred to as Phase II, allowing participants to use their own in-house methods. As regards the relative homogeneity of results obtained by different methodologies, the conclusion of Phase I was confirmed in Phase II, thus, justifying the use of
34
Table 3 Individual assay results (calibration against SPAM-1 reference) Assay
Serum potency expressed in ELU/ml cBRP calculated
cBRP Lab. IRR calculated
IRR Lab. In-house calculated
In-house stated
1
FHA FHA FHA PT PT PT PRN PRN PRN
1 2 3 1 2 3 1 2 3
138 154 150 33 39 38 44 * 43
144 152 157 34 39 38 36 34 38
226 238 245 23 22 22 57 * 53
219 231 235 26 25 24 56 52 53
14 167 15 459 16 892 4420 3975 3721 618 * 673
13 885 15 776 15 445 4234 3878 3447 628 764 715
2
FHA FHA FHA FHA PT PT PT PT PRN PRN PRN PRN
1 2 3 4 1 2 3 4 1 2 3 4
140 135 170 161 43 39 49 44 33 29 37 41
138 138 171 160 42 38 48 43 33 29 37 41
200 225 278 269 35 37 45 42 52 43 60 60
199 224 276 269 34 36 44 41 52 43 60 59
481 841 569 687 356 583 434 444 128 115 177 143
3
FHA FHA FHA PT PT PT PRN PRN PRN
1 2 3 1 2 3 1 2 3
150 181 176 44 42 40 35 38 41
150 181 176 44 42 40 35 38 41
295 290 296 38 40 47 55 55 53
295 290 296 38 40 47 55 55 53
4
FHA FHA FHA PT PT PT PRN PRN PRN FIM 2/3 FIM 2/3 FIM 2/3
1 2 3 1 2 3 1 2 3 1 2 3
206 68 95 * 46 55 80 21 16 81 36 46
192 65 78 31 30 25 31 11 8 46 26 19
303 145 175 * 45 57 78 45 37 57 31 34
306 147 174 22 43 41 54 28 34 42 34 16
Internal control calculated
Internal control Lab.
49
518 385 420 326 347 270 30 33 41
477 836 568 681 344 565 425 433 127 113 174 142
2307 2290 2985 2796 1047 1131 1323 1206 125 136 175 157
2294 2272 2970 2781 1010 1094 1297 1173 125 133 175 156
768 662 725 380 437 469 100 126 131
768 662 725 379 436 468 100 126 131
3196 2931 2994 1182 1196 1426 114 127 138
3196 2930 2994 1180 1194 1424 114 127 138
1022 302 406
1058 302 362 130 133 113 203 73 59 380 145 126
1060 279 385 * 208 169 209 85 68 339 145 168
1200 284 353 202 165 146 199 80 65 346 145 147
* 153 136 196 86 63 359 129 124
516 387 434 354 388 312 47 *
B. Poirier et al. / Biologicals 31 (2003) 25–38
Laboratory Antigen
Table 3
(continued)
Laboratory Antigen
Assay
Serum potency expressed in ELU/ml cBRP calculated
cBRP Lab. IRR calculated
IRR Lab. In-house calculated
PT PT PT
1 2 3
43 45 34
41 45 37
34 46 26
36 47 29
6
FHA FHA FHA PT PT PT PRN PRN PRN
1 2 3 1 2 3 1 2 3
124 146 119 34 35 34 23 24 28
124 146 119 34 35 34 23 24 28
200 234 195 23 26 24 36 38 37
200 234 195 23 26 24 36 38 37
7
FHA FHA FHA PT PT PT PRN PRN PRN FIM 2/3 FIM 2/3 FIM 2/3
1 2 3 1 2 3 1 2 3 1 2 3
88 171 245 32 39 73 29 * 44 50 55 101
* * * * * * * * * * * *
51 167 268 13 20 41 16 * 58 25 42 59
* * * * * * * * * * * *
8
FHA FHA FHA PT PT PT PRN PRN PRN FIM 2/3 FIM 2/3 FIM 2/3
1 2 3 1 2 3 1 2 3 1 2 3
156 126 147 41 42 36 26 28 25 67 50 50
149 117 143 39 40 37 26 27 25 68 49 54
237 280 281 29 34 40 29 53 47 59 60 62
237 277 285 27 33 39 27 51 48 50 59 64
1.28 1.24 1.24 433 634 531 331 389 331 88 90 104 235 1953 5350 82 93 249 197 * 1006 150 179 719 308 226 299 57 39 39 52 52 52 83 66 68
1.19 1.22 1.28
Internal control calculated
Internal control Lab.
64 58 60
63 59 63
433 634 531 331 389 331 88 90 104
2285 2902 2517 1166 1178 1160 92 94 115
2285 2902 2517 1166 1178 1160 92 94 115
* * * * * * * * * * * *
840 2160 5844 357 252 934 199 * 209 80 572 231
* * * * * * * * * * * *
300 222 287 56 39 38 54 50 53 86 61 67
208 138 190 32 22 25 4 4 5 281 197 217
202 117 177 33 22 24 5 9 4 289 178 252
B. Poirier et al. / Biologicals 31 (2003) 25–38
5
In-house stated
35
(continued)
Laboratory Antigen
9
36
Table 3
Assay
Serum potency expressed in ELU/ml cBRP Lab. IRR calculated
IRR Lab. In-house calculated
In-house stated
Internal control calculated
Internal control Lab.
129 144 142 * 34 * * * 28 50 49 56
* * * * * * * * * * * *
232 * 208 * 35 * * * 38 44 47 43
* * * * * * * * * * * *
* * * * * * * * * * * *
3115 * 3456 * 120 * * * 388 183 178 157
* * * * * * * * * * * *
131 * 91 9 * 9 45 33 32
136 88 104 9 10 9 49 31 32
* 12215 299 294 777
4470 5378 5382 7082 6077 10684 305 307 743
4022 3356 3412 793 698 683
FHA FHA FHA PT PT PT PRN PRN PRN FIM 2/3 FIM 2/3 FIM 2/3
1 2 3 1 2 3 1 2 3 1 2 3
FHA FHA FHA PT PT PT PRN PRN PRN
1 2 3 1 2 3 1 2 3
* 23 68 36 53
57 84 73 33 30 25 59 28 43
11
FHA FHA FHA PT PT PT
1 2 3 1 2 3
139 147 148 37 36 39
137 143 151 36 36 40
255 278 233 22 20 20
245 260 238 22 20 20
12
FHA FHA FHA PT PT PT PRN PRN PRN
1 2 3 1 2 3 1 2 3
* 170 * 24 24 25 * 30 *
156 173 163 26 24 25 30 30 36
* 283 * 64 * * * 45 47
309 272 264 63 51 62 54 45 52
10
52 * 78 34
1961 * 1830 * 101 * * * 44 146 152 142 5281 * 6542 6698
* 5044 * 467 324 375 * 321 255
74
456 14 12 14
96 128 97 577 421 428 10 11 12
3947 3280 3450 782 706 689
649 530 544 330 322 346
645 532 552 366 332 360
5979 5203 4904 709 555 559 312 280 247
* 5016 * 523 377 407 * 332 295
6844 5083 3930 746 543 555 269 329 291
* 95 591 *
B. Poirier et al. / Biologicals 31 (2003) 25–38
cBRP calculated
Table 3
(continued)
Laboratory Antigen
13
FHA FHA FHA PT PT PT PRN PRN PRN
Assay
1 2 3 1 2 3 1 2 3
Serum potency expressed in ELU/ml cBRP calculated
cBRP Lab. IRR calculated
IRR Lab. In-house calculated
In-house stated
Internal control calculated
Internal control Lab.
161 163 163 54 51 54 32 32 36
161 163 163 57 52 57 32 32 36
309 322 295 53 49 48 55 54 60
733 740 687 392 414 437 121 124 132
1299 1275 1256 460 441 477 74 84 83
1299 1275 1256 481 438 517 74 84 83
309 322 295 51 49 46 55 54 60
729 740 687 382 419 429 121 124 132
Calculated, AFSSAPS calculations; Lab, Laboratory calculations; *, Data not given or not quantifiable. B. Poirier et al. / Biologicals 31 (2003) 25–38 37
38
B. Poirier et al. / Biologicals 31 (2003) 25–38
in-house methods rather than that of a single standardised methodology for each pertussis component tested. In phase II, results (i.e. unitages of the sera expressed in reference to SPAM-1) calculated by AFSSAPS using a standard calculation methodology were similar to those reported by the participants. Results were overall comparable and exhibited a homogeneous variability among the laboratories. In order to assess that the proposed European standard (cBRP) could be used instead of the internal references, the positive control titres calculated relative to their corresponding internal reference (homologous situation) or the proposed European standard (heterologous situation) were compared. The most remarkable discrepancy observed between both situations was up to 1.7 log10 (Fig. 4) and seemed to be linked to the preliminary calibration of internal references. Indeed, it appeared in each case that unitages obtained when testing internal references versus SPAM-1 were different from those established in the procedures (Table 1). These results emphasise the need for a common reference [14]. The analysis of the outcome of this study has shown that the cBRP is suitable to be used as a reference antiserum. Therefore, the cBRP has been adopted by the Ph. Eur. commission during the 107th Session in June 2000 as B. pertussis mouse antiserum Ph. Eur. BRP batch 1 (EDQM product reference B1142000) with the following unitages (after reconstitution of the lyophilisate with 500 µl of distilled water): • • • •
Anti-PT: 39 ELU/ml Anti-FHA: 138 ELU/ml Anti-PRN: 34 ELU/ml Anti-Fim 2&3: 56 ELU/ml
Acknowledgements The organisers express their sincere thanks to all the participants for their contributions to this study. The study was run by the EDQM under the aegis of the Biological Standardisation Programme supported by the Council of Europe and the European Commission (project number BSP031). Appendix A. Participants R. Dobbelaer, G. Vanhooren, Institut Scientifique de la Sante´ Publique – Louis Pasteur, Brussels (Belgium)* M. Ducheˆne, E. Remy, Glaxo SmithKline Beecham Biologicals S.A., Rixensart (Belgium) G. Calver, N. Shakerchi, W. Wilson, Therapeutic Products Programme, Health Canada, Ottawa (Canada) B. Willumsen, Statens Serum Institut, Copenhagen (Denmark)
F. Fuchs, N. Bornstein, Agence Franc¸aise de Se´curite´ Sanitaire des Produits de Sante´, Lyon (France)* R. Bandet, A. Sabouraud, Aventis Pasteur, Marcy l’Etoile (France) G. Rautmann, B. Wild, C. Raphalen, EDQM, Council of Europe, Strasbourg, (France)* A. Zott, Paul Ehrlich Institut, Langen (Germany) P. Mastrantonio, Instituto Superiore di Sanita`, Roma (Italy) M. Duccio, N. Hug, Chiron – S.p.A., Siena (Italy) P. Newland, D. Xing, National Institute for Biological Standards and Control, Potters Bar (UK) J. Arciniega, B. Meade, Food and Drug Administration, Bethesda (USA) R. Gupta, Wyeth–Lederle Vaccines and Pediatrics, Pearl River (USA) *Laboratories involved in both Phase I and Phase II collaborative studies References [1] Corbel MJ, Xing DKL. The current status of acellular pertussis vaccines. J Med Microbiol 1997;46:817–8. [2] Corbel MJ, Xing DKL, Bolgiano B, Hockley D. Approaches to the control of acellular pertussis vaccines. Biologicals 1999; 27:133–41. [3] Corbel MJ, Xing DKL, Kreftenberg JG. Informal consultation with manufacturers and WHO Ad Hoc Working Group on mouse protection models for acellular pertussis vaccines. Biologicals 1999;27:189–93. [4] Xing D, Das R. International collaborative study: mouse protective models for acellular pertussis vaccines, September 2000 (unpublished report). [5] Mastrantonio P, Cerquetti M, Cardines R, Lande R, Ausiello CM, Cassone A. Immunogenicity issues in the quality control of the new acellular pertussis vaccines. Biologicals 1999;27:119–21. [6] European Pharmacopoeia. 4th ed. Pertussis vaccine (acellular, component, adsorbed), monograph 1356; Pertussis vaccine (acellular, co-purified, adsorbed), monograph 1595, 2002. [7] WHO guidelines for the production and control of the acellular pertussis component of monovalent or combined vaccines. WHO Tech Rep Ser 1998;878:57–76. [8] Andre´ M, Poirier B, Bornstein N, Marmonier D, El Zaouk A, Fuchs F. Key points for the development of mouse immunogenicity test as potency assay for acellular pertussis vaccines. Biologicals 2000;28(4):217–25. [9] BS/99.1901, WHO, 20 avenue Appia CH-1211 Geneva 27 Switzerland. [10] Wood DJ, Padilla A, Griffith E. WHO expert committee on Biological Standardization: highlights of the 50th meeting, October 1999. Biologicals 2000;28:199–206. [11] . Version 2.0. User’s manual, 103p. [12] . Manugistics. Version 3. User manual. [13] Gaines Das R, Xing D, Rigsby P, Newland P, Corbel M. International collaborative study: evaluation of proposed international reference reagent of pertussis antiserum (mouse). Biologicals 2001;29(2):137–48. [14] Fuchs F, Dobbelaer R. Collaborative study for the establishment of a European Pharmacopoeia biological reference preparation for Bordetella pertussis mouse antiserum for serological potency testing of acellular pertussis vaccines. Pharmeuropa Bio 2000; 2:25–42.