Validation of an ELISA method for quantification of the major Timothy grass pollen allergen Phl p 5a (BSP090).

Progress towards standardisation of allergen products has been made in recent years. Nevertheless, no standardised test method to quantify the allergen content of grass pollen allergen products is available at present. One aim of the BSP090 project was to validate a quantitative assay for a major Timothy grass (Phleum pratense) pollen allergen, Phl p 5. Qualification of a candidate ELISA system was performed with regard to range, robustness and cross-reactivity in preliminary studies. The assay specifically detected Phl p 5 with a quantification range from 3.9 ng/mL to 62.5 ng/mL. Suitability to quantify recombinant and natural Phl p 5 was further assessed in a collaborative study including 14 laboratories in Europe and the USA. Precision and accuracy of the assay was satisfactory with 93% of calculated Phl p 5 concentrations and 100% of total recoveries being within the ± 30% acceptance range. Similar results were obtained for spike recoveries, with exclusion of the lowest concentration spike, showing spike recoveries exceeding the acceptance range for six laboratories. Inter-assay (repeatability) and inter-laboratory (reproducibility) variability were satisfactory, in the format used in the present study. Robustness towards different statistical methods for data analysis was demonstrated. In conclusion, the assay can easily be established in routine testing and results of the preliminary testing and collaborative study support the proposal of the assessed Phl p 5-specific ELISA as a European Pharmacopoeia general method.

Council of Europe Black Sea Area Project: International Cooperation for the Development of Activities Related to Donation and Transplantation of Organs in the Region.

BACKGROUND: In 2011, the European Directorate for the Quality of Medicines & Healthcare of the Council of Europe launched a 3-year collaborative project to address the organ shortage and improve access to transplant health services in Council of Europe member states in the Black Sea area (Armenia, Azerbaijan, Bulgaria, Georgia, Moldova, Romania, Turkey, Ukraine, and the Russian Federation) through the development of safe and ethical donation and transplantation programs. OBJECTIVE: Support the development of donation and transplantation programs through close interstate cooperation between national health organizations and relevant stakeholders. METHODOLOGY: Several work packages (WP) were established: WP1, project coordination (European Directorate for the Quality of Medicines & Healthcare); WP2, development and implementation of an effective legislative and financial framework (Czech Republic and France); WP3, establishment of National Transplant Authorities (Italy and Portugal); and WP4, clinical practices (DTI Foundation). Data collection, surveys, and expert visits allowed for the collection of first-hand information from each participant country at national, regional, and hospital levels. RESULTS: Data analysis showed the positive impact of the project represented by a tendency to increase the total donation rates (per million people) in the participant countries (2011 vs 2013): Azerbaijan, +7.3; Armenia, -0.7; Georgia, +3.3; Bulgaria, +0.9; Moldova, +2.5; Ukraine:, +0.8; Romania, +2.3; and Turkey, +2.7. CONCLUSIONS: Increases in total donation rates are the result of a number of initiatives in the Black Sea area, including the stepwise implementation of legislative, organizational and institutional country-specific recommendations tailored by the CoE, efforts of the respective Ministries of Health in each country and synergism with other European projects in the region. These countries should invest further in implementing the recommendations that emerged from this project to improve their organ donation and transplantation programs and progress toward self-sufficiency.

Validation of ELISA methods for quantification of the major birch allergen Bet v 1 (BSP090).

To date, the potency of allergen products in Europe is expressed in manufacturer-specific units relative to a product-specific in-house reference. Consequently, cross-product comparability of allergen products from different manufacturers with respect to strength and efficacy is impossible. The Biological Standardisation Programme (BSP) project BSP090 addresses this issue via the establishment of reference standards in conjunction with ELISA methods for the quantification of major allergens in allergen products. Since the initiation of BSP090, the recombinant major allergen Bet v 1 has been adopted by the European Pharmacopoeia Commission as a Chemical Reference Substance (CRS). In parallel, two sandwich ELISA systems for quantification of Bet v 1 were found suitable in preliminary phases of BSP090 to be validated in a large collaborative study. In this study, the candidate ELISA systems were compared with respect to accuracy, precision and variability. Thirteen participating laboratories tested model samples containing the CRS as well as spiked and unspiked birch pollen extracts. Both in pre-testing and in the collaborative study, the 2 candidate ELISA systems confirmed their suitability to quantify recombinant and native Bet v 1. As no clear-cut decision for one of the ELISA systems could be made based on the results of the collaborative study, a post-study testing was performed. Bet v 1 content of 30 birch pollen allergen products was determined in parallel in both ELISA systems. Consequently, 1 candidate ELISA system was selected to be proposed as the future European Pharmacopoeia standard method for Bet v 1 quantification.

Calibration of the Ph. Eur. human coagulation Factor VIII concentrate BRP batch 5.

The European Pharmacopoeia Biological Reference Preparation (Ph. Eur. BRP) for Factor VIII Concentrate batch 5 was established through a collaborative study involving 14 laboratories organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM, Council of Europe) to be used as working standard for potency determination of human coagulation Factor VIII (FVIII) preparations. The potency of the BRP batch 5 was assigned with reference to the WHO 8th International Standard (IS) for FVIII Concentrate and the BRP batch 4. Participants were instructed to perform 3 independent Factor VIII potency assays following their own routine validated methods by the chromogenic assay as it is the assay prescribed by the European Pharmacopoeia. This publication reports the results obtained during the study. The consensus potency, 9.9 IU/ampoule (n = 14) when assessed against both standards, with inter-laboratory geometric coefficients of variation (GCV) of 3.2 % and 1.9 % against the WHO 8th IS and the BRP batch 4 respectively, was consistent with the expected value. The Ph. Eur. BRP batch 5 is a freeze-dried, plasma-derived concentrate. Based on accelerated degradation studies, the stability of the material is suitable as a reference preparation. The Ph. Eur. BRP batch 5 was adopted at the 151st session of the European Pharmacopoeia Commission in March 2015 and is available from the EDQM.

Collaborative study for the validation of an improved HPLC assay for recombinant IFN-alfa-2.

The current European Pharmacopoeia (Ph. Eur.) texts for Interferon (IFN)-alfa-2 include a nonspecific photometric protein assay using albumin as calibrator and a highly variable cell-based assay for the potency determination of the protective effects. A request was expressed by the Official Medicines Control Laboratories (OMCLs) for improved methods for the batch control of recombinant interferon alfa-2 bulk and market surveillance testing of finished products, including those formulated with Human Serum Albumin (HSA). A HPLC method was developed at the Medical Products Agency (MPA, Sweden) for the testing of IFN-alfa-2 products. An initial collaborative study run under the Biological Standardisation Programme (BSP; study code BSP039) revealed the need for minor changes to improve linearity of the calibration curves, assay reproducibility and robustness. The goal of the collaborative study, coded BSP071, was to transfer and further validate this improved HPLC method. Ten laboratories participated in the study. Four marketed IFN-alfa-2 preparations (one containing HSA) together with the Ph. Eur. Chemical Reference Substance (CRS) for IFN-alfa-2a and IFN-alfa-2b, and in-house reference standards from two manufacturers were used for the quantitative assay. The modified method was successfully transferred to all laboratories despite local variation in equipment. The resolution between the main and the oxidised forms of IFN-alfa-2 was improved compared to the results from the BSP039 study. The improved method even allowed partial resolution of an extra peak after the principal peak. Symmetry of the main IFN peak was acceptable for all samples in all laboratories. Calibration curves established with the Ph. Eur. IFN-alfa-2a and IFN-alfa-2b CRSs showed excellent linearity with intercepts close to the origin and coefficients of determination greater than 0.9995. Assay repeatability, intermediate precision and reproducibility varied with the tested sample within acceptable ranges. Test accuracy estimated by comparing the values obtained by the participants to the declared contents determined by the manufacturers was good despite the absence of a common reference preparation. In conclusion, the present study showed that the new method is suitable, reproducible and transferable. Proposals for the revision of Ph. Eur. texts are presented.

Standardization of allergen products: 3. Validation of candidate European Pharmacopoeia standard methods for quantification of major birch allergen Bet v 1.

BACKGROUND: The BSP090 project aims at establishing European Pharmacopoeia Reference Substances in combination with the corresponding ELISA methods for the quantification of major allergens in allergen products. Two sandwich ELISAs proved suitable for quantification of Bet v 1, the major birch pollen allergen, in preceding phases of BSP090. METHODS: Two Bet v 1-specific ELISA systems were compared with respect to accuracy and precision in a ring trial including 13 laboratories. Model samples containing recombinant rBet v 1.0101 as well as native birch pollen extracts were measured independently at least three times in each facility. The assessment was completed with a comparative quantification of Bet v 1 in 30 marketed birch allergen products in one laboratory, simulating the future use as reference method. RESULTS: In the collaborative study, both candidate ELISAs confirmed their suitability to quantify recombinant and native Bet v 1. ELISA-A showed higher precision and lower interlaboratory variability, yet ELISA-B exhibited slightly higher accuracy. Subsequent parallel measurement of Bet v 1 in a panel of 'real-life' birch allergen products indicated better repeatability of ELISA-B. Both systems detected substantial differences in Bet v 1 content between allergen products, but the effect was more pronounced using ELISA-B due to persistently higher values compared to ELISA-A. CONCLUSIONS: In the collaborative study, no deciding differences were observed between the two candidate ELISAs. Further comparison under conditions simulating the intended use combined with the criterion of long-term availability enabled the selection of one Bet v 1-specific ELISA for proposal as European Pharmacopoeia standard method.

Standardization of allergen products: 2. Detailed characterization of GMP-produced recombinant Phl p 5.0109 as European Pharmacopoeia reference standard.

BACKGROUND: The Biological Standardization Programme of the European Directorate for Quality of Medicines and Healthcare (EDQM) aims at the establishment of well-characterized reference standards based on recombinant allergens and validated assays for the quantification of major allergen content. The objective of this study was to examine the detailed physicochemical and immunological characterization of recombinant Phl p 5.0109, the second available allergen reference standard. METHODS: Recombinant Phl p 5.0109 PP5ar06007 was produced under GMP conditions and analyzed by an array of physicochemical and immunological methods for identity, quantity, homogeneity, and folding stability in bulk solution, as well as thermal denaturation, aggregation state, and biological activity when formulated for long-time storage. RESULTS: PP5ar06007 revealed as a highly homogeneous, monomeric, well-folded preparation of rPhl p 5.0109, as documented by mass spectrometry, SDS-PAGE, isoelectric focusing, size-exclusion chromatography with light scattering, circular dichroism, and infrared spectroscopy. Upon storage at +4°C, PP5ar06007 retained the monomeric state for at least 2 months. A protein quantity of 1.56 ± 0.03 mg/ml was determined by amino acid analysis in PP5ar06007, and its biological activity was shown to be comparable to natural Phl p 5 in terms of basophil activation and T-cell reactivity. CONCLUSIONS: Recombinant Phl p 5.0109 PP5ar06007 was characterized extensively at the physicochemical and immunological level. It revealed to be a highly stable, monomeric, and immunologically equivalent of its natural counterpart. PP5ar06007 is now available as European Pharmacopoeia allergen reference standard for grass pollen products.

The vaccines consistency approach project: an EPAA initiative.

The consistency approach for release testing of established vaccines promotes the use of in vitro, analytical, non-animal based systems allowing the monitoring of quality parameters during the whole production process. By using highly sensitive non-animal methods, the consistency approach has the potential to improve the quality of testing and to foster the 3Rs (replacement, refinement and reduction of animal use) for quality control of established vaccines. This concept offers an alternative to the current quality control strategy which often requires large numbers of laboratory animals. In order to facilitate the introduction of the consistency approach for established human and veterinary vaccine quality control, the European Partnership for Alternatives to Animal Testing (EPAA) initiated a project, the "Vaccines Consistency Approach Project", aiming at developing and validating the consistency approach with stakeholders from academia, regulators, OMCLs, EDQM, European Commission and industry. This report summarises progress since the project's inception.

Collaborative study for the establishment of the WHO 3(rd) International Standard for Endotoxin, the Ph. Eur. endotoxin biological reference preparation batch 5 and the USP Reference Standard for Endotoxin Lot H0K354.

An international collaborative study was organised jointly by the World Health Organization (WHO)/National Institute for Biological Standards and Control (NIBSC), the United States Pharmacopeia (USP) and the European Directorate for the Quality of Medicines & HealthCare (EDQM/Council of Europe) for the establishment of harmonised replacement endotoxin standards for these 3 organisations. Thirty-five laboratories worldwide, including Official Medicines Control Laboratories (OMCLs) and manufacturers enrolled in the study. Three candidate preparations (10/178, 10/190 and 10/196) were produced with the same material and same formulation as the current reference standards with the objective of generating a new (3(rd)) International Standard (IS) with the same potency (10 000 IU/vial) as the current (2(nd)) IS, as well as new European Pharmacopoeia (Ph. Eur.). and USP standards. The suitability of the candidate preparations to act as the reference standard in assays for endotoxin performed according to compendial methods was evaluated. Their potency was calibrated against the WHO 2(nd) IS for Endotoxin (94/580). Gelation and photometric methods produced similar results for each of the candidate preparations. The overall potency estimates for the 3 batches were comparable. Given the intrinsic assay precision, the observed differences between the batches may be considered unimportant for the intended use of these materials. Overall, these results were in line with those generated for the establishment of the current preparations of reference standards. Accelerated degradation testing of vials stored at elevated temperatures supported the long-term stability of the 3 candidate preparations. It was agreed between the 3 organisations that batch 10/178 be shared between WHO and EDQM and that batches 10/190 and 10/196 be allocated to USP, with a common assigned value of 10 000 IU/vial. This value maintains the continuity of the global harmonisation of reference materials and unitage for the testing of endotoxins in parenteral pharmaceutical products. Based on the results of the collaborative study, batch 10/178 was established by the European Pharmacopoeia Commission as the Ph. Eur. Endotoxin Biological Reference Preparation (BRP) batch 5. The same batch was also established by the Expert Committee on Biological Standardisation (ECBS) of WHO as the WHO 3(rd) IS for Endotoxin. Batch 10/190 was adopted as the USP Endotoxin Reference Standard, lot H0K354 and vials from this same batch (10/190) will serve as the United States Food and Drug Administration (USFDA) Endotoxin Standard, EC-7.

Establishment of the Ph. Eur. erythropoietin chemical reference substance batch 1.

The Erythropoietin (EPO) European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) batch 3 was calibrated in 2006 by in vivo bioassay and was used as a reference preparation for these assays as well as for the physicochemical methods in the Ph. Eur. monograph Erythropoietin concentrated solution (1316). In order to avoid the frequent replacement of this standard and thus reduce the use of animals, a new EPO Chemical Reference Substance (CRS) was established to be used solely for the physicochemical methods. Here we report the outcome of a collaborative study aimed at demonstrating the suitability of the candidate CRS (cCRS) as a reference for the physicochemical methods in the Ph. Eur. monograph. Results from the study demonstrated that for the physicochemical methods currently required in the monograph (capillary zone electrophoresis (CZE), polyacrylamide gel electrophoresis (PAGE)/immunoblotting and peptide mapping), the cCRS is essentially identical to the existing BRP. However, data also indicated that, for the physicochemical methods under consideration for inclusion in a revised monograph (test for oxidised forms and glycan mapping), the suitability of the cCRS as a reference needs to be confirmed with additional work. Further to completion of the study, the Ph. Eur. Commission adopted the cCRS as "Erythropoietin for physicochemical tests CRS batch 1" to be used for CZE, PAGE/immunoblotting and peptide mapping.

Establishment of hepatitis A vaccine (inactivated, non-adsorbed) BRP batches 2 and 3.

The current hepatitis A vaccine (HAV), inactivated, non-adsorbed, European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) is used for the in vitro potency assay of HAV as prescribed by the Ph. Eur. general chapter 2.7.14 Assay of hepatitis A vaccine. This reference preparation was calibrated in 2008 through an international collaborative study and was assigned a potency of 12 IU/mL. During use of this BRP it appeared to be inapplicable in certain cases due to a low nominal antigen content. Consequently, the European Directorate for the Quality of Medicines and HealthCare (EDQM) established replacement batches for this BRP, calibrated against the 1(st) WHO International Standard (IS) for HAV (inactivated), using the standard in vitro ELISA (enzyme-linked immunosorbent assay) method validated previously. The results of the study showed that the candidate BRPs were suitable for the intended purpose, and following completion of the study, they were adopted in November 2014 by the Ph. Eur. Commission as HAV (inactivated, non-adsorbed) BRP batches 2 and 3, with an assigned potency of 1350 IU/mL, for in vitro antigen content determination by ELISA. As the amount of material in each vial largely exceeds the amount required for the performance of a single assay, the BRPs are to be aliquoted by users as single-use aliquots and refrozen below -50 °C prior to their use as reference preparations.

[The network of official medicines control laboratories]. / Das Netzwerk der Official Medicines Control Laboratories.

Licensing, control and surveillance by competent authorities is the basis for ensuring efficacy, safety and quality of medicines in Europe. The control of the quality of medicines by national control laboratories, known as Official Medicines Control Laboratories (OMCLs) is an essential step in this process; it encompasses controls before and after granting a marketing authorisation. For certain groups of biomedical medicines (vaccines for human and veterinary use, medicines derived from human plasma) even each batch is controlled before it can be placed on the market. As single OMCLs would not be able to cope with their task, given the large number and diversity of medicines, in 1994 the OMCL network was founded upon initiative of the European Directorate for the Quality of Medicines & HealthCare, in close collaboration with the Commission of the European Union. Currently 68 OMCLs from 39 countries are part of the network. Prerequisite for the smooth operation of the OMCL network is the harmonisation of the quality management system of the individual OMCLs, based on the ISO 17025 standard, internal guidelines and the European Pharmacopoeia. Compliance with these standards is checked through regular audits, thus creating the basis for mutual recognition of test results. The collaboration in the OMCL network for the surveillance of the medicines market, the official control authority batch release and the fight against counterfeiting and illegal medicines enables OMCLs to keep pace with the developments in the field of medicines and to control the broad spectrum of medicines. In the 20 years since its start, the OMCL network has become a European success story.

[The EDQM Biological Standardisation Programme for the development of methods and reference preparations]. / Das Biological Standardisation Programme des EDQM zur Entwicklung von Methoden und Referenzpräparaten.

The Biological Standardisation Programme (BSP) of the European Directorate for the Quality of Medicines & HealthCare (EDQM) was founded in 1992 with the objective to provide the necessary tools for the quality controls prescribed by the European Pharmacopoeia (Ph. Eur.). The BSP accomplishes this task by establishing reference standards and materials, as well as standardised control methods. A key aspect of BSP's work on development of methods is the validation of methods which can replace Ph. Eur. tests involving animals. The current area of work includes vaccines (for human and animal use), medicines produced from human plasma, hormones, cytokines, allergens, as well as reference materials and methods for determination of impurities and contaminations. BSP closely collaborates with the World Health Organization (WHO) and national authorities; many reference standards are established in joint projects with WHO. Participants of studies for establishing of reference materials and methods are mainly national control laboratories and manufacturers. BSP has to date run 131 projects, whereby 121 reference materials were established. Method development was the objective of 38 projects, with 21 thereof aiming at replacement of animal tests. BSP is funded by the EDQM (Council of Europe) and by the European Commission. With its activities BSP makes a significant contribution to quality, safety and efficacy of biological medicinal products in Europe and beyond, and serves thereby health and well-being of human beings and animals.

Collaborative study for the establishment of the 3rd international standard for neomycin.

An international collaborative study was organised to establish the World Health Organization (WHO) 3(rd) International Standard (IS) for neomycin. Ten laboratories from different countries participated in the collaborative study. The potency of the candidate material, a freeze-dried preparation, was estimated by microbiological assays with sensitive micro-organisms. To ensure continuity between consecutive batches, the 2(nd) IS for neomycin was used as a standard. Based on the results of the study, the 3(rd) IS for neomycin was adopted at the meeting of the WHO Expert Committee on Biological Standardization (ECBS) in 2012 with an assigned potency of 19,050 IU per vial. The 3(rd) IS for neomycin is available from the European Directorate for the Quality of Medicines & HealthCare (EDQM).

Calibration of the human immunoglobulin BRPs for ACA and molecular size (batch 1) and for Fc function and molecular size (batches 1 & 2).

The current European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation batch 3 (BRP3) for Human Immunoglobulin was established in 2005. Stocks of this BRP are dwindling and a replacement batch is needed to serve as working standard in the tests for distribution of molecular size by HPLC, anticomplementary activity (ACA) and Fc function, in accordance with the requirements of the Ph. Eur. monographs Human normal immunoglobulin (0338) and Human normal immunoglobulin for intravenous administration (0918). The European Directorate for the Quality of Medicines & HealthCare (EDQM) carried out a project (BSP099) to establish replacement batches for this BRP. The project was run in 2 phases, a prequalification phase (Phase 1) and an international collaborative study (Phase 2) involving 19 laboratories. Three batches of candidate materials of various sizes, Samples A, B and C, were procured from 2 different manufacturers on the European market. Based on the results of the study, Sample A was shown to be suitable as a reference standard for the ACA test and for molecular size determination by HPLC, whereas Samples B and C were demonstrated to be suitable for the Fc function test and for the molecular size determination by HPLC. All 3 BRPs are to be used in conjunction with the monographs Human normal immunoglobulin (0338) and Human normal immunoglobulin for intravenous administration (0918). The BRPs were adopted by the Ph. Eur. Commission at its 141st session in November 2011 as official Ph. Eur. Human Immunoglobulin BRPs for ACA and molecular size Batch 1 (Sample A) and Fc function and molecular size Batch 1 and Batch 2 (Samples B and C respectively).

Collaborative study for the establishment of the 3rd international standard for dihydrostreptomycin.

An international collaborative study was organised to establish the World Health Organization (WHO) 3rd International Standard (IS) for dihydrostreptomycin. Eleven laboratories from different countries participated in the collaborative study. The potency of the candidate batch, a freeze-dried preparation, was estimated by microbiological assays with sensitive microorganisms. To ensure continuity between consecutive batches of the WHO IS, the 2nd IS for dihydrostreptomycin was used as standard. Based on the results of the study, the 3rd IS for dihydrostreptomycin was adopted at the meeting of the WHO Expert Committee on Biological Standardisation (ECBS) in 2011 with an assigned anti-microbiological activity of 19425 International Units (IU) per vial. The 3rd IS for dihydrostreptomycin is available from the EDQM.

Establishment of recombinant major allergens Bet v 1 and Phl p 5a as Ph. Eur. reference standards and validation of ELISA methods for their measurement. Results from feasibility studies.

The potency of allergen extracts is determined as total allergenic activity without consideration of their composition and the units differ from one manufacturer to another, making it very difficult to compare the different products. Recently, purified major allergens have been obtained by recombinant DNA technology and produced under Good Manufacturing Practice (GMP) conditions. In principle, such recombinant allergens could be established as reference standards and could help for the standardisation of the major allergen content of allergen extracts. Two recombinant major allergens, one from birch pollen, rBet v 1, and one from Timothy grass pollen, Phl p 5a, have been selected at the end of the CREATE programme as a potential starting point for the establishment as European Pharmacopoeia (Ph. Eur.) Reference Standards through a project run by the Biological Standardisation Programme (BSP) of the European Directorate for the Quality of Medicines & HealthCare (EDQM). To this end, bulk candidate recombinant materials, produced under GMP conditions, were procured from two European manufacturers and subsequently formulated and lyophilised. Four ELISA systems from three different manufacturers were included in the project, two for Bet v 1 and two for Phl p 5a with the aim of establishing reference methods for determination of the respective major antigens both in natural allergen extracts as well as in recombinant allergen products. The project was run in 3 phases: a preparatory and preliminary testing phase (feasibility phase or Phase 1), an extended feasibility phase carried out in 3 laboratories (Phase 2) to confirm the transferability of the methods and an international collaborative study with a large number of participating laboratories (Phase 3). This article describes the work done in Phase 1 and Phase 2, i.e. the physico-chemical and biological characterisation of the recombinant candidate reference standards, the assessment of their suitability for the intended purpose as well as the evaluation of the candidate ELISA systems. The results show that both candidate reference standards are suitable for the intended purpose. In addition, three out of the four ELISA systems that were included in the preliminary phase were found to be appropriate for further evaluation in the collaborative study which was organised in 2011. The results of the collaborative study will be published separately.

Calibration of the Ph. Eur. Biological Reference Preparation (BRP) for tetanus vaccine (adsorbed) batch 3.

A joint collaborative study was organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM) and the World Health Organization (WHO)/National Institute for Biological Standards and Control (NIBSC) to establish replacement batches for the European Pharmacopoeia (Ph. Eur.) Tetanus Vaccine (adsorbed) Biological Reference Preparation (BRP) batch 2 and for the WHO 3rd International Standard (IS) for Tetanus toxoid (adsorbed). Two freeze-dried stabilised tetanus vaccine (adsorbed) candidate preparations (Preparation A, 08/218 and Preparation B, 08/102) were calibrated against the current 3rd IS/BRP batch 2 (Preparation C) using challenge methods in guinea pigs and mice as described in the Ph. Eur. general chapter 2.7.8. Assay of tetanus vaccine (adsorbed). They were also assayed by serology methods. The WHO 2nd IS for Tetanus toxoid adsorbed (TEXA-2) was additionally included in the sample panel as Preparation D. Thirty-four laboratories (regulatory organisations and manufacturers) from 22 countries participated in the collaborative study. The majority of participants performed 2 independent challenge tests. Nine laboratories performed challenge assays in guinea pigs and 30 laboratories performed challenge assays in mice. Eight laboratories performed serology in guinea pigs and 1 laboratory performed serology in mice. For Preparation A, the geometric mean (GM) potency estimate (with 95 % confidence interval (CI)) in guinea pigs for all laboratories that provided valid results (n = 6) was 488.5 (354.2-673.6) IU/ampoule. For valid mouse assays (n = 25) the GM potency (with 95 % CI) was 259.8 (223.5-302.0) IU/ampoule. The inter-laboratory geometric coefficient of variation (GCV) was 36 % for guinea pig assays and 45 % for mouse assays. This compared favourably with the calibration of the 3rd IS/BRP batch 2 where the inter-laboratory GCV was 36 % and 42 % in guinea pigs and mice, respectively. For Preparation B, the GM potency estimate (with 95 % CI) in guinea pigs for all laboratories that provided valid results (n = 6) was 107.9 (64.1-181.7) IU/ampoule. For valid mouse assays (n = 24) the GM potency (with 95 % CI) was 147.9 (126.3-173.1) IU/ampoule. The inter-laboratory GCV was 64.3 % for guinea pig assays and 45.2 % for mouse assays. From the collaborative study, Preparation A appeared more suitable to be the replacement Ph. Eur. BRP as it is similar to the Tetanus vaccine (adsorbed) BRP batch 2, except for nature of the stabiliser. Preparation A was confirmed to have higher potency, readily detectable tetanus toxoid, and confirmed satisfactory stability and performance in challenge assays. Preparation A was adopted in January 2011 by the Ph. Eur. Commission as the Tetanus vaccine (adsorbed) BRP batch 3, with assigned potencies of 490 IU/ampoule in the guinea pig challenge assay and of 260 IU/ampoule in the mouse challenge assay. The same Preparation A was adopted in October 2010 as the WHO 4th IS for Tetanus toxoid (adsorbed), with the assigned activity of 490 IU/ampoule from guinea pig challenge assays. A follow-up study (reporting study) was organised by the EDQM to assess the impact of the potency assigned to the BRP batch 3 for mouse challenge assays on the outcome of batch release testing in Europe. Eight laboratories including official medicines control laboratories (OMCLs) and manufacturers reported the results of their routine testing, using the BRP batch 3 in addition to their regular reference preparation. For each tested product, participants calculated the potency relative to their routine reference and relative to the BRP batch 3. No common sample panel was distributed to participants. In total, data on 40 batches of different marketed tetanus vaccines were reported. Overall, a good concordance was observed between the potencies calculated relative to the BRP batch 2 and relative to the BRP batch 3. On average, the potency estimates were 10 % lower when expressed relative to the BRP batch 3. Cases of discrepant decisions for batch release were very limited and affected mainly batches with specifications close to the pharmacopoeial requirements. The reasons for differences in estimated potencies are discussed. The study showed that the use of the BRP batch 3 with an assigned potency of 260 IU/ampoule does not result in substantial change in the potency of different marketed products. This confirmed that the mouse challenge potency value assigned to the BRP batch 3 is suitable.

Collaborative study on influenza vaccine clinical trial serology - part 1: CHMP compliance study.

The Quality of Medicines & HealthCare (EDQM, Council of Europe) and the European Union (EU) Commission to evaluate the reproducibility of clinical serology results for seasonal influenza vaccines and to assess the impact of technical differences between laboratories on the compliance with the Committee for Human Medicinal Products (CHMP) criteria set by the European Medicines Agency (EMA). The study was run in 2 phases. The present article reports the 1st phase of the study, which aimed at evaluating the variability of the results obtained by 11 laboratories (5 national control laboratories and 6 influenza vaccine manufacturers) using their routine haemagglutination inhibition (HI) assay to test a common panel of clinical trial sera. The results confirmed the limited inter-laboratory reproducibility of the HI testing of influenza vaccine clinical trial samples. In some cases a good agreement was found between laboratories, while a systematic bias or a random scatter of results was observed in other cases. Analysis of estimated systematic bias confirmed that differences between laboratories can be significant (up to 16-fold) in some cases. Correction for this bias resulted in limited improvement. Differences between laboratories were found to result in discrepant decisions on marketing acceptance of vaccines or to decisions based on compliance to different criteria. The study showed that the seroconversion (SC) and mean fold increase (MFI) criteria are more robust against systematic over- or under-estimation of titres whereas the protection rate (PR) is very sensitive to this effect. The fundamental issues with the PR criteria are discussed.

Collaborative study on influenza vaccine clinical trial serology - part 2: reproducibility study.

A collaborative study was run by the Biological Standardisation Programme (BSP) under the aegis of the European Directorate for the Quality of Medicines & HealthCare (EDQM, Council of Europe) and the European Union (EU) Commission, to address the issue of the poor standardisation of serological assays used for the evaluation of seasonal influenza vaccines in Europe. The Phase 1 of the study focused on the compliance to Committee for Human Medicinal Products (CHMP) criteria by 6 manufacturers and 5 public laboratories. It confirmed the poor inter-laboratory correlation of haemagglutination inhibition (HI) test results. Phase 2 consisted in a reproducibility study examining the impact of extended method standardisation and the use of reference sera on inter-laboratory variation. Six manufacturers and 5 public laboratories contributed HI results, while the 5 public laboratories also performed single radial haemolysis (SRH) tests on the same sample panels. Results showed that method standardisation failed to significantly improve the inter-laboratory variation. Correction for pre-vaccination titres (Beyer correction) was found to have limited effect to improve the bias constituted by the Protection Rate (PR) criterion. The reasons underlying the difficulty in standardisation of HI and SRH tests are discussed and improved approaches for the compliance testing to CHMP criteria are suggested.