Kreuth IV: European consensus proposals for treatment of haemophilia with coagulation factor concentrates.

INTRODUCTION: This report summarizes recommendations relating to haemophilia therapy arising from discussions among experts from 36 European countries during the 'Kreuth IV' meeting in May 2016. AIM: The objective of the meeting was for experts in the field of haemophilia from across Europe to draft resolutions regarding current issues relating to the treatment of haemophilia. RESULTS: Hospitals providing clinical care for people with haemophilia and related disorders are strongly recommended to seek formal designation as either European Haemophilia Treatment Centres (EHTC) or European Haemophilia Comprehensive Care Centres (EHCCC). There should be agreed national protocols or guidelines on management of the ageing patient with haemophilia. The minimum consumption of factor VIII and IX concentrate in any country should be 4 IU and 0.5 IU per capita of general population respectively. Treatment for hepatitis C with direct-acting antiviral agents should be provided to all people with haemophilia on a priority basis. Genotype analysis should be offered to all patients with severe haemophilia. Genetic counselling, when given, should encompass the recommendation that genetic relatives of the affected person be advised to seek genetic counselling. People with inhibitors should have access to bypassing agents, immune tolerance and elective surgery. National or regional tenders for factor concentrates are encouraged. Outcome data including health related quality of life should be collected. Treatment with extended half-life factors should be individualized and protection against bleeding should be improved by increasing trough levels. Steps should be taken to understand and minimize the risk of inhibitor development. CONCLUSION: It is hoped that these recommendations will help to foster equity of haemophilia care throughout Europe.

Research in haemophilia B--approaching the request for high evidence levels in a rare disease.

Payers in European countries request studies with high levels of evidence for decision making also for rare diseases like haemophilia B (HB). The objective of the study was to determine the status quo of current studies in HB regarding the overall level of evidence generated. The methods used for performing the study were systematic literature research in EMBASE and MEDLINE, search terms 'HB' and 'factor IX' (FIX). The inclusion criteria were journal articles (JA), conference abstracts (CA), English language, published between January 2009 and March 2013, studies only; screening of titles, abstracts, full texts subsequently. ClinicalTrials.gov search: unpublished registered trials (RT) concerning HB or FIX. The analysis was performed on research topic, sponsor, recruitment status and study design. Screening of 1639 hits yielded 31 JA describing 35 studies, and 62 CA. FIX was subject of 21 studies (60.0%) and 29 CA (46.8%). Seven studies focused on various aspects of HB, six on haemophilia studies with separate HB data. Screening of 173 hits from ClinicalTrials.gov yielded 42 RT. Overall, 32 RT (76.2%) related to FIX. Measurement of health-related quality of life (HRQoL) was identified in none of these studies, four CA (6.5%), four RT (9.5%). Randomized study design was found in one study (2.9%), four RT (9.5%). Three studies (8.6%) and seven RT (16.7%) were prospective, observational and comparative. The majority of published clinical studies do not meet payers' expectations for evidence. Therefore, clinical investigation concepts addressing randomization, outcomes research including HRQoL and comparison of therapy options should be discussed. Refined statistical methods and exploitation of complementary real-life data sources may fill current evidence gaps concerning rare diseases.

Kreuth III: European consensus proposals for treatment of haemophilia with coagulation factor concentrates.

This report summarizes recommendations relating to haemophilia therapy arising from discussions among experts from 36 European countries during the Kreuth III meeting in April 2013. To optimize the organization of haemophilia care nationally, it is recommended that a formal body be established in each country to include the relevant clinicians, national haemophilia patient organization, health ministry, paying authority and (if appropriate) regulatory authorities. The minimum factor VIII consumption level in a country should be 3 I.U. per capita. Decisions on whether to adopt a new product should not be based solely on cost. Prophylaxis for children with severe haemophilia is already recognized as the optimum therapy. Ongoing prophylaxis for individual adults should also be provided when required based on clinical decision making by the clinician in consultation with the patient. Children with inhibitors who have failed, or who are not suitable for, immune tolerance therapy should be offered prophylaxis with bypassing agents. Single factor concentrates should be used as therapy wherever possible in patients with rare bleeding disorders. Orphan drug designation for a factor concentrate should not be used to hinder the development, licencing and marketing of other products for the same condition which have demonstrably different protein modification or enhancement.

Sexual risk behaviour and donor deferral in Europe.

One of the most controversial policies in blood transfusion worldwide is the permanent deferral from donating blood of men with sexual contacts to other men (MSM). This policy was implemented for safety reasons as sex between men is known to be a high risk factor for acquiring severe infectious diseases transmissible by blood transfusion. Sexual contacts among heterosexual persons may hold similar risks but a clear-cut discrimination between different individual risks is impossible. Nevertheless, the current blood donor deferral periods defined by European Union (EU) legislation depend on a distinction of different grades of risk with respect to sexual behaviour. Under the aegis of the Steering Committee on Blood Transfusion (CD-P-TS) of the Council of Europe (CoE), an international working group evaluated epidemiological and behavioural data, modelling studies on residual risk and spread of infections, and studies on adherence to donor selection criteria. The aim was to distinguish sexual behaviour of different risk categories. It was concluded, that existing data confirm that MSM and commercial sex workers (CSW) are groups at high risk. Any further grading lacks a scientific data base. Modelling studies indicate that adherence to deferral policies is of major relevance suggesting that good donor adherence may outweigh the small negative effects on blood safety postulated for changing from permanent to temporary deferral periods for high risk sexual behaviours. The fact that a considerable percentage of donors are MSM - despite the permanent deferral policy - demonstrates the need to increase donor understanding and adherence.

Determination of procoagulant activity in human normal immunoglobulin preparations for therapeutic use by FXIa chromogenic assay: Evaluation of test kit sensitivity, reference standard performance and product formulation effects on the FXIa assay.

In 2010, the reporting of thrombotic adverse events for one subcutaneous and certain intravenous immunoglobulins (IGs) raised some concerns. In Europe, regulatory bodies rapidly revised compendial specifications for therapeutic IGs to ensure they do not exhibit thrombogenic (procoagulant) activity (PCA). At the global level, a working group (GWG) was launched with the aim of assessing PCA measurement methods and limits, considering results obtained by human IG manufacturers during in-process controls. The GWG created three dedicated subgroups to investigate the FXIa chromogenic assay, the non-activated partial thromboplastin time (NAPTT) test and the thrombin generation assay (TGA). The European Directorate for the Quality of Medicines & HealthCare (EDQM) was responsible for co-ordinating the subgroup in charge of evaluating the FXIa chromogenic assay in a study that assessed the sensitivity and robustness of two commercial chromogenic FXIa test kits. The impact of IG product formulation on FXIa recovery and the suitability of PCA-containing IG products as potential reference standards/controls were also assessed. IG materials representative of marketed products were provided to four laboratories for a study that was carried out in two steps: 1) two chromogenic FXIa test kit manufacturers assessed the performance and determined optimal test conditions by their respective methods, 2) two OMCLs studied both kits using an optimised study design. Regarding sensitivity, the study results identified suitable dose-response intervals and limits with both chromogenic FXIa test kits. This allowed the establishment of dilution ranges for optimal detection of FXIa/PCA in 5 % and 10 % IG products in the range of 1-6 mIU/mL. However, careful optimisation of the sample dilutions was required (notably to avoid potential matrix effects) and the choice of the mode of data acquisition (kinetic or end-point method) contributed to sensitivity in routine use. Importantly, the composition of IG products was of minor concern for FXIa determination with both test kits. Potential reference materials evaluated in the study behaved as expected and could be useful should a separate reference standard to the FXIa WHO IS be deemed necessary in future.

Collaborative study for the characterisation of the BINACLE Assay for in vitro detection of tetanus toxicity in toxoids - Part 2.

Tetanus vaccines for human and veterinary use are produced by formaldehyde-induced inactivation of tetanus neurotoxin (TeNT) purified from Clostridium tetani cultures. Due to the high morbidity caused by exposure to TeNT it is essential that the quality control of tetanus vaccines includes testing for absence of tetanus toxin as prescribed by European Pharmacopoeia monographs 0452 and 0697. Currently this test is carried out in guinea pigs for each bulk of tetanus toxoid. To test the applicability of the in vitro BINACLE ("binding and cleavage") assay as an alternative method for the quality control of tetanus vaccines, two collaborative studies were run by the European Directorate for the Quality of Medicines & HealthCare under the aegis of the Biological Standardisation Programme. The first collaborative study indicated that the method allows sensitive TeNT detection. However, a clear conclusion could not be drawn due to the high variability of the results. To address the variability, the protocol was optimised and further standardised for the second study. The study results demonstrated good assay precision, both with respect to repeatability and reproducibility. Importantly, the limit of detection was 0.11 ng/mL TeNT in five out of nine laboratories and 0.33 ng/mL in four out of nine laboratories, suggesting that the BINACLE assay can detect TeNT with similar sensitivity as in vivo toxicity tests and can thus be taken into consideration as an alternative method to the current compendial in vivo test.

Collaborative study for the characterisation of the BINACLE Assay for in vitro detection of tetanus toxicity in toxoids - Part 1.

For several decades the European Pharmacopoeia monographs Tetanus vaccine (adsorbed) (0452) and Tetanus vaccine for veterinary use (0697) required that Specific toxicity and Absence of toxin and irreversibility of the toxoidof each bulk of tetanus toxoids had to be tested by an in vivo toxicity test in guinea pigs before it could be included in vaccines for human or veterinary use. In line with the 3Rs concept of replacing, reducing and refining animal experiments, an in vitro method for the detection of active tetanus neurotoxin (TeNT) has been developed at the Paul-Ehrlich-Institut (PEI, Germany). This method, the so-called BINACLE (binding and cleavage) assay, uses the receptor-binding and proteolytic properties of TeNT for the specific detection of active toxin molecules. Successful in-house validation studies as well as a small-scale transferability study had demonstrated that this method may represent a suitable alternative to the compendial in vivo toxicity test. As a follow up, an international collaborative study aimed at verifying the suitability of the BINACLE assay as a potential alternative to the guinea pig toxicity test for tetanus toxoids was organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM) under the aegis of its Biological Standardisation Programme (BSP). Within the framework of this study, coded BSP136, a feasibility phase - also referred to as Phase 1 - was run to select and qualify critical study reagents and samples and to assess the performance of the BINACLE Standard Operating Procedure developed by the project leaders. Then the international collaborative study aimed at evaluating the BINACLE, referred to as BSP136 Phase 2, was started. A total of 19 international laboratories (comprising vaccine manufacturers as well as national control laboratories) were supplied with a detailed assay protocol, critical reagents required for the assay, three samples consisting of three different bulk tetanus toxoids donated by major European vaccine manufacturers and one international standard toxoid. Each of the participants was asked to perform three independent BINACLE assays following the provided protocol. The statistical analysis of the results showed that most of the participating laboratories were able to perform the BINACLE assay according to the provided protocol. However, the results obtained by the participants varied widely, and not all the laboratories were able to achieve a sensitive detection of active TeNT. Multiple factors may have contributed to the elevated variability of the BSP136 study results. From an analysis of these factors, strategies were developed to help increase the standardisation of the BINACLE assay and obtain more consistent results in a follow-up validation study, BSP 136 Phase 3 (Part 2), for which the experimental phase took place in 2023. The present manuscript summarises the outcome of Phases 1 and 2, which constitute Part 1 of the BSP136 project.

Collaborative study for the establishment of Ph. Eur. Human immunoglobulin for electrophoresis BRP batch 4.

Due to the diminished stocks of the third batch of the European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) for Human immunoglobulin for electrophoresis, in 2020 the European Directorate for the Quality of Medicines & HealthCare (EDQM) initiated an international collaborative study for the establishment of a replacement batch. The study was run under the aegis of the Biological Standardisation Programme (BSP). Nineteen laboratories participated in the collaborative study to verify the suitability of the candidate reference preparations according to the Ph. Eur. monographs Human normal immunoglobulin for intravenous administration (0918), Human normal immunoglobulin for intramuscular administration (0338) and Human normal immunoglobulin for subcutaneous administration (2788) using the zone electrophoresis method with cellulose acetate and/or agarose as the testing medium. Capillary zone electrophoresis (CZE), a technique not yet included in monographs 0338, 0918 and 2788, was also used by some laboratories. The assignment of a value for immunoglobulin as a percentage of the total protein content could only be made for agarose electrophoresis and for CZE. The candidate preparation was found suitable for the intended purpose and was subsequently adopted by correspondence in May 2021 by the Ph. Eur. Commission as Human immunoglobulin for electrophoresis BRP batch 4 with an assigned range for immunoglobulin of 82.5 % to 87.8 % of the total protein content.

Collaborative study for the establishment of Ph. Eur. Human albumin (molecular size) Biological Reference Preparation batches 1, 2 and 3.

To comply with European Pharmacopoeia (Ph. Eur.) monograph Human albumin solution (0255), albumin solutions have to be tested for molecular-size distribution by size-exclusion chromatography (SEC). However, differences in interpretation of the test results continue to be observed among albumin manufacturers in Europe. A collaborative study was run by the European Directorate for the Quality of Medicines & HealthCare (EDQM), under the aegis of the Biological Standardisation Programme (BSP), to support the revision of Ph. Eur. monograph 0255 and to establish a Biological Reference Preparation (BRP) for use in the molecular-size distribution test. In 2019, Ph. Eur. Expert Group 6B proposed to include an analytical improvement of the SEC procedure in the monograph, which was then submitted for public enquiry. This publication describes the evaluation of three candidate BRPs to serve as a tool for both the system suitability test (SST) and albumin monomer and dimer peak identification according to the proposed revised methodology. Three Official Medicines Control Laboratories (OMCLs) involved in the official batch release of human albumin solution took part in the study. Based on the study results, the candidate BRPs were found suitable for purpose and were adopted by the Ph. Eur. Commission as Ph. Eur. Human albumin (molecular size) BRP batches 1, 2 and 3 concomitantly with the revised monograph Human albumin solution (0255) in November 2020.

Collaborative study for the validation of cell line assays for in-process toxicity and antigenicity testing of Clostridium septicum vaccine antigens - Part 2: Optimisation of cell line assays.

During the production of clostridial vaccines large numbers of mice are used for various in-process control tests. Replacement in vitro assays had been developed for the testing of the toxins and toxoids of several clostridial species, but none of these assays had been assessed in an international collaborative study. Under the common aegis of the European Partnership for Alternative Approaches to Animal Testing (EPAA) and of the European Directorate for the Quality of Medicines & HealthCare (EDQM), a project on clostridial vaccines for veterinary use was started as part of the EDQM-co-ordinated Biological Standardisation Programme (BSP). Within the framework of this project (coded BSP130) a collaborative study was organised to evaluate Vero cell-based alternative methods to the current mouse tests used to measure: i) the toxicity of Clostridium septicum toxin, ii) the absence of toxicity of C. septicum toxoid and iii) the antigenicity of C. septicum toxoid. The principal aims of the study were to determine the repeatability and reproducibility of the in vitro assays and to demonstrate concordance of the in vitro and current in vivo tests. The study results demonstrated good concordance, but the information gathered through the study (later on called Part 1) and the participants' workshop prompted the extension of the project in order to further optimise the in vitro protocols and improve their repeatability and reproducibility, which were comparable to but not better than those of the in vivo assays in Part 1. The 3 in vitro assays to be optimised in the extension of the BSP130 project were : i) the in vitro toxin neutralisation equivalence plus (TNE+), as a replacement for the in vivo minimum lethal dose (MLD) test for quantification of the toxicity of toxin; ii) the in vitro MLD, as a replacement for the in vivo MLD test for detection of residual toxicity associated with toxoid; iii) the in vitro total combining power (TCP), as a replacement for the in vivo TCP test for quantification of the antigenicity of toxoid. At this point, the Analytical Method Transfer Laboratory of Ceva-Phylaxia (Hungary), supported by the project management team, developed suitable SOPs for the 3 in vitro assays. These optimised methods were further assessed in BSP130 through a second international collaborative study (Part 2) aimed at defining repeatability and reproducibility in different laboratories and determining the levels of improvement compared with the original in vivo tests and the initial in vitro assays used in Part 1 of the project. Fourteen laboratories, comprising 4 public sector and 10 manufacturers' medicines control laboratories, from 11 countries participated in the collaborative Part 2 study, each testing 6 different C. septicum toxins and 6 C. septicum toxoids. Improved repeatability and reproducibility were observed for the optimised assays. The results of this study confirm the suitability of these assays for in-process control of C. septicum vaccines, with better repeatability and reproducibility than their in vivo equivalents. It is expected that, with appropriate minor changes and the use of relevant reagents, these optimised in vitro assays could be used not only for the assessment of C. septicum toxins and toxoids but for all cytotoxin-based clostridial antigens. The development and implementation of such in vitro assays would offer a great opportunity to significantly reduce animal usage, shorten the duration of QC test procedures and increase the precision of toxicity and antigenicity assays in clostridial veterinary vaccine in-process control. This would also provide more accurate and reproducible dosing of antigens in the final vaccine products, help to promote compendial acceptance and to proffer a basis for improved international harmonisation across this area of product testing.

Establishment of a candidate equine influenza Florida Clade 2 strain A/eq/Richmond/1/07 horse antiserum as Ph. Eur. Biological Reference Preparation/OIE International Reference Reagent.

Equine influenza (EI) is an important respiratory disease of horses, with welfare and economic consequences. Vaccination remains one of the most efficient prevention methods available. Equine influenza virus (EIV) is constantly evolving and consequently EI vaccines need to be updated on a regular basis. In 2010, the World Organisation for Animal Health (OIE) Expert Surveillance Panel (ESP) on EI provided a new recommendation for EI vaccine strain composition, including the incorporation of representative EIV strains of both Florida Clade 1 and Clade 2 sub-lineages (FC1 and FC2, respectively). In this context, the European Pharmacopoeia (Ph. Eur.) - OIE reference panel for EI had to be complemented by an antiserum raised in horses against the FC2 representative EIV strain A/eq/Richmond/1/07. An international collaborative study was organised and managed by the European Directorate for the Quality of Medicines and HealthCare (EDQM) within the framework of its Biological Standardisation Programme (BSP). The study aimed at evaluating a new candidate reference for use as a common OIE International Standard/Ph. Eur. Biological Reference Preparation (BRP) horse antiserum to FC2 EIV A/equine/Richmond/1/07. The standard was to be established using the SRH and HI tests for subsequent use in immunogenicity, efficacy and batch potency assay of EI vaccines as a Ph. Eur. BRP (Ph. Eur. monograph 0249) and for use in clinical diagnostic tests as an OIE-approved International Standard Reagent (OIE chapter 3.5.7). The collaborative study confirmed the suitability of the candidate and an SRH titre was assigned. The candidate was adopted as a BRP by the Ph. Eur. Commission and approved by the OIE Biological Standards Commission as an International Standard Serum in November 2017 and February 2018, respectively.

Collaborative study for the establishment of Etanercept Biological Reference Preparation Batch 1.

Three preparations of the human tumour necrosis factor (TNF) receptor II Fc fusion protein (TNFR II-Fc) Etanercept were formulated and lyophilised at the National Institute for Biological Standards & Control (NIBSC) prior to evaluation in a collaborative study for their suitability to serve as a World Health Organization (WHO) International Standard (IS)/European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) for the potency assay of Etanercept. Seven laboratories tested the preparations using an in vitro cell-based bioassay (TNF-α neutralisation) prescribed by the Ph. Eur. monograph on Etanercept (2895). The results of this study indicated that the candidate preparation, coded 13/204, established as the first IS for Etanercept with an assigned potency for TNF neutralisation activity of 10 000 IU per ampoule was also suitable to serve as Ph. Eur. BRP batch 1. The results were compared to those obtained with different cell-based neutralisation assays that were used by further laboratories in the context of establishing the 1st WHO IS for Etanercept. Based on these analyses, preparation 13/204 was adopted by the Ph. Eur. Commission as Etanercept BRP batch 1 with an assigned potency of 10 000 IU per ampoule.

Collaborative study for the establishment of Infliximab Biological Reference Preparation Batch 1.

Two preparations of the chimeric anti-Tumour Necrosis Factor (TNF) monoclonal antibody Infliximab were formulated and lyophilised at the National Institute for Biological Standards & Control (NIBSC) prior to evaluation in a collaborative study for their suitability to serve as a World Health Organization (WHO) International Standard (IS)/European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) for the potency assay of Infliximab. Twenty-six laboratories tested the preparations using different in vitro cell-based bioassays (TNF-α neutralisation, antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity) and binding assays. Amongst them, 19 laboratories performed cell-based bioassays. The results of this study indicated that the candidate preparation coded 16/170 was suitable to serve as an International Standard for Infliximab based on the data obtained for biological activity. This candidate standard was established in 2017 as the first International Standard for Infliximab with an assigned potency for TNF neutralisation activity of 500 IU per ampoule. In the same study, the suitability of preparation 16/170 of Infliximab to serve as the European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) for the Infliximab potency assay as described in the Ph. Eur. monograph on Infliximab concentrated solution (2928) was also evaluated. The corresponding analysis, based on the measurement of the inhibitory action of anti-human TNF (Infliximab) on the cytotoxic activity of TNF-alpha, was performed using data from a subset of 9 laboratories using the TNF-alpha-sensitive fibrosarcoma cell line WEHI-164. The results obtained were compared to those obtained from different cell-based neutralisation assays that were used by other laboratories in the context of establishing the 1st World Health Organization (WHO) International Standard (IS) for Infliximab. Based on the analyses, preparation 16/170 was adopted by the Ph. Eur. Commission in June 2018 as Infliximab BRP batch 1 with an assigned potency of 500 IU per ampoule.

Collaborative study for the validation of cell line assays for in-process toxicity and antigenicity testing of Clostridium septicum vaccine antigens - Part 1.

Large numbers of mice are used in testing during the production of Clostridial vaccines. Previous work has indicated that cell line assays could replace mouse tests for certain aspects of this testing. Replacement assays have been developed for the testing of the toxins and toxoids of several clostridial species but none of these assays have been assessed in an international collaborative study. Under the common aegis of the European Partnership for Alternative Approaches to Animal Testing (EPAA) and of the European Directorate for the Quality of Medicines & HealthCare (EDQM), collaborative study BSP130 was initiated to evaluate Vero cell based alternative methods to the current mouse tests used to measure the toxicity of Clostridium septicum toxin (the minimum lethal dose (MLD) test), the freedom from toxicity of C. septicum toxoid (the MLD test) and the antigenicity of C. septicum toxoid (the total combining power (TCP) test). The principal aims of BSP130 were to determine the repeatability and reproducibility of the in vitro assays and to demonstrate concordance of the proposed in vitro and current in vivo TCP and MLD tests. 11 laboratories from 7 countries participated in the collaborative study and each tested 6 toxins and 6 toxoids. The participants' Vero cell lines were up to 1 000 times more sensitive than the mouse strains. The MLD assay in mice and on Vero cells generally ranked the toxins in a similar order in most of the laboratories. The TCP assay in mice and on Vero cells also generally ranked the toxoids in a similar order in most of the laboratories. The results demonstrate that the repeatability and reproducibility of the in vitro Vero cell based assays are no worse than that of the in vivo assays and that they are easily transferable to other laboratories. The concordance correlations between the in vivo and in vitro methods were for the MLD assays ρc=0.961 (log-transformed values) and ρc=0.921 (non-log-transformed values) and for the TCP assays ρc=0.968 (log-transformed values) and ρc=0.980 (non log-transformed values). These correlations are excellent showing that the Vero cell assays can be used as alternatives to the mouse tests for the assessment of C. septicum toxin MLD and toxoid TCP values. This study can be used by vaccine manufacturing companies as a guide for applying the same approach to other clostridial toxins and toxoids.

Establishment of Ph. Eur. Bordetella pertussis mouse antiserum Biological Reference Preparation batches 2, 3 and 4.

A project aimed at establishing replacement batches for the European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) Bordetella (B.) pertussis mouse antiserum was started in 2013 under the aegis of the Biological Standardisation Programme (BSP) of the European Directorate for the Quality of Medicines & HealthCare (EDQM). This BRP is used for the immunogenicity assay in mice to assess the potency of acellular pertussis (aP) vaccines as described in Ph. Eur. general method 2.7.16. Assay of pertussis vaccine (acellular). In a preliminary phase of the project (referred to herein as BSP129 phase 1) a hyper-immune serum pool was produced in mice using a combined aP vaccine as immunogen. This pool was used to generate 3 freeze-dried candidate (c) B. pertussis anti-mouse serum BRP batches (cBRP2, cBRP3 and cBRP4). After the pre-qualification that showed their suitability as candidate batches, an international collaborative study (BSP129 phase 2) was carried out in order to standardise these 3 batches against the current BRP1 in terms of anti-PT, -FHA, -PRN and -FIM2/3 antibody contents. For the sake of continuity with the standardisation of BRP1, the corresponding WHO standard (1RR 97/642) was introduced as a second reference for the calibration of the 3 candidate BRPs. Eleven laboratories took part in phase 2. Ten of them performed the ELISA method they use routinely for aP vaccine batch release and one laboratory performed the Multiplex Immunoassay (MIA) as an alternative test. Four participants titrated the antibodies against all 5 pertussis antigens, 5 participants determined the antibody content against 3 antigens (PT, FHA, PRN), one participant titrated the antibodies against PT and FHA antigens and one laboratory determined the antibody content for the PT antigen only. Details of all ELISA methods used were analysed to evaluate their impact on the calibration of the cBRPs. The variability of the results in relation to the nature and methodology of the tests appeared rather limited. Discrepant titres of cBRPs were measured depending on the reference used: the use of the 1RR induced an overestimation (in 8 out of 11 laboratories) and a large inter-laboratory variation in the calculated titres. Regardless of the reference used, equivalency between the calculated titres of cBRP2 and cBRP3 was observed, whilst cBRP4 had systematically lower titres for all antibodies against the 5 acellular pertussis vaccine components. Based on these observations, it was decided to establish the candidate BRP batches against BRP1 and to assign the following potencies based on the mean values determined through centrally calculated results of the calibration assays performed by ELISA in BSP129 phase 2: For cBRP2 and cBRP3 Anti-pertussis toxin: 37 ELISA Units (ELU) per vial Anti-filamentous haemagglutinin: 114 ELU per vial Anti-pertactin: 44 ELU per vial Anti-fimbrial agglutinogens (FIM2/3): 25 ELU per vial For cBRP4 Anti-pertussis toxin: 32 ELU per vial Anti-filamentous haemagglutinin: 98 ELU per vial Anti-pertactin 38 ELU per vial Anti-fimbrial agglutinogens (FIM2/3):23 ELU per vial In February 2018, BRP2, BRP3 and BRP4 were adopted by correspondence by the Ph. Eur. Commission.

Production and characterisation of a candidate hyper-immune serum for the replacement of the Bordetella pertussis mouse antiserum Biological Reference Preparation.

For acellular pertussis (aP) vaccines, the current European Pharmacopoeia (Ph. Eur.) monograph Pertussis vaccine (acellular, component, adsorbed) (1356) requires an immunogenicity assay in mice or guinea pigs to assess the potency of each lot of vaccine (Ph. Eur. general method 2.7.16. Assay of pertussis vaccine (acellular)). This biological assay, carried out on the final bulk of the vaccine lot, is based on the measurement of the specific antibody response to the 5 antigenic components (pertussis toxin (PT), Fimbrial haemagglutinin (FHA), pertactin (PRN) and Fimbriae 2 and 3 (FIM2/3)) that are present in the combined aP vaccines. In the mouse assay, serum antibody levels are measured by ELISA. The immunogenicity of a vaccine under test is estimated versus a homologous reference vaccine and a reference antiserum e.g. the first Ph. Eur. Biological Reference Preparation for Bordetella (B.) pertussis mouse anti-serum (BRP1), established in 1998, is used to normalise the titre of antibodies (expressed in ELISA Units (ELU)/mL). In anticipation of the depletion of BRP1 stocks, a project was launched in 2013 by the Biological Standardisation Programme (BSP) of the European Directorate for the Quality of Medicines & HealthCare (EDQM) in order to establish a new standardised reference serum. The project, referred to herein as BSP129, was conducted in 2 phases: 1) the production and characterisation of a mouse serum pool (using a multicomponent aP vaccine marketed in Canada similar to the vaccine used in the BRP1 production as immunogen) and of candidate BRP batches (cBRPs) and 2) an international collaborative study aimed at calibrating the cBRPs in terms of antibody levels against PT, FHA, PRN and FIM2/3. This article presents the design and results of the first phase of the collaborative study to establish the optimal conditions for immunisation and bleeding of mice in order to produce a large pool of hyper-immune serum against the 5 antigens. After the characterisation of this pool, cBRP pilot lots were manufactured by freeze-drying diluted solutions of the hyper-immune serum pool. The pilot lots were then characterised in two Official Medicines Control Laboratories (OMCLs) for their antibody contents against aP vaccine antigens using in-house ELISA (based on methods developed by 2 European vaccine manufacturers) and Multiplex Immunoassay (MIA) methods. The antibody titres recovered demonstrated that a dilution factor of 1/40 could be considered for the scaled-up manufacture of candidate reference preparations (cBRPs). Three batches (15 000 vials) of cBRP were manufactured and fully characterised. In light of the data obtained, and although titration results between the ELISA methods were sometimes discrepant, it was agreed that the establishment study (phase 2) could be launched. Real-time and accelerated stability studies were also included in the first study phase to document the stability of the cBRPs in freeze-dried form and after reconstitution and storage at -20°C±5°C. The results showed that the stability of the freeze-dried cBRPs at usual storage and shipment temperatures is acceptable and that reconstituted cBRP solutions are stable for 12 months at -20°C±5°C. It could therefore be recommended to freeze small aliquots of the 1 mL solution obtained by the reconstitution of one BRP vial in order to store them for use in separate assays. With the application of this strategy, the stocks of the BRP1 replacement batches should cover the needs of OMCLs and manufacturers for at least the next decade.

International collaborative study to evaluate candidate reference reagents to standardize haemagglutination testing for anti-A and anti-B in normal intravenous immunoglobulin products.

BACKGROUND AND OBJECTIVES: The aim of the study was to evaluate, in an international collaboration, three lyophilized intravenous immunoglobulin (IVIG) preparations for their suitability to standardize and control haemagglutination testing for anti-A and anti-B in IVIG products. MATERIALS AND METHODS: Twenty-three laboratories tested candidate IVIG reference reagents consisting of a Positive control (07/306), a Negative control (07/308), and a specifically formulated Limit preparation (07/310) to define the maximum (e.g. pharmacopoeial) limits of anti-A and anti-B in IVIG products, where limits are applicable. Laboratories performed direct haemagglutination using papain-treated erythrocytes and/or indirect antiglobulin tests. RESULTS: For both methods, there was up to 16-fold variation in anti-A and anti-B titres, although there was good agreement over a two-fold titre range for anti-A and anti-B between laboratories for both 07/306 and 07/310 using the direct method. Comparative titration data for 07/306 and 07/310 indicated that the use of a 'Limit' reference reagent would facilitate identification of higher titre batches when the direct haemagglutination method is used. CONCLUSIONS: The establishment of preparations 07/306, 07/308 and 07/310 as reference reagents by the World Health Organization will facilitate global standardization of haemagglutination tests for anti-A and anti-B, ensure that such tests are sufficiently sensitive and specific, and facilitate identification of batches that exceed maximum recommended levels of anti-A and anti-B. The Commission of the European Pharmacopoeia and the United States Food and Drug Administration have adopted the same reference reagents including the maximal specifications defined by preparation 07/310.

[Understanding the role of the European public institutions in blood transfusion]. / Comprendre le rôle des institutions publiques européennes en transfusion sanguine.

In the context of increased demand of blood components, it is highly desirable to ensure development and implementation of established standards for ethical, organisational and regulatory fields in order to guarantee the sufficiency, quality and safety of blood components and their derivatives as well as the protection of donors and recipients. In Europe, the contributions of the European public institutions is fundamental for the achievement of these goals and for the harmonisation of practices inherent to transfusional medicine activities. The Council of Europe with its 47 member states constitutes the ideal platform to tackle these themes: for half a century, its public health and bioethics experts have contributed to the elaboration of conventions and recommendations which serve as a model to elaboration of national or European regulations relevant to this field.

Establishment of replacement batches for prekallikrein activator in albumin biological reference preparation.

A collaborative study was run by the European Directorate for the Quality of Medicines and HealthCare (EDQM) under the aegis of the Biological Standardisation Programme (BSP) to establish replacement batches of the current Prekallikrein activator in albumin Biological Reference Preparation (BRP) batch 1, the stocks of which were dwindling. Candidate BRP replacement batch 2 and batch 3 were assayed against the 2nd World Health Organization International Standard for Prekallikrein activator, human (2nd IS) and the Prekallikrein activator in albumin BRP batch 1. The candidate batches were manufactured from the same starting material as the current Biological Reference Preparation and the 2nd IS. They consisted of a 20 % solution of albumin lyophilised under the same conditions as the Prekallikrein activator in albumin BRP batch 1. Sixteen laboratories participated in the collaborative study and were requested to assay the candidates by their routine method, complying with the European Pharmacopoeia (Ph. Eur.) general method 2.6.15 for the determination of prekallikrein activator content. A central statistical analysis was performed at the EDQM using in-house calculations of prekallikrein activator contents provided by the participating laboratories. On the basis of the results of this study, which confirmed the assigned potency of 29 IU/vial of Prekallikrein activator in albumin BRP batch 1, the 2 candidate materials were assigned a potency of 30 IU/vial. The 2 candidates were adopted by the Ph. Eur. Commission in March 2008 as Ph. Eur. Prekallikrein activator in albumin Biological Reference Preparation batch 2 and batch 3.

Evaluation of two serological methods for potency testing of whole cell pertussis vaccines.

The European Pharmacopoeia (Ph. Eur.) and the World Health Organization (WHO) require the performance of extensive quality control testing including a potency test before a vaccine batch is released for human use. Whole cell pertussis (wP) vaccine potency is assessed by a mouse protection test (MPT) based on the Kendrick test. This test compares the vaccine dose necessary to protect 50% of mice against the effect of a lethal intracerebral dose of Bordetella pertussis and the dose of a suitable reference vaccine needed to give the same protection level. Due to the large variability in the results of this test and the severe distress which is inflicted on the many animals involved, its replacement by an alternative method is highly desirable. At the initiative of the European Directorate for the Quality of Medicines and HealthCare (EDQM) of the Council of Europe, in collaboration with the WHO and the In-vitro toxicology Unit/European Centre for the Validation of Alternative Methods (ECVAM) of the European Commission (EC) Joint Research Centre-Institute for Health and Consumer Protection (JRC-IHCP), wP vaccine specialists from all over the world were invited to present an overview of candidate alternatives at a symposium organised in Geneva (Switzerland) in March 2005. Although no alternative method was found suitable for immediate implementation of batch potency control, the Pertussis Serological Potency Test (PSPT), initially developed in mice and recently transferred to guinea pigs (gps), was identified as a model of interest. Using the PSPT in gps to test several components of combined vaccines such as Diphtheria-Tetanus-wP vaccines in the same animal series would allow further implementation of the European 3Rs policy to batch potency control, by additional method refinement and reduction of animal use. The present study evaluated 2 features of the serological response to wP vaccination: 1) the overall antibody response as measured by a "whole cell" ELISA (PSPT-wC-ELISA) which uses the B. pertussis 18323 challenge strain prescribed for the MPT to coat the assay plates and 2) the functional neutralising antibodies to pertussis toxin (PT, one of the main virulence factors of B. pertussis), as measured by the Chinese Hamster Ovary (CHO) cell assay. The results showed that 1) the gp model can be used for wP vaccine potency testing; 2) despite good repeatability and precision, the CHO cell assay did not generate results comparable to the MPT. Moreover, the CHO cell assay showed significant differences in the ability of wP vaccines to induce neutralising anti-PT antibodies, which did not correlate to the overall antibody response evaluated by PSPT-wC-ELISA; 3) comparable potencies were obtained in the MPT and the PSPT-wC-ELISA. This study, supported by the previous ones correlating the PSPT-wC-ELISA in mice with the MPT, confirms that PSPT-wC-ELISA in gps is a promising approach for batch release potency testing of wP vaccines for which consistency in production has already been demonstrated by the MPT. However, a large scale validation study is required prior to the adoption of PSPT-wC-ELISA as a compendial reference method for wP vaccines batch release control.