Collaborative Study for the Calibration of the Ph.Eur. Prekallikrein Activator in Albumin Biological Reference Preparation batches 8, 9 and 10.

An international collaborative study was organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM, Council of Europe) to calibrate replacement batches for the current European Pharmacopoeia (Ph. Eur.) Prekallikrein Activator (PKA) in albumin Biological Reference Preparation (BRP) whose stocks were dwindling. The study was run in the framework of the Biological Standardisation Programme (BSP) of the Council of Europe and the European Union (EU) Commission. Twenty-four laboratories from official medicines control authorities and manufacturers in Europe and outside Europe took part in the study. Three candidate replacement batches were produced with albumin solutions artificially spiked with a PKA concentrate to increase their PKA level. Participants were requested to evaluate the candidate batches against the 3rd World Health Organization (WHO) International Standard (IS) for Prekallikrein activator in albumin (16/364) using their routine assay method. The Ph. Eur. PKA in albumin BRP batch 7 (BRP7) was also included in the test panel to ensure the continuity of the consecutive BRP batches. The 3 candidate replacement batches were considered suitable for their intended use as BRPs. The study confirmed the stability of the PKA content of the current BRP7. Thermal stress study on the candidate batches confirmed the stability of their PKA activity. In December 2023, the Ph. Eur. Commission officially adopted the 3 candidate batches as Ph. Eur. PKA in albumin BRP batches 8, 9 and 10 with assigned potencies of 37 IU/vial, 33 IU/vial and 34 IU/vial, respectively. The activity of the 3 new batches of Ph. Eur. PKA in albumin BRP will be regularly monitored.

Collaborative study for the establishment of Human immunoglobulin (molecular size) BRP replacement batches 4, 5 and 6.

Human immunoglobulin products are used for the treatment of a number of diseases, such as primary or secondary immunodeficiencies and autoimmune conditions due to the complete absence of antibodies or the production of defective immunoglobulins. Quality control of human immunoglobulin products is essential to ensure therapeutic functionality and safety. This includes testing for Fc function and anticomplementary activity (ACA), as well as verification of appropriate molecular size distribution using size-exclusion chromatography as prescribed in the European Pharmacopoeia (Ph. Eur.) monographs 0338, 0918, 2788 and 1928. To this end, specific biological reference preparations (BRPs) must be used. Stocks of the Ph. Eur. Human immunoglobulin (molecular size) BRP were running low and therefore a collaborative study was run by the European Directorate for the Quality of Medicines & HealthCare (EDQM), under the aegis of the Biological Standardisation Programme, to calibrate replacement batches. Eighteen laboratories, including manufacturers and Official Medicines Control Laboratories, took part in the study. Three batches of candidate BRPs were assessed and compared to Ph. Eur. Human immunoglobulin (molecular size) BRP 3 to ensure continuity. Based on the study results, the candidate BRPs were adopted by the Ph. Eur. Commission as Ph. Eur. Human immunoglobulin (molecular size) BRP batch 4, 5 and 6.

Establishment of Ph. Eur. Hepatitis C Virus RNA for NAT testing BRP batch 2.

The European Pharmacopoeia (Ph. Eur.) monographs Human plasma for fractionation (0853) and Human plasma (pooled and treated for virus inactivation) (1646) require that plasma pools be tested for hepatitis C virus (HCV) RNA presence by nucleic acid amplification techniques (NAT) using a positive control at 100 IU/mL. HCV RNA for NAT testing BRP batch 1 was established in 1999 to this end. Due to dwindling stocks, the European Directorate for the Quality of Medicines & HealthCare (EDQM) organised a collaborative study to establish a replacement batch. The candidate material was produced as a lyophilised preparation of human plasma containing HCV genotype IA and calibrated against the 6th WHO International Standard for HCV RNA for NAT. Quantitative and qualitative HCV NAT assays based on real-time quantitative PCR techniques were used. Both types of assays were assessed separately. However, since no significant difference was observed between them, all results were pooled for the final potency assignment. Calculations based on Ct values were less variable than those based on end-point dilutions; they were thus used in the final combination. The combined overall mean potency was 959 IU/vial. An accelerated degradation study showed that the stability of the candidate material was satisfactory at the recommended long-term storage temperature, i.e. -20°C. The candidate BRP was established as Ph. Eur. HCV RNA for NAT testing BRP batch 2 by the Ph. Eur. Commission, with an assigned potency of 960 IU/vial. It will be available from the EDQM under catalogue number H0215000.

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 calibration of Ph. Eur. Heparin Low-Molecular-Mass for Assay Biological Reference Preparation batch 11.

European Pharmacopoeia (Ph. Eur.) monograph 0828 prescribes two in vitro assays to evaluate the biological activity of low-molecular-mass heparin products. These assess the capacity of low-molecular-mass heparins to accelerate the inhibition of factor Xa and factor IIa by antithrombin. A reference standard calibrated in International Units (IU) such as the Heparin low-molecular-mass for assay Biological Reference Preparation (BRP), is required to express results. Due to low stocks, the Biological Standardisation Programme (BSP) of the Council of Europe and the European Union launched an international collaborative study to establish a replacement batch for the current Heparin low-molecular-mass BRP (BRP10). Thirteen Official Medicines Control Laboratories and manufacturers contributed data to calibrate a candidate batch against the World Health Organization (WHO) 3rd International Standard for Heparin, low molecular weight (11/176; 3rd IS) using chromogenic assays. The overall anti-Xa and IIa activities of the candidate BRP against the 3rd IS based on the participants' calculations and on central calculation at the European Directorate for the Quality of Medicines & HealthCare (EDQM) were very close. The inter-laboratory variation for both assays was between 2.8 % and 7.5 %. The study data confirmed the assigned activities of BRP10. Based on the results of this collaborative study, in December 2019 the Ph. Eur. Commission adopted the candidate BRP as Heparin low-molecular-mass for assay BRP batch 11 with assigned anti-Xa activity of 110 IU/mL and anti-IIa activity of 37 IU/mL.

Collaborative study for the establishment of Human immunoglobulin for anticomplementary activity BRP replacement batches 3, 4, 5 and 6.

Human immunoglobulin products are used for the treatment of a number of diseases, such as primary or secondary immunodeficiencies and autoimmune conditions due to the complete absence of antibodies or the production of defective immunoglobulins. Quality control of human immunoglobulin products is essential to ensure therapeutic functionality and safety. This includes testing for Fc function and anticomplementary activity (ACA), as well as verification of appropriate molecular size distribution using size-exclusion chromatography as prescribed in the European Pharmacopoeia (Ph. Eur.) monographs 0338, 0918, 2788 and 1928. To this end, specific biological reference preparations (BRPs) must be used. Stocks of the Ph. Eur. Human immunoglobulin for anticomplementary activity BRP were running low and therefore a collaborative study was run by the European Directorate for the Quality of Medicines & HealthCare (EDQM), under the aegis of the Biological Standardisation Programme, to calibrate replacement batches. Six laboratories, including manufacturers and one Official Medicines Control Laboratory, took part in the study. Several batches of candidate BRPs were calibrated against Ph. Eur. Human immunoglobulin for anticomplementary activity BRP batch 2 to ensure continuity. Based on the study results, the candidate BRPs were adopted by the Ph. Eur. Commission as Ph. Eur. human immunoglobulin for anticomplementary activity BRP batch 3, 4, 5 and 6.

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 calibration of Ph. Eur. Heparin Sodium Biological Reference Preparation batch 4.

An international collaborative study was organised under the aegis of the Biological Standardisation Programme (BSP) of the Council of Europe and the European Union to calibrate a replacement batch for the European Pharmacopoeia (Ph. Eur.) Heparin sodium Biological Reference Preparation (BRP). Seventeen laboratories contributed data to value assign a candidate batch (cBRP4) in International Units (IU) against the WHO 6th International Standard for Unfractionated Heparin using chromogenic and sheep plasma clotting assays according to Ph. Eur. texts 2.7.5. on unfractionated heparin and 0878 on human antithrombin III. The continuity of consecutive batches of BRP was evaluated by including BRP3 in the set of test samples. The central analysis of the study data showed good precision and reproducibility of both chromo-genic and clotting assays among laboratories. Based on the study data, the Ph. Eur. Commission adopted cBRP4 as Ph. Eur. Heparin sodium BRP4 with assigned activities of 985 IU/mL for anti-IIa assays, 995 IU/mL for anti-Xa assays and 1035 IU/mL for sheep clotting assays.

Calibration of the Ph. Eur. human coagulation Factor VIII Concentrate BRP batch 6.

The European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) for human coagulation Factor VIII (FVIII) Concentrate is used as working standard for potency determination of human coagulation FVIII preparations by chromogenic assay. BRP batch 5 was established in 2015 and its stocks were running low. Therefore, the European Directorate for the Quality of Medicines & HealthCare (EDQM) initiated a project (BSP156) for the calibration of a replacement batch. The potency of BRP batch 6 was assigned during an international collaborative study involving 16 laboratories worldwide, with reference to the WHO 8th International Standard (IS) and BRP batch 5. Participants were instructed to perform 3 independent FVIII potency assays following their own routine validated methods for the chromogenic assay, which is the assay prescribed by the Ph. Eur. As an outcome of the study, Ph. Eur. human coagulation FVIII Concentrate BRP batch 6 was assigned a consensus potency of 9.9 IU/ampoule for the chromogenic assay. The Ph. Eur. BRP batch 6 is a freeze-dried, plasma-derived concentrate. Based on accelerated degradation studies, the stability of the material is suitable for a reference preparation. The Ph. Eur. BRP batch 6 was adopted at the 167th session of the Ph. Eur. Commission in June 2020 and is available from the EDQM under product code H0920000.

Establishment of Pertussis toxin BRP batch 2 for CHO clustering assay.

Recently, the Chinese hamster ovary (CHO) cell-based clustering assay replaced the in vivo Histamine Sensitisation Test (HIST) in mice in European Pharmacopoeia (Ph. Eur.) general chapter 2.6.33. 'Residual pertussis toxin' as the recommended method to test for residual pertussis toxin in acellular pertussis vaccine intermediates. To support the standardised CHO clustering assay, availability of a reference standard is critical. Ph. Eur. pertussis toxin Biological Reference Preparation (BRP) batch 1 was first calibrated in International Units in 2008 for the HIST and subsequently also calibrated for the CHO clustering assay in 2017. However, its stocks were dwindling and needed to be replaced. In an effort to maintain adequate supply, a project (BSP141) was initiated by the European Directorate for the Quality of Medicines & HealthCare (EDQM), under the aegis of the Biological Standardisation Programme, to establish a second pertussis toxin BRP (BRP2). Candidate material was manufactured ad hoc by an acellular pertussis vaccine manufacturer and an optimal formulation for long-term stability was defined. Exhaustive in-process and post-production controls demonstrated that the material was fit for its intended purpose and therefore a collaborative study for calibration and stability assessment of the candidate material was organised, which included 10 laboratories worldwide. As a result of the study, the candidate material was established as Ph. Eur. Pertussis toxin BRP batch 2 with a potency of 130 IU/vial for the CHO clustering assay. Unopened vials must be stored at −20°C. The BRP may be used for up to two weeks after reconstitution if appropriately handled and stored at 2­8°C.

Calibration of the WHO 5th IS for Blood Coagulation Factor IX, Concentrate and Ph. Eur. Human Coagulation Factor IX Concentrate Biological Reference Preparation Batch 3 and investigation of the suitability of an IS as potency standard for purified full-length recombinant FIX.

A joint World Health Organization (WHO) - European Directorate for the Quality of Medicines & HealthCare (EDQM) study was run to calibrate the WHO 5th International Standard (IS) for Blood Coagulation Factor IX (FIX), Concentrate, and European Pharmacopoeia (Ph. Eur.) Human Coagulation Factor IX concentrate Biological Reference Preparation (BRP) Batch 3. The suitability of the 4th IS as a potency standard for purified full-length recombinant FIX (rFIX) was also investigated. Forty-nine laboratories contributed data for the calibration of 2 plasma-derived FIX candidates, relative to the 4th IS, from clotting and chromogenic assays. The intra-laboratory variability was reasonably low; the inter-laboratory variation was lower for sample B (14/148) than for sample C (14/162). Although there were no discrepancies between clotting and chromogenic assays, a significantly lower potency was obtained for sample C with clotting assays when buffer rather than FIX-deficient plasma was used as pre-diluent. A significant assay discrepancy was observed with estimates for the 4th IS for Blood Coagulation Factors FII, VII, IX, X, Plasma against the 4th IS, resulting in a clotting to chromogenic activity ratio of 1.11. The study also investigated the comparability of the plasma-derived concentrate standard with the rFIX products and considered the establishment of an IS for rFIX. The 3 rFIX products currently licensed were represented in this study. Data from 49 laboratories for 2 rFIX candidates were received, with additional results for another full-length rFIX test sample returned by 6 laboratories. The intra-laboratory variability when the rFIX samples were assayed against the 4th IS was acceptably low. Although the full-length rFIX could be assayed against the plasma-derived 4th IS and provided statistically valid results, there were large discrepancies among the clotting assays using different APTT reagents. The inter-laboratory variability of the chromogenic assays was similarly high. There were also significant clotting and chromogenic assay discrepancies. The data from the present study indicate that a recombinant standard for rFIX products will minimise assay discrepancies and improve inter-laboratory agreement. However, they also underline that the value assignment of the 1st rFIX IS needs careful consideration. The Expert Committee on Biological Standardization (ECBS) of WHO was therefore not requested to consider the establishment of an IS for rFIX. In order to ensure continued harmonised standards, sample B (14/148) was established as the WHO 5th IS for Blood Coagulation Factor IX, Concentrate, and as Ph. Eur. Human Coagulation Factor IX, concentrate BRP Batch 3 with the functional activity of 10.5 IU/ampoule.

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.

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.

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.

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 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 establishment of erythropoietin BRP batch 5.

The European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) for erythropoietin (EPO) is used as a working standard for potency determination of EPO preparations by in vivo bioassay as prescribed in Ph. Eur. monograph 1316 'Erythropoietin concentrated solution'. BRP batch 4 (BRP4) was calibrated in 2014 and its stocks are depleted. The European Directorate for the Quality of Medicines and HealthCare (EDQM) thus endorsed a project (BSP147) to calibrate a replacement batch in International Units against the 3rd WHO International Standard (IS) for erythropoietin, recombinant, for bioassay (11/170). The amount of material contained in the vial of BRP4 greatly exceeded the amount needed for one bioassay, sometimes leading to considerable waste. It was thus decided to prepare a candidate material with a lower EPO content. The collaborative study involved eight laboratories in Europe, the USA and Australia. Based on the outcome of the study, the Ph. Eur. Commission adopted the proposed standard as Erythropoietin BRP batch 5 in June 2018 for use as a reference preparation solely for the polycythaemic and normocythaemic mouse bioassays, with an assigned potency of 2000 IU/ampoule. Furthermore, the potency of BRP batch 4 was confirmed during the study thus warranting a good continuity of the International Unit.

Calibration of pertussis toxin BRP batch 1 in a standardised CHO cell-based clustering assay.

The European Pharmacopoeia (Ph. Eur.) pertussis toxin (PT) Biological Reference Preparation (BRP) is used as a working standard for safety testing of acellular pertussis vaccines as prescribed in the Ph. Eur. monographs 1356 "Pertussis vaccine (acellular, component, adsorbed)" and 1595 "Pertussis vaccine (acellular, co-purified, adsorbed)". The BRP was calibrated in 2006 in the murine histamine sensitisation test (HIST) against the World Health Organization (WHO) 1st International Standard (IS) for PT. In recent years, there have been increasing efforts to replace the in vivo test with in vitro methods. The Chinese hamster ovary (CHO) cell clustering assay has been used for many years by manufacturers to monitor residual PT activity in detoxified non-adjuvanted bulks. More recently a standardised protocol has been developed for this assay and a PT reference preparation was needed. Due to low stocks, the WHO 1st International Standard for Pertussis Toxin (JNIH-5) needed to be replaced and therefore a joint study between the European Directorate for the Quality of Medicines & HealthCare (EDQM) and WHO was initiated to calibrate the PT BRP for the CHO clustering assay and to replace the IS. The collaborative study involved 14 laboratories from Europe, North America and Asia. The outcome of the study confirmed that the BRP is suitable for use as a reference preparation in the CHO clustering assay. The material was assigned a potency of 1360 IU per vial for the CHO clustering assay.

Collaborative study for the establishment of human immunoglobulin BRP replacement batches.

Human immunoglobulin products are used for the treatment of a number of diseases, such as primary or secondary immunodeficiencies and autoimmune conditions due to the complete absence of antibodies or the production of defective immunoglobulins. Quality control of human immunoglobulin products is essential to ensure therapeutic functionality and safety. This includes testing for Fc function and anticomplementary activity (ACA), as well as verification of appropriate molecular size distribution using size-exclusion chromatography as prescribed in the European Pharmacopoeia (Ph. Eur.) monographs 0338, 0918, 2788 and 1928. To this end, specific biological reference preparations (BRPs) must be used. Stocks of these BRPs were running low and therefore a collaborative study was run by the European Directorate for the Quality of Medicines & HealthCare (EDQM), under the aegis of the Biological Standardisation Programme, to calibrate replacement batches. Seventeen laboratories, including manufacturers and Official Medicines Control Laboratories, took part in the study. Several batches of candidate BRPs were calibrated against human immunoglobulin (ACA and molecular size) BRP batch 1 and human immunoglobulin (Fc function and molecular size) BRP batch 1 to ensure continuity. Based on the study results, the candidate BRPs were adopted by the Ph. Eur. Commission as Ph. Eur. human immunoglobulin for anticomplementary activity BRP batch 1 and batch 2, Ph. Eur. human immunoglobulin for Fc function BRP batch 1 and batch 2 and Ph. Eur. human immunoglobulin (molecular size) BRP batch 2 and batch 3.

Establishment of the Ph. Eur. Hepatitis A virus RNA for NAT testing BRP batch 1.

Detection of viral contamination in plasma donations is critical to prevent transmission of infectious diseases. The European Pharmacopoeia (Ph. Eur.) monograph 1646 'Human plasma (pooled and treated for virus inactivation)', requires that plasma pools used for the manufacture of this product be tested, among others, for the presence of hepatitis A virus RNA by nucleic acid testing (NAT) using a positive control containing 100 International Units (IU) of hepatitis A virus (HAV) RNA per mL. To this end, the European Directorate for the Quality of Medicines & HealthCare (EDQM, Council of Europe) organised an international collaborative study under the aegis of the Biological Standardisation Programme, for the establishment of the 1st Biological Reference Preparation (BRP) for HAV RNA for NAT testing. A freeze-dried candidate material was thus prepared and calibrated against the WHO 2nd International Standard for HAV for NAT (00/562) in a study in which thirteen European and North American laboratories including Official Medicines Control Laboratories (OMCLs), manufacturers of plasma-derived products, producers of in vitro diagnostic kits and a blood transfusion centre participated. Based on the outcome of the study, an HAV RNA content of 40 000 IU/vial (corresponding approximately to 4.6 log10 IU/vial) was assigned to the BRP, which was adopted by the Ph. Eur. Commission in March 2016 as Ph. Eur. hepatitis A virus RNA for NAT testing BRP batch 1.