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.

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.

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 2–8°C.

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.

Erythropoietin (EPO) is a monomeric, highly glycosylated, protein hormone (molecular size around 30-35 kD), produced mainly in adult kidneys, which acts principally on red blood cell progenitors and precursors to promote red cell production. Therapeutic EPO products are widely used biotherapeutics. They are mainly produced by recombinant DNA technology in mammalian cells and their biological activity is closely linked to the degree of N-glycan sialylation. Determination of the sialic acids\' content and complexity by glycan mapping therefore appears critical to ensure the quality and efficacy of the EPO therapeutic products. The European Directorate for the Quality of Medicines & HealthCare organised a study (BSP144) under the aegis of the Biological Standardisation Programme to assess N-glycan mapping tests with the aim of incorporating a standard method into the European Pharmacopoeia monograph \'Erythropoietin concentrated solution\' (1316). The use of a \'reagent panel\' consisting of six EPO preparations with a range of iso-electric properties facilitated comparison between laboratories and methodologies. Based on the study results, a robust and repeatable HPAEC-PAD chromatographic method was identified and work to introduce it in the monograph as an example method has been initiated.

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.

The European Pharmacopoeia (Ph. Eur.) monograph 1316 \'Erythropoietin concentrated solution\' prescribes that the dimer content of therapeutic erythropoietin (EPO) preparations must not exceed 2% as determined by Size-Exclusion Chromatography (SEC). This report describes the evaluation of a candidate Chemical Reference Substance (cCRS) to serve as system suitability reference material for the qualification of SEC systems used to assess dimer and oligomer content in EPO solutions. The study organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM) was performed with the participation of six European laboratories which tested the candidate material and the EPO for physicochemical tests CRS batch 1. The candidate material was shown to be a suitable reference material for the determination of the resolving capability of the SEC system for separation of dimer and higher oligomers from monomeric EPO. The cCRS was adopted by the Ph. Eur. Commission as Erythropoietin for SEC system suitability CRS batch 1 following consideration of the report. The importance of the resolving capability of the SEC system, as defined by the peak ratios or the peak-to-valley resolution, together with the asymmetry of the peaks eluted, and the linear response of the UV detector were all seen as critical parameters. Therefore, the monograph Erythropoietin concentrated solution (1316) was revised concomitantly to take account of the CRS and to set acceptance criteria for these critical parameters..

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.

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.

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.