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 establishment of replacement batches of Ph. Eur. Heparin Low-Molecular-Mass for Calibration CRS.

An international collaborative study was run within the framework of the Biological Standardisation Programme (BSP) of the Council of Europe and the Commission of the European Union to establish replacement batches for European Pharmacopoeia (Ph. Eur.) Heparin Low-Molecular-Mass (LMM) for calibration Chemical Reference Substance batch 3 (CRS3) used for the characterisation of LMM heparins by high performance size-exclusion chromatography. Two candidate batches (A, cCRS4 and B, cCRS5) were filled using the same material as the existing official calibrants, adopted with either an assigned number-average molecular mass (Mna) or a broad standard table (BST). Fifteen laboratories evaluated the suitability of these candidate batches for use as calibrants with the pharmacopoeial dual refractive index/ultraviolet (RI/UV) detector calibration method, as well as with a modified mobile phase and the BST calibration method. Seven preparations of LMM heparin were tested. The results confirmed that the proposed batches are suitable for use with the same characteristic Mna as CRS3 and with the BST established for the World Health Organization (WHO) 2nd International Standard (IS). The BST calibration method gave comparable results to the RI/UV method, while showing better reproducibility, being easier to perform and requiring no calibrant with UV absorbance. The modified mobile phase had no impact on the calculated values while improving separation between the calibrant and salt peaks. The two candidate batches were adopted as Ph. Eur. Heparin LMM for calibration CRS batches 4 and 5, respectively, with the assigned Mna value of 3800 and a BST. In anticipation of the depletion of the calibrant required for use with the RI/UV method, and taking into account the unlikely procurement of a new lot of suitable starting material, it was recommended to include the BST method in Ph. Eur. monograph 0828, Heparins, low-molecular-mass. In order to improve peak separation, it was also recommended to include the use of ammonium acetate solution as mobile phase in the monograph, both for the Ph. Eur. RI/UV and the proposed BST calibration methods. Further to this study, Ph. Eur. monograph 0828 was revised to replace the RI/UV method by the BST method. This contributed to the harmonisation of methods across regions, thereby facilitating a concerted global action for the development and establishment of the next batches of calibrants for the quality control of LMM heparins.

Validation of a qPCR method for determination of viral genome titres of AAV2-based vector preparations.

The viral genome titre is universally used for the dosing of adeno-associated virus (AAV)-based vectors used for gene therapy. To standardise this determination, the development of a common method would be valuable to facilitate comparison of viral doses used in the clinic and in the subsequent quality control of the products. A collaborative study was initiated by the Gene Therapy Working Group of the General European Official Medicines Control Laboratories Network in order to validate a qPCR-based method targeting the ITR2 sequence common to a broad variety of AAV vectors, independently from the serotype of the capsid or from the specific transgene. Five preparations of AAV vectors from various serotypes, including the AAV2/2 (RSS2) and AAV2/8 (RSS8) Reference Standard Stocks (American Type Culture Collection, USA) were used in the study. A plasmid carrying the ITR2 sequence was used to prepare standard curves. Its digestion outside the ITR regions facilitated melting of the hairpin ITR sequence during PCR, allowing better accessibility to the DNA polymerase. The results show that this qPCR method is satisfactory in terms of accuracy and precision. The reproducibility is also acceptable when compared with other similar studies, as it was shown previously that titres obtained by qPCR generally show higher inter-laboratory variability. The use of RSS2 or RSS8 as normalisation control in each assay demonstrated a promising help to identify potential sources of variation in a given laboratory or to smooth out inter-laboratory variations, thus improving reproducibility.

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.

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

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

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.

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.

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.

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.

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.

Consideration on a few aspects of the stability studies post licensure.

Stability studies represent a significant workload for both manufacturers and regulatory reviewers and therefore a careful selection of the study design and of the stability indicator test is required to make sure that the study will provide the relevant information. Moreover, alternatives to the existing regulatory approaches should be favoured in order to better or quicker analyze the impact of a specific change. In this article, examples of alternative approaches are given. For the stability monitoring study, emphasis is put on the bulk stability program. Concerning comparability stability study, two case studies illustrating how accelerated stability studies could be used to support manufacturing changes are presented.