Collaborative study for the establishment of Ph. Eur. Biological Reference Preparation for Human tetanus immunoglobulin batch 2.

This publication describes the outcome of a project to develop a replacement European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) for Human tetanus immunoglobulin (TIg) as well as for the World Health Organization (WHO) International Standard (IS) for Tetanus Immunoglobulin, Human. Bulk TIg was kindly provided by a European manufacturer and was used to prepare the candidate standard. The candidate standard was freeze-dried and calibrated in an international collaborative study jointly co-ordinated by the Medicines & Healthcare products Regulatory Agency (MHRA) and the European Directorate for the Quality of Medicines & HealthCare (EDQM, Council of Europe). The results of this study show that there was good agreement between laboratories for the potency estimates obtained for the candidate standard relative to the current WHO IS/Ph. Eur. BRP. The study also demonstrated that the candidate standard is suitable for use in Ph. Eur. assays for potency testing of TIg products and there was good agreement in the potency estimates obtained using the different assay methods included in the study. Accelerated degradation studies performed at the MHRA over a period of 4 years suggest that the freeze-dried candidate standard will be very stable. The candidate standard was established as Ph. Eur. BRP for Human tetanus immunoglobulin, batch 2 with an assigned potency of 45 IU/ampoule. The same preparation was also adopted by the WHO Expert Committee on Biological Standardization (ECBS) to serve as the WHO 2nd IS for Tetanus Immunoglobulin, Human (13/240).

Collaborative study for the establishment of Ph. Eur. Biological Reference Preparation for Human tetanus immunoglobulin batch 2.

This publication describes the outcome of a project to develop a replacement European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) for Human tetanus immunoglobulin (TIg) as well as for the World Health Organization (WHO) International Standard (IS) for Tetanus Immunoglobulin, Human. Bulk TIg was kindly provided by a European manufacturer and was used to prepare the candidate standard. The candidate standard was freeze-dried and calibrated in an international collaborative study jointly co-ordinated by the Medicines & Healthcare products Regulatory Agency (MHRA) and the European Directorate for the Quality of Medicines & HealthCare (EDQM, Council of Europe). The results of this study show that there was good agreement between laboratories for the potency estimates obtained for the candidate standard relative to the current WHO IS/Ph. Eur. BRP. The study also demonstrated that the candidate standard is suitable for use in Ph. Eur. assays for potency testing of TIg products and there was good agreement in the potency estimates obtained using the different assay methods included in the study. Accelerated degradation studies performed at the MHRA over a period of 4 years suggest that the freeze-dried candidate standard will be very stable. The candidate standard was established as Ph. Eur. BRP for Human tetanus immunoglobulin, batch 2 with an assigned potency of 45 IU/ampoule. The same preparation was also adopted by the WHO Expert Committee on Biological Standardization (ECBS) to serve as the WHO 2nd IS for Tetanus Immunoglobulin, Human (13/240).

Assay discrepancies using human coagulation factor VIII chromogenic kits: Results from a plasma-derived factor VIII collaborative study (BSP112).

Chromogenic assay discrepancies were reported at General European Official Medicines Control Laboratories Network (GEON) meetings by laboratories testing FVIII-products. The objectives of the present investigation were to carry out a controlled collaborative study to examine these reports and to delineate the reasons for these discrepancies by assessing affected and unaffected FVIII products. The laboratories followed a strict study protocol, which included assessing their own individual observed factor X (FX) activation times, i.e. the time to reach 50% of maximal FX activation (T1/2), for each chromogenic kit. This measurement was used, in parallel with the kit manufacturers' prescribed FX activation times, to assess the performance of the chromogenic potency assays on FVIII test products. This study confirmed a significant discrepancy between Coatest® and Coamatic® kits and between Siemens and Coamatic® kits when the kit manufacturers' prescribed T1/2 incubation times were followed. Coamatic® kits tended to produce higher potencies than the Coatest® or Siemens kits. Furthermore, FX activation assays revealed marked differences between individual laboratories for all three chromogenic kits in the observed T1/2 incubation times, which also did not correspond to the prescribed T1/2 incubation times. The resulting differences in potency between kits, in some cases, were significantly reduced when using the actual observed T1/2 incubation times instead of the prescribed T1/2 incubation times. The study showed that FVIII potency discrepancies can occur between chromogenic kits. To compensate for this, laboratories should ideally perform FX activation curves for each new chromogenic kit in order to determine the correct observed T1/2 incubation times, which can then be used to determine FVIII potencies in therapeutic concentrates.

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.

Development and verification of an enzyme-linked immunosorbent assay for the quantification of toxoid A and toxoid B from Clostridioides difficile.

Clostridioides difficile (C. difficile) is the most common cause of nosocomial antibiotic associated diarrhoea. The incidence of C. difficile infection (CDI) has been rising worldwide over the last 20 years with consequent rises in morbidity, mortality and healthcare costs, although the incidence has fallen in the UK over the last few years. Confirmation of diagnosis and early intervention are critical to the management of CDI. The standard treatment for CDI is the administration of antibiotics. However, vaccination has been recognized as the most cost-effective treatment for the prevention and possible long-term protection against CDI episode. There are several promising vaccine candidates in various stages of development. Many of these vaccines have displayed good efficacy for CDI under laboratory conditions or in clinical trials. With the emergence of vaccines against C. difficile, here we describe the development and verification of an Enzyme Linked Immunosorbent Assay (ELISA) that can be used for the quality control testing of candidate vaccines against C. difficile through the measurement of vaccine antigen content. Verification of the assay was performed by assessment of specificity, sensitivity, intermediate precision and relative accuracy. The ELISAs were specific for the toxoids being detected and the detection limit of the assay for toxoid A was 4.88 ng/mL and 3.91 ng/mL for toxoid B. The geometric coefficients of variation for intermediate precision did not exceed 25% and relative accuracy was within 77-130%. We therefore conclude that the ELISA described here is sufficiently sensitive, specific, precise and accurate for use for the quality control testing of candidate C. difficile vaccines.

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.

The effect of increasing the sulfation level of chondroitin sulfate on anticoagulant specific activity and activation of the kinin system.

Oversulfated chondroitin sulfate (OSCS) was identified as a contaminant in certain heparin preparations as the cause of adverse reactions in patients. OSCS was found to possess both plasma anticoagulant activity and the ability to activate prekallikrein to kallikrein. Differentially sulfated chondroitin sulfates were prepared by synthetic modification of chondroitin sulfate and were compared to the activity of OSCS purified from contaminated heparin. Whilst chondroitin sulfate was found to have minimal anticoagulant activity, increasing sulfation levels produced an anticoagulant response which we directly show for the first time is mediated through heparin cofactor II. However, the tetra-sulfated preparations did not possess any higher anticoagulant activity than several tri-sulfated variants, and also had lower heparin cofactor II mediated activity. Activation of prekallikrein was concentration dependent for all samples, and broadly increased with the degree of sulfation, though the di-sulfated preparation was able to form more kallikrein than some of the tri-sulfated preparations. The ability of the samples to activate the kinin system, as measured by bradykinin, was observed to be through kallikrein generation. These results show that whilst an increase in sulfation of chondroitin sulfate did cause an increase in anticoagulant activity and activation of the kinin system, there may be subtler structural interactions other than sulfation at play given the different responses observed.

An International reference reagent for the detection of anti-human neutrophil antigen-1a.

BACKGROUND AND OBJECTIVES: Testing for neutrophil antibodies has become more common as awareness of transfusion-related acute lung injury (TRALI) has increased. However, unlike other areas of blood cell antibody testing, there are no certified reference reagents available with which laboratories can determine the sensitivity of detection of their assays. This report describes the production and evaluation of a freeze-dried preparation of human plasma, code 09/284, containing anti-human neutrophil antigen-1a (anti-HNA-1a) for use as a minimum sensitivity reagent. MATERIALS AND METHODS: One-millilitre of aliquots of plasma containing anti-HNA-1a were freeze-dried in glass ampoules. To characterize the material, 24 laboratories took part in an international collaborative study. The participants evaluated doubling dilutions of the material using their in-house routine assays and recorded the highest dilution in which the antibody could be detected. RESULTS: When diluted 1 in 4, most laboratories were able to detect the anti-HNA-1a in the material, and the participants agreed that this was an appropriate level to set as the minimum sensitivity required. CONCLUSIONS: In October 2011, the WHO Expert Committee on Biological Standardization approved the material 09/284 as an International Reference Reagent for the detection of anti-HNA-1a.

International reference reagents to standardise blood group genotyping: evaluation of candidate preparations in an international collaborative study.

BACKGROUND AND OBJECTIVES: The aim of the study was to evaluate, in an international collaboration, four lyophilised genomic DNA preparations, selected from genotyped and phenotyped donors by the study organisers, for their suitability to standardise and control blood group genotyping procedures for common ancestral Caucasian and Black African alleles. MATERIALS AND METHODS: Twenty-nine laboratories performed 'blind' testing of replicated ampoules of the candidate reference reagents, RBC1 (10/232), RBC4 (10/236), RBC5 (10/238) and RBC12 (10/234), using a range of genotyping procedures, most commonly classical PCR using allele or sequence specific primers. RESULTS: The majority of laboratories reported blood group genotypes in accordance with those determined by the study organisers and the serological phenotypes. Despite an overall high level of accuracy in genotyping, the identified errors and inconsistencies, and the limited genotyping capabilities of many laboratories, confirmed the need for validated reference materials to control test procedures. CONCLUSIONS: The establishment of RBC1, RBC4, RBC5 and RBC12 as World Health Organization Reference Reagents will facilitate international standardisation of blood group genotyping and ensure that such tests are sufficiently sensitive and specific.

An international reference reagent for the detection of RHD and SRY DNA in plasma.

BACKGROUND AND OBJECTIVES: The determination of foetal RHD genotype using foetal DNA contained in the maternal circulation is increasingly used to manage pregnancies at risk of haemolytic disease of the foetus and newborn (HDFN) caused by maternal anti-D. The test is becoming increasingly reliable, and routine clinical services have been established in some centres. However, laboratories currently have no reference materials with which to determine the performance of their tests. This report describes the production and evaluation of a freeze-dried preparation of human plasma, code 07/222, containing RHD and SRY sequences which can be used as a minimum sensitivity reagent. MATERIALS AND METHODS: RhD-positive male plasma was diluted in an excess of RhD-negative female plasma, and 1 ml aliquots were freeze-dried in glass ampoules. To characterise the material, 19 laboratories took part in an international collaborative study. The participants evaluated dilutions of the material using their in-house routine assays and recorded the highest dilution where the genes could be detected. RESULTS: When diluted 1 in 2, most laboratories were able to detect the presence of RHD and SRY sequences in the material and the participants agreed that this was an appropriate level to set as the minimum sensitivity required. CONCLUSIONS: In October 2010, the WHO Expert Committee on Biological Standardization approved the material 07/222 as an International Reference Reagent for the detection of RHD and SRY DNA in plasma.

An international collaborative study to establish the WHO 1st international standard for alpha-1-antitrypsin.

BACKGROUND AND OBJECTIVES: The aim was to establish the 1st International Standard (IS) for alpha-1-antitrypsin (AAT) to standardise potency assignment of therapeutic products, calibrated in moles and mg active AAT in line with product labelling practice. Assigning total protein and antigen values to the IS was also investigated. MATERIALS AND METHODS: The active concentration of four candidate AAT preparations was determined in an international collaborative study by inhibition of trypsin (calibrated by active-site titration). Total protein and antigen content were determined for each candidate using local methods and in-house standards, and a common AAT preparation. The total protein content of the IS was also determined by amino acid analysis. Potency determination of recombinant and transgenic materials against the IS was investigated in a follow-up study. RESULTS: Data analysis for potency determination indicated no statistical difference between any of the candidates, or between the results for recombinant and plasma-derived products. Total protein content of the IS determined by amino acid analysis was consistent with the potency value. The variability in the total protein and antigen results for the other candidates was reduced when the data were recalculated relative to the IS. CONCLUSIONS: Candidate C (05/162) was established by the WHO Expert Committee on Biological Standardization (ECBS) in 2006 as the WHO 1st IS for AAT with a potency of 243 nmoles (12·4 mg) active inhibitor per ampoule. In 2008, ECBS approved the IS for potency determination of recombinant material and assigned a total protein and antigen value of 12·4 mg.

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

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

Phrenic nerve-hemidiaphragm as a highly sensitive replacement assay for determination of functional botulinum toxin antibodies.

Botulinum neurotoxins induce a prolonged muscle paralysis by specifically blocking the release of neuronal transmitters from peripheral nerve junctions. Potency testing of toxin and antitoxin therapies is entirely dependent on mouse lethality bioassay which is associated with extreme suffering of large numbers of animals to ensure high precision. The mouse phrenic nerve-diaphragm assay is an ex vivo assay that closely mimics in vivo respiratory paralysis offering substantial refinement and reduction in the number of animals used. A range of botulinum antitoxin standards, one licenced product and experimental antitoxins were tested for neutralising potency using ex vivo hemidiaphragm assay and compared with in vivo determined activities. Overall, there was an excellent agreement between neutralising activity detected by the two assay systems and for each toxin serotype using only 4-7 replicates for each product (almost perfect concordance for type A antitoxins: ρ = 0.997, and substantial concordance for type B antitoxins: ρ = 0.991 and type E antitoxins: ρ = 0.964, respectively). These findings confirm that the mouse nerve-diaphragm preparation can provide a functional ex vivo replacement assay for specific, sensitive and precise assessment of toxin and antitoxin activity.