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This paper describes a double antigen ELISA (DAE) for rapid, specific and reliable assessment of the antitetanus immune status of horses and sheep. Compared with the indirect ELISA, the double antigen ELISA has the advantage of species-independent testing of sera. Thanks to its test design, it is more specific since the detected antibodies are forced to bind tetanus toxoid twice. In addition, it is very sensitive to tetanus antibodies, enabling the detection of low antibody titres, in range which is relevant for the assessment of the protective status (tetanus toxin neutralising antibodies). The detection limit of the DAE for tetanus antibodies is in the order of 10(-4) EU/ml. A comparison of in vitro results of individual sera with in vivo titres showed that horse sera with titres of 0.04 and 0.05 EU/ml in the DAE showed titres of > 0.05 IU and 0.034 IU/ml respectively during in vivo testing thus indicating good agreement. For tested sheep sera which were rated > 0.05 IU/ml in vivo, the corresponding titre in the DAE was 0.24 EU/ml. Clear tetanus antitoxin establishment of protective ELISA limits requires further comparative examination of sera with low titres (< 1.0 EU/ml) in the double antigen ELISA and the toxin neutralisation test. With the double antigen ELISA, efficacy can be determined for marketing authorisation procedures of tetanus vaccines ad us. vet. As a consequence, the toxin neutralisation test (still being the standard method of choice for quantifying tetanus toxin neutralising antitoxin titres) could be replaced, since it requires too great a number of animals per test and involves considerable suffering for the animals. The test described here reduces the use of mice and guinea pigs within vaccine efficacy testing. In addition, it involves less exposure of the laboratory personnel to toxin.

Treatment of respiratory allergies can be performed with allergen-specific immunotherapy using allergen extracts. These products are biologicals with an extremely complex and variable composition. Only a few components are of major importance for the disease, the so-called major allergens. At present, standardisation of allergen extracts is dominated by techniques that aim at establishing their overall IgE-binding potencies using pooled sera of allergic patients. Each company in the market uses its own type of units to express potencies, thus hampering comparability. Another disadvantage is that the major allergen composition is not determined. Most companies have introduced assays for the measurement of major allergens in their quality control systems, but these data are not yet used for labelling purposes. The need to include major allergen content in standardisation protocols is now widely accepted. To support future labelling on the basis of major allergen content the European Union has funded the multidisciplinary multicentre project CREATE. This project aims at developing international certified references for the most important major respiratory allergens and at evaluating the performance of available ELISA for their measurement. The project will facilitate expression of potencies by active ingredient (major allergen) content and will allow direct comparison of competitor products.

An International Collaborative Study was organized to replace the current World Health Organization (WHO) International Standard (IS) for Prekallikrein Activator (PKA) and to establish a European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP). The project was jointly organized by the European Directorate for the Quality of Medicines (EDQM) and the National Institute for Biological Standards and Control (NIBSC) to identify and calibrate suitable materials that could act as an IS and a Ph. Eur. BRP. The current IS for PKA (82/530) is popular and stocks are declining rapidly, therefore necessitating calibration of a replacement. A Ph. Eur. BRP is needed, as PKA control on the finished product is part of the Official Control Authority Batch Release (OCABR) of Human Albumin. The current IS, 82/530 is a 5 per cent albumin solution spiked with purified PKA. However, during planning stages it was decided that the replacement IS (and BRP) should be made from a 20 per cent albumin preparation containing a significant level of PKA as the current IS is used to measure PKA in albumin and high levels are more likely to be encountered in more concentrated 20 per cent solutions. A suitable material was sourced by the EDQM and filled into ampoules at NIBSC and vials by the EDQM. Both preparations were included in the collaborative study that involved 31 laboratories from 17 countries. Another important goal of this study was to investigate the influence of the prekallikrein substrate (PKS) on PKA determination in albumin solutions following earlier concerns that variability amongst PKS prepared in-house could significantly affect PKA determinations. Laboratories were requested to perform their routine assays following Ph. Eur. guidelines and recommendations on doses, replication and randomization were also provided to study participants. Participants were requested to use material A (the current IS, 82/530) to perform at least 4 assays to determine PKA levels in sample B (NIBSC ampouled material, candidate IS, 02/168), sample C (EDQM material in vials candidate Ph. Eur. BRP Batch 1), and sample D (an ampouled preparation of 2.5 per cent albumin containing a lower level of PKA). A commercial substrate was provided for participants to perform half the assays and the remaining assays were to be performed using the laboratories' in-house substrate (where available). Collation of participants' results showed that samples B and C had the same level of PKA of 29 IU/ampoule, the concentration anticipated from development studies. Importantly, there was no significant difference between the PKA level obtained using the commercial substrate provided and the laboratories' own in-house substrate. Previous observations on lyophilized preparations of PKA indicate that the enzyme is very stable. Detailed investigations conducted in this study show that the PKA in albumin used to make samples B and C is very stable and suitable for long-term storage as a r

Thirty laboratories participated in a collaborative study to calibrate replacements for the 1st International Standard for Low Molecular Weight Heparin and the European Pharmacopoeia Low-molecular-mass heparin for assay Biological Reference Preparation. Two freeze-dried materials and one liquid preparation were included in the study. All three samples gave excellent intra- and inter-laboratory variations (majority of mean % geometric coefficient of variation < 10 %) when assayed against the 1st International Standard by both anti-Xa and anti-IIa assays. There were no major differences found between potency estimates using all methods and that obtained using European Pharmacopoeia method only. Overall, this study showed that the differences between the candidates are marginal. Based on the results of the study Sample B, 01/608 was established as the 2nd International Standard for Low Molecular Weight Heparin. Sample A, 01/592 and sample C, the liquid preparation, were established as replacements for the European Pharmacopoeia 'Low-molecular-mass heparin for assay' Biological Reference Preparation.

A quantification assay for the Haemagglutinin-Neuraminidase (HN) protein of Newcastle Disease Virus (NDV) has been developed at CIDC-Lelystad as a candidate in vitro potency test for inactivated Newcastle disease (ND) vaccines. In studies performed at CIDC-Lelystad, a high correlation was demonstrated between the results of this candidate in vitro potency assay and the results of the serological potency assay (European Pharmacopoeia monograph 0870; test A). Furthermore, a high correlation between the serological data (Haemagglutination Inhibition-antibody titres) and clinical protection after challenge was demonstrated. Correlation between in vivo and in vitro potency assays was confirmed in a collaborative pre-validation study. In the pre-validation study three Official Medicines Control Laboratories (OMCLs) determined both the NDV-HN antigen content and the in vivo potency (vaccination-serology and vaccination-challenge) of 6 vaccine batches. The conclusion of the pre-validation study was that a large-scale collaborative study should be organised to validate the in vitro method and the suitability of the reference preparation. This report describes the outcome of this study. In brief, 14 laboratories (8 OMCLs and 6 vaccine manufacturers) determined the NDV-HN antigen content of 9 different vaccines in 3 independent tests. The vaccine batches were produced by 5 different manufacturers and represent a quantitative range of ND antigen content. One vaccine batch with insufficient potency and one poultry vaccine not containing NDV were included. Statistical evaluation of the results indicated that the antigen content could be determined with high precision. A good repeatability as well as reproducibility was found. Furthermore all laboratories found a similar ranking of the vaccines, based on the antigen content. Comparison of the antigen content and the in vivo potency of a series of vaccines with relatively low potencies indicated that a threshold relative antigen level of 7.0 antigen units per dose would discriminate between vaccine batches with sufficient and insufficient potency. An in vitro assay with this threshold level for antigen content did not result in any false positive results and only a limited number of false negative results in the BSP055 study. We conclude that the in vitro measurement of the antigen content of inactivated ND-vaccines with the proposed method is a reliable alternative potency assay that could be included as a new method in monograph 0870 on ND-vaccines.

9 laboratories from 7 countries including both laboratories from the public and private sector participated in a collaborative study organised under the aegis of the European Directorate for the Quality of Medicines Biological Standardisation Programme in order to establish batch 4 of the European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) for rabies vaccine (inactivated) for veterinary use. Establishment of Ph. Eur. BRP batch 4 was necessary in order to replace Ph. Eur. BRP batch 3, the stocks of which were dwindling. 8 laboratories provided results. Ph. Eur. BRP batch 4 was calibrated against the 5th International Standard for inactivated rabies vaccine in International Units (IU) using the vaccination challenge method of the Ph. Eur. monograph 0451. The International Standard (IS), Ph. Eur. BRP batch 4 and batch 3 are all freeze-dried vaccines prepared by beta-propiolactone inactivation of the Pitman Moore strain of rabies. Based on the results of the study, a potency of 11 IU/vial was assigned to Ph. Eur. BRP batch 4 for rabies vaccine (inactivated) for veterinary use. Nevertheless, it was noted that the vaccination challenge assay used as the "golden standard" for potency determination of inactivated rabies vaccines for veterinary use is a crude assay requiring the use of a large number of animals. Evidence from this study and from the collaborative study to establish Ph. Eur. BRP batch 3 suggests that the assay is difficult to perform and provides highly variable results. The validation of a suitable in vitro alternative is therefore highly recommended, as is the possible improvement of the in vivo assay, which will most likely remain the "golden" standard.

The preparation and establishment of the 2nd European Pharmacopoeia (Ph. Eur.) Biological Reference Preparation (BRP) for erythropoietin was the goal of a project run within the framework of the European Biological Standardisation Programme. The project, coded BSP062, was carried out between October 2002 and July 2003. The candidate preparation (cBRP2) was prepared in a similar manner to the first BRP batch (BRP1), as follows: -50:50 (weight/weight) blending of the two erythropoietin preparations currently available on the European market (epoietin-alpha and epoietin-beta), -lyophilisation using a protein-free carrier formulation to allow use of the standard for both biological and physico-chemical assay methods, -each vial contains approximately 250 microg erythropoietin. The cBRP2 was analysed in a collaborative study, carried out with the following aims: -to calibrate cBRP2 by in vivo bioassay in terms of the International Standard for erythropoietin, and assign a unitage, -to demonstrate continuity of unitage with BRP1, -to evaluate the suitability of cBRP2 to serve as a reference material for physico-chemical tests of erythropoietin. The collaborative study involved 14 laboratories both from Europe, and from Australia, Canada, South-Korea and the United States of America. Participants carried out biological and physicochemical assays on the candidate BRP batch 2, using BRP 1 and the 2nd World Health Organization (WHO) International Standard (IS) for recombinant erythropoietin as the reference standards. It was demonstrated that: -an assigned potency of 32,500 U per vial would maintain continuity between BRP1 and BRP2 in terms of the IS for erythropoietin, -the replacement batch was appropriate for use as erythropoietin BRP in the context of the control of erythropoietin concentrated solutions according to the Ph. Eur. monograph 1316. In July 2003, the Ph. Eur. Commission established the proposed standard as 'Erythropoeitin BRP batch 2' for use as a reference preparation for the polycythaemic and normocythaemic mouse bioassay, with an assigned potency of 32,500 U/vial, the identification by capillary zone electrophoresis (CZE), by polyacrylamide gel electrophoresis, immunoblotting and peptide mapping and as a reference for checking the system suitability of size exclusion chromatographic procedures used in the test for 'Dimers and related substances of higher molecular mass'.

In the European Pharmacopoeia, the monographs on somatropin, somatropin bulk solution and somatropin for injection prescribe a number of tests including "related substances", "dimer and related substances of higher molecular weight" and "isoform distribution" which are intended to control the levels of impurities in the substance. The aim of this study was to verify the robustness of a new method by capillary electrophoresis and to compare its performance with that of the existing test for "isoform distribution" by isoelectric focusing. It was demonstrated that the capillary electrophoresis method was superior to the method of isoelectric focusing. The interest of the CZE method consists in the resolution of related impurities that might be process specific and/or generated by the expression system.

Human Growth hormone (hGH, somatotrophin) is a 22 kDa, 191 amino-acid single chain protein produced by somatroph cells of the anterior pituitary gland. It is the major physiological regulator of growth, and deficiencies in growth hormone levels have long been recognized as the underlying cause of growth disorders (dwarfism). The ability of exogenous hGH to restore normal rates of growth in both human and animal models of growth retardation has long been recognized and the use of hGH in therapy goes back several decades. Initial preparations were prepared by extraction and purification from cadaveric pituitary tissue, and since 1984, hGH has been prepared by recombinant Deoxyribosenucleic acid (rDNA) technology. As is usually the case with "biologicals", characterization of the drug substance depended on a combination of physico-chemical and biological methods, and the hGH molecule became well characterized fairly early in its life as a drug. Indeed, by 1980 the major degradation forms and structural variants of the hGH molecule had been described and reviewed. Little satisfactory progress had been made in refining biological assays for hGH, and, although in vitro assays were described, potency-defining assays remained dependant on the whole body growth response in rats, and were both invasive and imprecise. In the early 1990's a series of collaborative studies on analysis of recombinant hGH (somatropin) established that available bioassays were much less selective that physico-chemical methods in detecting and quantifying structural degradation, and 1994 saw an international consensus to replace the bioassays with physico-chemical analytical methods for the routine batch release of somatropin. During the last decade in most markets somatropin has, unusually for a protein, been subject to batch release and control dependent entirely on physico-chemical analysis, without the routine use of any form of bioassay. During that time there has been a continuous development and refinement of methods, and the identification of a range of structural variants and degradation products of the molecule. The present review sets out to summarise the current knowledge on physico-chemical analytical methods for somatropin, and the structural variants that have been identified and characterized. A survey of available biological analytical methods is beyond the scope of this review, as is consideration of the earlier pituitary preparations and the recombinant 192 amino-acid methionyl form of the molecule (somatrem), although it is likely that many of the methods and variants described would be equally applicable to somatrem.

Phase I of BSP034 collaborative study was extended in two laboratories to include correlation of serology with in vivo toxin neutralisation test (TNT) using 2 separate sets of 20 serum pools, produced in-house. The study investigated the extent to which the in vitro methods for diphtheria antibodies, Vero cell assay and diphtheria enzyme-linked immunosorbent assay for diphtheria antitoxin (D-ELISA), can detect neutralising antibodies by comparison with TNT in guinea pigs. The study was also performed to compare the antibody neutralising potency obtained in relation to guinea pig (GP) or equine (DI) antitoxin standard. In addition, the study provided an opportunity to compare ELISA for tetanus antitoxin (T-ELISA) and TNT assay for detection of anti-tetanus antibodies, from the same set of serum pools. The data obtained show that antitoxin potency obtained by Vero cell assay, D-ELISA and T-ELISA using the same GP standard, highly correlated with neutralising potency as determined in respective TNT assays. Vero cell assay with DI provided estimates that also correlated with neutralising potency, but were of significantly lower titre. Since reference to DI standard is widely used in serodiagnosis, as well as in clinical studies where diphtheria antitoxin titres obtained in the Vero cell method are taken as surrogate markers for vaccine efficacy, it should be investigated if a similar difference is also observed for human serology.