An international collaborative study of the effect of active pertussis toxin on the modified Kendrick test for acellular pertussis vaccines.

Speculation that the Japanese modified intra-cerebral challenge assay, which is used in several countries for control of acellular pertussis vaccines, depends on the presence of small amounts of active pertussis toxin led to an assumption that it may not be appropriate for highly toxoided or genetically detoxified vaccines. Consequently, at the recommendation of a World Health Organisation AD Hoc Working Group on mouse protection models for testing and control of acellular pertussis vaccine, the effect of pertussis toxin on the modified intra-cerebral challenge assay (modified Kendrick, MICA) was evaluated in an international collaborative study. Results of this study showed that for genetically detoxified vaccines both with and without active pertussis toxin the MICA clearly distinguished mice vaccinated with acellular vaccines from unvaccinated mice and gave a significant dose-response relationship. However, vaccine samples containing active pertussis toxin (5 or 50 ng/single human dose) appeared to be more potent than the equivalent sample without active pertussis toxin. Similar results were also given by two respiratory infection models (intranasal and aerosol) included in the study. The results also indicated that the effect of pertussis toxin may vary depending on mouse strain.

Planning for reduction.

Reduction is one of the Three Rs which can be readily achieved in practice. This can be done by careful consideration of the experimental strategy and the implementation of good experimental design. Moreover, strategic planning leads to 'best' scientific practice and can have a positive impact on both refinement and replacement. The FRAME Reduction Steering Committee has developed a flow chart for an overall strategy for planning and conducting biomedical research. This, and important planning considerations for each of the steps proposed, are discussed. The strategy involves taking an initial overview and undertaking related background research, then planning a sequence of experiments expected to give satisfactory results with the least animal use and minimal severity, choosing an efficient design for each experiment in the sequence, reviewing the results of one experiment before progressing to the next, and conducting an overall analysis at the end of the programme. This approach should minimise animal use and maximise the quality of the resultant scientific output.

Characterization of reference materials for human antiserum to pertussis antigens by an international collaborative study.

Enzyme-linked immunosorbent assay (ELISA) has been widely used to evaluate antibody responses to pertussis vaccination and infection. A common reference serum is essential for the standardization of these assays. However, no internationally recognized reference serum is available. At the request of the Expert Committee on Biological Standardization (ECBS) of the World Health Organization (WHO), a set of four candidate international standards has been prepared. These candidate materials have been assessed for suitability and compared to the widely used U.S. reference pertussis antiserum (human) lot 3, lot 4, and lot 5 by 22 laboratories from 15 countries in an international collaborative study. Laboratories measured immunoglobulin G (IgG) and IgA antibodies to pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), and fimbriae (Fim2&3) using their established immunoassays. The results of this study showed each of the four candidates to be suitable as an international standard. With the agreement of the participants, a recommendation has been made to the ECBS that the candidate material coded 06/140 be established as the First International Standard for pertussis antiserum (human), with the following assigned international units (IU): IgG anti-PT, 335 IU/ampoule; IgA anti-PT, 65 IU/ampoule; IgG anti-FHA, 130 IU/ampoule; IgA anti-FHA, 65 IU/ampoule; IgG anti-PRN, 65 IU/ampoule; and IgA anti-PRN, 42 IU/ampoule. No formal units have been proposed for anti-Fim2&3 because most assays used a mixture of fimbrial antigens. In addition, the candidate material coded 06/142 has been proposed as a WHO working preparation for characterization of assay systems.

Transferability of dermal temperature histamine sensitization test for estimation of pertussis toxin activity in vaccines.

All current acellular pertussis vaccines (ACVs) contain detoxified pertussis toxin (PT) as a major component. An essential part of the safety evaluation of these vaccines, required by regulatory authorities, is to monitor their active PT content and to check for reversion to toxicity of the detoxified PT. Although various in vitro tests are under investigation, the only practicable means for detecting active PT at present is the histamine sensitization test. The methods given in the European Pharmacopoeia and in the US Pharmacopoeia are based on recording a binary response to histamine challenge (using a lethal end point). A more sensitive method based on measurement of rectal temperature is given in the Japanese Minimum Requirements for Biological Products. More recently, a refinement of this method based on dermal temperature measurement has been developed for ACVs in combination with diphtheria and tetanus vaccines (DTaP). We show that this method also can be used for more complex combination vaccines and is readily transferable. Furthermore use of dermal temperature provides a more precise quantitative estimate of toxin activity than the binary response, leading to an increase in information from a specified number of animals, or allowing a reduction in the number of animals required. We suggest that, pending the development of an alternative in vitro replacement method, the temperature based method may serve as an intermediate solution to the estimation of PT activity giving a precise estimate with reduction in animal numbers.

Physicochemical and biological assays for quality control of biopharmaceuticals: interferon alpha-2 case study.

A selection of physicochemical and biological assays were investigated for their utility in detecting changes in preparations of Interferon alpha-2a and Interferon alpha-2b (IFN-alpha 2a, IFN-alpha 2b), which had been subjected to stressed conditions, in order to create models of biopharmaceutical products containing product-related impurities. The stress treatments, which included oxidation of methionine residues and storage at elevated temperatures for different periods of time, were designed to induce various degrees of degradation, aggregation or oxidation of the interferon. Biological activity of the stressed preparations was assessed in three different in vitro cell-based bioassay systems: a late-stage anti-proliferative assay and early-stage assays measuring reporter gene activation or endogenous gene expression by quantitative real time Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). Relevant physicochemical methods such as SDS-PAGE, reverse phase (RP) chromatography, size-exclusion chromatography (SEC) and dynamic light scattering (DLS), proved their complementarity in detecting structural changes in the stressed preparations which were reflected by reductions in biological activity.

Thermal stability of the WHO international standard of interferon alpha 2b (IFN-alpha 2b): application of new reporter gene assay for IFN-alpha 2b potency determinations.

A World Health Organization requirement for biological standards is that they should exhibit long-term stability at their recommended storage temperature. Thermal stability is usually predicted in accelerated thermal degradation studies, where ampoules of the lyophilized standard are stored at elevated temperatures for relatively short times before testing. To confirm the predicted thermal stability of the 2nd international standard of human interferon alpha 2b (IFN-alpha2b; 95/566), we tested the potency of the ampouled contents of this standard after 9 years storage at the customary storage temperature of -20 degrees C in comparison with ampoules of the IS which had been stored continuously at temperatures ranging from -150 degrees C to 56 degrees C. Since IFN-alpha2b potency estimates derived from the results of antiviral assays (AVA) showed high within-assay variability, we investigated a novel reporter gene assay (RGA) based on induction of secreted alkaline phosphatase (SEAP) for comparability and precision of such estimations. We show that this RGA generated comparable estimates with overall lower variation. Additionally, the SEAP conversion of p-nitrophenyl phosphate to yellow product could be followed kinetically. Absorbance readings were shown to increase with time in proportion with increasing concentration of IFN-alpha2b. When the time-dependent increments of absorbance were plotted graphically, the slopes of lines corresponded to concentration. This approach enabled single dilutions of IFN samples, identical in molecular structure to an IFN-alpha2b standard, to be used for potency estimates by interpolation of slope value against those of the standard at fixed concentrations. It appears attractive for high through-put potency testing of various R&D IFN-alpha2b samples.

Multicentre evaluation of stable reference whole blood for enumeration of lymphocyte subsets by flow cytometry.

BACKGROUND: Clinical indications for lymphocyte subset enumeration by flow cytometry include monitoring of disease progression and timing of therapeutic intervention in infection with human immunodeficiency virus. Until recently international standardisation has not been possible due to a lack of suitable stable reference material. METHODS: This study consisted of two trials of a stabilised whole blood preparation. Eleven participants were sent two standard protocols for staining plus gating strategy and asked to report absolute counts for lymphocyte subsets. RESULTS: No significant difference was detected between the two methods when results from the two assays and all partners were pooled. Significant differences in results from the different partners were observed. However, representative mean counts were obtained for geometric means, geometric coefficient of variation, and 95% confidence interval for CD3 910 cells/mul, 9%, and 888 to 933, respectively), CD4 (495 cells/mul, 12%, and 483 to 507), and CD8 (408 cells/mul, 13%, and 393 to 422). CONCLUSION: We have introduced a stabilised blood preparation and a well-characterized biological standard. The availability of this reference material greatly simplifies the validation of new techniques for CD4(+) T-cell enumeration and the expansion of external quality assurance programmes for clinical laboratories, including those that operate in resource-restricted environments. (c) 2005 Wiley-Liss, Inc.

A comparison of vials with ampoules for the storage of biological reference materials.

Lyophilization is a key strategy in the stabilization of biological materials. Protection of the lyophilized material from an oxidizing atmosphere is essential if stability is to be maintained under long term storage. Vials of lyophilized albumin closed by two methods and ampoules of albumin have been examined for moisture and oxygen ingress after storage both under conditions of stress for two months and under thermally accelerated conditions for one year. The results show that the gas and moisture contents of ampoules do not detectably change even under conditions of stress, in contrast to vials for which this study shows clearly detectable moisture ingress and suggests some oxygen ingress. This is consistent with the results of other studies. Thus, although vials may be satisfactory under constrained conditions of temperature and storage for limited periods of time, and are presently used satisfactorily for some working standards, it would be prudent to continue to use ampoules for storage of international reference materials which are intended for indefinite storage and for which stability is an essential requirement.

"Statistics" is not a sausage machine: a statistician's viewpoint and some comments on experimental design.

Any experiment involving the use of animals which is not well-planned, meticulously carried out, and scrupulously analysed, is unethical. Planning, or good experimental design, followed by analysis appropriate for the design, will help to ensure the optimal use of animals. Thus, collaboration between biologist and statistician, especially at the planning and analysis stages, is one of the best ways of achieving an ethical and successful experiment. However, genuine communication is necessary for any collaboration, and this requires time and patience, on the part of both biologist and statistician. Although the three fundamental principles of experimental design, replication, randomisation and local control, are straightforward in theory, there is substantial scope for misunderstanding and misinterpretation in practice. Each experiment presents unique and interacting biological and statistical problems, and both the right design and the correct analysis should be decided on a case-by-case basis.

Monocyte activation test for pro-inflammatory and pyrogenic contaminants of parenteral drugs: test design and data analysis.

An optimised test designed for an in vitro monocyte activation test for pro-inflammatory and pyrogenic contaminants of parenteral drugs is described, together with ways to address the inherent variability of such assays in which cells are cultured using 96-well plates. The test preparation is cultured with peripheral blood mononuclear cells (PBMNC) and the contaminants in the test article stimulate the release from the cells of the endogenous pyrogenic cytokine interleukin-6 (IL-6). The test system is in use within the pharmaceutical industry and at a national control authority for detecting pro-inflammatory and pyrogenic contaminants, including 'rabbit-negative' and 'LAL-negative' non-endotoxin pyrogens. Products tested include small molecules, biologicals and vaccines. The PBMNC/IL-6 monocyte activation test has been approved by the US FDA as an 'end-product release test' and also is being used for in-process testing.

Role for standards in assays of botulinum toxins: international collaborative study of three preparations of botulinum type A toxin.

The biological activity of therapeutic preparations of botulinum type A toxin is currently expressed in units defined on the basis of the median lethal intraperitoneal dose of that preparation in mice at 72 h, the LD50 dose. In this study we describe the comparison, by ten laboratories in five countries, of three different formulations of botulinum type A toxin using the mouse lethality test, and also using the relative activities of the preparations. The results of this study show that use of a standard preparation and expression of relative potency gives substantially greater consistency between and within laboratories than when mouse LD50 unit is used to define activity of botulinum toxin.

A rapid 'one-plate' in vitro test for pyrogens.

A rapid, 'one-plate' monocyte-activation test is described for detecting endotoxin and non-endotoxin pyrogens in parenteral medicinal products. The one-plate test offers useful gains over conventional 'two-plate' (cell culture plate+ELISA plate) tests in terms of its limit of detection, robustness, speed and cost. The 'one-plate' test is likely to be applicable to a wide range of products because it allows less time for product interference in the test. The 'one-plate' test utilises pyrogen-free anti-cytokine (interleukin (IL)-6 or tumour necrosis factor alpha (TNFalpha)) antibodies (Ab), coated and stabilised onto (pyrogen-free) 96-well plates. Monocytes/monocytic cells, endotoxin (lipopolysaccharides, LPS) standard or sample and (pyrogen-free) second (labelled) Ab are cultured together (usually for 2-4 h) on the Ab-coated plate and then the plate is washed and the ELISA completed. There is no transfer from one plate to another and no (further) incubations of (released) cytokine with, first, coating Ab and, then, developing Ab since these steps have already taken place during the initial cell culture. The rapid, 'one-plate' test is readily automated. The preferred readout is IL-6, which gives a limit of detection of 0.015 endotoxin units (EU)/ml with peripheral blood mononuclear cell (PBMNC), 0.03 EU/ml with diluted whole blood and 0.05 EU/ml with a monocytic cell line (MONO MAC 6).