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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.

The European Directorate for the Quality of Medicines (EDQM) supplies Chemical Reference Substances (CRS) for Interferon (IFN) alfa-2a (CRS I0320300) and for IFN alfa-2b (CRS I0320301) for specified physicochemical tests. However, no information is provided as to their biological activity. In contrast, the World Health Organization (WHO) provides the 2nd International Standards (IS) for IFN alfa-2a (code 95/650) and for IFN alfa-2b (code 95/566), with activity defined in International Units (IU) for calibration of biological activity of preparations of IFN. We have compared the EDQM CRSs with the WHO ISs in two bioassay systems, one measuring the anti-proliferative activity in the Daudi cell line and the other measuring a reporter gene activation in an A549 cell line. In each of these assay systems, the CRSs gave dose - response relations, which were similar to those for the WHO ISs. Estimates of relative activity for each CRS, in terms of the respective IS, showed specific biological activity for the CRSs of the same order as the nominal specific activity for the ISs. However, the estimates of relative activity were not consistent between the two assays systems, emphasizing the need for calibration within each system, if the CRS were to be used as a working standard for bioassays. For structure-activity studies, both physicochemical and biological activity characterisation are required for the same biopharmaceutical preparation. CRS I0320300 and CRS I0320301 may prove useful as working standards for some bioassay systems.

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.

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., (Copyright 2004 Elsevier B.V.)

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).

Two methods for predicting the specific in vivo bioactivity of recombinant follicle stimulating hormone (FSH), based on quantitative measures of isoform distribution by isoelectric focusing (IEF)(1) and by capillary zone electrophoresis (CZE)(2) respectively, have been subjected to an international collaborative study by six laboratories from six countries. Both methods were used to estimate the predicted bioactivities of four preparations of follitropin beta, coded FSH A-D, differing widely in their isoform compositions and specific bioactivities. The mean predicted estimate of potency by IEF and CZE for each FSH preparation by each laboratory was within 80-125% of its potency estimated by bioassay, except for the mean estimates by CZE of that for FSH A by one laboratory and of that of FSH D by another. Each of the six laboratories using the IEF method, and each of the five laboratories using the CZE method were able to rank these FSHs according to their bioactivities, namely FSH B>FSH C>FSH A>FSH D. All laboratories were able to use both IEF and CZE to discriminate between FSH A and C, with bioactivities within 76-132% of one another. Four of six laboratories were able to use IEF, and two of five laboratories were able to use CZE, to discriminate between FSH B and C, with bioactivities within 89-112% of one another. This suggests that the accuracy and precision of both these methods should be sufficient to discriminate between FSHs which would meet or fail European Pharmacopoeia requirements for this type of hormone, since these stipulate that estimates of potency should fall between 80-125% of its stated potency. Using in most cases duplicate estimates in two independent assays, and excluding Laboratory 4, the pooled intra-laboratory geometric coefficient of variation (GCV) was about 4% for both IEF and CZE, and the inter-laboratory GCV was about 7% for IEF and about 10% for CZE. The use of one FSH preparation as a standard, with its specific activity as an assigned value, reduced the inter-laboratory variability of estimates for the remaining FSHs by both methods. This increased the accuracy of the predicted estimates of bioactivity for these remaining FSHs in terms of their approximation to the values for their bioactivities estimated by bioassay. These data therefore suggest that both these methods, and particularly IEF, are sufficiently accurate, precise and robust to be used for predicting the bioactivity of batches of follitropin beta, and especially if used with a standard preparation., (Copyright 2002 The International Association for Biologicals. Published by Elsevier Science Ltd. All rights reserved.)

A pre-validation study was carried out, by six laboratories from six countries, of two physicochemical methods for predicting the in vivo biological potency of recombinant follicle stimulating hormone (follitropin beta), based on quantitative measures of isoform distribution by isoelectric focusing (IEF) and by capillary zone electrophoresis. Each of these methods was used to estimate the predicted bioactivities of four preparations of follitropin beta differing widely in their isoform compositions and specific bioactivities. The results of this study indicate that these methods, and particularly IEF, are transferable between laboratories, and produce results which are sufficiently accurate, precise, and reproducible, for them to be used for predicting the bioactivity of follitropin beta, especially if used with a standard preparation. The performance of these two methods for predicting the bioactivity of other types of follicle stimulating hormone, such as follitropin alfa, would need to be assessed separately, and might involve quantitatively different relationships between the responses measured in the physicochemical method and the bioactivities of preparations estimated by bioassay.

Underlying the design of any assay and further, interpretation of the results, are multitudes of assumptions and implications, ranging from 'biological' assumptions about the nature of the assay system and its response to the materials assayed to 'statistical' assumptions about the form of the dose-response relationship and the distribution of the response data. As far as possible, assays should be designed and analysis carried out to assess these assumptions. Implications for the individual assay are discussed, since this is where all studies of assays necessarily begin. Consideration is then extended to implications for the combination of data and results of several assays.

During the course of an experiment using animals, many variables (e.g., age, body weight at several times, food and water consumption, hematology, and clinical biochemistry) and other characteristics are often recorded in addition to the primary response variable(s) specified by the experimenter. These additional variables have an important role in the design and interpretation of the experiment. They may be formally incorporated into the design and/or analysis and thus increase precision and power. However, even if these variables are not incorporated into the primary statistical design or into the formal analysis of the experiment, they may nevertheless be used in an ancillary or exploratory way to provide valuable information about the experiment, as shown by various examples. Used in this way, ancillary variables may improve analysis and interpretation by providing an assessment of the randomization process and an approach to the identification of outliers, lead to the generation of new hypotheses, and increase generality of results or account for differences in results when compared across different experiments. Thus, appropriate use of additional variables may lead to reduction in the number of animals required to achieve the aims of the experiment and may provide additional scientific information as an extra benefit. Unfortunately, this type of information is sometimes effectively discarded because its potential value is not recognized. Guidelines for use of animals include, in addition to the obligation to follow humane procedures, the obligation to use no more animals than necessary. Ethical experimental practice thus requires that all information be properly used and reported.

Hepatocyte growth factor/scatter factor (HGF/SF) is a potent paracrine growth factor with motogenic, mitogenic and morphogenic activities, and a potential therapeutic role in hepatic and renal disease, as well as diagnostic and prognostic applications. It is synthesised as an inactive, single-chain precursor that is cleaved by serine proteases to give a biologically active, heterodimeric form. To develop World Health Organization (WHO) International Standards (IS) for HGF/SF, candidate preparations of the two forms were assessed in a multicentre study in which they were compared with local standards by bioassay and immunoassay. Among laboratories, there was a wide variation in the estimates of potencies of the candidate standards in terms of in-house reference preparations, but between-assay and within-assay variabilities were low within laboratories. In some assay systems, the precursor and heterodimer showed different responses. Since both molecular forms are widely used in current assay systems, this suggested that a reference preparation was required for each form of the HGF/SF molecule. Accordingly, the Expert Committee on Biological Standardization of WHO established the heterodimeric material (96/564) as the first IS for HGF/SF, human, recombinant, with an assigned unitage of 4000 IU/ampoule and, for the purpose of immunoassay calibration, a nominal HGF/SF content of 4 microg/ampoule. The precursor preparation (96/556) was established as the first IS for HGF/SF (precursor) with an assigned unitage of 2000 IU/ampoule and, for the purpose of immunoassay calibration, a nominal HGF/SF (precursor) content of 4 microg/ampoule. The preparations can be obtained upon written request to the National Institute for Biological Standards and Control (NIBSC, PO Box 1193), by e-mail (standards@nibsc.ac.uk) or ordered at http://www.nibsc.ac.uk.

A preparation of recombinant insulin-like growth factor-II (IGF-II) (NIBSC code 96/538) was compared with local standards in bioassays and immunoassays by eight laboratories in four countries to assess its suitability for use as a World Health Organisation (WHO) reference reagent. Estimates of relative potencies for the bioassays gave a geometric mean of 1.04 (0.94--1.16) microg of local standard per microg of 96/538. Estimates of relative immunological activities by immunoassay gave a geometric mean of 1.15 (0.94--1.38) microg of local standard per microg of 96/538. The study provided evidence that a common standard for rhIGF-II would be helpful and that 96/538 was sufficiently stable to serve as a reference reagent. Accordingly 96/538 was established as the First WHO Reference Reagent for IGF-II, human, recombinant, and assigned a unitage of 5000 units per ampoule and on the basis of the immunoassay results a nominal mass content of 5 microg per ampoule., (Copyright 2001 Harcourt Publishers Ltd.)

FSH has a key role in the development and function of the reproductive system and is widely used both diagnostically and therapeutically in developmental and reproductive medicine. The accurate measurement of FSH levels, in patients for diagnosis and monitoring and in therapeutic preparations for clinical use, is essential for safe and successful treatment. Historically, FSH was defined on the basis of classical in vivo endocrine activity, and early therapeutic preparations were calibrated using in vivo bioassays. There was early recognition that reference preparations were required for calibration if the results from different laboratories were to be comparable. In response to the perceived need, the World Health Organization established the first standard for such preparations in 1959. Subsequent developments in biotechnology have led to recognition that there is no single molecule that can be uniquely defined as FSH, and that FSH can induce a range of biological activities. Several highly purified standards for FSH are now available, but discontinuity and heterogeneity of estimates of FSH activity in terms of these standards made using in vitro assays and binding assays have been noted. It is thus essential that any measurement of FSH include specification both of the standard with which the measured FSH is compared and the assay method used for that comparison.

Assay evaluation and validation is essential to ensure that assays are sufficiently specific and provide estimates with sufficient precision for the required purposes. This must be an on-going process, and assays should therefore be designed to permit some degree of both direct and indirect measurement of intra- and inter-assay variation. Quality control procedures may contribute relevant information, but may not be sufficient. Results obtained by two laboratories using as nearly as possible identical reagents and samples in an ELISA for anti-pertussis antibodies are described. These results illustrate aspects of assay performance which may be overlooked once a formal "validation exercise" has been carried out and assays are in routine use. This study also illustrates the information, in addition to that available from in-house studies, which may be provided by appropriately designed inter-laboratory studies, such as the collaborative studies carried out for characterization and calibration of reference materials and standards., (Copyright 1999 The International Association for Biologicals.)

Ensuring the reliability and precision of assay results requires careful attention to assay design. In this case study we describe validation studies of an in vitro assay for botulinum neurotoxin type A based on its endopeptidase activity towards immobilised synthetic substrate. This assay, in common with many in vitro assays, is sensitive to changes in reagents and assay conditions and is time dependent. In addition, the toxin is not stable in solution. Differences in estimates of potency, resulting from positional factors, which are not significant in individual assays, are shown to be consistent and statistically significant over a longer series of assays. This study emphasizes that assay validation should not be viewed as a single step in assay development but must be considered as a continuing process if assay results are to be reliable and reproducible.

An ampouled preparation of acute phase serum rich in serum amyloid A protein (SAA) was evaluated in seven laboratories in six countries for its suitability to serve as the international standard for immunoassay of SAA. A variety of different immunoassays were used. On the basis of the results reported here and with the authorization of the Expert Committee on Biological Standardization of the World Health Organization (WHO) this preparation (coded 92/680) was established as the first international standard of SAA.

Epoetin alfa and beta are the two forms of recombinant DNA-derived erythropoietin (rEPO), both synthesized in Chinese hamster ovary cells, which are used for the treatment of erythropoietin (EPO)-responsive anaemias. Several batches of each of these rEPOs were compared for differences in their EPO isoform compositions by isoelectric focusing (IEF) and in a range of lectin-binding assays, and for differences in their EPO activities by in-vivo and in-vitro mouse bioassays and by immunoassay. Epoetin beta was found to differ from epoetin alfa in containing: (a) a greater proportion of more basic isoforms, (b) a greater proportion of EPO binding to Erythrina cristagalli agglutinin (which binds N-glycans with nonsialylated outer Gal beta1-4GlcNAc moieties), and (c) isoforms with higher in-vivo:in-vitro bioactivity ratios. Epoetin beta also contained slightly more than epoetin alfa of EPO binding to Lycopersicon esculentum agglutinin (which binds N-glycans containing repeating Gal beta1-4GlcNAc sequences), to the leucoagglutinin of Phaseolus vulgaris (which binds tetraantennary and 2,6-branched triantennary N-glycans) and to Agaricus bisporus agglutinin (which binds Gal beta1-3GalNAc containing O-glycans). No differences were found between the two rEPOs in their binding to a further five lectins. The differences between the isoform composition of epoetin alfa and beta, and the smaller inter-batch differences appear to be due to differences in glycosylation. The higher murine in-vivo:in-vitro bioactivity ratio of epoetin beta compared to epoetin alfa could not be explained in terms of differences in their degrees of sialylation, but was consistent with differences in their pharmacokinetics and pharmacodynamics observed in human subjects. There have been no reports that epoetin alfa differs from epoetin beta in its clinical efficacy, but the differences between epoetin alfa and beta in some analytical systems suggest that there might be a need for separate international standards for these two types of rEPO.

Assays have been developed for the isoforms of erythropoietin (EPO) based on their binding to eight different lectins. These assays were used to compare the isoform compositions of two preparations of human urinary EPO (uEPO) and four preparations of recombinant DNA-derived human EPO (rEPO), which had been shown to differ in their biological and immunological properties and in their isoform composition as judged by isoelectric focusing and electrophoresis. Agarose-bound Ricinus communis agglutinin I (RCA), Erythrina cristagalli agglutinin (ECA), Maackia amurensis leukoagglutinin (MAL), Sambucus nigra agglutinin (SNA), Lycopersicon esculentum agglutinin (LEA), concanavalin A (Con A), Phaseolus vulgaris agglutinin-L4 (L-PHA) and Agaricus bisporus agglutinin (ABA) were used to bind EPO isoforms possessing: N-glycans containing non-sialylated outer Gal beta 1-4GlcNAc (RCA and ECA), NeuAc alpha 2-3Gal beta 1-4GlcNAc (MAL), NeuAc alpha 2-6Gal (SNA), or repeating Gal beta 1-4GlcNAc sequences (LEA); biantennary N-glycans (Con A); tetraantennary and 2,6-branched triantennary N-glycans (L-PHA); and O-glycans containing NeuAc alpha 2-6GalNAc (SNA) and Gal beta 1-3GalNAc (ABA). Free EPO was measured by mouse spleen cell bioassay or immunoassay. Estimates from most lectin-binding assays were reproducible between assays and batches of lectin-agarose, although batches of MAL- and ABA-agarose, and to a lesser extent LEA-agarose, differed in their EPO-binding. Lectin-binding assays showed differences between the isoform compositions of all EPOs, including the two Chinese hamster ovary cell-derived rEPOs, with RCA- and ECA-binding assays being the most discriminating. Lectin-binding estimates provided evidence that uEPO differs from these rEPOs in its lower content of isoforms with biantennary N-glycans and higher content of those with multiantennary N-glycans, and in its lower content of isoforms with N-glycans possessing repeating Gal beta 1-4GlcNAc sequences and of those with O-glycans containing Gal beta 1-3GalNAc. Lectin-binding estimates also indicated that, contrary to some reports, uEPO possesses Gal beta 1-3GalNAc-containing O-glycans but not NeuAc alpha 2-6GalNAc-containing O-glycans or NeuAc alpha 2-6Gal-containing N-glycans. Most groups of lectin-bound EPO isoforms did not differ in their relative bioactivities and immunoreactivities. However, estimates for ABA-bound EPO isoforms suggested that O-glycans might influence the bioactivity of EPO differently to its immunoreactivity. Furthermore, the bioactivities of some ECA-bound EPO isoforms were higher, and those of some of the MAL-bound EPO isoforms lower, than their immunoreactivities, consistent with the reported enhancement of EPO in vitro bioactivity by desialylation.

The First International Standard for Inhibin, Human Recombinant, (ISI), a lyophilized preparation of rDNA-derived human 32 kDa Inhibin A in ampoules coded 91/624, was evaluated by international collaborative study for its suitability to serve as an International Standard. This study, which involved 15 laboratories in nine countries, included a variety of in vitro bioassays and immunoassays. The ISI was compared with two other lyophilized preparations of human recombinant inhibin, the International Standard for Porcine inhibin (ISP) and preparations of human follicular fluid inhibin. Predicted loss of activity based on estimates of potency of contents of ampoules which had been stored under conditions of accelerated thermal degradation indicated that the ISI has satisfactory stability. On the basis of the results of this study, the ISI was deemed suitable to serve as a standard for in vitro bioassays and immunoassays and was established by the Expert Committee on Biological Standardization of the World Health Organization as the First International Standard for inhibin, recombinant human, with an assigned unitage of 150,000 International Units per ampoule. This unitage maintains an approximate continuity of units with the ISP.

Biological assays for tumour necrosis factor (TNF) are primarily based on its cytotoxic effect in tumour cell lines, and many of these bioassays are carried out using microtitre plates. Many immunoassays for TNF also routinely use microtitre plates. Data from an international collaborative study, carried out by twenty participants in nine countries, each of whom evaluated seven different ampouled preparations of human tumour necrosis factor alpha (hTNF-alpha), one ampouled preparation of human tumour necrosis beta (hTNF-beta) and one ampouled preparation of mouse tumour necrosis factor alpha (mTNF-alpha) provided a unique opportunity for a broadly based evaluation and comparison of assay designs and results. The results of this evaluation can be applied to a wide range of in vitro assays based on cell cytotoxicity or proliferation. The results of this evaluation indicated that although it is difficult to achieve control of all factors which contribute to the variability of assay responses, assays may be designed to provide measures of the variation due to some factors and to improve reliability of estimates of relative potency.

An international collaborative study was performed to investigate the reproducibility of influenza serological techniques. Participants in seven laboratories representing five countries measured antibody to A/Sichuan/2/87 (H3N2), A/Taiwan/1/86 (H1N1) and B/Beijing/1/87 influenza viruses in 11 human sera and three postinfection ferret sera. Two different serological techniques were used, haemagglutination inhibition (HI) and single-radial haemolysis (SRH) and, although each technique was reproducible within laboratories, variability between laboratories was higher for HI (maximum variability 32-fold; geometric coefficient of variation, GCV, 112%) than for SRH (maximum variability 3.8-fold; GCV 57%). The use of a standard serum allowed direct comparison of HI and SRH data and, for each technique, a standard serum improved inter-laboratory agreement. For influenza A viruses there was a correlation between HI and SRH antibodies (correlation coefficient approximately 0.9). An HI titre of 1:40 in human sera corresponded to an SRH titre of 19-33 mm2. The results of the study indicate that two sera would be expected to contain different antibody levels if their HI titres differed by > fourfold and SRH areas differed by > 50%. Both SRH and HI possessed equivalent sensitivity for measurement of antibody to influenza A viruses but SRH was more sensitive for detection of antibody to influenza B viruses. The study provided valuable information about standardization of antibody assays.

Five enzyme materials (gamma-glutamyltransferase, alkaline phosphatase, creatine kinase, alanine aminotransferase and prostatic acid phosphatase) are currently certified using a reference method. Furthermore, feasibility studies have been performed for four other enzymes (aspartate aminotransferase, lactate dehydrogenase, amylase and lipase). They indicated that these enzymes can be purified and stabilized, but the materials have not yet been certified. This shows that the most important enzymes in clinical laboratories can be purified, and stabilized, without significant alteration of their catalytic properties. By carefully choosing a matrix, the commutability of these enzyme preparations and patients' samples between some methods, including routine methods, may be preserved. Thus, these materials can be used to calibrate the routine methods in terms of the corresponding reference methods after commutability has been verified. Current studies suggest that this objective can be reached, provided three criteria are satisfied: i) the calibrated and reference methods must be of equal specificity; ii) the enzyme calibrator should be, as closely as possible, identical to the human analyte enzyme in its native matrix (eg serum); iii) and the inter-method ratio should be constant (within the limits of experimental error) for the enzyme calibrator and for all patients' samples.

An immunologic crossreactant of erythropoietin seemed to develop and persist in serum samples from a patient during treatment and remission of idiopathic aplastic anemia. It had a steeper slope to radioimmunoassay log-dose response lines and a larger molecular size than erythropoietin. On fractionation of serum, the apparent crossreactant was bound by staphylococcal Protein A at pH 7.5 and recovered by elution from it at pH 3.0. Adsorption of serum from the patient, and from one of two similarly affected children, with rabbit IgG linked to agarose appeared to remove completely the apparent crossreactant. These treated sera gave radioimmunoassay log-dose response lines essentially parallel to that given by the International Reference Preparation (IRP) for erythropoietin and estimates of immunoreactive erythropoietin appropriate to the normal hemoglobin concentrations. The apparent crossreactant of erythropoietin is thus accounted for by heterophilic antibodies to rabbit IgG. These developed in the patient following treatment with rabbit antilymphocyte globulin but seem to have arisen spontaneously in the children. Thus iatrogenic and idiopathic antibodies to rabbit IgG interfered in a radioimmunoassay for erythropoietin in serum through their ability to react with the radioimmunoassay anti-erythropoietin antiserum raised in rabbits.

The second International Standard for Human Pituitary LH (in ampoules coded 80/552; 2nd IS) and LH 81/535 (prepared in the same way as the 2nd IS from the same LH preparation) were compared with the International Reference Preparation of Human Pituitary LH for Immunoassay (IRP 68/40) by 19 laboratories in 11 countries, using in-vivo and in-vitro bioassays, a receptor assay and immunoassays. Geometric mean estimates of the LH content of the 2nd IS (with 95% fiducial limits) in terms of IRP 68/40 were: 34.6 (29.1-41.0) IU/ampoule by in-vivo bioassays; 35.8 (27.0-47.4) IU/ampoule by in-vitro bioassays; 58.6 IU/ampoule by one receptor assay; and 36.8 (35.5-38.1) IU/ampoule by immunoassays. The close agreement between the relative activities of the 2nd IS and IRP 68/40 in the wide range of assay systems studied appears to reflect the fact that both standards contain highly purified LH with similar isoform compositions as judged by isoelectric focusing. Estimates of the LH content of LH 81/535 in terms of IRP 68/40 and in terms of the 2nd IS tended to be lower than those for the 2nd IS across all methods, but the differences were not statistically significant. The 2nd IS was found to be as suitable as IRP 68/40 as a standard for the in-vitro bioassay and immunoassay of LH in the two serum samples studied. However, the mean estimates of serum LH in terms of either of these standards were more than 150% larger by in-vitro bioassays than by immunoassays and more than 50% larger by one-site than by two-site immunoassays.(ABSTRACT TRUNCATED AT 250 WORDS)

The biological potency of calcitonins in clinical use in long-term treatment of Paget's disease of bone and, increasingly, in osteoporosis is usually expressed international units defined by the relevant World Health Organization international reference preparation. The international reference preparations for porcine and human calcitonins were ampouled in 1970 and stocks are now exhausted. Replacement standards were ampouled in 1989 and have been evaluated and calibrated by an international collaborative study comprising 16 laboratories in 12 countries. Evaluations included high-performance liquid chromatography and in vitro bioassay; calibration of each new ampouled preparation in terms of its international reference preparation was by in vivo rat hypocalcaemia bioassay. On the basis of the results of the study and with the agreement of the participants, replacement standards were established by the Expert Committee on Biological Standardization of the World Health Organization in 1991: the international standard for porcine calcitonin (ampoule code 89/540), with an assigned potency of 0.8 international units per ampoule, and the international standard for human calcitonin, with an assigned potency of 17.5 international units per ampoule. Both international standards appeared to be sufficiently stable to serve as the international standards for in vivo biological assays. Comparison of the two species of calcitonin in the same hypocalcaemia assay showed that they were approximately equipotent when the doses were given intravenously but that the human peptide was four- to sixfold more potent than porcine calcitonin when doses were given subcutaneously, emphasizing the need to compare "like with like".

Three ampouled preparations of interleukin-6 (IL-6) were evaluated by 12 laboratories in seven countries for their suitability to serve as the international standard of IL-6. The preparations were assayed using in vitro bioassays and immunoassays. On the basis of the results reported here, with the agreement of the participants in the study and with the authorization of the Expert Committee on Biological Standardization (ECBS) of the World Health Organization (WHO) one of the preparations (coded 89/548) was established as the international standard of IL-6.

Objective: Thyroxine binding globulin is the major thyroid hormone binding and transport protein of the plasma, and its quantitative estimation is therefore of clinical importance. The objectives of the present study were to prepare and ampoule a preparation of thyroxine binding globulin suitable to serve as an international standard, to confirm the suitability of the preparation in a multi-centre collaborative study, and to assign an ampoule content., Design: The collaborative study was designed with the following aims: (1) to compare the candidate preparation with local standards; (2) to calibrate the preparation with local standards and to confirm the assigned ampoule content; (3) to assess the stability of the ampouled preparation., Measurements and Results: Thyroxine binding globulin was purified by a combination of affinity and conventional chromatography. Analysis by gel electrophoresis, N-terminal sequencing and electrospray mass spectroscopy showed the preparation to be > 99% pure and to have a structure consistent with the known structure of thyroxine binding globulin. Thyroxine binding globulin assays performed in the multi-centre collaborative study indicated that the preparation behaved as thyroxine binding globulin when compared to local standards and that the ampoule content was consistent with that assigned on the basis of physicochemical measurements of protein concentration., Conclusions: The thyroxine binding globulin preparation coded 88/638 was established by the Expert Committee on Biological Standardization of the World Health Organization as the International Standard for Thyroxine Binding Globulin, 30.0 International Units per ampoule, with the additional information that for the preparation in 88/638, 1 IU is equivalent to 1 microgram. The preparation is available from the National Institute for Biological Standards and Control.

The First International Standard (IS) for Antitetanus Immunoglobulin, Human (coded 26/488) was established by the WHO Expert Committee on Biological Standardization in October 1992 on the basis of an extensive pharmaceutical evaluation and an international collaborative study. Fifteen laboratories from 15 countries performed the toxin neutralization assay in vivo in the study. Twelve laboratories also performed an alternative in vitro assay. The new International Standard is a purified human immunoglobulin containing antibodies which are able to neutralize tetanus toxin. This new standard replaces the Second International Standard for Tetanus antitoxin, Equine, for testing of human antitetanus immunoglobulin preparations used clinically as well as for titration of human serum samples for tetanus antitoxin. The new International Standard was assigned a potency of 120 International Units (IU) of Tetanus Antitoxin per ampoule on the basis of its calibration in terms of the International Unit defined by the Second International Standard for Tetanus Antitoxin, Equine by toxin neutralization assay in vivo. The same preparation was also established as the first European Pharmacopoeia Standard for Antitetanus Immunoglobulin, Human by the European Pharmacopoeia Commission, Biological Standardization Programme in March 1993.

A lyophilized preparation of inhibin from porcine ovarian follicular fluid, ampoule code 86/690, was made internationally available as a research standard for in vitro bioassays in 1987. A study involving ten participants in eight countries assessed the stability and suitability of this research standard to serve as an international standard. Each of the participants used in vitro assays, the majority of which depended upon the inhibition of release of follicle-stimulating hormone from dispersed rat anterior pituitary cells. The research standard 86/690 was compared with coded ampoules of 86/690 stored under conditions of accelerated thermal degradation and with inhibins from different species. Intra- and interlaboratory variation for estimates of potency of a coded duplicate ampoule of the research standard provided the basis for comparisons of non-identical inhibins, but the fourfold variability of potency estimates for identical ampoules was such that no conclusions about the differences seen for non-identical inhibins could be made. Predictions of stability from consensus estimates of potency of ampoules that have undergone accelerated thermal degradation indicated that the research standard had satisfactory stability. On the basis of this study, the research standard 86/690 was deemed sufficiently stable and suitable to serve as a standard for in vitro bioassays and was established by the World Health Organization Expert Committee on Biological Standardization as the First International Standard for Porcine Inhibin. The possible presence, in biological extracts (standard or sample), of other bioactive proteins, such as activin and follistatin, complicates the quantitative interpretation of bioassay data. A standard of highly purified human inhibin is now required as a standard for immunoassays used for clinical research purposes; sufficient quantities of recombinant human inhibins have recently been donated for ampouling and evaluation by bio- and immunoassay in the subsequent phase of the standardization of inhibins.

The International Standard (IS) for Recombinant DNA-Derived (rDNA) Erythropoietin (EPO) (in ampoules coded 87/684) and three other rDNA EPO preparations in ampoules coded 87/690, 87/696 and 88/574 respectively, were compared with two preparations of highly purified human urinary (HU) EPO and the 2nd International Reference Preparation of Human Urinary Erythropoietin for Bioassay (2nd IRP) by 26 laboratories in 11 countries using a wide range of in-vivo and in-vitro bioassays and immunoassays. These EPO preparations were also compared by electrophoresis and isoelectric focusing. Estimates of EPO content in terms of the 2nd IRP by all in-vivo bioassay methods gave combined unweighted geometric means (with 95% fiducial limits) of: 86 (75-99) IU/ampoule for the IS, 81 (70-94) IU/ampoule for 87/690, 58 (48-71) IU/ampoule for 87/696 and 120 (100-143) IU/ampoule for 88/574. Mean estimates of EPO content in terms of the 2nd IRP by in-vitro bioassays (except receptor assays) were larger than, and those by immunoassays were similar to, the mean estimates by in-vivo bioassays. The use of purified rDNA or HU EPO as standards in place of the 2nd IRP reduced the inter-laboratory variability of estimates of purified EPO preparations by in-vivo and in-vitro bioassays and by immunoassays, and reduced the variability of overall mean estimates for each of these preparations between the three types of method. The inter-laboratory variability of immunoassay estimates of human serum EPO was similar whether the 2nd IRP or one of the purified EPOs was used as standard. Significant differences in in-vivo and in-vitro biological, immunological and physicochemical properties were found between these four rDNA EPO preparations and between them and the HU EPO in the two purified preparations and in the 2nd IRP. There were also differences between the immunoreactivities of the two serum EPO samples included in the study, and between them and the immunoreactivities of the purified EPOs. The differences between rDNA EPOs appeared to be related to differences between the cells used for their biosynthesis, but may also be the result of differences in purification methods and of inter-batch variations. Significant differences in assay specificity were observed within each of the three general types of method. The specificity of the in-vivo bioassays was influenced by the route of hormone administration. The specificities of the mouse spleen cell in-vitro bioassays differed from that of the mouse spleen receptor-binding assay.(ABSTRACT TRUNCATED AT 400 WORDS)

Certain brain gut-peptides are known to either stimulate or inhibit gastric acid secretion in several species after direct injection into the central nervous system. However there is inconsistency of published results on the gastric acid secretory response to some of these peptides after peripheral administration in different experimental systems. Seven peptides, namely neurotensin (NT), substance P, cholecystokinin (CCK), thyrotropin releasing hormone (TRH), human calcitonin (hCT), rat calcitonin-gene-related peptide (rCGRP) and bombesin, all known to modulate gastric acid secretion after central administration, were initially screened for activity after peripheral (subcutaneous) injection of 10 micrograms/kg body weight in a single rat model. Peptides showing an effect were retested at lower doses. Despite the inherent variability of the gastric acid secretory response in the non-anaesthetized pylorus ligated rat, a standardized experimental design confirmed that reproducible and statistically valid results could be obtained. The technical feasibility of using a one hour collection period as might be appropriate for short acting peptides was demonstrated by the significant dose dependent inhibitory activity of salmon calcitonin. In this model, NT and substance P had no significant effect on either volume or concentration of acid secreted, CCK showed a slight stimulation of acid output, and TRH, hCT, rCGRP and bombesin all inhibited acid output; CGRP and bombesin were active at 10 and 100-fold lower doses. The potent and inhibitory activity of bombesin in this system is in disagreement with other publications reporting no effect or variable stimulatory effect in rats. Time and dose dependent responses in our rat system indicate that this apparent discrepancy may be explained by the short duration of action of bombesin.

This report describes the results of a collaborative study organized by a joint working group of IFCC and WHO and involving nine manufacturers of TSH immunometric assay kits. The study was designed to determine whether a calibrator with a common matrix gives better between-laboratory agreement for calibration of serum samples than the various kit calibrators, and to assess various materials for their suitability for use as common matrices. Kit calibrators or calibrators consisting of the IRP for TSH made up in two common matrices, (1) serum from patients with untreated thyrotoxicosis or (2) serum taken from subjects treated with suppressive doses of triiodothyronine, gave similar results for the between-laboratory variation of estimates of TSH concentration for a range of serum samples. Dose-response curves for the two calibrators in 'common' matrices were similar to one another and to those for the kit calibrator. However, the occurrence of non-specific serum effects is shown by the comparison of results for these calibrators with results for calibrators made up in a third common matrix, serum treated with wheat germ lectin. Dose-response curves for this calibrator were dissimilar to those for the other calibrators and between-laboratory variation for estimates in terms of this latter calibrator showed a substantial increase. Moreover, although the between-laboratory variances for estimates of the TSH concentration in terms of each of these calibrators (except those made up in serum treated with in the wheat germ lectin) were similar for any one sample from five hyperthyroid patients, the variances were not consistent between samples, even for samples with similar mean TSH concentrations. These results suggest that a major factor in the between-laboratory variation, especially in the region near 'zero dose', is sample-related, and is caused by particular samples interacting differently with different assay systems. In general, it would appear that for the well controlled 'ultra-sensitive' TSH immunometric assay kits, included in this study, between-laboratory agreement of estimates of the TSH concentration in serum samples is not likely to be substantially improved by use of a common matrix for the standards.

Three ampouled preparations of interleukin-1 alpha (IL-1 alpha) and three ampouled preparations of interleukin-1 beta (IL-1 beta) were evaluated by 13 laboratories in six countries for their suitability to serve as international standards for these materials. The preparation were assayed in in vitro and in vivo bioassays, radioreceptor assays and immunoassays. On the basis of the results reported here, with the agreement of participants in the study and with the authorization of the Expert Committee on Biological Standardization (ECBS) of the World Health Organization (WHO), one of the preparations of IL-1 alpha and one of the preparations of IL-1 beta were established as international standards for these materials. Further, since the relative activities of IL-1 alpha and IL-1 beta were dependent upon the assay systems used, it was decided that reference should be made to units of IL-1 alpha activity or IL-1 beta activity rather than to IL-1 activity.

The tissue distribution and route of clearance of human recombinant interleukin 1 alpha (IL 1 alpha) injected intravenously in rats was studied. The plasma half-life was approximately 2.5 min, and this was increased after nephrectomy, the kidney being the major organ through which the IL 1 alpha was excreted. Two iodinated fragments of IL 1 alpha, of approximately 5 and 9 kDa, were excreted by the kidneys whereas only intact, 17-kDa IL 1 alpha was detected in plasma, suggesting that the protein was being degraded after uptake by the kidney. The results of in vivo experiments in which surface endopeptidase-24.11 was inhibited with phosphoramidon and in vitro experiments in which rat kidney homogenates were incubated with radiolabeled IL 1 alpha suggest that the cytokine was endocytosed and then hydrolysed by lysosomal proteinases.

Regulatory specifications in most countries require that the potency of salmon calcitonin (sCT) clinical products be expressed in International Units (IU) defined by the World Health Organization (WHO) International Standard. The first ampouled standard was prepared in 1972 and has been distributed world-wide since then. A batch of ampoules to serve as the replacement standard is now required. Other piscine calcitonins, eel calcitonin (eCT) and an amino-suberic acid analogue of eCT (Asu1-7 eCT) are now clinical products in some countries and international standards are required for these peptides which are similar to, but not identical with, sCT. This paper describes the preparation of three new ampouled standards and their biological calibration by international collaborative study comprising 17 participants from 10 countries. Following the recommendations in the final report of the collaborative study, the 2nd International Standard (IS) for sCT, the 1st IS for eCT and the 1st IS for Asu1-7 eCT were recently established by WHO, each with an assigned potency in IU, and are now available for issue.

It is well established that salmon calcitonin (sCT), given either by central (intracerebroventricular) or peripheral (parenteral) injection is a potent inhibitor of gastric acid secretion. It is generally believed that this effect of sCT is mediated by the central nervous system based on reports that the effective peripheral dose is up to 1000 times greater than the effective dose administered centrally. We have reexamined this hypothesis by carrying out a number of independent experiments in two laboratories on the effect of sCT, given either by intracerebroventricular or by subcutaneous injection, on basal gastric acid secretion in unanaesthetized rats. Statistical evaluation of the data showed the reproducibility of the effective inhibitory dose ranges of sCT despite the inherent variability of the pylorus ligated rat system (Shay test). The effective dose range for sCT given centrally was between three- and ten-fold lower than that for peripherally administered sCT. There is no published evidence that a significant amount of peripherally administered sCT passes through the blood-brain barrier to relevant areas of the brain and this is confirmed in our study by autoradiography of serial sections of brain following intravenous administration of radioiodinated sCT. It therefore appears that the inhibitory effect of sCT, given centrally or peripherally, may not necessarily be mediated by a common pathway in the central nervous system. Our results also show that the inhibitory effect of peripherally administered sCT is lost when rats are treated with cysteamine to deplete somatostatin, thus implicating somatostatin as a peripheral mediator.

Although dilution of materials is generally accepted to involve some error, workers often assume that this error may be discounted. However, comparing solutions of two identical materials diluted in the same way shows that dilution errors can cause a considerable departure from the expected identity. I describe this effect and its dependence on the errors at various stages of the dilution process.

We describe the program SCAN which has been designed for use in conjunction with analysis of variance of immunoassay or bioassay data and which may also be used with more general one-way analysis of variance. Data within treatment groups are examined for outliers and normality of distribution. Overall statistics for normality and homogeneity of variance are also computed. Contributions of individual groups to these statistics are tabulated to permit the detection of aberrant groups. Means and variances are available in a form which facilitates detection of any response error relationship.

This paper reports the comparison of recently-developed chromatographic assays (using high performance liquid chromatography) with the present British Pharmacopoeial methods for the combined formulation of ergometrine and oxytocin. The estimates using high performance liquid chromatography were in good agreement with both the Pharmacopoeial determinations and nominal contents. The variability of estimates for ergometrine was similar for both procedures, whereas the variability of estimates of oxytocin content by chromatographic assay was significantly less than the variability of estimates by the Pharmacopoeial bioassay.

The preparation and nature of the International Reference Preparation of Tetracosactide for Bioassay (IRP; in ampoules coded 80/590) are described. The IRP was studied by six laboratories in five countries using in-vivo and in-vitro bioassays and various physicochemical methods. The bulk (1-24)corticotrophin-tetracosapeptide (batch 000179) from which the IRP was prepared contained 10.4% (w/w) acetic acid and 8.3% (w/w) water; its (1-24)corticotrophin-tetracosapeptide content was estimated to be 71.7% (w/w) by amino acid analysis, 74.2% (w/w) by high performance liquid chromatography (HPLC) and 77.5% (w/w) by spectrophotometry. (1-24)Corticotrophin-tetracosapeptide accounted for more than 90% (w/w) of the total peptide in the IRP as judged by HPLC, thin-layer chromatography, carboxymethyl-cellulose chromatography, isoelectric focusing (IEF) and electrophoresis. The homogeneity of the peptide in the IRP was similar by all methods to that in batch 000179 from which it was prepared. The (1-24)corticotrophin-tetracosapeptide content of the IRP (with 95% confidence limits), in terms of batch 000179, was found to be 491 micrograms/ampoule by HPLC and spectrophotometry, 473 (433-513) micrograms/ampoule by IEF and 505 (473-539) micrograms/ampoule by the in-vitro rat adrenocortical cell assay. A comparison in the same bioassay system of the IRP with a laboratory house standard of (1-24)corticotrophin-tetracosapeptide, which originated from a different manufacturer, gave similar results. Accelerated thermal degradation studies of the IRP by adrenocortical cell assay, HPLC and IEF suggested that more than 99.9% of its original content of (1-24)corticotrophin-tetracosapeptide would remain after 10 years under normal storage conditions of -20 degrees C in the dark. Bioassay estimates of samples of the IRP which had undergone significant degradation were higher than estimates by HPLC, indicating that molecular species other than (1-24)corticotrophin-tetracosapeptide contributed to their corticotrophic activity. The corticotrophic activity of the IRP was demonstrated by cytochemical bioassay and by in-vivo bioassays as well as by the adrenocortical cell assay. After consideration of these data, the Expert Committee on Biological Standardization of the World Health Organization established the ampoule d preparation, coded 80/590, as the International Reference Preparation of Tetracosactide for Bioassay and assigned to it a potency of 490 i.u./ampoule; thus the i.u. is represented by 1 microgram (1-24)corticotrophin-tetracosapeptide.