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.

"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.

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).

Role of ancillary variables in the design, analysis, and interpretation of animal experiments.

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.

Physicochemical methods for predicting the biological potency of recombinant follicle stimulating hormone: an international collaborative study of isoelectric focusing and capillary zone electrophoresis.

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.