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Any biopharmaceutical must obviously conform to final product potency specifications. Such specifications are usually expressed in terms of ‘units of activity’ per vial of product (or per therapeutic dose, or per mg of product). A number of different approaches may be undertaken to determine product potency. Each exhibits certain advantages and disadvantages.
Bioassays represent the most relevant potency-determining assay, as they directly assess the biological activity of the biopharmaceutical. Bioassay involves applying a known quantity of the substance to be assayed to a biological system which responds in some way to this applied stimulus. The response is measured quantitatively, allowing an activity value to be assigned to the substance being assayed.
All bioassays are comparative in nature, requiring parallel assay of a ‘standard’ preparation against which the sample will be compared. Internationally accepted standard preparations of most biopharmaceuticals are available from organizations such as the World Health Organization (WHO) or the US Pharmacopoeia.
An example of a straightforward bioassay is the traditional assay method for antibiotics. Bioassays for modern biopharmaceuticals are generally more complex. The biological system used can be whole animals, specific organs or tissue types, or individual mammalian cells in culture.
Bioassays of related substances can be quite similar in design. Specific growth factors, for example, stimulate the accelerated growth of specific animal cell lines. Relevant bioassays can be undertaken by incubation of the growth factor-containing sample with a culture of the relevant sensitive cells and radiolabelled nucleotide precursors. After an appropriate time period, the level of radioactivity incorporated into the DNA of the cells is measured. This is a measure of the bioactivity of the growth factor.
The most popular bioassay of erythropoietin (EPO) involves a mouse-based bioassay [EPO stimulates red blood cell (RBC) production, making it useful in the treatment of certain forms of anaemia; Chapter 6]. Basically, the EPO-containing sample is administered to mice, along with radioactive iron (57Fe). Subsequent measurement of the rate of incorporation of radioactivity into proliferating RBCs is undertaken (the greater the stimulation of RBC proliferation, the more iron is taken up for haemoglobin synthesis).
One of the most popular bioassays for interferons is termed the ‘cytopathic effect inhibition assay’. This assay is based upon the ability of many interferons to render animal cells resistant to viral attack. It entails incubation of the interferon preparation with cells sensitive to destruction by a specific virus. That virus is then subsequently added, and the percentage of cells that survive thereafter is proportional to the levels of interferon present in the assay sample. Viable cells can assimilate certain dyes, such as neutral red. Addition of the dye, followed by spectrophotometric quantitation of the amount of dye assimilated, can thus be used to quantitfy percentage cell survival. This type of assay can be scaled down to run in a single well of a microtitre plate. This facilitates automated assay of a large number of samples with relative ease.
While bioassays directly assess product potency (i.e. activity), they suffer from a number of drawbacks, including:
• Lack of precision. The complex nature of any biological system, be it an entire animal or an individual cell, often results in the responses observed being influenced by factors such as the metabolic status of individual cells, or (in the case of whole animals) sub-clinical infections, stress levels induced by human handling, etc.
• Time. Most bioassays take days, and in some cases weeks, to run. This can render routine bioassays difficult, and impractical to undertake as a quick QC potency test during downstream processing.
• Cost. Most bioassay systems, particularly those involving whole animals, are extremely expensive to undertake.
Because of such difficulties, alternative assays have been investigated, and sometimes are used in conjunction with, or instead of, bioassays. The most popular alternative assay systems are the immunoassays.
Immunoassays employ monoclonal or polyclonal antibody preparations to detect and quantify the product. The specificity of antibody-antigen interaction ensures good assay precision. The use of conjugated radiolabels (radioimmunoassay; RIA) or enzymes (enzyme immunoassay: EIA) to allow detection of antigen-antibody binding renders such assays very sensitive. Furthermore, when compared to bioassays, immunoassays are rapid (undertaken in minutes to hours), inexpensive and straightforward to undertake.
The obvious disadvantage of immunoassays is that immunological reactivity cannot be guaranteed to correlate directly with biological activity. Relatively minor modifications of the protein product, while having a profound influence on its biological activity, may have little or no influence on its ability to bind antibody.