Product analysis - Pharmaceutical Biotechnology: Concepts and Aapplications - page 178

peutic dose, or per milligram 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 that 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 United States Pharmacopeia.
An example of a straightforward bioassay is the traditional assay method for antibiotics. This
usually entailed measuring the zone of inhibition of microbial growth around an antibiotic-con-
taining disc, placed on an agar plate seeded with the test microbe. Bioassays for modern biop-
harmaceuticals are generally more complex. The biological system used can be whole animals,
specifi c organs or tissue types, or individual mammalian cells in culture.
Bioassays of related substances can be quite similar in design. Specifi c growth factors, for ex-
ample, stimulate the accelerated growth of specifi c 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 bio-
activity of the growth factor.
The most popular bioassay of EPO involves a mouse-based bioassay (EPO stimulates red
blood cell production, making it useful in the treatment of certain forms of anaemia; Chapter 10).
Basically, the EPO-containing sample is administered to mice along with radioactive iron ( 57 Fe).
Subsequent measurement of the rate of incorporation of radioactivity into proliferating red blood
cells is undertaken. (The greater the stimulation of red blood cell proliferation, the more iron
taken up for haemoglobin synthesis.)
One of the most popular bioassay for interferons is termed the 'cytopathic effect inhibi-
tion 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 specifi c 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 fol-
lowed by spectrophotometric quantitation of the amount of dye assimilated can thus be used
to quantitate 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 large numbers of samples with
relative ease.
Although 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 indi-

vidual cell, often results in the responses observed being infl uenced by factors such as metabolic
status of individual cells, or (in the case of whole animals) subclinical infections, stress levels
induced by human handling, etc.