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Current viral assays fall into one of three categories:
• assays based on viral DNA probes;
Generation of antibodies that can recognize and bind to specific viruses is straightforward. A sample of live or attenuated virus, or a purified component of the viral caspid, can be injected
into animals to stimulate polyclonal antibody production (or to facilitate monoclonal antibody production by hybridoma technology). Harvested antibodies are then employed to develop specific immunoassays which can be used to routinely screen test samples for the presence of that specific virus. Immunoassays capable of detecting a wide range of viruses are available commercially. The sensitivity, ease, speed and relative inexpensiveness of these assays render them particularly attractive.
An alternative assay format entails the use of virus-specific DNA probes. These can be used to screen the biopharmaceutical product for the presence of viral DNA. The assay strategy is similar to the dot-blot assays used to detect host cell derived DNA contaminants, as discussed earlier.
Viral bioassays of various different formats have also been developed. One format entails incubation of the final product with cell lines sensitive to a range of viruses. The cells are subsequently monitored for cytopathic effects or other obvious signs of viral infection.
A range of mouse, rabbit or hamster antibody production tests (MAPs, RAPs or HAPs) may also be undertaken. These bioassays entail administration of the product to a test animal. Any viral agents present will elicit the production of anti-viral antibodies in that animal. Serum samples (withdrawn from the animal approximately 4 weeks after product administration) are screened for the presence of antibodies recognizing a range of viral antigens. This can be achieved by enzyme immunoassay, in which immobilized antigen is used to screen for the virus-specific antibodies. These assay systems are extremely sensitive, as minute quantities of viral antigen will elicit strong antibody production. A single serum sample can also be screened for antibodies specific to a wide range of viral particles. Time and expense factors, however, militate against this particular assay format.
In addition to those already discussed, biopharmaceutical products may harbour other contaminants, some of which may be intentionally added to the product-stream during the initial stages of downstream processing. Examples could include buffer components, precipitants (ethanol or other solvents, salts, etc.), proteolytic inhibitors, glycerol, anti-foam agents, etc. In addition to these, other contaminants may enter the product during downstream processing in a less controlled way. Examples could include metal ions leached from product-holding tanks/pipework, or breakdown products leaking from chromatographic media. The final product containers must also be chosen carefully. They must be chemically inert, and be of suitable quality to eliminate the possibility of leaching of any substance from the container during product storage. For this reason high-quality glass vials are often used.
In some instances it may be necessary to demonstrate that all traces of specific contaminants have been removed prior to final product filling. This would be true, for example, of many proteolytic inhibitors added during the initial stages of downstream processing to prevent proteolysis by endogenous proteases. Some such inhibitors may be inherently toxic, and many could (inappropriately) inhibit endogenous proteases of the recipient patient.
Demonstration of the absence (from the product) of breakdown products from chromatographic columns may be necessary in certain instances. This is particularly true with regard to some affinity chromatography columns. Various chemical-coupling methods may be used to attach affinity ligands to the chromatographic support material. Some such procedures entail the use of toxic reagents which, if not entirely removed after coupling, could leach into the product. In some cases, ligands can also subsequently leach from the columns, particularly after
THE DRUG MANUFACTURING PROCESS 183
sustained usage or over-vigorous sanitation procedures. Improvements in the chemical stability of modern chromatographic media have, however, reduced such difficulties, and most manufacturers have carried out extensive validation studies regarding the stability of their products.
Sophisticated analytical methodologies facilitate detection of vanishingly low levels of many contaminants in biopharmaceutical preparations. The possibility exists, however, that uncharacterized contaminants may persist, remaining undetected in the final product. As an additional safety measure, finished products are often subjected to ‘abnormal toxicity’ or ‘general safety’ tests. Standardized protocols for such tests are outlined in various international pharmacopoeias. These normally entail parenteral administration of the product to at least five healthy mice. The animals are placed under observation for 48 h, and should exhibit no ill-effects (other than expected symptoms). The death or illness of one or more animals signals a requirement for further investigation, usually using a larger number of animals. Such toxicity testing represents a safety net, designed to expose any unexpected activities in the product which could compromise the health of the recipient.