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Contaminant-clearance validation studies are of special significance in biopharmaceutical manufacture. As discussed in the previous section, downstream processing must be capable of removing contaminants such as viruses, DNA and endotoxin from the product stream. Contaminant-clearance validation studies normally entails spiking the raw material (from which the product is to be purified) with a known level of the chosen contaminant, and subjecting the contaminated material to the complete downstream processing protocol. This allows determination of the level of clearance of the contaminant achieved after each purification step, and the contaminant reduction factor for the overall process.
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Viral clearance studies, for example, are typically undertaken by spiking the raw material with a mixture of at least three different viral species, preferably ones that represent likely product contaminants, and for which straightforward assay systems are available. Loading levels of up to 1 x 1010 viral particles are commonly used. The cumulative viral removal/inactivation observed should render the likelihood of a single viral particle remaining in a single therapeutic dose of product being greater than one in a million.
A similar strategy is adopted when undertaking DNA clearance studies. The starting material is spiked with radiolabelled DNA and then subjected to downstream processing. The level of residual DNA remaining in the product stream after each step can easily be determined by monitoring for radioactivity.
The quantity of DNA used to spike the product should ideally be somewhat in excess of the levels of DNA normally associated with the product prior to its purification. However, spiking of the product with a vast excess of DNA is counter-productive in that it may render subsequent downstream processing unrepresentative of standard production runs.
For more comprehensive validation studies, the molecular mass profile of the DNA spike should roughly approximate to the molecular mass range of endogenous contaminant DNA in the crude product. Obviously, the true DNA clearance rate attained by downstream processing procedures (e.g. gel-filtration) will depend to some extent on the molecular mass characteristics of the contaminant DNA.
Other manufacturing procedures requiring validation include cleaning, decontamination and sanitation (CDS) procedures developed for specific items of equipment/processing areas. Of particular importance is the ability of such procedures to remove bioburden. This may be assessed by monitoring levels of microbial contamination before and after application of CDS protocols, to the equipment item in question.
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Sources of biopharmaceuticals and upstream processing