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Microbial and viral contaminants
Finished-product biopharmaceuticals, along with other pharmaceuticals intended for parenteral administration, must be sterile (the one exception being live bacterial vaccines). The presence of microorganisms in the final product is unacceptable for a number of reasons:
• parenteral administration of contaminated product would likely lead to the establishment of a severe infection in the recipient patient;
• microorganisms may be capable of metabolizing the product itself, thus reducing its potency. This is particularly true of protein-based biopharmaceuticals, as most microbes produce an array of extracellular proteases;
• microbial-derived substances secreted into the product could adversely effect the recipient’s health. Examples include endotoxin secreted from Gram-negative bacteria, or microbial proteins which would stimulate an immune response.
Terminal sterilization by autoclaving guarantees product sterility. Heat sterilization, however, is not a viable option in the case of biopharmaceuticals. Sterilization of biopharmaceuticals by filtration, followed by aseptic filling into a sterile final-product container, inherently carries a greater risk of product contamination. Finished-product sterility testing of such preparations thus represents one of the most critical product tests undertaken by QC. Specific guidelines relating to sterility testing of finished products are given in international pharmacopoeias.
Biopharmaceutical products are also subjected to screening for the presence of viral particles prior to final product release. Although viruses could be introduced, e.g. via infected personnel during downstream processing, proper implementation of GMP minimizes such risk. Any viral particles found in the finished product are most likely derived from raw material sources. Examples could include HIV or hepatitis viruses present in blood used in the manufacture of blood products. Such raw materials must be screened before processing for the presence of likely viral contaminants.
A variety of murine (mouse) and other mammalian cell lines have become popular host systems for the production of recombinant human biopharmaceuticals. Moreover, most
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monoclonal antibodies used for therapeutic purposes are produced by murine-derived hybridoma cells. These cell lines are sensitive to infection by various viral particles. Producer cell lines are screened during product development studies to ensure freedom from a variety of pathogenic advantageous agents, including various species of bacteria, fungi, yeasts, mycoplasma, protozoa, parasites, viruses and prions. Suitable microbiological precautions must subsequently be undertaken to prevent producer cell banks from becoming contaminated with such pathogens.
Removal of viruses from the product stream can be achieved in a number of ways. The physicochemical properties of viral particles differ greatly from most proteins, ensuring that effective fractionation is automatically achieved by most chromatographic techniques. Gel-filtration chromatography, for example, effectively separates viral particles from most proteins on the basis of differences in size.
In addition to chromatographic separation, specific downstream processing steps may be undertaken which are specifically aimed at removal or inactivation of viral particles potentially present in the product stream. Significantly, many are ‘blanket’ procedures, equally capable of removing known or potentially likely viral contaminants, and any uncharacterized/undetected viruses. Filtration through a 0.22 mm filter effectively removes microbial agents from the product stream, but fails to remove most viral types. Repeat filtration through a 0.1 mm filter appears more effective in this regard. Alternatively, incorporation of an ultrafiltration step (preferably at the terminal stages of downstream processing) also proves effective.
Incorporation of downstream processing steps known to inactivate a wide variety of viral types provides further assurance that the final product is unlikely to harbour active virus. Heating and irradiation are amongst the two most popular such approaches. Heating the product to 40-60°C for several hours inactivates a broad range of viruses. Many biopharmaceuticals can be heated to such temperatures without being denatured themselves. Such an approach has been used extensively to inactivate blood-borne viruses in blood products. Exposure of product to controlled levels of UV radiation can also be quite effective, while having no adverse effect on the product itself.
A range of assay techniques may be used to detect and quantify viral contaminants in both raw materials and finished biopharmaceutical products. No generic assay exists that is capable of detecting all viral types potentially present in a given sample. Viral assays currently available will detect only a specific virus, or at best a family of closely related viruses. The strategy adopted, therefore, usually entails screening product for viral particles known to be capable of infecting the biopharmaceutical source material. Such assays will not normally detect newly evolved viral strains, or uncharacterized/unknown viral contaminants. This fact underlines the importance of including at least one step in downstream processing which is likely to indiscriminately inactivate or remove viruses from the product. This acts as a safety net.