Product analysis - Pharmaceutical Biotechnology: Concepts and Aapplications - page 199

• Microbial-derived substances secreted into the product could adversely affect the recipient's

health. Examples include endotoxin secreted from Gram-negative bacteria, or microbial pro-
teins that 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
fi ltration, followed by aseptic fi lling into a sterile fi nal-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. Specifi c guidelines relating to
sterility testing of fi nished products are given in international pharmacopoeias.
Biopharmaceutical products are also subjected to screening for the presence of viral particles
prior to fi nal product release. Although viruses could be introduced, for example, via infected per-
sonnel during downstream processing, proper implementation of GMP minimizes such risk. Any
viral particles found in the fi nished 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 monoclonal anti-
bodies 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, yeast, 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 physi-
cochemical properties of viral particles differ greatly from most proteins, ensuring that effective
fractionation is automatically achieved by most chromatographic techniques. Gel-fi ltration chro-
matography, for example, effectively separates viral particles from most proteins on the basis of
differences in size.
In addition to chromatographic separation, downstream processing steps may be undertaken
that are specifi cally aimed at removal or inactivation of viral particles potentially present in the
product stream. Signifi cantly, many are 'blanket' procedures, equally capable of removing known
or potentially likely viral contaminants and any uncharacterized/undetected viruses. Filtration
through a 0.22 µm fi lter effectively removes microbial agents from the product stream, but fails to
remove most viral types. Repeat fi ltration through a 0.1 µm fi lter is more effective in this regard.
Alternatively, incorporation of an ultrafi ltration step (preferably at the terminal stages of down-
stream processing) also proves effective.
Incorporation of downstream processing steps known to inactivate a wide variety of viral types
provides further assurance that the fi nal product is unlikely to harbour active virus. Heating and ir-
radiation are amongst the two most popular such approaches. Heating the product to between 40 and
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 exten-
sively 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.