Product analysis - Pharmaceutical Biotechnology: Concepts and Aapplications - page 198

primarily upon the presence of active oncogenes in the genome of several producer cell types (e.g.
monoclonal antibody production in hybridoma cell lines). Parenteral administration of DNA con-
taminants containing active oncogenes to patients is considered undesirable. The concern is that
uptake and expression of such DNA in human cells could occur. There is some evidence to suggest
that naked DNA can be assimilated by some cells at least, under certain conditions (Chapter 14).
Guidelines to date state that an acceptable level of residual DNA in recombinant products is of the
order of 10 pg per therapeutic dose.
DNA hybridization studies (e.g. the 'dot blot' assay) utilizing radiolabelled DNA probes allows de-
tection of DNA contaminants in the product, to levels in the nanogram range. The process begins with
isolation of the contaminating DNA from the product. This can be achieved, for example, by phenol
and chloroform extraction and ethanol precipitation. The isolated DNA is then applied as a spot (i.e. a
'dot') onto nitrocellulose fi lter paper, with subsequent baking of the fi lter at 80 C under vacuum. This
promotes (a) DNA denaturation, yielding single strands, and (b) binding of the DNA to the fi lter.
A sample of total DNA derived from the cells in which the product is produced is then radiola-
belled with 32 P using the process of nick translation. It is heated to 90

C (promotes denaturation,
forming single strands) and incubated with the baked fi lter for several hours at 40 C. Lowering
the temperature allows reannealing of single strands via complementary base-pairing to occur.
Labelled DNA will reanneal with any complementary DNA strands immobilized on the fi lter.
After the fi lter is washed (to remove non-specifi cally bound radiolabelled probe) it is subjected to
autoradiography, which allows detection of any bound probe.
Quantifi cation of the DNA isolated from the product involves concurrent inclusion in the dot
blot assay of a set of spots, containing known quantities of DNA, and being derived from the pro-
ducer cell. After autoradiography, the intensity of the test spot is compared with the standards.
In many instances there is little need to incorporate specifi c DNA removal steps during down-
stream processing. Endogenous nucleases liberated upon cellular homogenization come into di-
rect contact with cellular DNA, resulting in its degradation. Commercial DNase's are sometimes
added to crude homogenate to reduce DNA-associated product viscosity (Chapter 6). Most chro-
matographic steps are also effective in separating DNA from the product stream. Ion-exchange
chromatography is particularly effective, as DNA exhibits a large overall negative charge (due to
the phosphate constituent of its nucleotide backbone; Chapter 3).

7.6.4 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 fi nal 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 ar-
ray of extracellular proteases.