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The molecular mass of most biopharmaceuticals is considerably less than 100 kDa (Table
3.28). The proteins would thus elute from gel-filtration columns much later than contaminating
THE DRUG MANUFACTURING PROCESS 179
Table 3.28. The molecular mass of some polypeptide biopharmaceuticals. Many are glycosylated, thereby exhibiting a range of molecular masses due to differential glycosylation
Protein Molecular Protein Molecular Protein Molecular
mass (kDa) mass (kDa) mass (kDa)
IFN-a 20-27 TNF-a 52* EGF 6
IFN-b 20 GM-CSF 22 NGF 26
IFN-g 20-25 G-CSF 21 Insulin 5.7
IL-2 15-20 EPO 36 hGH 22
IL-1 17.5 TPO 60 FSH 34
IL-12 30-35 IGF-1 7.6 LH 28.5
*Biologically active, trimeric form. IFN = interferon; IL=interleukin; TNF = tumour necrosis factor; GM-CSF = granulocyte macrophage colony stimulating factor; G-CSF = granulocyte colony stimulating factor; EPO = erythropoietin; TPO = thrombopoietin; IGF = insulin-like growth factor; EGF = epidermal growth factor; NGF = nerve growth factor; hGH = human growth hormone; FSH = follicle stimulating hormone; LH = leuteinizing hormone
endotoxin aggregates. Should the biopharmaceutical exhibit a molecular mass approaching or exceeding 100 kDa, effective separation can still be achieved by inclusion of a chelating agent, such as EDTA, in the running buffer. This promotes depolymerization of the endotoxin aggregates into monomeric (20 kDa) form.
Additional techniques capable of separating biomolecules on the basis of molecular mass (e.g. ultrafiltration) may also be used to remove endotoxin from the product stream.
The clinical significance of DNA-based contaminants in biopharmaceutical products remains unclear. Many traditional biological-based preparations, especially those such as vaccines produced in cell culture systems, have been found to consistently contain host cell-derived DNA. No adverse clinical effects related to the presence of such DNA has been reported.
The concerns relating to the presence of DNA in modern biopharmaceuticals focuses 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 contaminants containing active oncogenes to patients is considered undesirable. The concern is that uptake and expression of such DNA in human cells could transform those cells, leading to cancer. There is some evidence to suggest that naked DNA can be assimilated by some cells at least, under certain conditions (Chapter 11). 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 detection of DNA contaminants in the product, to levels in the nanogram (ng) 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 filter paper, with subsequent baking of the filter at 80°C under vacuum. This promotes (a) DNA denaturation, yielding single strands, and (b) binding of the DNA to the filter.
A sample of total DNA derived from the cells in which the product is produced is then radiolabelled with 32P using the process of nick translation. It is heated to 90°C (promotes
denaturation, forming single strands) and incubated with the baked filter for several hours at 40°C. Lowering the temperature allows re-annealing of single strands via complementary base-pairing to occur. Labelled DNA will re-anneal with any complementary DNA strands immobilized on the filter. After the filter is washed (to remove non-specifically bound radiolabelled probe) it is subjected to autoradiography, which allows detection of any bound probe.
Quantification 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 derived from the producer cell. After autoradiography, the intensity of the test spot is compared with the standards.
In many instances there is little need to incorporate specific DNA removal steps during downstream processing. Endogenous nucleases liberated upon cellular homogenization come into direct contact with cellular DNA, resulting in its degradation. Commercial DNases are sometimes added to crude homogenate to reduce DNA-associated product viscosity. Most chromatographic 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).