Viral clearance studies are required for pharmaceuticals derived from human and/or animal sources such as recombinant proteins produced in eukaryotic cell lines, human blood products and vaccines, and even for some critical class III medical devices. It is mandatory to demonstrate that steps in the manufacturing process are capable of inactivating or removing potential viral contaminants. For this, a laboratory-scale (downscale) of the process step is developed and challenged with different model virus solutions. The viral concentrations are quantitatively determined in the feed material and the relevant product fraction. The ratio of both defines the reduction in virus and specifies the viral inactivation or viral removal capacity of the investigated process step…
Tag: <span>qpcr</span>
Therapeutic proteins manufactured in cellular systems contain residual DNA derived from host cell substrates used in production. Risk assessment of the residual host cell DNA is necessary, as some of these DNA sequences may be potentially infectious or oncogenic. Oncogenic potential lies in transmission of the activated oncogenes to subjects receiving the product, thereby inducing oncogenic events. Therefore, it becomes essential for drug manufacturers to show clearance of genomic DNA (oncogenic sequences as well) throughout production processes and to confirm low levels of residual DNA in the final drug substance. This study attempted to estimate the oncogenes in the total residual DNA using a highly sensitive, specific, and robust method—quantitative polymerase chain reaction (qPCR). Routinely, total residual DNA is estimated using either the 18S ribosomal (r)DNA gene or Alu equivalent multicopy gene sequence as qPCR targets. We have determined the copy numbers of these qPCR targets along with the oncogene (Ras gene) and housekeeping genes (ACTB and GAPDH) and established a ratio of their presence in protein samples. Another objective of the study was to estimate the level of oncogenes from several in-process step samples in the manufacturing and purification process and check the clearance of total residual DNA including oncogenes. Upon quantification, the proportions of oncogenes present were one tenth of the quantified residual DNA levels (Ras gene:18S RNA) in the purification stage samples, providing information that the therapeutic protein product was safe from the presence of oncogenes in residual DNA by a factor of ten…