Risk Assessment of Residual Genomic DNA in Therapeutic Proteins Using Gene Copy Number Application

by Rahul Fadnis, PhD, Reena Nr, and Rajeev Soni, PhD
Volume 13, Issue 1 (Spring 2014)

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...

Fadnis R, Nr R, Soni R. Risk Assessment of Residual Genomic DNA in Therapeutic Proteins Using Gene Copy Number Application. BioProcess J, 2014; 13(1): 32-40. http://dx.doi.org/10.12665/J131.Fadnis.

Posted online April 23, 2014.