Residual DNA analysis in biologics development: Review of measurement and quantitation technologies and future directions

Abstract Residual DNA (rDNA) is comprised of deoxyribonucleic acid (DNA) fragments and longer length molecules originating from the host organism that may be present in samples from recombinant biological processes. Although similar in basic structural base pair units, rDNA may exist in different sizes and physical forms. Interest in measuring rDNA in recombinant products is based primarily on demonstration of effective purification during manufacturing, but also on some hypothetical concerns that, in rare cases, depending on the host expression system, some DNA sequences may be potentially infectious or oncogenic (e.g., HIV virus and the Ras oncogene, respectively). Recent studies suggest that a sequence known as long interspersed nucleotide element‐1 (LINE‐1), widely distributed in the mammalian genome, is active as a retrotransposon that can be transcribed to RNA, reverse‐transcribed into DNA and inserts into a new site in genome. This integration process could potentially disrupt critical gene functions or induce tumorigenesis in mammals. Genomic DNA from microbial sources, on the other hand, could add to risk of immunogenicity to the target recombinant protein being expressed, due to the high CpG content and unmethylated DNA sequence. For these and other reasons, it is necessary for manufacturers to show clearance of DNA throughout production processes and to confirm low levels in the final drug substance using an appropriately specific and quantitative analytical method. The heterogeneity of potential rDNA sequences that might be makes the testing of all potential analytes challenging. The most common methodology for rDNA quantitation used currently is real‐time polymerase chain reaction (RT‐PCR), a robust and proven technology. Like most rDNA quantitation methods, the specificity of RT‐PCR is limited by the sequences to which the primers are directed. To address this, primase‐based whole genome amplification is introduced herein. This paper will review the recent advancement in rDNA quantitation and recent findings regarding potential risks of immunogenicity, infectivity, and oncogenicity of rDNA. Biotechnol. Bioeng. 2012; 109:307–317. © 2011 Wiley Periodicals, Inc.