In the current biotherapeutical industry, different host cells, such as CHO, hybridoma & engineered bacteria strains in the bio-reactors can be utilized for the manufacturing of protein therapeutics. But limited by current purification techniques, low levels of host DNA, in different sizes and physical forms, may still remain in the final products even after effective purification during manufacturing. Residual DNA (rDNA) defined as the sum total of deoxyribonucleic acid (DNA) and fragments present in biological samples derived from recombinant host cells during expression, are considered to be a risk factor due to oncogenic or infectivity potential, and may trigger unpredictable immune responses after dosing, even only ppm level left in final therapeutical products.
The potential risks associated with rDNA are infectivity (through viruses like HIV), oncogenicity (through oncogenes like Ras), immunogenicity (through CpG rich-but-poorly methylized sequences from bacteria), and mutagenesis (through transposons, retrotransposons and DNA recombination). So for the biotherapeutics of clinical utilization, the regulatory authorities require to closely monitor the clearance of residual DNA in bioprocesses and confirm the acceptable level of residual DNA in final drug substances. Initially World Health Organization recommended the total rDNA should not exceed 100 pg/dose (WHO, 1987), and later the limit was modified to 10ng/dose. This differs from host cell proteins (HCPs), handled case by case.
There are several quantitative methods that have been developed for rDNA quantification. The classic approaches, can be categorized to 3 types: PicoGreen method, Southern Blot-like hybridization and Threshold technique, which have their own advantages and limitations. Considering the key role of rDNA testing in biotherapeutic manufacturing, a sensitive, fast, and cost-efficient method is needed, and the booming PCR-based quantitative technique has proven to be a promising alternative approach in the biotechnology industry. Because with the PCR reaction, a sequence-specific DNA template would be amplified to billions of copies within 1~2h, and the signals detected in this early exponential phase is highly proportional to the quantity of starting DNA, which allows real-time detection of extremely low levels of DNA.
Among the technical solutions in real-time PCR, three are more popular and all based on the use of fluorescent dyes as below:
- SYBR Green
Based on double-strand DNA binding dyes
- Dye-Primer based Signaling
- TaqMan Technique
Currently most popular technique in the industry
Similar to the analysis of HCPs, rDNA is also taken as a host related impurity, so the demonstration of rDNA clearance to an acceptable level is an important step in biotherapeutic development. Creative Proteomics can help you to make it with our professional technical staff, and the experience in bioanalytical works.