Most biological agents enter the body directly without passing through the gastrointestinal tract, so in addition to biological activity, relevant authorities have very strict requirements on impurities in drugs. Among them, the host cells residual DNA has always been the focus of regulatory agencies because of its special potential safety risks.
Although recombinant protein drugs, antibody drugs, vaccines and other products in biological products have undergone strict purification processes, there may still be residual host cell DNA in the products. Such residual DNA may have infectious or tumorigenic risks, for example, the residual DNA may carry the HIV virus or the Ras oncogene. Host Cell DNA Testing is a tool to accurately quantify the amount of host cell DNA residues in biologics using qpcr and other methods.
The Purpose of Host Cell Residual DNA Detection
Confirm that the purification process is reasonable and can effectively remove the host cell residual DNA.
Confirm that the impurity content in the product meets the standard requirements.
Residual Host Cell DNA Testing Method
Considering the potential safety risks of host cell residual DNA, it is important to establish sensitive, accurate, specific and durable methods for the quantitative determination of residual DNA. Establishing appropriate host cell residual DNA assays can help monitor the production process and ensure the safety and quality of biological products. The main assays for residual DNA are as follows：
DNA probe hybridization
Some studies have shown that the detection results of hybridization method are quite different from the real host cells residual DNA content, and the method is unstable and the detection time is relatively long.
Fluorescent staining
This method is also a total DNA detection method, but the fluorescent signal is susceptible to interference and has poor specificity, so environmental DNA contamination must be avoided, and all materials and reagents must be DNA-free.
Threshold method
This method quantitatively detects ssDNA to calculate the total amount of DNA in a sample, is limited to detecting DNA fragments less than 800 bp, and may be inhibited by high concentrations of DNA (1 ng/ml), and is also susceptible to short DNA fragments (20–80 bp) ) and lack stability.
Quantitative PCR for Residual Host Cell DNA Testing
At present, and real-time quantitative PCR is currently the best, which is specific, sensitive, rapid and can achieve high throughput detection. Establishing appropriate methods for detecting residual DNA of host cells can help monitor the production process and ensure the safety and quality of biological products.
Residual Host Cell DNA Testing workflow
By designing specific primers and fluorescently labeled probes for exogenous DNA or adding sensitive fluorescent dyes to the reaction system, changes in the amount of specific amplification products can be reflected in real time by continuously monitoring changes in the fluorescence values in the reaction system. When the fluorescence intensity released during the reaction reaches a preset threshold, the number of PCR cycles of the system has a linear relationship with the logarithm of the amount of initial DNA template. Using DNA standard substance of known concentration and according to the above relationship, constructing a standard curve and quantitatively analyzing the specific template, the residual amount of exogenous DNA in the test sample can be determined.