What is Host Cell Residual DNA?
Biological products cannot contain foreign substances, especially host DNA, otherwise immune rejection is likely to occur, thereby posing a threat to life safety. Thus, drug regulatory agencies in various countries have strict limits on host cell DNA residual amounts, and pharmacopoeias of various countries have provided several classic detecting methods for residual DNA quantification, including threshold methods, hybridization methods and real-time quantitative PCR.
Host cell residual DNA is DNA fragment from host cells that may exist in biological products. Host cell residual DNA in biological products produced by passage cell lines may carry tumor or virus-related genes and has potential risks. For example, residual DNA may carry the HIV virus or the Ras oncogene. LINE-1 sequences distributed in mammalian cell genomes may be inserted into chromosomes as retrotransposons, and this insertion may affect the performance of key gene functions, such as activating oncogenes or inhibiting tumor suppressor genes.
In addition, microbial-derived genomic DNA is rich in CpG and unmethylated sequences, increasing the risk of immunogenicity of recombinant protein drugs in vivo. The current findings suggest that the tumorigenic risk of residual DNA is lower compared to the infectious risk, but considering that tumorigenicity experiments are done in animals and infectious experiments are done at the cellular level, perhaps neither risk should be taken lightly. Therefore, there are strict standards for the detection of host DNA residues in biologics.
Residual DNA in Biological products production
What is host cell line?
The growth and replication of viruses need to depend on living organisms or biological tissues and cells, and the cells used to culture viruses in vitro are collectively referred to as cell matrices. Cells are the main raw materials for the production of biological products such as vaccines, and their types, generations, and growth characteristics will directly affect the quality and yield of products, especially their safety. Currently, there are a variety of animal cell matrices used for the production of vaccines and other biologics, such as primary cells, passaged cells, human diploid cells, etc.
Residual DNA
The host cells for the production of biological products are not the same, and the potential harm caused by the residual DNA of the host cells is also different. Although these DNAs have the same basic structural unit, their fragments have different lengths and exist in different physical forms, which can cause a variety of consequences after entering the human body.
Oncogenicity of Residual DNA
The main oncogenic mechanism of residual DNA is the introduction of dominant oncogenes, such as MYC and RAS, which can directly transform normal cells, causing some cells to differentiate into tumorigenic cells. Other oncogenic mechanisms may be mutations caused by the insertion of residual DNA from the host cell, which is less likely to occur due to the low probability of integration of small fragments of DNA into the genome of the inoculator, and the correspondingly low probability of activating a proto-oncogene or suppressing an oncogene even if integration happens to occur at a critical location. Theoretically, the DNA of the passaged cell line has the potential ability to make other cells grow out of control and produce oncogenic activity, so it is necessary to carry out quality control on the amount of DNA residues.
Infectious of Residual DNA
Residual DNA is infectious because of the possible presence of infectious viral genomes in cellular DNA, which can amplify and produce infectious viral particles through replication and transcription, and therefore the risk of DNA infectivity may be higher than the risk of carcinogenicity. When developing biologics derived from continuous cell lines, DNA infectivity needs to be considered to assess the safety of residual DNA in host cells.
Residual Host Cell DNA Limit
The WHO, US FDA and European Pharmacopoeia currently recommend that risk assessment should take into account the properties of the cell matrix and the use of the biologic product. When tumorigenic cell lines are used, more stringent control of DNA residues is required to ensure product safety. During the manufacture of some products, it is difficult to reduce DNA residues without altering the potency of the product, and DNA alkylating agents such as β-propanolactone can be used to alter the structure and size of residual DNA, thereby reducing the associated risks.
Current WHO and US FDA guidelines recommend residual DNA in finished products to be no higher than 10 ng/dose and no greater than 200 bp in length. The guidelines issued by the U.S. FDA state that the residual DNA limit for host cells of biologics is 100 pg/dose, while for high-dose biologics such as monoclonal antibodies, the residual DNA is not higher than 10 ng/dose depending on the source of the residual DNA and the route of administration.
Most of the residual DNA limits for biological products stipulated in the European Pharmacopoeia are no more than 10 ng/dose, but the residual DNA limits for individual vaccines are more stringent, for example, the residual DNA in inactivated hepatitis A vaccine should not exceed 100 pg/dose and the residual DNA in hepatitis B vaccine should not exceed 10 pg/dose.
Creative Proteomics has optimized the qPCR method for the detection of CHO residual DNA, which can specifically and quickly detect the residual DNA in the intermediate, semi-finished and finished products during the development and production of biological products such as antibody drugs and recombinant protein vaccines.
References
- Li, S. M., Bai, F. L., Xu, W. J., Yang, Y. B., An, Y., Li, T. H., ... & Wang, W. F. (2014). Removing residual DNA from Vero-cell culture-derived human rabies vaccine by using nuclease. Biologicals, 42(5), 271-276.
- World Health Organization. (1987). Acceptability of cell substrates for production of biologicals: report of a WHO study group [meeting held in Geneva from 18 to 19 November 1986].
- Grachev, V., Magrath, D., Griffiths, E., & Petricciani, J. C. (1998). WHO requirements for the use of animal cells as in vitro substrates for the production of biologicals (requirements for biological substances No. 50). Biologicals, 26(3), 175-193.
- Food and Drug Administration. (1993). Points to consider in the characterization of cell lines used to produce biological products [2022-04-04].
- Food and Drug Administration. (1997) Points to consider in the manufacture and testing of monoclonal antibody products for human use [2022-04-04].
- European Pharmacopoeia Commission. European pharmacopoeia 9. 8[M]. Strasbourg: European Directorate for the Quality of Medicines & HealthCare, 2019.
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