Host Cell Residual DNA: Understanding its Significance and Detection Methods

Host Cell Residual DNA (rcDNA) refers to fragments of DNA derived from host cells that may be present in biological products. These products must not contain foreign substances, particularly host DNA, to avoid immune rejection and potential threats to life safety. Regulatory agencies worldwide have imposed strict limits on the amount of rcDNA, and various pharmacopoeias have outlined several reliable methods for residual DNA quantification, including threshold methods, hybridization methods, and real-time quantitative PCR.

The Potential Risks of Host Cell Residual DNA:

Host cell lines used in the production of biological products may harbor genes associated with tumors or viruses, presenting potential risks. For instance, rcDNA may carry the HIV virus or the Ras oncogene. The insertion of LINE-1 sequences, found in mammalian cell genomes, into chromosomes as retrotransposons can impact the functionality of essential genes, potentially activating oncogenes or inhibiting tumor suppressor genes.

Moreover, microbial-derived genomic DNA rich in CpG and unmethylated sequences increases the risk of immunogenicity in recombinant protein drugs within the human body. While current evidence suggests that the tumorigenic risk of residual DNA is lower than the infectious risk, caution should be exercised considering that tumorigenicity experiments are conducted in animals, and infectious experiments are performed at the cellular level. Consequently, both risks should be taken seriously, necessitating stringent standards for detecting host DNA residues in biologics.

Understanding Host Cell Lines:

Host cell lines, collectively referred to as cell matrices, are essential for the growth and replication of viruses. These cells, used for in vitro virus cultivation, significantly impact the quality, yield, and safety of biological products, particularly vaccines. Various animal cell matrices, such as primary cells, passaged cells, and human diploid cells, are currently employed in the production of vaccines and other biologics.

Residual DNA and its Implications:

The potential harm caused by residual DNA varies depending on the host cells used for biological product production. Although these DNAs share the same basic structural unit, their fragments differ in length and physical form, leading to diverse consequences upon entering the human body.

Oncogenicity of Residual DNA:

Residual DNA's main oncogenic mechanism involves the introduction of dominant oncogenes, such as MYC and RAS, which can directly transform normal cells, leading to the formation of tumorigenic cells. Other oncogenic mechanisms may result from mutations caused by the insertion of residual DNA fragments from the host cell. However, the probability of integration of small DNA fragments into the recipient's genome is low, reducing the likelihood of activating proto-oncogenes or suppressing oncogenes. Nevertheless, the potential of passaged cell line DNA to induce uncontrolled cell growth and exhibit oncogenic activity necessitates quality control measures for DNA residue amounts.

Infectious Potential of Residual DNA:

Residual DNA can be infectious due to the presence of viral genomes within cellular DNA. Such genomes can undergo replication and transcription, leading to the production of infectious viral particles. Consequently, the risk of DNA infectivity may surpass the risk of carcinogenicity. Therefore, when developing biologics derived from continuous cell lines, assessing DNA infectivity is crucial to ensure the safety of residual DNA in host cells.

Limiting Residual Host Cell DNA:

The World Health Organization (WHO), U.S. Food and Drug Administration (FDA), and European Pharmacopoeia recommend risk assessment based on cell matrix properties and product use. Stricter control of DNA residues is required when using tumorigenic cell lines to ensure product safety. In some cases, reducing DNA residues without compromising product potency can be challenging. DNA alkylating agents, such as β-propanolactone, can be employed to modify the structure and size of residual DNA, thereby reducing associated risks.

The current guidelines from the WHO and FDA stipulate that residual DNA in finished products should not exceed 10 ng/dose, with a length no greater than 200 bp. FDA guidelines specify that the residual DNA limit for host cells in biologics is 100 pg/dose. However, high-dose biologics, such as monoclonal antibodies, should have residual DNA not exceeding 10 ng/dose, depending on the source of the residual DNA and the route of administration.

Although most residual DNA limits set by the European Pharmacopoeia for biological products do not exceed 10 ng/dose, individual vaccines have more stringent requirements. For example, inactivated hepatitis A vaccine should not contain residual DNA exceeding 100 pg/dose, and hepatitis B vaccine should have residual DNA not exceeding 10 pg/dose.

Detection of CHO Residual DNA:

Creative Proteomics has optimized the qPCR method for detecting CHO residual DNA. This method enables specific and rapid detection of residual DNA in intermediate, semi-finished, and finished products during the development and production of biological products such as antibody drugs and recombinant protein vaccines.