Biotherapeutics produced in host cells must undergo monitoring for residual host cell DNA during manufacturing and in the final product. Host cell DNA is associated with the risk of infection, oncogenicity, immunogenicity, and mutagenesis.
Biotherapeutics such as Adeno-associated virus (AAV) vectors are typically produced in host cells derived from mammalian cell lines such as HEK293. Plasmids containing the AAV vector genome and helper genes required for viral replication were transfected into these host cells. When AAV vectors are harvested, there is always a risk of host cell residuals contaminating the therapeutic product. Monitoring and controlling the levels of host cell DNA impurities are crucial to ensure the quality and safety of the final AAV product for patient administration.
The risk to the human body increases as the length of residual host cell DNA fragments increases. Therefore, during the purification process, DNA is intentionally fragmented using nucleases. However, nuclease treatment may not eliminate certain impurities, possibly because they are encapsulated within the capsid.
Host Cell Oncogenes
Some host cells utilized in biopharmaceutical processes may host oncogenic viruses. For example, HEK293 was immortalized in 1973 by the integration of an adenoviral fragment, including the genes encoding for E1A and E1B proteins. The expression of these proteins enables continuous culturing of HEK293 by inhibiting apoptosis and interfering with transcription and cell cycle control pathways. HEK293T cells have an SV40 large T antigen integrated into their genome, allowing them to produce recombinant proteins and retroviruses in higher amounts. HeLa cells harbor the HPV E6 and E7 oncogenes.
Infection Risk
Continuous cell lines like CHO and HEK cells might have been exposed to adventitious viruses that potentially contaminated them. If the cell line is contaminated with retroviruses, viral particles could be present in the final product, which could infect patients upon administration.
Immunogenicity Risk
Host cell DNA or protein can trigger an immune response upon administration.
Mutagenesis Risk
Integrating host cell DNA, including potential oncogene sequences or retrotransposon sequences like LINE-1, into the patient’s genome through biotherapeutics can lead to cancer development.
Regulatory Requirements
Routine testing for residual DNA is necessary for products derived from continuous mammalian cell lines, such as HEK293. The production process needs to be established so that at given purification steps, those impurities are removed consistently and reproducibly to acceptable levels. Analytical assays like qPCR or dPCR must be in place to monitor impurities and ensure the levels are under set limits.
The primary goal of fragmentation is to disrupt the integrity of elements like oncogenes and eliminate infectious units such as integrated retroviruses and other functional sequences. It’s important to note that the AAV capsid shields the packaged nucleic acid from nuclease digestion, making removing or further reducing its size impossible.
Current guidelines recommend that residual cell-substrate DNA levels should not exceed ≤10 ng per dose, with a median DNA size of 200 bp or less.
PCR assays for host cell DNA detection
Host cell DNA residuals can be detected and quantified using qPCR or dPCR. We use assays targeting oncogenes (E1A, E1B, SV40 large T antigen, or any other gene of interest). One approach is detecting different-sized fragments of a gene, like 18S rRNA, found in multiple copies within the HEK293 genome. At least one of the amplicons is longer than 200 bp. This approach allows for detecting how much residual host DNA the sample contains that is 200 bp or longer and how fragmented the DNA is.
Optimized Production
We develop, optimize, qualify, and validate assays for qPCR and dPCR. In addition to your GMP laboratory, our short lead times and fast analyses allow for swift replies on when further optimization of your purification protocols is needed and provide confidence in your product before GMP analysis. Our assays help with deviation control and verify the GMP results for safe product delivery. We have two decades of experience in molecular analyses such as qPCR and dPCR. We operate from a state-of-the-art laboratory with high-throughput instrumentation, focusing on advanced therapies and biotherapeutics.