Host cell DNA residual assays

Quality assessment of gene therapy products involves evaluating impurities not intended to be part of the final product. These impurities can be categorized as cellular impurities or process-related impurities, encompassing various elements such as reagents, media components, host cell components, residual DNA, viral vectors, vector-associated impurities, CRISPR-Cas9 ribonucleoproteins (RNPs), RNP-associated impurities, and other materials associated with viral production.

The risks associated with host cell DNA are potential infection and oncogenicity risks. The rep gene, utilized in vector production, possesses helicase and endonuclease activities that may increase mutation rates and promote vector integration in transduced cells.

Among DNA impurities, viral oncogenes represent a significant concern theoretically associated with an increased risk of oncogenesis. These genes are typically compact enough to fit within the viral capsid. Cell lines containing viral oncogenes are often used to produce AAV vectors. For instance, HEK 293 cells carry adenovirus E1, HEK 293T cells have adenovirus E1 and SV40 large T antigen, and HeLa cells harbor human papillomavirus E6 and E7. Plasmids containing the adenovirus E4 region, known for encoding proteins inhibiting the tumor suppressor p53 gene, are frequently used in triple transfection.

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, certain impurities may persist even after nuclease treatment, potentially because they are encapsulated within the capsid.

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.

Host cell DNA detection employs quantitative PCR methods like digital PCR. A representative segment, such as the 18S rRNA gene found in multiple copies within the HEK 293 cell genome, can serve as a target to identify host cell DNA. Ideally, we measure multiple amplicons of varying sizes to quantify a gene in more than one genome copy. 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.