An Introduction to Assay Methods for Residual Host Cell DNA in Biological Products

Host cells are the cornerstone of biological products, and mammalian cell lines have now become the most widely used hosts for the expression and preparation of biological products. Common host cells include Chinese Hamster Ovary (CHO) cells, Vero cells from African green monkey kidneys, and Human Embryonic Kidney (HEK 293) cells.

Risk of Residual Host Cell DNA

Carcinogenicity

The primary carcinogenic mechanism of residual host cell DNA is the introduction of dominant oncogenes, such as MYC and RAS. These dominant oncogenes can directly transform normal cells, causing some normal cells to differentiate into tumor cells. Insertion mutations of residual host cell DNA are also potential carcinogenic factors.

Infectivity

Residual host cell DNA may contain infectious viral genomes, which can produce infectious viral particles through replication and transcription amplification. Therefore, the infectivity risk of residual host cell DNA might be higher than its carcinogenic risk.

Immunogenicity

Because genomic DNA from microbial sources is rich in CpG and unmethylated sequences, it increases the immunogenicity risk of recombinant protein drugs in vivo. For example, CpG-rich sequences from bacteria may trigger immune responses mediated by TLR (Toll-like receptors).

Detection Methods of Residual Host Cell DNA

DNA Probe Hybridization

In this method, the exogenous DNA in the test sample is denatured to single strands and adsorbed onto a solid-phase membrane. Under certain conditions, it can re-hybridize with complementary single-stranded DNA probes labeled with markers to form double-stranded DNA. However, the detection results of the hybridization method have significant discrepancies with the actual residual host cell DNA content, and the method is unstable with relatively long detection times.

Fluorescent Staining

This method uses double-stranded DNA fluorescent dyes that specifically bind to double-stranded DNA to form complexes, producing a strong fluorescent signal when excited at a wavelength of 480 nm. The fluorescent signal at 520 nm is detected using a fluorometer. The fluorescence intensity is proportional to the DNA concentration. However, this method's fluorescent signal is easily interfered with and has poor specificity, requiring the avoidance of environmental DNA contamination, and all materials and reagents used must be DNA-free.

Quantitative PCR

Currently, the most conventional method for detecting host cell DNA (HCD), this technique involves real-time monitoring of the PCR process through fluorescent signals to quantitatively analyze the template. Based on chemical principles, it can be divided into two types: the TaqMan probe method and the SYBR dye method. 

The TaqMan probe method uses gene-specific oligonucleotide probes labeled with fluorescent dyes during the PCR reaction to detect the product, while the SYBR dye method adds an excess of fluorescent dye to the PCR reaction system. The dye specifically incorporates into the DNA double strand and emits a fluorescent signal. The TaqMan probe method increases specificity through the probe recognition step, while the SYBR dye method is simpler and more straightforward.