Part One: Biologics and the Problem with Residual DNA

Part One: Biologics and the Problem with Residual DNA

As opposed to traditional drug production, the production of biological products (biologics) requires the involvement of human, animal or microbial cells. Biologics are some of the most effective and cutting-edge therapeutics, such as vaccines and antibodies that are produced through recombinant protein technologies. Biologics produced in cell culture contain unique impurities, including host cell proteins and host cell DNA. Therefore, the World Health Organization (WHO), the European Pharmacopoeia, and the US Food and Drug Administration (FDA) have established strict regulatory guidelines to guarantee product quality in the drug substance.  

Residual host DNA is a cellular impurity that arises from the use of host cells in the production of biologics.

A highly sensitive method of DNA quantification is known as quantitative real-time polymerase chain reaction (qPCR) which offers a sensitive and reliable method to detect host cell DNA. With each qPCR experiment, it is highly recommended to conduct a standard curve of target DNA to accurately quantify any detectable signals obtained from samples. The value of DNA per reaction can be back calculated to determine the amount of DNA per dose of biologic. qPCR assays detect cell-specific DNA targets (specificity) and have a dynamic range of up to six orders of magnitude detection (sensitivity). In addition, DNA extractions can be performed from complex matrices ensuring a high-quality DNA template across a broad range of sample types, from in-process samples to bulk drug substance.  

Currently, the different qPCR detection chemistries used enable rapid and specific quantitation of host cell DNA, often below the picograms (pg) level. qPCR chemistries for residual DNA use either sequence-dependent, fluorescent-based probes, such as TaqMan®, or sequence-independent, intercalating fluorescent dyes, such as SYBR® green, to quantify the amount of contaminating DNA.

Current qPCR Chemistries

Fluorescent-quencher probes
With PCR, two short DNA sequences (primers) are needed to amplify the target DNA. During the steps of PCR, primers will hybridize to target sequences on opposing strands on the DNA molecule. The sequence-dependent fluorescent probe hybridizes to a complimentary sequence between the primer binding sites. Probes will utilize a fluorophore on the 5’ end of the DNA probe coupled with a quencher on the 3’ end of the DNA probe. The intramolecular interaction of the fluorophore and quencher maintains a low level of fluorescence ensuring that very little fluorescence is detected in the absence of target DNA. During the PCR steps, the exonuclease activity of the DNA polymerase degrades the DNA sequence of the fluorescent probe resulting in separation of the fluorophore and quencher. Now, the fluorophore is free to emit fluorescence, upon excitation, and the magnitude of the fluorescent signal is proportional to the amount of free fluorophore.   

Intercalating fluorescent dyes
In contrast to fluorescent-quencher probes, fluorescent dyes intercalate into the double-stranded DNA molecule and when excited, emit fluorescence. As with probe chemistries, two DNA primers are required for amplification of the target DNA; however, no additional sequence-dependent molecular is added. Instead, the fluorescent dye is the reporter molecule and is added at the optimized concentration. During amplification of DNA, there is an increased amount of intercalating dye that binds to the double-stranded DNA. This results in an increase of fluorescent signal that is proportional to the amount of amplified DNA.  

Probes vs. intercalating dyes
These two qPCR chemistries are the most popular and both have advantages and disadvantages. For fluorescent probes, a benefit is that the sequence specificity is inherent in the method. This reduces the likelihood of false-positives. In addition, there is no need to perform post run analysis of the PCR product. With intercalating dyes, this approach is more cost effective than sequence-dependent fluorescent probes. However, a caveat is that intercalating dyes will detect any double-stranded DNA molecule and this may result in an increased likelihood of false positives. Furthermore, post run analysis, i.e. a melting curve analysis, is required to confirm amplification of the target DNA. With both approaches, it is important to optimize reactions to ensure quality results.

qPCR for Regulatory Compliance and Quality Products

The detection of host cell impurities is a critical step in the production of biopharmaceutical products. In addition to potential safety issues associated with extraneous host cell DNA, the regulatory guidance for products produced in cell culture specifies that DNA content in the final product should be as low as possible. FDA has determined the maximum amount of allowable residual host DNA is 100pg per therapeutic dose. Quantification of residual DNA is important to address product quality and to ensure regulatory compliance. qPCR offers an effective means to ensure both quality and compliance.

Thank you for your interest in learning more about biologics. At Alcami, our subject matter experts leverage knowledge and experience to assess the level of host cell impurities in biopharmaceutical products. Stay tuned for Part Two: Cell-Based Bioassays.