How To De-Risk Cell And Gene Therapies

Dr Yvonne Collins explores the technical advances that are enabling safer biomanufacturing

As the number of cell and gene therapies approved and entering clinical trials grows, so does the need for standardisation and robust quality control (QC). Compliance with QC standards is a basic requirement for products manufactured under good manufacturing practices (GMP), which aims to minimise risks such as contamination.

When manufacturing cell and gene therapies, there are many potential sources of contamination, including residual host cell DNA (HCD), mycoplasma and other microorganisms. Compared with conventional pharmaceuticals, there is also a limited amount of final product available for QC testing, and they have a drastically shortened shelf life.

To boost effectiveness and ensure patient safety, manufacturers must identify and mitigate the risk of contamination along the entire bioprocessing workflow. Many turn to quantitative PCR (qPCR), but it cannot deliver absolute quantification as it relies on standard curves, creating variability and bias. Regulators have recognised the need for new approaches, and the industry is responding with novel innovations in analytics, such as digital PCR (dPCR).

Digital Pcr Takes Quality Control To The Next Level

dPCR is increasingly becoming the technology of choice for robust manufacturing and QC primarily because it ensures absolute quantification without standard curves. Using microfluidic nanoplate technology, the QIAcuity digital PCR system integrates sample partitioning, thermocycling and imaging into one system. This results in a simplified workflow, faster time to results, higher throughput and enhanced multiplexing. Well-designed dPCR assays offer precise and sensitive contaminant testing of mycoplasma and residual host cell DNA, ensuring the potency, purity and safety of the therapeutic product.

Residual Host Cell DNA

Residual host cell DNA (HCD), often carried over during viral vector production, poses considerable safety concerns, including oncogenicity, infectivity and immunogenicity. Guidance issued by the FDA and World Health Organization states that biotherapeutics developers “limit the amount of HCD for continuous non-tumorigenic cells to less than 10 ng/dose and the DNA size to below approximately 200 base pairs.”

To comply with this guidance and deliver a safe product with the appropriate therapeutic dose, manufacturers need to accurately quantify the amount of residual HCD. Here, qPCR, a common method for monitoring residual HCD, has drawbacks, including amplification bias, nonspecific signals, and lack of reproducibility. Furthermore, qPCR requires time- and labour-intensive sample extraction and calibration standards for quantification.

Nanoplate digital PCR presents a robust alternative with higher precision and sensitivity of detection at a lower template input range. Residual DNA (resDNA) quantification assays detect femtogram levels of HCD from CHO and E.coli in a single reaction, even when PCR contaminants and other inhibitory reagents are present. Moreover, multicopy target assays ensure the results are not affected by the fragmentation of HCD. What’s particularly attractive is the digestion- and purification-free workflow, which reduces hands-on time.


When producing adeno-associated virus (AAV)-mediated therapies, the use of live cell culture to grow AAVs opens up the possibility of potential mycoplasma contamination. Mycoplasma are difficult to detect, spread quickly, resistant to antibiotics and pathogenic to humans, rendering the entire production workflow vulnerable to failure.

Until recently, qPCR was the “gold standard” for detecting process- or product-related impurities, identifying mycoplasma in a day, but estimating its levels using a standard curve approach. Although this was considered an improvement over traditional culture-based tests, it is still not sensitive enough.

Nanoplate dPCR offers a powerful approach to rapidly identify contamination down to a single copy number and discriminate between live and dead microbes within complex gene therapy formulations. It supports advanced raw material and in-process testing, allowing more effective detection of microbial contamination and offering users 100% certainty about a product.

Sterility Testing

Bioburden detection is critical for offsetting the risk of microbial contamination in the final product. However, delays in testing can cause significant bottlenecks. Nanoplate dPCR speeds up bioburden detection and product release with higher throughput. Dedicated assays detect the total amount of viable microorganisms in or on a medical device, container, or component using multiplexed dPCR. This provides absolute quantification results and serves as an early warning system for possible production problems that could lead to inadequate sterilisation.

End-To-End Solutions

The QIAcuity digital PCR system uses GMP-compliant software that meets the stringent requirements of U.S. FDA 21 CFR Part 11 regulations. This provides a secure environment for the maintenance, verification and tracking of all electronic records generated by the software during the biomanufacturing and QC process.

Although today’s biomanufacturers are making great strides in discovering novel therapeutics, safety is still a concern, and they remain vigilant in keeping their products free of contaminants and impurities. Technological advancements such as nanoplate digital PCR offer a big leap forward for safe and effective biomanufacturing.

Dr Yvonne Collins is with Qiagen

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