Speeding Up Safety: How ddPCR Is Changing Mycoplasma Testing
Find out how ddPCR is reshaping contamination risk management with rapid mycoplasma detection in biopharma quality control.
Cell-based therapies and other advanced biologics have transformed the pharmaceutical landscape, but they’ve also introduced new challenges for quality control (QC). One persistent threat to the safety and efficacy of therapeutic products is the risk of mycoplasma contamination.
As advanced therapy medicinal products (ATMPs) with short shelf lives become more prevalent, the need for faster, more reliable contamination detection methods has grown. To meet these demands, quality control labs are turning to molecular tools like droplet digital PCR (ddPCR), which offers faster turnaround, higher sensitivity and better resistance to PCR inhibitors compared to traditional culture methods.
Technology Networks recently spoke with Dr. Denise Teber, a scientific expert in molecular biology at Charles River Laboratories, to learn more about how ddPCR is being used to improve contamination risk management.
What is driving the demand for rapid and reliable mycoplasma detection?
Denise Teber, PhD (DT):
Mycoplasma contaminations are a constant threat for cell-based production processes. They lack a cell wall and belong to one of the smallest classes of bacteria known. These attributes make them difficult to filter out during pharmaceutical manufacturing. Mycoplasma contaminations during the production process can alter product quality, and some species are able to infect humans.
Furthermore, the change of drug products is also driving the demand for faster methods, which require a smaller sample volume, especially since the introduction of ATMPs to the market. Cell therapeutics, for example, have a short shelf life and thus require faster testing than the culture method can deliver. This is critical to be able to provide the therapeutic product to the patients in time.
In general, for commercial batch release, prolonged storage times due to incomplete QC testing is accompanied by additional costs for each day the production batch cannot be released.
KR:
What role does ddPCR play in improving contamination risk management strategies?
DT:
The laboratory work for ddPCR can be completed within one day; it can also be used to implement testing at additional product stages to enable an earlier detection of mycoplasma in the production process, which in the end could save time and money. Furthermore, in contrast to qPCR methods, ddPCR is even more sensitive (2 genome copies/reaction or 1 CFU/mL can be detected) and less prone to PCR inhibitors. Thus, matrix effects of the sample material, which can lead to PCR inhibition, have a less significant impact on the detection of mycoplasma and related mollicutes. Therefore, the risk of false negative results is further minimized.
KR:
What are the advantages of PCR-based screening over traditional methods?
DT:
Significantly shorter turnaround times and smaller sample volumes are required. AAV products are often produced on a much smaller scale than e.g., antibody products. It is desirable to reduce the sample volume, which has to be submitted to QC testing as much as possible.
PCR-based methods can also detect a broader range of mollicutes species if various primer–probe sets are applied. Some species do not grow under standard culture conditions, which is why the indicator culture method is applied in addition. This method however has a lower sensitivity than the culture method for some species. While PCR-based methods usually detect more than 100 species, the traditional methods only detect those species in favor of the culture conditions.
KR:
How does rapid ddPCR mycoplasma testing integrate into existing QC pipelines?
DT:
It can replace the traditional methods (culture and indicator cell method) after suitable method validation. A comparability study might be required in addition. If a generic validation is in place, the number of mycoplasma species that need to be included in the product-specific validation can be reduced. A risk assessment considering the product, the production process and the introduced raw materials should be performed to choose the correct reference strains to be included in the product-specific validation.
KR:
Have you received any feedback from early adopters in biopharma QC labs?
DT:
Not yet, we are still in the validation phase for the ddPCR method, but after a suitable product-specific validation, our current qPCR method has already been accepted by regulatory bodies. Nucleic acid amplification techniques (NATs), like qPCR and ddPCR, are mentioned as a suitable alternative by both the European Pharmacopoeia (chapter 2.6.7) and the United States Pharmacopeia (chapter 63). The applied NAT needs to be validated and able to detect at least 10 CFU/mL or less to be used as a replacement for both traditional methods. In addition, the robustness of the method should be evaluated and specificity testing should be performed to exclude unspecific detection by the applied NAT of closely related bacterial species like Clostridium, Lactobacillus or Streptococcus.
