Digital PCR Applications: Residual DNA and Mycoplasma Detection for Cell and Gene Therapy Development

Biopharmaceutical products like gene therapies, cell-based vaccines, and similar biotherapeutics are produced in mammalian or bacterial host cells and as a result, residual DNA is a common contaminant in these products. The presence of residual DNA in biopharmaceutical products can pose a risk to delay in product development and regulatory submission. Therefore, it is essential to detect and quantify residual DNA in these products accurately. Government agencies like the U.S. Food and Drug Administration and the World Health Organization regulate optimal levels for consumer safety.

Digital PCR (dPCR) offers a precise and accurate way to provide absolute quantification of these targets. The use of dPCR for residual DNA detection offers several advantages over traditional methods, such as qPCR or Southern blotting. These advantages include:

• Increased sensitivity: dPCR can detect low levels of target DNA with high sensitivity, making it ideal for detecting residual DNA in biopharmaceutical products that have undergone extensive purification.
• Improved precision and accuracy: dPCR reduce the variability in amplification efficiency, which improves the precision and accuracy of quantification compared to traditional methods.
• Ability to detect a broad range of target DNA sizes: dPCR can detect a broad range of target DNA sizes, including small fragments that may be missed by traditional methods.
• Reduced false positives: dPCR is less prone to false positives than traditional methods, as it reduces the effects of sample matrix components that can interfere with PCR amplification.

Overall, dPCR is a highly sensitive and specific method for residual DNA detection in biopharmaceutical products. It offers several advantages over traditional methods, including increased sensitivity, improved precision and accuracy, and the ability to detect a broad range of target DNA sizes. Therefore, it is an essential tool for biopharmaceutical manufacturers and researchers involved in biologics development.

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Mycoplasma contamination is a significant concern in cell and gene therapy research, as it can affect analytical evaluation. Mycoplasma is a type of bacteria that lacks a cell wall and is known for its small size and ability to pass through filters. It can contaminate cell cultures and affect the growth and function of cells, leading to altered gene expression and potentially compromising the development cycle of the final product. Mycoplasma detection is therefore a critical step in the quality control process for cell and gene therapy. Digital PCR (dPCR) is a highly sensitive and precise method of target quantification and for mycoplasma detection, it is becoming an increasingly popular tool in the industry.

Digital PCR performs absolute quantification by compartmentalizing a typical qPCR reaction into many thousands of micro reactions before amplifying to endpoint. Since each microchamber contains a single molecule or just a handful of molecules containing the target DNA, the absolute quantity of original molecules can be determined using Poisson statistics. Compared to traditional methods, dPCR offers improved sensitivity and precision and can detect low levels of mycoplasma DNA even in complex sample matrices. The use of dPCR for mycoplasma detection is expected to become increasingly prevalent in the industry as the demand for high-quality cell and gene therapy research products continues to grow.

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