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Droplet Digital PCR Can Detect Mycoplasma During Gene Therapy Production
Article

Droplet Digital PCR Can Detect Mycoplasma During Gene Therapy Production

Droplet Digital PCR Can Detect Mycoplasma During Gene Therapy Production
Article

Droplet Digital PCR Can Detect Mycoplasma During Gene Therapy Production

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Nearly one-third of cell lines across the world are contaminated with bacteria of the genus mycoplasma. Long a nuisance to molecular biologists, these bacteria affect virtually all aspects of cell physiology and often impact research results. But now that gene therapies are entering the clinic, mycoplasma threatens research results and patients’ lives.

Today, most gene therapies use adeno-associated virus (AAV) as their nucleic acid delivery vehicle. AAVs are grown in cell culture, and since mycoplasma bacteria are commonly found in cell cultures, gene therapy batches are susceptible to contamination. These bacteria can reach patients if they are not detected and screened out early in gene therapy production.

One mycoplasma species, M. pneumoniae, is pathogenic. This species causes approximately
2 million cases of bacterial pneumonia and 100,000 hospitalizations each year. Some biologists refer to it as the “crabgrass” of bacteria because it is pervasive, hard to detect, and hard to treat in patients.

As more patients receive treatment with AAVs, this bacteria species threatens to impact even more lives. Therefore, biomanufacturers need sensitive tools to test for mycoplasma throughout the development and manufacturing process to ensure the bacteria do not reach patients.

Challenges in detecting and removing Mycoplasma

Mycoplasma
bacteria live everywhere, from animal-derived cell culture media to lab coats, easily evading detection. They measure only 2-3 µm across, too small to be seen through a standard light microscope lens. They are so small that they could appear in cell culture supernatant in concentrations of up to 107 cells per mL without affecting the culture’s appearance.

Even upon detection, mycoplasma bacteria are difficult to remove from cell cultures. Because they are Gram negative, they are resistant to the beta-lactam antibiotics that scientists typically use to clear cell lines of bacterial contamination. Mechanical separation is also a challenge because their small size makes it easy for them to pass through filters.

Scientists have instead resorted to direct detection of mycoplasma as their only hope of screening out contaminated gene therapy batches. Traditionally, scientists have used
various methods to detect these bacteria—using broth or agar to test for colony growth, staining or labeling their genetic material, or looking for mycoplasma proteins. But these methods can take up to a month to deliver results, which can severely slow down manufacturing. Quantitative PCR (qPCR) can detect mycoplasma in a day, but the tool uses a standard curve to estimate mycoplasma levels in a sample, which creates variability that reduces the technique's sensitivity.

Droplet digital PCR as a mycoplasma detection tool

Droplet digital PCR (ddPCR) technology can also deliver results quickly, but unlike qPCR, ddPCR measures mycoplasma levels directly, making it more sensitive.

ddPCR technology involves partitioning a 10-µL nucleic acid sample into 20,000 uniform 1-nL droplets and running a separate reaction in each one. With each droplet containing only one or a few nucleic acid strands, this technique enables a researcher to perform endpoint PCR thousands of times on individual DNA strands simultaneously. Droplets containing the target sequence will emit a strong fluorescent signal upon PCR amplification, while droplets that do not contain the sequence will only emit weak fluorescence. Using Poisson statistics, the software counts the number of positive versus negative droplets to derive the concentration of target nucleic acids in the original sample.

ddPCR assays use probe-based chemistry and employ three primers, decreasing the occurrence of non-specific DNA amplification compared to qPCR. By quantifying mycoplasma DNA in this way, biomanufacturers can be more certain about the presence or absence of Mycoplasma in their samples and deliver safer gene therapies.

In a
recent study, researchers tested ddPCR technology’s ability to detect mycoplasma with sensitivity and specificity. The team examined three prototypical species found in nature: A. laidlawii, M. pneumoniae, and M. hyorhinis. They found the limit of detection (LOD) for all three was consistently low: the LOD for A. laidlawii was 4.19 genome copies (GC)/well, the LOD for M. pneumoniae was 6.29 GC/well, and that for M. hyorhinis was 5.63 GC/well. In fact, in a direct comparison, ddPCR technology detected A. laidlawii standards at 1 colony forming unit/mL, while qPCR did not.

These researchers also tested for cross-reactivity by comparing the detection of three mycoplasma species to the detection of three control species: C. sporogenes, L. acidophilus, and S. bovis. They confirmed that their ddPCR assay only detects mycoplasma.

A growing need for mycoplasma testing in gene therapy development

As gene therapies enter the clinic, so do some of the contamination challenges that plague laboratory research. For example, since some mycoplasma species cause illness in people, gene therapies must be screened for mycoplasma before they are administered to patients. Despite the complexity of gene therapy development, today’s biomanufacturers have access to the tools they need to be assured of the quality of their treatments. ddPCR technology is one of these tools, and it will likely play a major role in ensuring gene therapies are safe and effective for everyone.

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