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28 Pharmaceutical Technology BIOLOGICS AND STERILE DRUG MANUFACTURING EBOOK 2021 P h a r mTe c h . c o m Analytics ultimately reduce the effectiveness of the ther- apy and put patients at risk. A high-risk con- taminant that is especially likely to appear in AAV-mediated gene therapies is bacteria of the genus Mycoplasma. The sensitivity of droplet digital PCR (ddPCR) helps manufacturers de- tect Mycoplasma contamination in AAV-based gene therapy products. Mycoplasma is prevalent and persistent Mycoplasma is a genus of small, antibiotic-resis- tant bacteria commonly found in cell cultures. By one estimate, Mycoplasma bacteria are pres- ent in approximately 30% of cell lines across the globe (7,8). It is diff icult to trace their origin, and is similarly diff icult to avoid contamina- tion—t hese bacteria are present ever y where, from animal-derived cell-culture media to the breath and clothing of laboratory personnel (9). Additionally, because they measure only 2–3 µm across, they cannot be seen using standard light microscopy and may appear in numbers of up to 107 cells per milliliter of cell culture super- natant without affecting the appearance of the culture (7). Despite their size, Mycoplasma bac- teria signif icantly impact cell physiolog y and can affect results (7). While bacteria belonging to the Mycoplasma genus have been a source of concern in research labs for decades, they have recently become more of a threat to human health due to the growing popu larit y of gene t herapies. Because Myco- plasma is prevalent in cell cultures, they are fre- quently found in the cell lines used to produce AAV vectors for therapy; if not removed promptly, they can ultimately reach patients. One Myco- plasma species, Mycoplasma pneumoniae, can cause respiratory infections in humans: every year in the United States, this species is to blame for approximately two million cases of bacterial pneumonia and 100,000 hospitalizations (10). Pa- tients undergoing treatment with gene therapies can sometimes lack the strength to fight off a My- coplasma infection; therefore, it is especially criti- cal that gene therapies are tested for the presence of Mycoplasma at multiple points throughout the development and manufacturing process. To detect Mycoplasma, scientists have tradi- tionally used methods such as monitoring for colony growth with broth or agar, staining or labeling nucleic acids, or searching for bacterial gene products. However, these tests are slow, tak- ing up to four weeks to deliver results (11, 12). Quantitative polymerase chain reaction (qPCR) is an alternative testing method that detects My- coplasma DNA in cell cultures in one day, but this strategy has its own limitations. qPCR can- not quantif y Mycoplasma levels direct ly and therefore cannot deliver an absolute count of Mycoplasma genomes. qPCR measures the number of PCR amplifica- tion cycles it takes for a f luorescent readout of DNA quantity to reach a certain threshold. This number is converted to DNA concentration using a standard curve of a presumably known quantity The accelerated pace of gene therapies is a sign of enhancements in our understanding of genetics and viral engineering as well as innovation in manufacturing processes.