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32 Pharmaceutical Technology BIOLOGICS AND STERILE DRUG MANUFACTURING EBOOK 2021 P h a r mTe c h . c o m ddPCR was consistently low, at 6.29 GC/well for M. pneumoniae, 4.19 GC/well for A. laidlawii, and 5.63 GC/ well for M. hyorhinis (23). The team also tested their ddPCR assay for cross-reactivity to assess its tendency to produce false positives. To do this, the researchers used ddPCR assays de- signed to quantify Mycoplasma species in sam- ples containing three control species: Clostridium sporogenes, Lactobacillus acidophilus, and Strep- tococcus bovis. They confirmed that their ddPCR assay was highly specif ic, detecting only the Mycoplasma species (23). As the above studies demonstrate, technologies such as ddPCR could enable AAV developers to detect Mycoplasma species with better sensitiv- ity and specificity than other methods. Yet My- coplasma detection is only one quality control measure AAV developers need to adopt. Other quality control challenges in AAV development AAV development cannot be completely controlled, and, therefore, several aspects of the process must be carefully monitored. First, since AAV vectors are grown in living cells, cellular components such as DNA and proteins can make it into a final batch and create an additional health hazard, so they must also be detected and removed. A batch might also contain empty AAV capsids; in some cases, up to 90% of the capsids may be empty (24). This requires manufacturers to increase the deliv- ery volume to capture the number of active vectors needed to create an effective dose, but increased volumes can cause problems when therapies need to be delivered in small spaces such as the brain and spinal cord (25). These empty vectors, there- fore, must also be detected and removed. Finally, the AAV titer in the final batch can vary. Titer correlates with potency, which means variabilit y ma kes it diff icult to dose patients correctly. ddPCR can quantify AAV viral titer more accurately than qPCR, making it suitable for use in this aspect of AAV-based gene ther- apy development too (26). AAV development for gene therapies is complex, but with the proper qua lit y cont rol tools, ma nufacturers ca n be more confident that their products will consis- tently deliver safe, high-quality treatments to the patients who need them. References 1. T. Friedmann and R. Roblin, Science 175 (4025) 949–955 (1972). 2. FDA, "FDA Approves Novel Gene Therapy to Treat Patients with a Rare Form of Inherited Vision Loss," Press Release, Dec. 18, 2017. 3. US National Library of Medicine, Search Engine, ClinicalTrials. gov, accessed March 1, 2020. 4. FDA, "Statement from FDA Commissioner Scott Gottlieb, M.D. and Peter Marks, MD, PhD, Director of the Center for Biolog- ics Evaluation and Research on New Policies to Advance Devel- opment of Safe and Effective Cell and Gene Therapies," FDA Statement, Jan. 15, 2019. 5. C. Quinn, et al., Value Health 22 (6) 621–626 (2019). 6. M. Davidsson, et al., Sci Rep 10, 21532 (2019). 7. L. Nikfarjam and P. Farzaneh, Cell J 13, 203–212 (2012). 8. G. Bolske, Zentralbl Bakteriol Mikrobiol Hyg A 269 (3) 331–340 (1988). 9. K. Jadhav, "Mycoplasma Contamination: Where Does It Come From and How to Prevent It," blog.addgene.org, Oct. 8, 2020. 10. K. Waites, et al., Clin Microbiol Rev 30 (3) 747–809 (2017). 11. H.G. Drexler and C.C. Uphoff, Cytotechnology 39 (2) 75–90 (2002). 12. S.E. Armstrong, et al., Biologicals 38 (2) 211–213 (2010). 13. B. Gattinger, et al., Vet. Med. Austria 95, 22–27 (2008). 14. N. Mehle, et al., Anal Bioanal Chem 410, 3815–3825 (2018). 15. Y. Maheshwari, et al., PLoS ONE 12, e0184751 (2017). 16. M. Jahne, et al., Water Res 169, 115213 (2020). 17. R. Gonzalez, et al., Water Res, 186 (1) e11629 (2020). 18. C. Alteri, et al., PLoS ONE 15 (9) e0236311 (2020). 19. S. Naser, et al., World J Gastroenterol 20 (23) 7403–7415 (2014). 20. R. Sharp, et al., Front Cell Infect Microbiol 8, 11 (2018). 21. M. Ricchi, et al., Front Microbiol 8,1174 (2017). 22. N. Song, et al., Emerg Microbes Infect. 7 (1) 78 (2018). 23. M. Scherr, et al., "Vericheck ddPCR Mycoplasma Detection Kit: Probe-based Mycoplasma Detection to Reduce False-Positives Results," Bio-Rad Mycoplasma Poster, accessed Feb. 3, 2021. 24. J. Grieger, et al., Mol Ther. 24 (2) 287–97 (2016). 25. J.A. Hernandez Bort, "Challenges in the Downstream Process of Gene Therapy Products," www.americanpharmaceuticalre- view.com, June 25, 2019. 26. D. Dobnik, et al., Front Microbol. 10, 1570 (2019) PT. Analytics