Issue link: https://www.e-digitaleditions.com/i/1302985
58 Pharmaceutical Technology Regulatory Sourcebook October 2020 P h a r mTe c h . c o m Regulatory Guidance surement by some method will be replaced, espe- cially early in product development and clinical material assessment. However, having multiple orthogonal methods applied to determine AAV product potency would support acceptance of modifications, improving the reliability of infec- tious titer quantitation (4). Summary Viral vector products, and rAAV in particular, may be a heterogeneous mixture of empty and partial capsids, noninfectious particles (containing DNA, but do not result in detectable in-vitro DNA ampli- fication), and infectious particles (complete vectors that enter the cell and in-vitro DNA amplification and transgene expression is detectable). Particles that do not result in expression/ampli- fication are considered product-related impurities that can impact product efficacy and immunoge- nicity, and thus must be quantified (3). Produc- tion conditions and purification processes can dramatically impact the levels of these impurities. While there is some debate around the impact of these impurities on product performance (4), the regulatory guidance identif ying these par- ticles as contaminants suggests attempts should be made to at least reduce, if not eliminate, non- transgene expressing particles. To do that ef- fectively, accurate and precise measurements of these particles must be available. As described previously, extensive efforts are being made to apply a variety of instruments and applications toward achieving that objec- tive, but it will take additional effort to iden- tify and refine standardized methods that can be universally applied across the range of AAV vectors being developed, as has been done for other biopharmaceutical products. Because of their complexity and heterogeneity, it is likely that an analytical matrix still may be required even when acceptable standardized methods are available. As directed by regulatory guidance, product developers should consider the development and application of orthogonal methods to character- ize their AAV-vectored gene therapies. This ap- proach will ensure the proper selection of and specifications for CQAs. References 1. FDA, "FDA Continues Strong Support of Innovation in Devel- opment of Gene Therapy Products," News Release, fda.gov, January 28, 2020. 2. FDA, Guidance for Industry, Chemistry Manufacturing and Control (CMC) Information for Human Gene Therapy Investi- gational New Drug Applications (INDs), fda.gov, (CBER, Janu- ary 2020). 3. FDA, Guidance for Industry, Potency Tests for Cellular and Gene Therapy Products, fda.gov, (CBER, January 2011) 4. FDA, Guidance for Industry, Considerations for the Design of Early-Phase Clinical Trials of Three Cellular and Gene Therapy Products, fda.gov (CBER, June 2015). 5. J.L. Shirley, Mol Ther 28(3):709-722 (March 4, 2020). 6. J.F. Wright, Biomedicines, 2: 80-97 (2014). 7. J.A. Allay, S. Sleep, S. Long , et al, Hum. Gene Ther. 22(5): 595- 604 (2011). 8. X. Fu, W.C. Chen, C. Argento et al. Hum Gene Ther Methods 30(4):144–152 (2019). 9. T. Li, T. Gao et al., Determination of Full, Partial and Empty Capsid Ratios for Adeno-Associated Virus (AAV) Analysis, SCIEX, Brea, CA. Accessed on Jun., 10, 2020 10. F. Dorange and C. Le Bec. Cell Gene Therapy Insights 4(2), 119- 129 (2018). 11. J.M. Sommer, P.H. Smith P.H. et al., Mol Ther 7(1):122– 128 (2003). 12. M. Chen, A. Purchel, AN1617: Quantifying Quality Attributes of AAV Gene Therapy Vectors by SEC-UV-MALS-dRI, Wyatt Technology, Santa Barbara, CA. Accessed on June 10, 2020. 13. B. Burnham, S. Nass, E. Kong et al., Hum Gene Ther Methods 26(6):228–242 (2015). 14. S.D.Fuerstenau, W.H. Benner, Rapid Commun. Mass Spectrom. 9, 1528–1538 (1995). 15. E.E. Pierson, D.Z. Keifer, A. Asokan, M.F. Jarrold, Anal. Chem. 88, 6718–6725 (2016). 16. C.H. Arnaud, C&EN, 36, 23-25 (2019). 17. D. Dobnik, et al. Frontiers in Microbiology, July 17, 2019. 18. Z. Zhen, et.al. Hum Gene Ther. 15:709 (2004). 19. W.D. Cheung, "Relative Infectivity as an Alternative to the TCID50 Assay," Presentation at the European Gene Therapy Analytical Summit, March 2020. 20. A. Francois, M. Bouzelha et al., Mol Ther, Meth and Clin Dev. 10(9) (2018). PT