Issue link: https://www.e-digitaleditions.com/i/1300450
www.biopharminternational.com October 2020 BioPharm International eBook 17 ular methods already described, a not he r i mp or t a nt p a r a me te r for estimation of potency is the ability of the particle preparation to deliver the transgene to cells; referred to as the product's "infec- tious titer." The combination of VG and infectious titer are applied to derive the virus particles:infectious units ratio referenced in the CMC guidance. Determination of the infectious titer applied to QC testing of AAV dr ug substance and dr ug prod- uct generally follows the TCID50 method described by Zhen et al. (18). Although this method was developed using qPCR, ddPCR is also an option. Importantly, the accuracy of the TCID50 method can be inf luenced by the pres- ence of aggregates and impurities, increasing assay variability (18,19). For this reason, other methods have been developed and evalu- ated with varying success at dis- criminating bet ween infectious particles and non-infectious con- t rols. T hese include infec t ious center assays that add complexity by use of membrane transferred nucleic acid and probe hybridiza- tion, and a method that applies permissive cell lines to eliminate the need to use helper virus (20). More recently, a relative infec- tivity method has been proposed as an alternative to the TCID50 method for use in screening early AAV vector candidates that also eliminates the need for a helper virus (19). The variability associ- ated with infectivity methods may be further exacerbated when mea- sures of potency progress to expres- sion or functional activity assays, as these measure transgene activ- ity downstream from the trans- duction events quantitated by PCR methods. As a result, it is unlikely that infectious titer measurement by some method will be replaced, especially early in product develop- ment and clinical material assess- ment. However, having multiple orthogonal methods applied to determine AAV product potency would support acceptance of mod- ifications, improving the reliability of infectious titer quantitation (4). SUMMARY Viral vector products, and rAAV in particular, may be a heteroge- neous mixture of empty and par- tial 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/amplification are con- sidered product-related impurities that can impact product efficacy and immunogenicit y, and thus must be quantified (3). Production conditions and purification pro- cesses can dramatically impact the levels of these impurities. While there is some debate around the impac t of t hese impu r it ies on product performance (4), the regu- latory guidance identifying these particles as contaminants suggests attempts should be made to at least reduce, if not eliminate, non-trans- gene expressing particles. To do that effectively, accurate and pre- cise measurements of these particles must be available. As described previously, exten- sive effor ts are being made to apply a variety of instruments and applications toward achieving that objective, but it will take additional effort to identify and refine stan- dardized methods that can be uni- versally applied across the range of AAV vectors being developed, as has been done for other biopharma- ceutical 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. Product developers should consider the development a nd applicat ion of or t hogona l methods to characterize their AAV- vectored gene therapies to ensure proper selection of and specifica- tions for CQAs. REFERENCES 1. FDA, "FDA Continues Strong Support of Innovation in Development 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 Investigational New Drug Applications (INDs), fda.gov, (CBER, January 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). BP Regulatory Sourcebook Regulatory Guidance