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54 Pharmaceutical Technology Regulatory Sourcebook October 2020 P h a r mTe c h . c o m terfering particles in these products can inf luence the product's efficacy in alleviating the targeted disease or deficiency and can dramatically impact the safety and longevity of the product after ad- ministration (5,6). Unique properties of individual vectors have inf luenced the choice of vectors applied to vari- ous disease/deficiency targets. AAV vectors have been chosen for gene delivery because they are able to transduce multiple cell types and tissues. In addition, the genes they introduce do not in- tegrate into the host's genomic DNA; gene expres- sion is durable, and wild type AAVs are associated with only weak, transient innate and adaptive im- mune responses. However, pre-existing immunity in patients is present to many AAV capsid sero- types, and the presence of neutralizing antibodies to the capsid serotype, the route of administra- tion, and the dosage applied can inf luence prod- uct effectiveness, durability, and safety (5). Because cellular immunity against the viral cap- sid has also been observed in some clinical trials, immunosuppression is becoming standard prac- tice (6). Questions remain regarding how serotype, vector design, manufacturing parameters, and even target gene overexpression might inf luence clinical responses. These clinical observations and the complexity of rAAV products underscore the importance of having well-defined and robust manufacturing processes and analytical methods in place that define their physical, chemical, molecular, and biological properties. Analytical methods applied to establishing those parameters vary widely, as do their sensitivity and limitations. Method-specific variation observed in the data generated further confounds the presence of these impurities with clinical observations (6). Improving the accuracy and precision of methods applied to characterize these products is particularly critical to improv- ing the understanding of their safety and effec- tiveness prior to and resulting from clinical trials. Characterizing product impurities such as empty or partially filled capsids, or defective particles that contain the transgene but fail to deliver it to a cell, are therefore increasingly important in understanding and establishing a product's CQAs. Capsid characterization The production of AAV vectors can result in capsid generation in which 90% contain only some parts of the transgene, or completely lack the trans- gene. These partial and empty capsids lack thera- peutic benefit and may elicit unwanted immune responses (7,8). Therefore, determining the ratio of full, partial, and empty capsids is considered a CQA for AAV-vectored products. This ratio is expected to correlate with efficacious dosage and safety and must be closely monitored to ensure lot- Regulatory Guidance The brevity of the expected analyses for characterizing and quantitating these impurities belies the complexity of these products, their manufacture, and the challenges of measuring these impurities with the accuracy and precision that have been possible for other drug products.