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www.biopharminternational.com October 2020 BioPharm International eBook 15 these products can influence the product's efficacy in alleviating the targeted disease or deficiency and can dramatically impact the safety and longevity of the product after administration (5,6). Unique properties of individual vectors have influenced the choice of vectors applied to various dis- ease/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 integrate into the host's genomic DNA; gene expression is durable, and wild type AAVs are associated with only weak, tran- sient innate and adaptive immune responses. However, pre-existing immunity in patients is present to many AAV capsid serotypes, and the presence of neutralizing antibod- ies to the capsid serotype, the route of administration, and the dosage applied can influence product effec- tiveness, durability, and safety (5). B e c au s e c e l lu l a r i m mu n it y against the viral capsid has also been observed in some clinical tri- als, immunosuppression is becom- ing standard practice (6). Questions remain regarding how serotype, ve c tor desig n, ma nu fac t u r i ng parameters, and even target gene overexpression might influence clin- ical responses. These clinical observations and the complexity of rAAV products underscore the importance of hav- ing well-defined and robust manu- facturing 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. M e t h o d - s p e c i f i c v a r i a t i o n observed in the data generated further confounds the presence of these impurities with clinical obser- vations (6). Improving the accuracy and precision of methods applied to characterize these products is par- ticularly critical to improving the understanding of their safety and effectiveness prior to and resulting from clinical trials. Characterizing product impurities such as empty or partially filled capsids, or defec- tive particles that contain the trans- gene, but fail to deliver it to a cell, are therefore increasingly import- ant in understanding and establish- ing 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 transgene. These partial and empty capsids lack therapeutic benefit and may elicit unwanted immune responses (7,8). Therefore, deter- mining 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-to-lot consistency. Various tech- niques have been developed and applied to characterize AAV vector capsid content ( Table I). Initial methods indirectly deter- mined the full and non-full (both partial and empty) AAV capsid ratio using quantitative polymerase chain reaction (qPCR) to quantitate viral genomes present and the total cap- sid quantified by capsid specific enzy me-linked immunosorbent assay (ELISA). Because the ELISA also quantitates capsid-free proteins, this approach has the potential to misrepresent the actual ratio of full: non-full capsid (7,9,10). Transmission electron microscopy (TEM) TEM allows direct visualization of full, empty, and partial capsids. However, the method is highly labo- rious and subjective (7), making it less suitable for current good manu- facturing practice (cGMP) testing. Spectrophotometry Spectrophotometry can estimate viral capsid content, based on char- acterizing the expected absorbance of the viral capsid and genome to derive their extinction coefficients, by applying the 260/280 ratio to quantif y the DNA and proteins in a solution (8). The number of empty capsids in purified vector preparations reduces the A260/ A280 ratio in a predictable man- ner. The method is quick and easy to perform and utilizes common laboratory equipment. However, it requires highly purified and con- centrated (>5x1011 vg/mL) vector, and accuracy can be reduced by impurities associated with more complex compositions (8). As a result, this method is generally only applied for analytical testing during early product development (11). Anion-exchange high-perfor- mance liquid chromatography Because full capsids have lower isoelectric point (pI) values than the empty capsids due to the neg- ative charge of DNA present in the capsid, anion-exchange high-per- formance liquid chromatography (AEX–HPLC) and capillary isoelec- Regulatory Sourcebook Regulatory Guidance The brevity of ... analyses for characterizing and quantitating impurities belies the complexity of these products and their manufacture.