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14 BioPharm International eBook October 2020 www.biopharminternational.com impact of these impurities on prod- uct quality and performance. This article will highlight the characterization of: • Empty and partial viral parti- cle contaminants relative to the quantity of full capsids. • Quantitation of the total num- ber of genomes present relative to the viral particles infectivity (i.e., those that are capable of deliver- ing the gene of interest to cells). The CMC guidance recommends t hat mu lt iple produc t cha rac- teristics be evaluated prior to and during early clinical trials in order to identify a product's critical qual- ity attributes (CQAs) and biological properties towards obtaining "rel- evant and meaningful" informa- tion about product potency (1). This conforms with earlier guidance on potency determination, which acknowledged that "no single test … can adequately measure those prod- uct attributes that predict clinical efficacy" and so recommends appli- cation of an assay matrix (3). The new guidance also builds upon an early clinical trial design guidance, which recognizes that cell and gene therapy (CGT) products have unique features relative to other drug products that make the CMC and preclinical data less predictive of safety and potency observed in clini- cal trials (4): • Expectation of prolonged biologi- cal activity • Potential for immunogenicity. The "product-related" impurities to be measured are identified as "defective interfering"; "non-in- fectious"; "empty capsid particles"; and "recombinant virus contam- inants" that are to be reported as ratios (e.g., full:empty or virus par- ticles:infectious units). The brevity of the expected analyses for char- acterizing 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 possi- ble for other drug products, includ- ing biotherapeutics. The presence of partially filled capsids, resulting from encapsidation of fragmented genomes or non-transgene-related DNA contaminants, adds to this complexity. Analyses applied toward quan- tifying the viral particles for both genomes present and their effi- ciency at delivering their genetic payload to cells are impor tant because they are applied in early development to support clinical testing and guide early clinical tri- als. This review provides a limited overview of product implications that arise from the associated prod- uct-related impurities found in rAAV vector gene therapy products. It also reviews the range and rela- tive performance of the technical applications used to characterize these impurities. RELEVANCE TO AAV VECTORS The features present in viral-vectored gene therapy products take advan- tage of the evolved effectiveness of viral agents at transducing cells and delivering genes and genetic ele- ments that can establish long-term expression of transgenes. However, because the human immune system has evolved to identify and respond to viral agents, the vectors them- selves, their recombinant nucleic acid contents, and the product-re- lated impurities associated with these vectors have the potential to impact both potency and safety. Observations from clinical trials of vectored gene therapy products demonstrate that the presence of defective or interfering particles in Regulatory Sourcebook Regulatory Guidance Table I. Summary, capsid content methods. Method Capsid detection* Run time Throughput Comments ELISA/ qPCR F,E Hours High Two separate methods, Indirect calculation TEM F,E ~ 1 day Low Subjective results requiring human interpretation UV Spec F,E Min High Requires very pure preparations along with knowledge of extinction coefficient of capsid AEX- HPLC F,E 30 min High Reproducible and robust, HPLC commonly used in regulatory environment cIEF F,E,P α <1 hour High Requires very pure preparations due to UV detection SEC- MALS F,E 30 min High Provides capsid concentration and degree of aggregation AUC F,E,P ~6 hours Low Fully characterizes partial capsids. Large samples size requirement CDMS F,E,P ~2 hours [15] Low Fully characterizes full, partial, empty capsids. Currently not commercially available * F=Full, E=Empty, P=Partial, α=Partial. not fully characterized. ELISA/qPCR is Enzyme-Linked Immunoassay/ quantitative polymerase chain reaction; TEM is Transmission Electron Microscopy; UV Spec is Ultraviolet Spectroscopy; AEX-HPLC is Anion-Exchange-High-Performance Liquid Chromatography; cIEF is Capillary Iso- Electronic Focusing; SEC-MALS is Size-Exclusion Chromatography with Multiple Angle Light Scattering; AUC is Analytical Ultrcentrifugation; CDMS is Charge Detection Mass Spectrometry.