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16 BioPharm International eBook October 2020 www.biopharminternational.com tric focusing (cIEF) allow for char- acterization of vector capsids. The AEX–HPLC method benefits from the native fluorescence of aromatic amino acids and avoids interfer- ence from UV absorption of light at 280nm by packaged viral DNA, improving the accuracy of quanti- fication over other methods. AEX– HPLC analyses are fast and allow high throughput, making them a good fit for Quality Control (QC) labs (12), but doesn't resolve par- tially filled capsids. cIEF has higher resolving power than AEX–HPLC for distinguishing full and partial capsids but uses UV detection at 280nm, which can be a problem with impure samples (13). Size-exclusion chromatography with multi-angle light scattering (SEC–MALS) SEC–MALS also resolves full and empty capsids by hydrodynamic volume and size separation and determination of the mass and molar mass of the capsid and DNA. This allows calculation of the cap- sid content, and provides total cap- sid concentration and aggregation estimates. Although also limited by its inability to resolve partial cap- sids, it is otherwise well suited to QC laboratories (12). Analytical ultracentrifugation (AUC) AUC separates the A AV capsids by their sedimentation proper- ties and allows characterization of full, empty, and partially full viral capsids. Analysis is performed with in-situ monitoring, and can quantify and characterize capsid preparations independent of the serotype, DNA size, or form pres- ent. Disadvantages include lack of 21 Code of Federal Regulations Part 11 compliant software, the need for large sample size, low through- put, and a long run time (12). Charge detection mass spectrometry (CDMS) CDMS is a single ion technique that differs from conventional MS because it simultaneously detects the mass-to-charge ratio (m/z) by mea- suring the velocity of an ion with known electrostatic energy, and charge (z) by utilizing a sensitive amplifier, allowing the mass of each ion to be determined (14). CDMS has shown the ability to resolve cap- sids that contain the entire vector genome from those that contain par- tial and empty capsids (15). While no commercial systems specific for CDMS are currently available, there are commercial instruments whose use can be modified (16). In the authors' experience, AUC is currently the preferred method for performing these analyses, but the application of multiple orthog- onal methods is generally accept- able for early phase clinical trials. The apparent utility of CDMS and new/modified instrumentation capa- ble of supporting this analysis has the potential to either augment or replace AUC. POTENCY DETERMINATION Early approaches to quantitate vec- tor genome (VG) included dot blot DNA assays, southern blot, ultra- violet (UV) spectrometry, and flu- orometry. More recently, real-time quantitative PCR (RT-qPCR) and digital droplet PCR (ddPCR) have emerged as the industry standards. The precision of each approach can be influenced to varying degrees by several A AV product-related factors, including secondary struc- tures of the AVV genome, process impurities (e.g., residual DNA or protein), and buffer matrix. RT-qPCR and ddPCR have inher- ent distinctions. While qPCR func- tions over a wider dynamic range and is more economical, ddPCR offers key advantages. One import- ant benefit is that ddPCR provides absolute quantification of vector genomes, whereas qPCR uses indi- rect quantitation relative to a refer- ence standard, which may or may not be available or properly repre- sent the rAAV product. Additional ddPCR advantages result from droplet compartmen- talization of individual DNA mol- ecules that reduces potential for inhibitory effects of sample matrix or impurities, and droplets concen- trate the target sequence. When c omp a r e d , dd P C R h a s show n increased accuracy and precision (16). It should be noted that VG titers derived from these methods have been observed to differ sig- nificantly when applied to indi- vidual vector lots, and that "viral structure, aggregates, and impu- rities" and results obtained can "be altered by sample preparation" (17). While determination of vec- tor particle quantity can be derived by the physicochemical and molec- Regulatory Sourcebook Regulatory Guidance Clinical observations and the complexity of recombinant adeno-associated virus (rAAV) products underscore the importance of having well-defined and robust manufacturing processes and analytical methods in place.