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56 Pharmaceutical Technology Regulatory Sourcebook October 2020 P h a r mTe c h . c o m Regulatory Guidance ods. AEX–HPLC analyses are fast and allow high throughput, making them a good fit for QC labo- ratories (12), but doesn't resolve partially filled cap- sids. cIEF has higher resolving power than AEX– HPLC for distinguishing full and partial capsids but does utilize UV detection at 280nm, which can be problematic with impure samples (13). S i z e - e xc l u s i o n c h r o m a t o g r a p hy w i t h m u l t i -a n g l e light scattering (SEC–MALS). SEC–MALS a lso re- solves 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 capsid con- tent, provides total capsid concentration and aggregation estimates. Although also limited by its inability to resolve partial capsids, it is otherwise well suited to QC laboratories (12). Analytical ultracentrifugation (AUC). AUC separates the AAV capsids by their sedimentation proper- ties and allows characterization of full, empty, and partially full viral capsids. Analysis is per- formed with in-situ monitoring and can quantify and characterize capsid preparations independent of the serotype, DNA size, or form present. Dis- advantages include the lack of 21 Code of Fed- eral Regulations Part 11 compliant software, the need for large sample size, low throughput, 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 measuring 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 capsids that contain the entire vector genome from those that contain partial and empty capsids (15). While no com- mercial systems specific for CDMS are currently available, there are commercial instruments whose use can be modified (16). In the authors' experi- ence, AUC is currently the preferred method for performing these analyses, but the application of multiple orthogonal methods is generally accept- able for early phase clinical trials. The apparent utility of CDMS and new/modified instrumenta- tion that is capable of supporting this analysis has the potential to either augment or replace AUC. Potency determination Early approaches to quantitate vector genome (VG) included dot blot DNA assays, southern blot, ultraviolet (UV) spectrometry, and f luorometry. 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 inf luenced to varying de- grees by several AAV product-related factors, in- cluding secondary structures of the AVV genome, process impurities (e.g., residual DNA or protein), and buffer matrix. RT-qPCR and ddPCR have inherent distinc- tions. While qPCR functions over a wider dy- namic range and is more economical, ddPCR of- 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.