Comprehensive Size Distribution and Composition Analysis of Adeno-Associated Virus Vector by Multiwavelength Sedimentation Velocity Analytical Ultracentrifugation

Abstract

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Correlating physicochemical and biological properties to define critical quality attributes of a recombinant AAV vaccine candidate

“…The percent full-capsids (~36%) in the AC3 preparation was determined by TEM and SV-AUC, however, TEM analysis cannot currently distinguish empty vs. partially-filled capsids 87 . SV-AUC is thus the current "gold standard" to evaluate the relative proportions of full, partially-filled and empty capsids as well as aggregates 64,88,89 , yet this is a highly specialized technique, so efforts to identify alternative, more easily implemented analytical approaches (e.g., chromatographic, electrophoretic and mass spectrometry) are ongoing 76 . Such compositional analyses of rAAV preparations are performed to monitor lot-to-lot consistency, yet reports of their effects on gene transduction or in vivo performance are limited 64,90 .…”

“…SV-AUC is thus the current "gold standard" to evaluate the relative proportions of full, partially-filled and empty capsids as well as aggregates 64,88,89 , yet this is a highly specialized technique, so efforts to identify alternative, more easily implemented analytical approaches (e.g., chromatographic, electrophoretic and mass spectrometry) are ongoing 76 . Such compositional analyses of rAAV preparations are performed to monitor lot-to-lot consistency, yet reports of their effects on gene transduction or in vivo performance are limited 64,90 . From a rAAV vaccine perspective, a possible correlation between a higher full/empty capsid ratio with heightened immune responses was reported 91 .…”

Assessment of quality attributes for adeno‐associated viral vectors

“…Furthermore, partial capsids will contain undesired DNA, such as truncated vector, HCD, or residual plasmid DNA, and could therefore be a concern for safety due to possible genotoxic or immunogenic effects. Burnham et al, 2015;Fu et al, 2019;Maruno et al, 2021) among others (Gimpel et al, 2021). Charge detection mass spectrometry (CDMS) is an emerging technique that has been reported to be able to resolve empty, full, and partially filled capsids (Pierson et al, 2016).…”

“…Both the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) guidelines specify the need to control empty particle number (EMA, 2018; US Department of Health and Human Services, FDA, Center for Biologics Evaluation and Research, 2020). Many different methods can be used to quantify empty:full ratio including Transmission Electron Microscopy (TEM; Dobnik et al, 2019; Horowitz et al, 2013), VG/capsid titer ratio, anion‐exchange high‐performance liquid chromatography (AEX‐HPLC; Fu et al, 2019; Lock et al, 2012; C. Wang et al, 2019), absorbance ratio at 260/280 nm (Sommer et al, 2003), size‐exclusion chromatography multiangle light scattering (SEC‐MALS; Chen & Purchel, 2021), and Sedimentation Velocity Analytical Ultra Centrifugation (SV‐AUC; Burnham et al, 2015; Fu et al, 2019; Maruno et al, 2021) among others (Gimpel et al, 2021). Charge detection mass spectrometry (CDMS) is an emerging technique that has been reported to be able to resolve empty, full, and partially filled capsids (Pierson et al, 2016).…”

Reverse-Phase Ultra-Performance Chromatography Method for Oncolytic Coxsackievirus Viral Protein Separation and Empty to Full Capsid Quantification

“…Conventionally, the virus capsid concentration and/or capsid empty/full ratio are measured using low-throughput techniques, such as analytical ultracentrifugation, Cryo-EM. 15–17 Polymerase chain reaction techniques that measure the genome titer or immuno-binding assays, such as ELISA, have also been employed to assess oncolytic particles. These assays are highly variable and labor intensive.…”