Analytical characterization of full, intermediate, and empty AAV capsids

Abstract

Manufacturing of recombinant adeno-associated virus (AAV) vectors produces three types of capsids: full, intermediate, and empty. While there are different opinions about the impact of intermediate and empty capsids on safety and efficacy of AAV products, they are generally considered impurities because they are not the intended fully intact vector product. The presence of these impurities could impact product efficacy due to potential competition with fully packaged AAVs for cellular transduction, as well as have potential implications to patient safety due to increased capsid load during dosing. To determine the impact of intermediate capsids on potency, an AAV preparation was separated into fractions enriched for full, intermediate, or empty capsids. Using a matrix of in vitro (infectivity, gene expression, biological activity) and in vivo potency assays to determine potency as a function of capsid content, our results indicate that while intermediate capsids contribute to the vector genome titer of the product and are equally as infectious as full capsids, they do not contribute to the potency of the AAV product. This study confirms the criticality of reducing and controlling the level of intermediate capsids to ensure a more efficacious AAV product.

Fig. 1. Capsid content determination by AUC.

AUC sedimentation distribution plots for rAAV samples pre- and post-preparative ultracentrifugation, n = 1 for the assay. A OXBS1 AEX product (representative of DS), B OXBS1-F pool, C OXBS1-I pool, and D OXBS1-E pool. The percentage of full capsids (F), intermediate capsids (I) and empty capsids (E) are shown in each figure.

Fig. 2. Capsid content determination by CDMS.

CDMS mass distribution plots for rAAV samples post-preparative ultracentrifugation. Tables below the figure detail the identified peak, mass range (MDa) and % relative abundance, n = 1 for the assay. A OXBS1-F, B OXBS1-I, and C OXBS1-E.

Fig. 3. Characterization of VP purity, VP1:VP2:VP3 ratio, and post-translational modifications of full, intermediate, and empty capsids by CE-SDS and LC-MS/MS.

A VP purity and VP ratio of OXBS1-F, OXBS1-I, and OXBS1-E were determined by CE-SDS. B, C Peptide mapping of the VP proteins present in OXBS1-F, OXBS1-I, and OXBS1-E pools was determined by LC-MS/MS. B Sequence coverage was confirmed according to the primary amino acid sequence using BioPharma Finder™ software (Thermo Fisher Scientific) and C post-translational modifications were identified using both BioPharma Finder™ and Skyline. N = 1 for the assay. VP Viral Protein, I aspartic acid isomerization, D deamidation, S succinimide, P phosphorylation, M methylation.

Fig. 4. Measurement of VG titer, capsid titer, process-related impurities, and residual sequence expression in HeLa cells.

A VG titer and capsid titer for OXBS1-F, OXBS1-I, and OXBS1-E pools were determined by ddPCR and ELISA, respectively. B Levels of plasmid-derived DNA impurities (Rep/Cap, Helper plasmid, KanR; left y-axis) and host cell DNA (right y-axis) were determined by ddPCR and qPCR, respectively. Results for each target were normalized to 1E + 13 capsids to allow direct comparison between samples. C mRNA expression of GOI and KanR was measured across a dose range of MOIs for OXBS1-F and OXBS1-I in HeLa cells by RT-qPCR. Each point represents the average of n = 2 wells. A no RT control (dotted lines) was run for each to confirm the assay is specifically measuring gene expression.

Fig. 5. Evaluating the impact of intermediate capsids on in vitro and in vivo potency.

A Infectivity (VG/IU; left y-axis), transgene expression (%RGE; right y-axis) and biological activity (%RP; right y-axis)= were determined for OXBS1-F (black bars) and OXBS1-I (blue bars) samples using the nominal VG titer to determine potency as a function of vector genome. N = 1 for each sample. B Transgene expression was determined in vitro (%RGE; black bars) and in vivo (%RGE; blue bars) for OXBS1-F, OXBS1-I, and OXBS1-E samples using the nominal capsid titer to determine potency as a function of capsid content. % Full capsids were determined by AUC (purple bars). N = 1 for all assays. C Transgene expression was determined in liver tissue 5 weeks-post infusion of mice with 9E13 capsids/kg of OXBS1-F, OXBS1-I, or OXBS1-E samples. Formulation buffer was used as a vehicle control. GOI copies per ng RNA was determined by ddPCR. One-way analysis of variance (ANOVA) was used to determine any statistically significant differences between the means of the capsid groups. Error bars are SEM. N = 4 mice per cohort.