During a December 2021 presentation, Matthew Lotti (senior research associate in analytical development at Ultragenyx) highlighted difficulties with determining adenoassociated virus (AAV) capsid titers during gene therapy production. With support from Gyros Protein Technologies, Lotti spoke about his team’s development of a method for measuring AAV titers using a Gyrolab xPand immunoassay system.

Lotti’s Presentation
Simple analytical methods such as UV spectrophotometry measure capsid titers most effectively after virions have undergone purification. To test samples from earlier process stages, Ultragenyx previously used enzyme-linked immunosorbent assay (ELISA) kits, but such assays are expensive to run and take four to five hours to generate results. ELISAs also feature a narrow dynamic range (8 × 10⁶ vp/mL to 5 × 10⁸ vp/mL). Because unpurified samples average between 10¹² vp/mL and 10¹⁴ vp/mL, such materials require significant dilution — and that can diminish assay accuracy.

Ultragenyx worked with Gyros Protein Technologies to develop a method that could measure AAV total capsid titers quickly and accurately while reducing dilution requirements. The team implemented a Gyrolab xPand system, which supports five compact discs (CDs), each enabling parallel processing of 96 or 112 samples in a single automated run. The sample volume — 20 nL to 4000 nL, as defined by the microstructures in the different CD types — determines the sensitivity. Samples pass through a 15-nL affinity capture column within the CD, in which target molecules bind to biotinylated capture reagents previously bound to the streptavidin-coated particles of the affinity column. In a simple “sandwich” assay format, fluorophore-labeled detection reagent is added. Each column in the CD is scanned, and florescence data are generated.

Lotti highlighted that the Gyrolab xPand system can perform titer assays in less than two hours. The instrument covers a broad standard dynamic range (7 × 10⁶ vp/mL to 5 × 10¹² vp/mL), requires small sample volumes, and analyzes more samples per run than an ELISA can. All those capabilities, he explained, could reduce screening costs considerably.

The Development Journey: To establish proof of concept, Lotti’s team analyzed in-process AAV8 samples using a Gyrolab AAVX titer kit (Gyros Protein Technologies), which includes a 1,000-nL CD and capture and fluorescent detection reagents at appropriate concentrations. The dynamic range of standard curves was unacceptable when using undiluted dyes. Further dilution of the detection reagent to concentrations of 1:3, 1:9, and 1:27 yielded sufficient curves, although the high background reduces the accuracy of measurement for samples with low AAV concentration.

Lotti’s team sought to adjust the method’s dynamic range, shifting the low end to 10⁸ vp/mL to accommodate samples with high AAV concentrations. Analysts performed a multiparameter study to compare two disc formats (200-nL and 1,000-nL CDs), two buffers (Gyrolab AAVX titer sample dilution buffer and Rexxip buffer, both from Gyros Protein Technologies), and a range of detection concentrations (full strength and dilution factors of 1:3 or 1:10 for the 200-nL and 1,000-nL CDs). Runs performed in the 200-nL format using Gyrolab AAVX titer sample dilution buffer and undiluted detection reagent showed the broadest dynamic range, highest precision, and best sensitivity. Samples loaded in those conditions also exhibited dilutional linearity. Ultragenyx now applies those conditions for all its capsid titer assays.

The company continues to explore potential applications — for instance, the measurement of empty:full capsid ratios, which are usually obtained by analytical centrifugation (AUC) of drug substance samples. Ultragenyx incorporates the ratio of genome titer by quantitative polymerase chain reaction (qPCR) and Gyrolab titer results to generate surrogate measurements for empty:full capsid ratios of unpurified sample and in-process samples. Monitoring full-capsid enrichment guides process decisions. The assay also can provide valuable data for column-breakthrough studies and media screenings.

Questions and Answers
How could this titer assay be optimized? Further adjustments could help to extend the method’s dynamic range and prevent matrix effects, and a positive control could be developed to help analysts monitor the assay’s performance over time.

What other assays might Ultragenyx develop using the Gyrolab xPand system? The titer method described above was developed alongside an impurity assay. In 2022, Ultragenyx will assess two or three other such tests for AAV vectors.

The recorded webcast is available to view on-demand: Watch Now.