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Biolayer interferometry for adeno-associated virus capsid titer measurement and applications to upstream and downstream process development.
Leibiger, Thomas M; Remmler, Luke A; Green, Erica A; Lee, Kelvin H.
Afiliação
  • Leibiger TM; University of Delaware, Department of Chemical and Biomolecular Engineering, Newark, DE, USA.
  • Remmler LA; University of Delaware, Department of Chemical and Biomolecular Engineering, Newark, DE, USA.
  • Green EA; University of Delaware, Department of Chemical and Biomolecular Engineering, Newark, DE, USA.
  • Lee KH; University of Delaware, Department of Chemical and Biomolecular Engineering, Newark, DE, USA.
Mol Ther Methods Clin Dev ; 32(3): 101306, 2024 Sep 12.
Article em En | MEDLINE | ID: mdl-39220638
ABSTRACT
Faster and more accurate analytical methods are needed to support the advancement of recombinant adeno-associated virus (rAAV) production systems. Recently, biolayer interferometry (BLI) has been developed for high-throughput AAV capsid titer measurement by functionalizing the AAVX ligand onto biosensor probes (AAVX-BLI). In this work, an AAVX-BLI method was evaluated using Octet AAVX biosensors across four rAAV serotypes (rAAV2, -5, -8, and -9) and applied in an upstream and downstream processing context. AAVX-BLI measured the capsid titer across a wide concentration range (1 × 1010-1 × 1012 capsids/mL) for different rAAV serotypes and sample backgrounds with reduced measurement variance and error compared to an enzyme-linked immunosorbent assay (ELISA) method. Biosensors were regenerated for repeated use, with lysate samples showing reduced regeneration capacity compared to purified and supernatant samples. The AAVX-BLI method was applied in a transfection optimization study where direct capsid titer measurement of culture supernatants generated a representative response surface for the total vector genome (VG) titer. For rAAV purification, AAVX-BLI was used to measure dynamic binding capacity with POROS CaptureSelect AAVX affinity chromatography, showing resin breakthrough dependence on the operating flow rate. Measurement accuracy, serotype and sample background flexibility, and high sample throughput make AAVX-BLI an attractive alternative to other capsid titer measurement techniques.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Idioma: En Revista: Mol Ther Methods Clin Dev Ano de publicação: 2024 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Idioma: En Revista: Mol Ther Methods Clin Dev Ano de publicação: 2024 Tipo de documento: Article