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Development of an icIEF assay for monitoring AAV capsid proteins and application to gene therapy products.
He, Xiaoping Z; Powers, Thomas W; Huang, Sisi; Liu, Zhenjiu; Shi, Heliang; Orlet, John D; Mo, Jim J; Srinivasan, Saipraveen; Jacobs, Steven; Zhang, Kun; Runnels, Herbert A; Anderson, Melissa M; Lerch, Thomas F.
Afiliação
  • He XZ; Pfizer Inc., Analytical Research and Development, Chesterfield, MO, USA.
  • Powers TW; Pfizer Inc., Analytical Research and Development, Chesterfield, MO, USA.
  • Huang S; Pfizer Inc., Analytical Research and Development, Chesterfield, MO, USA.
  • Liu Z; Pfizer Inc., Analytical Research and Development, Chesterfield, MO, USA.
  • Shi H; Pfizer Inc., Analytical Research and Development, Chesterfield, MO, USA.
  • Orlet JD; Pfizer Inc., Analytical Research and Development, Chesterfield, MO, USA.
  • Mo JJ; Pfizer Inc., Analytical Research and Development, Chesterfield, MO, USA.
  • Srinivasan S; Pfizer Inc., Analytical Research and Development, Chesterfield, MO, USA.
  • Jacobs S; Pfizer Inc., Analytical Research and Development, Chesterfield, MO, USA.
  • Zhang K; Pfizer Inc., Analytical Research and Development, Chesterfield, MO, USA.
  • Runnels HA; Pfizer Inc., Analytical Research and Development, Chesterfield, MO, USA.
  • Anderson MM; Pfizer Inc., Analytical Research and Development, Chesterfield, MO, USA.
  • Lerch TF; Pfizer Inc., Analytical Research and Development, Chesterfield, MO, USA.
Mol Ther Methods Clin Dev ; 29: 133-144, 2023 Jun 08.
Article em En | MEDLINE | ID: mdl-37025949
ABSTRACT
Adeno-associated virus (AAV) gene therapy vectors, which contain a DNA transgene packaged into a protein capsid, have shown tremendous therapeutic potential in recent years. Methods traditionally used in quality control labs, such as high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE), do not provide a complete understanding of capsid viral protein (VP) charge heterogeneity. In the present study, we developed simple, one-step sample preparation and charge-based VP separation using imaged capillary isoelectric focusing (icIEF) for monitoring AAV products. The robustness of the method was confirmed through a design of experiments (DoE) exercise. An orthogonal reverse-phase (RP) HPLC method coupled with mass spectrometry was developed to separate and identify charge species. Additionally, capsid point mutants demonstrate the capability of the method to resolve deamidation at a single site on the viral proteins. Finally, case studies using two different AAV serotype vectors establish the icIEF method as stability indicating and demonstrate that increases in acidic species measured by icIEF correlate with increased deamidation, which, we show, results in decreased transduction efficiency. The addition of a rapid and robust icIEF method to the AAV capsid analytical toolkit enables development and consistent manufacturing of well-characterized gene therapy products.
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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: 2023 Tipo de documento: Article País de afiliação: Estados Unidos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Idioma: En Revista: Mol Ther Methods Clin Dev Ano de publicação: 2023 Tipo de documento: Article País de afiliação: Estados Unidos