Derivation of a Clonal HEK293 Suspension Cell Line for High Yield AAV Production
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Abstract
Cells are critical components of biotherapeutic productions and implementation of clonal cell lines is highly recommended in manufacturing processes. Cell line clonality mitigates the genetic heterogeneity inherent to most biological cell lines. Andelyn Biosciences utilizes a robust suspension platform for AAV production using a clonal HEK293 suspension cell line developed from an adherent HEK293 parental line. We subjected an existing adherent line of HEK293 to a selection series with decreasing serum concentrations before switching it to growth in serum-free media. These processes resulted in a family of suspension-adapted, serum-free HEK293 cell lines that were the parental lines for subsequent isolation of single-cell clones. Clonal selection was performed with the Solentim Verified In-Situ Plate Seeding system which delivers Day 0 assurance of clonality. Clonal cell lines were initially assessed for AAV production in 125mL shake flasks. Promising candidates were further assessed with the Ambr® 250 modular and then subjected to 2L yield assessments. The final candidate was selected from a panel of six comparable clones. It was scaled to a 50L production with AAV Serotype A using an Allegro STR50 bioreactor and purified to final fill. At all stages of production, AAV yields exceeded those produced from the commercially available control cell line. We will discuss further optimization of this newly developed cell line, including viral titers of AAV serotypes with our production platform.
Download our ESGCT 2022 Poster
Get exclusive access to our ESGCT 2022 poster! Enter your email to download the poster and stay informed on how we’re pushing the boundaries of cell and gene therapy for a better future.
Abstract
Cells are critical components of biotherapeutic productions and implementation of clonal cell lines is highly recommended in manufacturing processes. Cell line clonality mitigates the genetic heterogeneity inherent to most biological cell lines. Andelyn Biosciences utilizes a robust suspension platform for AAV production using a clonal HEK293 suspension cell line developed from an adherent HEK293 parental line. We subjected an existing adherent line of HEK293 to a selection series with decreasing serum concentrations before switching it to growth in serum-free media. These processes resulted in a family of suspension-adapted, serum-free HEK293 cell lines that were the parental lines for subsequent isolation of single-cell clones. Clonal selection was performed with the Solentim Verified In-Situ Plate Seeding system which delivers Day 0 assurance of clonality. Clonal cell lines were initially assessed for AAV production in 125mL shake flasks. Promising candidates were further assessed with the Ambr® 250 modular and then subjected to 2L yield assessments. The final candidate was selected from a panel of six comparable clones. It was scaled to a 50L production with AAV Serotype A using an Allegro STR50 bioreactor and purified to final fill. At all stages of production, AAV yields exceeded those produced from the commercially available control cell line. We will discuss further optimization of this newly developed cell line, including viral titers of AAV serotypes with our production platform.
Download our ESGCT 2022 Poster
Get exclusive access to our ESGCT 2022 poster! Enter your email to download the poster and stay informed on how we’re pushing the boundaries of cell and gene therapy for a better future.
Abstract
Cells are critical components of biotherapeutic productions and implementation of clonal cell lines is highly recommended in manufacturing processes. Cell line clonality mitigates the genetic heterogeneity inherent to most biological cell lines. Andelyn Biosciences utilizes a robust suspension platform for AAV production using a clonal HEK293 suspension cell line developed from an adherent HEK293 parental line. We subjected an existing adherent line of HEK293 to a selection series with decreasing serum concentrations before switching it to growth in serum-free media. These processes resulted in a family of suspension-adapted, serum-free HEK293 cell lines that were the parental lines for subsequent isolation of single-cell clones. Clonal selection was performed with the Solentim Verified In-Situ Plate Seeding system which delivers Day 0 assurance of clonality. Clonal cell lines were initially assessed for AAV production in 125mL shake flasks. Promising candidates were further assessed with the Ambr® 250 modular and then subjected to 2L yield assessments. The final candidate was selected from a panel of six comparable clones. It was scaled to a 50L production with AAV Serotype A using an Allegro STR50 bioreactor and purified to final fill. At all stages of production, AAV yields exceeded those produced from the commercially available control cell line. We will discuss further optimization of this newly developed cell line, including viral titers of AAV serotypes with our production platform.