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Platforms

Yposkesi produces AAV and lentiviral vectors based on transfection processes. We offer two process platforms for each of these two products based either on adherent cells or on suspension cells.

  • 293 cell thawing
  • Cell expansion
  • Transfection
  • Vector production
  • Purification
  • Formulation
  • Fill & finish

Lentiviral process platforms

Both lentiviral processes use the same process steps and share the same cell type (293T). The upstream and transfection steps differ:

Adherent process :

  • Cell expansion takes place in shake-flask and Cell Factory 10 devices / Hyperstack devices. At that point, transfection is carried out using calcium phosphate procedure. Two harvests are performed and pooled: one after 24 hours and the other after 48 hours with a benzonase treatment. Recovery and purification of the lentiviral vector follows the steps as shown below. Fill and Finish operations (sterile filtration, vial filling, capping, crimping, container integrity testing, visual inspection and labeling) are performed ‘on-line’ to avoid any product loss caused by freezing.

Suspension process :

  • Cell expansion is performed in suspension in shake-flasks before transfer to the Single-Use-Bioreactor, using Serum-Free media. Cell expansion occurs up to the scale of one or two 200L bioreactors. Transfection is performed using PEI or a modified ‘optimized’ PEI. After 48hrs and the benzonase treatment, the product undergoes the same purification as outlined below steps. Fill and Finish operations are also performed on-line to avoid any product loss caused by freezing.

Yposkesi’s transfection process for lentiviral vector production in suspension is patented

  • Cell thawing
  • Cell amplification
  • Transfection
  • Harvest
  • Clarification
  • IEX Chromatography
  • UF/Diafiltration
  • Sterilizing, filtration & aseptic filling

AAV process platforms

Both AAV processes use the same process steps. The upstream and transfection steps differ:

Adherent process :

  • A 293 non T-cell expansion takes place in shake-flask and Cell Factory 10 devices up to a scale of 24 Cell Factory 10 units. At this point, transfection is carried out using a calcium phosphate procedure. One harvest is performed after 48hrs with benzonase treatment. Recovery and purification of the AAV vector follows the steps as shown below. The product is frozen before Fill-Finish operations.

Suspension process :

  • Two options are available concerning the cell substrate to be used: either a HEK293 cell or a HEK293T YPK+ cell clone. The HEK293T YPK+ cells is a high productivity clone isolated from a parental HEK293T cell bank. Cell expansion is performed in suspension in shake-flasks before transfer to a Single-Use-Bioreactor, using Serum-Free media. Cell expansion is performed up to the scale of one or two 200L bioreactors. Transfection is carried out using PEI or a modified ‘optimized’ PEI. After 48hrs and benzonase treatment, the product undergoes the same purification steps. The product is frozen before Fill-Finish operations.

  • Cell thawing
  • Cell amplification
  • Transfection
  • Harvest
  • Clarification
  • IEX Chromatography
  • UF / Diafiltration
  • Sterilizing filtration & Asptic filling

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