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September 01, 2011 (Vol. 31, No. 15)

Transient Transfection of Suspension Cells

Optimization of Key Parameters Essential for Robust Protein Production

  • Reagent Concentration

    Reagent-to-DNA ratio is arguably the most influential parameter in transient transfection. Insufficient or excess reagent quantities can impede DNA delivery. The electrostatic interactions between the cationic transfection reagent and negatively charged plasmid DNA facilitate the plasma membrane binding and internalization of the transfection complex. However, excess positive charge is typically associated with cellular toxicity and necessitates a titration of transfection reagent-to-DNA ratios to determine peak protein expression.

    Figure 2 compares three different levels of TransIT-PRO and PRO Boost Reagent (0.5 µL–2.0 µL per µg of DNA); the highest luciferase expression is obtained with TransIT-PRO and PRO Boost Reagents at a concentration of 1.0 µL each per 1.0 µg of DNA.

  • Media Formulation

    Click Image To Enlarge +
    Figure 3. Protein yield is dependent on media formulation and transfection method. FreeStyle CHO-S cells were adapted to five representative growth media. Cells were transfected using the TransIT-PRO and PRO Boost Reagent (1:1:1), Reagent P (4:1), or Reagent F (1:1) transfection reagents. Transfections were performed in 24-well deep well shaker blocks using 1 µg DNA per milliliter of culture and 0.5 x 106 cells/mL at the time of transfection. Human IgG1 was quantitated from day 5 clarified supernatants and analyzed by a standard sandwich ELISA. Error bars represent the standard deviation of triplicate wells.

    Transfection efficiencies are significantly influenced by the culture growth media, and there are many commercially available serum-free complete growth media for biotherapeutic protein production. Since these media formulations are proprietary, it may be necessary to test several media formulations to find one that is complementary with the desired transfection method.

    Figure 3 illustrates suspension CHO cells that have been adapted to five different commercially available media formulations. Cells adapted to the respective media were transfected with a plasmid encoding a human IgG1 antibody construct using multiple transfection methods including: TransIT-PRO Transfection Kit, Reagent P, and Reagent F (Figure 3).

    Secreted antibody titers varied by greater than 10-fold, depending on the media type, within a given transfection methodology. Additionally, not all transfection technologies exhibited broad-spectrum media compatibility. The highest antibody titers were obtained when transfections were performed with the TransIT-PRO Transfection Kit in combination with BD Select™ CD 1000 medium (BD Biosciences).

    High-efficiency transient transfection of suspension CHO-derived cells enables efficient manufacture of complex biotherapeutics. Notably, protein yield is highly dependent on the intrinsic properties of the recombinant protein; two antibody constructs of the same subtype can produce vastly different protein titers. When comparing two or more transfection methods, perform transfections on the same day with the same plasmid DNA construct and the same batch of cells. This allows cellular and experimental variables to be minimized.

    New transfection methods such as the TransIT-PRO Transfection Kit allow researchers to obtain higher protein titers in a straightforward and consistent manner. Further optimization of key transfection parameters enables researchers to obtain high transfection efficiencies with sufficient protein yields to accelerate drug discovery.

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