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. 2024 Aug 16;19(8):e0308547.
doi: 10.1371/journal.pone.0308547. eCollection 2024.

Optimization of culture condition for Spodoptera frugiperda by design of experiment approach and evaluation of its effect on the expression of hemagglutinin protein of influenza virus

Fatemeh Alizadeh  1   2 , Hamideh Aghajani  2 , Fereidoun Mahboudi  1   2 , Yeganeh Talebkhan  1 , Ehsan Arefian  3 , Sepideh Samavat  2 , Rouhollah Raufi  2
Affiliations

Optimization of culture condition for Spodoptera frugiperda by design of experiment approach and evaluation of its effect on the expression of hemagglutinin protein of influenza virus

Fatemeh Alizadeh et al. PLoS One. .

Abstract

The baculovirus expression vector system (BEVS) is a powerful tool in pharmaceutical biotechnology to infect insect cells and produce the recombinant proteins of interest. It has been well documented that optimizing the culture condition and its supplementation through designed experiments is critical for maximum protein production. In this study, besides physicochemical parameters including incubation temperature, cell count of infection, multiplicity of infection, and feeding percentage, potential supplementary factors such as cholesterol, polyamine, galactose, pluronic-F68, glucose, L-glutamine, and ZnSO4 were screened for Spodoptera frugiperda (Sf9) cell culture and expression of hemagglutinin (HA) protein of Influenza virus via Placket-Burman design and then optimized through Box-Behnken approach. The optimized conditions were then applied for scale-up culture and the expressed r-HA protein was characterized. Optimization of selected parameters via the Box-Behnken approach indicated that feed percentage, cell count, and multiplicity of infection are the main parameters affecting r-HA expression level and potency compared to the previously established culture condition. This study demonstrated the effectiveness of designing experiments to select and optimize important parameters that potentially affect Sf9 cell culture, r-HA expression, and its potency in the BEVS system.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Pareto charts for the Plackett-Burman design.
The charts demonstrate the order and effect of each parameter on (A) r-HA expression level and (B) Viable cell count.
Fig 2
Fig 2. The comparative analysis of 23 Placket-Burman-designed experiments on main responses.
(A) Cell count and (B) r-HA expression. Error bars represent the SD of three independent experiments.
Fig 3
Fig 3. Comparative evaluation of HA expression level within 54 designed RSM experiments.
The highest expression level has been achieved under conditions described in experiment#14 (Black bar). Error bars represent the SD of three independent experiments.
Fig 4
Fig 4
Response surface 3D plots represent the interactions between r-HA expression level and (A) Feed percentage and TOI; (B) Feed percentage and MOI; (C) CCI and MOI.
Fig 5
Fig 5. Evaluation of HA expression.
(A) r-HA expression level in different culture conditions (Control condition in bioreactor, optimized condition in shake flask, and optimized condition in bioreactor: 71, 239, and 205 μg/ml, respectively); (B) SDS-PAGE (non-reduced samples): M: Mw marker; #1: Standard HA protein; #2: Cell lysate from control culture condition; #3: Cell lysate from optimized culture condition; #4: Eluate of lectin resin from control culture condition; #5: Eluate of lectin resin from the optimized condition. **** represents P <0.0001. Error bars show the SD of triplicate runs.
Fig 6
Fig 6. Recombinant HA potency assay in control and RSM-optimized conditions compared to the standard HA antigen.
See this image and copyright information in PMC

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