Selection and optimization of transfection enhancer additives for increased virus-like particle production in HEK293 suspension cell cultures

Abstract

The manufacturing of biopharmaceuticals in mammalian cells typically relies on the use of stable producer cell lines. However, in recent years, transient gene expression has emerged as a suitable technology for rapid production of biopharmaceuticals. Transient gene expression is particularly well suited for early developmental phases, where several potential therapeutic targets need to be produced and tested in vivo. As a relatively new bioprocessing modality, a number of opportunities exist for improving cell culture productivity upon transient transfection. For instance, several compounds have shown positive effects on transient gene expression. These transfection enhancers either facilitate entry of PEI/DNA transfection complexes into the cell or nucleus or increase levels of gene expression. In this work, the potential of combining transfection enhancers to increase Gag-based virus-like particle production levels upon transfection of suspension-growing HEK 293 cells is evaluated. Using Plackett-Burman design of experiments, it is first tested the effect of eight transfection enhancers: trichostatin A, valproic acid, sodium butyrate, dimethyl sulfoxide (DMSO), lithium acetate, caffeine, hydroxyurea, and nocodazole. An optimal combination of compounds exhibiting the highest effect on gene expression levels was subsequently identified using a surface response experimental design. The optimal consisted on the addition of 20 mM lithium acetate, 3.36 mM valproic acid, and 5.04 mM caffeine which increased VLP production levels 3.8-fold, while maintaining cell culture viability at 94%.

Quality Assessment of Virus-Like Particles at Single Particle Level: A Comparative Study

“…Uranium strongly reacts with phosphate and amino groups and typically stains proteins, nucleic acids and lipid membranes [44]. Thus, VLPs were observed as spherical electrodense structures surrounded by a bright corona (white arrows) that might correspond to structured Gag-eGFP monomers surrounded by the lipid membrane [36,45]. As expected, the presence of baculoviruses (BV) (black arrows) was detected along with VLPs (white arrows) in infected Sf9 samples.…”

Efficient Development of Stable Recombinant Chinese Hamster Ovary (rCHO) Cell Lines to Produce Antibodies by Using Dimethyl Sulfoxide (DMSO) in Electroporation

“…Transfection methods using cationic polymers and lipids have been developed for the transfection of adherent CHO host cells [5,6] whereas the electroporation is widely used for transfection of suspension-adapted CHO host cells [7]. A number of compounds, so called transfection enhancers, can enhance transgene expression in various cell types when they are used as additives in transfection procedures [8]. Among these compounds, dimethyl sulfoxide (DMSO) can increase efficiency of transgene expression when it is used as an additive in electroporation of various host cells (HL60, TR146, Cos-7, and L132) [9].…”

Immortalizing Mesenchymal Stromal Cells from Aged Donors While Keeping Their Essential Features

“…The histone deacetylase inhibitor valproic acid (VPA, 2propyl-valeric acid) could be used for transgene expression induction [37,38]. VPA not only prevents the transcriptional silencing of genes [39] but also reduces cell proliferation [39,40] and might even exert a negative [39] or positive effect on cell viability, depending on the concentration and cell type [37,40]. Thus, the concentration needs to be adjusted in order to enhance transgene expression without affecting cell viability and proliferation.…”

“…After spinoculation, MSCs were incubated for 4 hours at 37°C, and after, retroviral supernatants were replaced by a fresh culture medium with different concentrations of VPA (Cayman Chemical Company, Ann Arbor, Michigan, USA) [37] (Table 2). After three days, the culture medium was replaced by a selection culture medium containing 2.5 μg/ml puromycin (Thermo Fisher Scientific) or 75 μg/ml 2.3.…”