Calcium-phosphate mediated DNA transfer into HEK-293 cells in suspension: control of physicochemical parameters allows transfection in stirred media. Transfection and protein expression in mammalian cells

This is the first report of transient transfection of suspended cells with purified plasmid DNA in bioreactors or spinner flasks. DNA/calcium phosphate complexes were pumped or injected directly into stirred cultures of the immortalized human embryo kidney cell line 293 (HEK-293) which had been adapted to growth in suspension. We identified culture conditions suitable for this approach and modified the protocol for the generation of precipitate complexes, based on our earlier work. In order to stabilize the DNA-vehicle-complex in the culture medium, we identified pH ranges and ion-concentrations which prevent dissolution or aggregation of the precipitate particles. Such conditions maintained suspended fine particles in spinners or bioreactors for up to 6 hr. During that period, cells and precipitate complexes interacted sufficiently to allow DNA transfer and subsequent expression of recombinant protein. In a simple 5 day batch process, with a starting density of 0.3 × 10⁶ cells mL^-1, about 0.5 mg L^-1 of a recombinant tissue plasminogen activator variant was observed.     

Effects of the dilution rate on cell cycle distribution and PEI-mediated transient gene expression by CHO cells in continuous culture

In this study, a continuous culture system was applied to mammalian cells on large scale, and polyethyleneimine (PEI) mediated transient gene expression (TGE). PEI MAX 40,000 was chosen as a superior reagent from three types of PEI. The cell cycle distribution of cells in batch and continuous cultures was determined, in which the effects of cell cycle distribution on transfection efficiency, post-transfection proliferation and recombinant prothrombin expression were evaluated. Compared with cells from end-log and plateau phase in batch culture, cells from mid-log phase possessed a larger fraction of S and G2/M phase cells and a smaller fraction of G1 phase cells. In the continuous culture, the fraction of cells in the S and G2/M phases increased and the fraction of cells in the G1/G0 phase decreased with increasing dilution rates. Cells from the continuous culture run at highest dilution rate had excellent proliferation, transfection efficiency and protein expression. These results were confirmed by transfecting cells synchronized to different phases. The G2/M arrested cells exhibited a nearly 10-fold increase in recombinant human prothrombin production relative to that of non-dividing cells. The use of continuous culture for large scale transfection demonstrated a better cell physiological state for TGE process.     

Recombinant protein production by large-scale transient gene expression in mammalian cells: state of the art and future perspectives

The expansion of the biologics pipeline depends on the identification of candidate proteins for clinical trials. Speed is one of the critical issues, and the rapid production of high quality, research-grade material for preclinical studies by transient gene expression (TGE) is addressing this factor in an impressive way: following DNA transfection, the production phase for TGE is usually 2-10 days. Recombinant proteins (r-proteins) produced by TGE can therefore enter the drug development and screening process in a very short time--weeks. With "classical" approaches to protein expression from mammalian cells, it takes months to establish a productive host cell line. This article summarizes efforts in industry and academia to use TGE to produce tens to hundreds of milligrams of r-proteins for either fundamental research or preclinical studies.     

Enhancement of transient gene expression by fed-batch culture of HEK 293 EBNA1 cells in suspension

Enhanced green fluorescence protein (GFP) and erythropoietin (EPO) were used as reporters to assess and improve transient gene expression in HEK 293 EBNA1 cells. The production of EPO only lasted 3 days and reached 18.1 mg/l in suspension cultures in 1 l batch bioreactors. However, GFP expression examined in well-plate experiments persisted for 12 days in transfected cells but decreased rapidly within the next 15 days. These results suggest that the retaining of a plasmid in cells may not be a limiting factor for protein expression in large-scale transient transfection. To improve cell maintenance and protein expression, a fed-batch culture was performed using an enriched medium, a mixture of equal volumes of 293 SFM II medium and a 5 × amino acid solution prepared based on DMEM/F12 medium formula. EPO reached 33.6 mg/l, representing 86% increase over that of the batch culture. Moreover, the total amount of EPO produced was increased by 165% in view of the volume increase in the fed-batch culture. The serum-free medium used in this work enables cells growing well and transfection without medium change. Thus, the process reported here is simple and easy to scale up.     

Serum-free suspensin large-scale transient transfection of CHO cells in WAVE bioreactors

Here, we report the development of a large-scale transient expression platform utilizing Chinese hamster ovary (CHO) cells. The majority of recombinant proteins and antibodies that are produced for preclinical models and clinical trials are expressed in stably transfected CHO cells. A protocol for transient transfection of CHO cells that is rapid, reproducible, and cost-effective would therefore streamline the process from research to development and help avoid any potential host species induced variation in the molecule of interest. CHO cells were adapted to grow in serum-free suspension conditions in spinner flask cultures in a proprietary in-house developed growth medium. In developing this transient transfection protocol, the parameters optimized included the transfection reagent of choice, the cell density at the time of transfection, the plasmid DNA concentration, and the transfection reagent concentration. Using this optimized protocol, we have expressed recombinant proteins, including antibodies, at an expression level of up to 9.4 mg/L. We also report transient transfections from 500 mL working volume (w.v.) up to 20 L w.v. in a WAVE^TM bioreactor. Using this optimized protocol, it is possible to rapidly (within 10 d) produce up to 100 mg of recombinant protein for further study.     

High-density transfection with HEK-293 cells allows doubling of transient titers and removes need for a priori DNA complex formation with PEI

Recombinant proteins are of great commercial and scientific interest. Yet, most production methods in mammalian cells involve the time- and labor-consuming step of creating stable cell lines. Production methods based on transient gene expression are advantageous in terms of speed and versatility; yet, depending on the transfection protocol, transient transfection faces some bottlenecks such as a priori complex formation, limitations in terms of transfection and production media used and the need for medium exchange prior to and/or after transfection. Published protocols for transfection of suspension-adapted HEK-293 cells with polyethyleneimine have shown great promise in overcoming some of these bottlenecks, but still require a priori complex formation for optimal yields and limit the choice of transfection and production media. Here, we report successful in situ transfection of suspension-adapted HEK-293 cells with 25-kDa linear polyethyleneimine at densities up to 20 x 10(6) cells/mL in complex media followed by production at lower cell densities (1 x 10(6) cells/mL). After concentrating cells to such high densities, transfection of HEK-293 cells becomes possible in most commonly used media and is not restricted to a specific medium. Furthermore, there is no need to make transfection complexes a priori, a step that prevents inline sterile filtration of the DNA bulk for transfection, an important consideration when scaling processes up to 100 or 1,000 L. Finally, transfecting HEK-293 cells at high density in complex media is superior to existing transfection protocols and doubles yields of recombinant protein obtainable by transient gene expression.     

Co-transfection and tandem transfection of HEK293A cells for overexpression and RNAi experiments

pIRES2-EGFP was employed and a non-target shRNA expressing plasmid was constructed to simulate overexpression and RNAi (RNA interference) experiments. Transfection of pIRES2-EGFP into HEK293A cells by cationic lipids VigoFect demonstrated that transfection efficiency increased in a dose-dependent manner with amount of DNA plasmid used, and optimal transfection time and cell density should be identified to reach a compromise of higher transfection efficiency and lower toxicity. Co-transfection experiments indicated that the two co-transfected plasmids were equivalently delivered into the same cells, and the co-transfection efficiency was rarely affected by cell density and proportion of the two plasmids. However, plasmid-receipted cells seemed indisposed to accept plasmid again during the second transfection, and very low co-transfection efficiency was observed in tandem transfection.     

Sex hormones affect the production of recombinant Factor IX in CHO and HEK-293 cell lines

Recombinant human Factor IX (rFIX) was cloned in a mammalian expression vector and transfected into CHO and HEK-293. Treatment with 10⁻⁹ M methyl testosterone increased rFIX production by 30–50% in CHO and HEK clones. However, 10⁻⁹ M 17β-oestradiol increased production of rFIX by ~50% in CHO-F7 clone and decreased production by 48% and 37% in CHO-F8 and HEK-F2-6, respectively. Progesterone treatment inhibited rFIX production in both cell lines. Production of rFIX can thus be increased by sex hormone treatment and therefore used to enhance biotechnological production in mammalian cells.     

Design of Experiment in CHO and HEK transient transfection condition optimization

Transient gene expression (TGE) is a well-established enabling technology for rapid generation of recombinant proteins, with Human Embryonic Kidney (HEK) and Chinese Hamster Ovary (CHO) cell lines and polyethyleneimine (PEI) as the transfection reagent being its most popular components. However, despite considerable progress made in the field, volumetric titers can still be a limiting factor causing the manipulation of increasing quantities of culture media and DNA. Here, we report a systematic analysis of TGE conditions and their influence on yields and protein quality. Guided by Design of Experiments (DoE), we conclude that TGE yields with one test antibody can be maximized by a parallel increase of cell density - 2.4 to 3.0 × 10(6)cells/mL - and PEI concentration - 24 to 30 mg/L - while maintaining a 1:1 ratio of heavy chain and light chain encoding plasmids. Interestingly, we also show that in these conditions, DNA concentration can be maintained in the 1mg/L range, thereby limiting the need for large DNA preparations. Our optimized settings for PEI-mediated TGE in HEK and CHO cells evaluated on several proteins are generally applicable to recombinant antibodies and proteins.     

High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells

A scalable transfection procedure using polyethylenimine (PEI) is described for the human embryonic kidney 293 cell line grown in suspension. Green fluorescent protein (GFP) and human placental secreted alkaline phosphatase (SEAP) were used as reporter genes to monitor transfection efficiency and productivity. Up to 75% of GFP-positive cells were obtained using linear or branched 25 kDa PEI. The 293 cell line and two genetic variants, either expressing the SV40 large T-antigen (293T) or the Epstein–Barr virus (EBV) EBNA1 protein (293E), were tested for protein expression. The highest expression level was obtained with 293E cells using the EBV oriP-containing plasmid pCEP4. We designed the pTT vector, an oriP-based vector having an improved cytomegalovirus expression cassette. Using this vector, 10- and 3-fold increases in SEAP expression was obtained in 293E cells compared with pcDNA3.1 and pCEP4 vectors, respectively. The presence of serum had a positive effect on gene transfer and expression. Transfection of suspension-growing cells was more efficient with linear PEI and was not affected by the presence of medium conditioned for 24 h. Using the pTT vector, >20 mg/l of purified His-tagged SEAP was recovered from a 3.5 l bioreactor. Intracellular proteins were also produced at levels as high as 50 mg/l, representing up to 20% of total cell proteins.     

Investigation of relationship between EBNA-1 expression level and specific foreign protein productivity in transient gene expression of HEK293 cells

In an attempt to determine the relationship between the Epstein–Barr virus nuclear antigen-1 (EBNA-1) expression level and specific foreign protein productivity (q_p), EBNA-1-amplifed HEK293 cells, which achieved a higher EBNA-1 expression level than that achieved by HEK293E cells, were established using dihydrofolate reductase (dhfr)-mediated gene amplification. Compared with a control culture in a null pool, Fc-fusion protein production by transient transfection in the EBNA-1-amplified pool showed a significant improvement. q_p was linearly correlated with the EBNA-1 expression level in the transient transfection of EBNA-1-amplified clones, as indicated by the correlation coefficient (R² = 0.7407). The Fc-fusion protein production and q_p in a transient gene expression-based culture with EBNA-1-amplified HEK293 cells, E-amp-68, were approximately 2.0 and 3.2 times, respectively, higher than those in a culture with HEK293E cells. The increase in q_p by EBNA-1 amplification mainly resulted from an enhancement in the amount of replicated DNA and level of mRNA expression but not an improved transfection efficiency. Taken together, it was found that EBNA-1 amplification could improve the therapeutic protein production in an HEK293 cell-based transient gene expression system.     

Miniaturization of gene transfection assays in 384- and 1536-well microplates

The miniaturization of gene transfer assays to either 384- or 1536-well plates greatly economizes the expense and allows much higher throughput when transfecting immortalized and primary cells compared with more conventional 96-well assays. To validate the approach, luciferase and green fluorescent protein (GFP) reporter gene transfer assays were developed to determine the influence of cell seeding number, transfection reagent to DNA ratios, transfection time, DNA dose, and luciferin dose on linearity and sensitivity. HepG2, CHO, and NIH 3T3 cells were transfected with polyethylenimine (PEI)–DNA in both 384- and 1536-well plates. The results established optimal transfection parameters in 384-well plates in a total assay volume of 35 μl and in 1536-well plates in a total assay volume of 8 μl. A luciferase assay performed in 384-well plates produced a Z′ score of 0.53, making it acceptable for high-throughput screening. Primary hepatocytes were harvested from mouse liver and transfected with PEI DNA and calcium phosphate DNA nanoparticles in 384-well plates. Optimal transfection of primary hepatocytes was achieved on as few as 250 cells per well in 384-well plates, with CaPO₄ proving to be 10-fold more potent than PEI.     

Development and improvement of a serum-free suspension process for the production of recombinant adenoviral vectors using HEK293 cells

Human Embryonic Kidney 293 (HEK293) cells were adapted into a serum-free suspension medium through steps of gradual serum weaning for the production of adenoviral (AdV) gene therapy vectors. The presence of sodium heparin in the medium formulation reduced cell clumping dramatically in suspension culture. The adapted cells were ready to grow either in serum-containing medium as an attached culture or in serum-free medium in suspension culture. A scalable production process was developed in shake flasks and was then evaluated in stirred tank bioreactors. This process includes a growth phase in batch-mode followed by a production phase involving medium perfusion and supplementation. Fortification with calcium chloride post viral inoculation resulted in an increase in virus production by at least one fold. Addition of stimulating agents such as sodium butyrate, N-acetyl-L-cysteine (NAC), dimethyl sulfoxide(DMSO), or ethyl alcohol post infection was shown to further improve virus production in a dose-dependent manner. The serum-free suspension process described here should be suitable for the manufacturing of other E1-deleted AdV vectors and could potentially be used for the production of recombinant proteins by HEK293 cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

Improving heterologous protein expression in transfected Drosophila S2 cells as assessed by EGFP expression

Drosophila melanogaster S2 cells were co-transfected with plasmid vectors containing the enhanced green fluorescent protein gene (EGFP), under the control of metallothionein promoter (pMt), and the hygromycin selection gene, in view of establishing parameters for optimized gene expression. A protocol of transfection was worked out, leading after hygromycin selection, to ∼90% of S2MtEGFP fluorescent cells at day 5 after copper sulfate (CuSO₄) induction. As analyzed by confocal microscopy, S2MtEGFP cell cultures were shown to be quite heterogeneous regarding the intensity and cell localization of fluorescence among the EGFP expressing cells. Spectrofluorimetry kinetic studies of CuSO₄ induced S2MtEGFP cells showed the EGFP expression at 510 nm as soon as 5 h after induction, the fluorescence increasing progressively from this time to attain values of 4.6 × 10⁵ counts/s after 72 h of induction. Induction with 700 μM of CuSO₄ performed at the exponential phase of the S2MtEGFP culture (10⁶ cells/mL) led to a better performance in terms of cell growth, percent of fluorescent cells and culture intensity of fluorescence. Sodium butyrate (NaBu) treatment of CuSO₄ induced S2MtEGFP cell cultures, although leading to a loss of cell culture viability, increased the percent of EGFP expressing cells and sharply enhanced the cell culture fluorescence intensity. The present study established parameters for improving heterologous protein expression in stably transfected Drosophila S2 cells, as assessed by the EGFP expression.     

SEAP expression in transiently transfected mammalian cells grown in serum-free suspension culture

A transient transfection process was established using a novel 'in-house' developed transfection reagent, Ro-1539. It allows rapid production of large quantities of various recombinant proteins. Here we describe the transient expression of the secreted human placental alkaline phosphatase (SEAP) by HEK293EBNA and CHO cells in serum-free suspension culture. Unexpectedly, high expression levels of SEAP (150 μg/ml) were found 3–4 days post-transfection when placental alkaline phosphatase (AP) was used as the reference enzyme. To confirm these data, an SDS–PAGE analysis was performed and the visible SEAP protein band (MW of 65 kDa) was compared with co-migrated purified placental AP protein as reference. The scanning analysis of the gel showed that SEAP, a truncated form of AP, has a higher specific activity than the purified placental AP. A correction factor was introduced permitting a direct comparison of placental AP activity with the expression levels of SEAP. Scale-up of the transfection system from spinner flask to bioreactor was simple and straightforward, resulting in similar yields of SEAP. Finally, the effectiveness of Ro-1539 was compared to that of other transfection reagents. This revised version was published online in July 2006 with corrections to the Cover Date.