Large-scale transient transfection of serum-free suspension-growing HEK293 EBNA1 cells: peptone additives improve cell growth and transfection efficiency

Large-scale transient transfection of mammalian cells is a recent and powerful technology for the fast production of milligram amounts of recombinant proteins (r-proteins). As many r-proteins used for therapeutic and structural studies are naturally secreted or engineered to be secreted, a cost-effective serum-free culture medium that allows their efficient expression and purification is required. In an attempt to design such a serum-free medium, the effect of nine protein hydrolysates on cell proliferation, transfection efficiency, and volumetric productivity was evaluated using green fluorescent protein (GFP) and human placental secreted alkaline phosphate (SEAP) as reporter genes. The suspension growing, serum-free adapted HEK293SF-3F6 cell line was stably transfected with an EBNA1-expression vector to increase protein expression when using EBV oriP bearing plasmids. Compared to our standard serum-free medium, concomitant addition of the gelatin peptone N3 and removal of BSA slightly enhanced transfection efficiency and significantly increased volumetric productivity fourfold. Using the optimized medium formulation, transfection efficiencies between 40-60% were routinely obtained and SEAP production reached 18 mg/L(-1). To date, we have successfully produced and purified over fifteen r-proteins from 1-14-L bioreactors using this serum-free system. As examples, we describe the scale-up of two secreted his-tagged r-proteins Tie-2 and Neuropilin-1 extracellular domains (ED) in bioreactors. Each protein was successfully purified to >95% purity following a single immobilized metal affinity chromatography (IMAC) step. In contrast, purification of Tie-2 and Neuropilin-1 produced in serum-containing medium was much less efficient. Thus, the use of our new serum-free EBNA1 cell line with peptone-enriched serum-free medium significantly improves protein expression compared to peptone-less medium, and significantly increases their purification efficiency compared to serum-containing medium. This eliminates labor-intensive and expensive chromatographic steps, and allows for the simple, reliable, and extremely fast production of milligram amounts of r-proteins within 5 days posttransfection.     

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.     

Fortification of a protein-free cell culture medium with plant peptones improves cultivation and productivity of an interferon-γ-producing CHO cell line

A strong tendency is currently emerging to remove not only serum but also any product of animal origin from animal cell culture media during production of recombinant proteins. This should facilitate downstream processing and improve biosafety. One way consists in the fortification of protein-free nutritive media with plant protein hydrolysates. To investigate the effects of plant peptones on mammalian cell cultivation and productivity, CHO 320 cells, a clone of CHO K1 cells genetically modified to secrete human interferon-gamma (IFN-gamma), were first adapted to cultivation in suspension in a protein-free medium. Both cell growth and IFN-gamma secretion were found to be equivalent to those reached in serum-containing medium. Eight plant peptones, selected on the basis of their content in free amino acids and oligopeptides, as well as molecular weight distribution of oligopeptides, were tested for their ability to improve culture parameters. These were improved in the presence of three peptones, all having an important fraction of oligopeptides ranging from 1 to 10 kDa and a small proportion of peptides higher than 10 kDa. These peptones do not seem to add significantly to the nutritive potential to basal protein-free nutritive medium. Nevertheless, supplementation of an oligopeptide-enriched wheat peptone improved cell growth by up to 30% and IFN-gamma production by up to 60% in shake-flask experiments. These results suggest that the use of plant peptones with potential growth factor-like or antiapoptotic bioactivities could improve mammalian cell cultivation in protein-free media while increasing the product biosafety.     

Transfected insect cells in suspension culture rapidly yield moderate quantities of recombinant proteins in protein-free culture medium

Methodology to rapidly express milligram quantities of recombinant proteins through the Lipofectin-mediated transfection of insect cells in small-scale, protein-free suspension culture is presented. The transfection phase in suspension culture was first optimized using the green fluorescence protein coupled with FACs analysis to examine the effect of variables such as the transfection media, duration, and cell density on transfection efficiency and expression level. The recombinant protein production phase was optimized using secreted alkaline phosphatase (SEAP) as a reporter protein to evaluate the cell seeding density and harvest time. Using this method, 5 secreted, 2 intracellular, and 1 chimeric protein were expressed at levels ranging from 6 to 50 mg/L. Furthermore, the ability to purify over 2 mg of His(6)-tagged SEAP by immobilized metal affinity chromatography from 50 mL insect cell culture medium to greater than 95% purity was also demonstrated. This method is suitable for scale-up and high-throughput applications.     

Transient Transfection Factors for High-Level Recombinant Protein Production in Suspension Cultured Mammalian Cells

The efficient transfection of cloned genes into mammalian cells system plays a critical role in the production of large quantities of recombinant proteins (r-proteins). In order to establish a simple and scaleable transient protein production system, we have used a cationic lipid-based transfection reagent-FreeStyle MAX to study transient transfection in serum-free suspension human embryonic kidney (HEK) 293 and Chinese hamster ovary (CHO) cells. We used quantification of green fluorescent protein (GFP) to monitor transfection efficiency and expression of a cloned human IgG antibody to monitor r-protein production. Parameters including transfection reagent concentration, DNA concentration, the time of complex formation, and the cell density at the time of transfection were analyzed and optimized. About 70% GFP-positive cells and 50-80 mg/l of secreted IgG antibody were obtained in both HEK-293 and CHO cells under optimal conditions. Scale-up of the transfection system to 1 l resulted in similar transfection efficiency and protein production. In addition, we evaluated production of therapeutic proteins such as human erythropoietin and human blood coagulation factor IX in both HEK-293 and CHO cells. Our results showed that the higher quantity of protein production was obtained by using optimal transient transfection conditions in serum-free adapted suspension mammalian cells.     

Cell growth arrest by nucleotides,nucleosides and bases as a tool for improved production of recombinant proteins

Arresting cell growth and thus decreasing cell division potentially lessens the chance for genetic drift in the cell population; this would be of utmost importance for the consistent production of biopharmaceuticals during long periods. The drawback of the addition of well-known synchronizing agents, such as chemotherapeutics, is that they cause a disproportionate accumulation of cellular constituents, leading to cell death. The use of compounds that are naturally synthesized by the cell, as is the case of nucleotides, nucleosides, and bases (Nt/Ns/B), is shown in this work to be a promising tool. The addition of purines and pyrimidines was tested using a CHO cell line producing the secreted form of the human placental alkaline phosphatase enzyme (SEAP). From the chemical alternatives tested, AMP was the most promising compound for protein production improvement; it reduced cell growth and maintained the culture with high cell viability for long periods, while increasing SEAP specific productivity 3-fold. The use of CHO and BHK mammalian cells producing Factor VII and the use of a insect cell line (Sf9) showed that the effect of AMP addition seems to be independent of the r-protein and cell line. With the addition of AMP, accumulation of cells at the S phase was accompanied by an increase of the protein specific productivity. Addition of known synchronizing drugs (aphidicolin and doxorubicin) and application of environmental cell growth arrest strategies (depletion of nutrients and byproduct accumulation) showed also to effectively arrest CHO cell growth. A careful look onto cell cycle distribution in the different scenarios created, shows whether it is important to consider r-protein expression dependency upon cell cycle in process optimization and operation strategies.     

Comparative recombinant protein production of eight insect cell lines

Summary A recombinantAutographa californica baculovirus expressing secreted alkaline phosphatase (SEAP) gene was used to evaluate the expression of a secreted glycoprotein in eight insect cell lines derived fromSpodoptera frugiperda, Trichoplusia ni, Mamestra brassicae andEstigmene acrea. Because cell density was found to influence protein production, SEAP production was evaluated at optimal cell densities for each cell line on both a per cell and per milliliter basis. On a per cell basis, theT. ni-derived BTI-TN-5B1-4 cells produced a minimum of 20-fold more SEAP than theS. frugiperda-derived Sf9 or Sf21 cell lines and a minimum of 9-fold more than any of the other cell lines growing in serum-containing medium. On a per milliliter basis, BTI-TN-5B1-4 cells produced a minimum of fivefold more SEAP than any of the other cell lines tested. Using cell lines that were adapted to serum-free medium, SEAP yields were the same or better than their counterparts in serum-containing medium. At 3 days postinoculation, extracellular SEAP activity ranged from 59 to 85% of total SEAP activity with cell lines grown in serum-free and serum-containing media.     

The use of peptones as medium additives for the production of a recombinant therapeutic protein in high density perfusion cultures of mammalian cells

Protein hydrolysates as substitutes for serum havebeen employed by many in cell culture mediumformulation, especially with the shift to low proteinor protein-free media. More recently, vegetablehydrolysates have also been added as nutritionalsupplements to fortify the amino acid content in smallpeptide form for batch and fed-batch fermentations. Several of these new hydrolysates (peptones of soy,rice, wheat gluten etc.) were tested as protein-freemedium supplements for the production of a recombinanttherapeutic protein. Multiple peptone-supplemented,continuous perfusion bioreactor experiments wereconducted, varying dilution rates and basal mediumcomposition over the various runs. Cell specificrates and product quality studies were obtained forthe various peptones and compared with peptone-freemedium. The potential for peptones to decreaseintrinsic and proteolytic degradation of the productwas also investigated.It was found that peptones confer a nutritionalbenefit, especially at low dilution rates, for therecombinant BHK cell line used in this investigation.The specific productivity increased 20–30% comparedto the peptone-free controls. However, this benefitwas also fully delivered by using fortified medium inplace of the peptone-enriched media. Therefore, whilepeptones may be considered as useful medium additiveswhen development time is limited, their addition maybe avoided by systematic medium development ifpermitted by the time line of the project.     

High cell density fed batch and perfusion processes for stable non-viral expression of secreted alkaline phosphatase (SEAP) using insect cells: comparison to a batch Sf-9-BEV system

The development of insect cells expressing recombinant proteins in a stable continuous manner is an attractive alternative to the BEV system for recombinant protein production. High cell density fed batch and continuous perfusion processes can be designed to maximize the productivity of stably transformed cells. A cell line (Sf-9SEAP) expressing high levels of the reporter protein SEAP stably was obtained by lipid-mediated transfection of Sf-9 insect cells and further selection and screening. The expression of the Sf-9SEAP cells was compared with the BEVS system. It was observed that, the yield obtained in BEVS was similar to the batch Sf-9SEAP at 8 and 7 IU/mL, respectively. The productivity of this foreign gene product with the stable cells was enhanced by bioprocess intensification employing the fed-batch and perfusion modes of culture to increase the cell density in culture. The fed batch process yielded a maximum cell density of 28 x 10(6) cells/mL and 12 IU/mL of SEAP. Further improvements in the productivity could be made using the perfusion process, which demonstrated a stable production rate for extended periods of time. The process was maintained for 43 days, with a steady-state cell density of 17-20 x 10(6) cells/mL and 7 IU/mL SEAP. The total yield obtained in the perfusion process (394 IU) was approximately 22 and 8 times higher than that obtained in a batch (17.6 IU) and fed batch (46.1 IU) process, respectively.     

Combined approach of NMR and chemometrics for screening peptones used in the cell culture medium for the production of a recombinant therapeutic protein

Soy peptones or soy hydrolysates are widely used as key medium additives in serum-free cell culture processes for industrial production of therapeutic recombinant proteins. The heterogeneous nature of these vegetable-derived materials can lead to substantial lot- to-lot variability in cell culture processes. In this study, we demonstrated the feasibility of nuclear magnetic resonance (NMR) spectroscopy in combination with chemometrics in rapid screening peptone lots in order to optimize efficiency and consistency of large-scale protein production. This report is the first that shows a correlation between the intrinsic NMR spectral characteristics of complex heterogeneous materials and product titer using chemometrics.     

Extracellular purine and pyrimidine catabolism in cell culture

The presence of purines and pyrimidines bases, nucleosides, and nucleotides in the culture medium has shown to differently affect the growth of a Chinese hamster ovary (CHO) cell line producing the secreted form of the human placental alkaline phosphatase enzyme (SEAP; Carvalhal et al., Biotech Prog. 2003;19:69-83). CHO, BHK, as well as Sf9 cell growth was clearly reduced in the presence of purines but was not affected by pyrimidines at the concentrations tested. The knowledge about the mechanisms by which nucleotides exert their effect when present outside the cells remains very incomplete. The catabolism of both extracellular purines and pyrimidines was followed during the culture of CHO cells. Purines/pyrimidines nucleotides added at a concentration of 1 mM to the culture medium decreased to negligible concentrations in the first 2 days. Purine and pyrimidine catabolism originated only purinic and pyrimidic end-products, respectively. The comparison between AMP catabolism in serum-free cultures (CHO cells expressing Factor VII and Sf9 cells) and in cultures containing serum (CHO cells expressing SEAP and BHK cells expressing Factor VII) showed that AMP extracellular catabolism is mediated by both cells and enzymes present in the serum. This work shows that the quantification of purines and pyrimidines in the culture medium is essential in animal cell culture optimization. When using AMP addition as a chemical cell growth strategy for recombinant protein production improvement, AMP extracellular concentration monitoring allows the optimization of the multiple AMP addition strategy for a prolonged cell culture duration with high specific productivity.     

Continuous culture of CHO-K1 cells producing thrombomodulin and estimation of culture conditions

Recombinant Chinese hamster ovary (CHO-K1) cells expressing human soluble thrombomodulin (rsTM) were cultured in a continuous culture system with a fluidized-bed reactor. Cells were grown in a medium containing 1% serum for 10 d, and then cultured in a serum-free medium. The protein production rate increased remarkably in the serum-free culture, with a decrease in the lactate production rate. This suggests that CHO-K1 cells exhibit different physiological characteristics in response to serum removal from the medium, which resulted in a higher rsTM concentration (about 60 mg/l). A procedure for estimating protein productivity was developed using experimental glucose and lactate measurements. In this procedure, cell density was estimated from the glucose consumption rate, and the specific protein (rsTM) production rate was obtained from the ratio of lactate production/glucose consumption (ΔL/ΔG). Since the cell density and protein productivity in repeated batch culture were well estimated, the procedure was applied to continuous culture in a fluidized-bed bioreactor culture. The estimation procedure was also found to be effective in this continuous culture using the models derived from the repeated batch culture.     

Cell growth stimulating effect of <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> spores and their potential application for Chinese hamster ovary K1 cell cultivation

In this work, water-soluble extracts of Ganoderma lucidum spores (Gls), a Chinese medicinal herb that possesses cell growth stimulating function, were found to be an effective growth factor for Chinese hamster ovary (CHO) cell cultivation. The Gls extract was prepared and supplemented to CHO K1 cell culture media with various serum levels. Our results obtained from both the static culture and the spinner-flask suspension culture showed that use of small-amount Gls extract effectively promoted cell growth and suppressed cell apoptosis induced by serum deprivation with normal cell cycle maintained in a low-serum medium. The low-serum medium containing 1 % (v/v) fetal bovine serum (FBS) and 0.01 % (w/v) Gls extract showed a comparable performance on both cell growth and fusion protein productivity with the conventional CHO culture medium containing 10 % (v/v) FBS and a commercial serum-free medium. This is the first study of the potential of Gls extracts for use as an alternative cell growth factor and nutrient for CHO cells. The findings have presented a new approach to economic cultivation of CHO cells for therapeutic protein production.     

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 cultivation of anchorage-dependent cells on microcarrier: Effective production of human macrophage colony-stimulating factor

For the purpose of establishing a large scale production process of biologically active substances by cultivation of anchorage-dependent mammalian cells, basic studies were carried out on the following items; establishment of a new cell line and derivation of high productivity; construction of optimal serum-free medium; optimization of cultivation method using microcarrier in serum-free medium; and establishment of purification process. The cell line, TRC-29SF, used in this study was newly established from human renal carcinoma with a function of producing macrophage colony-stimulating factor constitutively. Improvement of M-CSF productivity upon TRC-29SF cell line was performed by M-CSF gene amplification with dhfr-MTX system and by truncation of membrane-binding amino acid sequence by recombinant DNA technique. Two kinds of serum-free media, IPEG-85 and IREG-89, were formulated for the growth of TRC-29SF cell and its transformant, respectively. A new cell-adhesion method which permits homogeneous attachment to microcarrier in short term was developed by equalising the sedimentation velocity between cells and microcarrier by addition of 7% Ficoll into the medium. High cell density perfusion culture of TRC-29SF cells was achieved by microcarrier method using IPEG-85 medium, and final cell density reached over 10⁷ cells/ml. Based on the results obtained, long-term perfusion cultures were performed using Mn10-5 and Mn10-5/R600 cell lines, which were created by M-CSF gene transfection and amplification. We found that the productivity of M-CSF per cell began to decrease from the end of logarithmic growth phase. Long-term cultivation with high productivity was accomplished by perfusing medium containing 2 mM sodium butyrate. Purification process for M1-CSF from the culture supernatant of transformed cell line was also established.     

Serum-free cultivation of anchorage-dependent cells on microcarrier: Effective production of human macrophage colony-stimulating factor

For the purpose of establishing a large scale production process of biologically active substances by cultivation of anchorage-dependent mammalian cells, basic studies were carried out on the following items; establishment of a new cell line and derivation of high productivity; construction of optimal serum-free medium; optimization of cultivation method using microcarrier in serum-free medium; and establishment of purification process. The cell line, TRC-29SF, used in this study was newly established from human renal carcinoma with a function of producing macrophage colony-stimulating factor constitutively. Improvement of M-CSF productivity upon TRC-29SF cell line was performed by M-CSF gene amplification with dhfr-MTX system and by truncation of membrane-binding amino acid sequence by recombinant DNA technique. Two kinds of serum-free media, IPEG-85 and IREG-89, were formulated for the growth of TRC-29SF cell and its transformant, respectively. A new cell-adhesion method which permits homogeneous attachment to microcarrier in short term was developed by equalising the sedimentation velocity between cells and microcarrier by addition of 7% Ficoll into the medium. High cell density perfusion culture of TRC-29SF cells was achieved by microcarrier method using IPEG-85 medium, and final cell density reached over 10⁷ cells/ml. Based on the results obtained, long-term perfusion cultures were performed using Mn10-5 and Mn10-5/R600 cell lines, which were created by M-CSF gene transfection and amplification. We found that the productivity of M-CSF per cell began to decrease from the end of logarithmic growth phase. Long-term cultivation with high productivity was accomplished by perfusing medium containing 2 mM sodium butyrate. Purification process for M1-CSF from the culture supernatant of transformed cell line was also established.     

A functional high‐content miRNA screen identifies miR‐30 family to boost recombinant protein production in CHO cells

The steady improvement of mammalian cell factories for the production of biopharmaceuticals is a key challenge for the biotechnology community. Recently, small regulatory microRNAs (miRNAs) were identified as novel targets for optimizing Chinese hamster ovary (CHO) production cells as they do not add any translational burden to the cell while being capable of regulating entire physiological pathways. The aim of the present study was to elucidate miRNA function in a recombinant CHO‐SEAP cell line by means of a genome‐wide high‐content miRNA screen. This screen revealed that out of the 1, 139 miRNAs examined, 21% of the miRNAs enhanced cell‐specific SEAP productivity mainly resulting in elevated volumetric yields, while cell proliferation was accelerated by 5% of the miRNAs. Conversely, cell death was diminished by 13% (apoptosis) or 4% (necrosis) of all transfected miRNAs. Besides these large number of identified target miRNAs, the outcome of our studies suggest that the entire miR‐30 family substantially improves bioprocess performance of CHO cells. Stable miR‐30 over expressing cells outperformed parental cells by increasing SEAP productivity or maximum cell density of approximately twofold. Our results highlight the application of miRNAs as powerful tools for CHO cell engineering, identified the miR‐30 family as a critical component of cell proliferation, and support the notion that miRNAs are powerful determinants of cell viability.     

Peptone,a low-cost growth-promoting nutrient for intensive animal cell culture

The effect of addition of peptone to serum-free and serum supplemented media for the growth of hybridoma cells in various systems was studied. Supplementation of defined medium with either proteose peptone or meat peptone resulted in significant increases in cell number and specific monoclonal antibody production in batch culture system. Other peptones were either inactive or less effective. In continuous culture, using medium supplemented with new born calf serum, the addition of peptone resulted in 125% and 150% increases in cell and antibody concentrations respectively. Similar increase in cell number (128%) was also obtained in spin-filter perfusion culture when medium was supplemented with peptone. By comparison, the substitution of a defined 1xMEM amino acids mixture resulted in only a 50% increase. At higher perfusion rates the cell number maintained in steady state using peptone supplement could be increased to 1.3×10⁷ cells ml^–1 while the serum concentration was reduced from 5% to 1% at a perfusion rate of 2.5 volumes per day.     

Transient production of recombinant proteins by Chinese hamster ovary cells using polyethyleneimine/DNA complexes in combination with microtubule disrupting anti-mitotic agents

We have developed a simple and robust transient expression system utilizing the 25 kDa branched cationic polymer polyethylenimine (PEI) as a vehicle to deliver plasmid DNA into suspension-adapted Chinese hamster ovary cells synchronized in G2/M phase of the cell cycle by anti-mitotic microtubule disrupting agents. The PEI-mediated transfection process was optimized with respect to PEI nitrogen to DNA phosphate molar ratio and the plasmid DNA mass to cell ratio using a reporter construct encoding firefly luciferase. Optimal production of luciferase was observed at a PEI N to DNA P ratio of 10:1 and 5 mug DNA 10(6) cells(-1). To manipulate transgene expression at mitosis, we arrested cells in G2/M phase of the cell cycle using the microtubule depolymerizing agent nocodazole. Using secreted human alkaline phosphatase (SEAP) and enhanced green fluorescent protein (eGFP) as reporters we showed that continued inclusion of nocodazole in cell culture medium significantly increased both transfection efficiency and reporter protein production. In the presence of nocodazole, greater than 90% of cells were eGFP positive 24 h post-transfection and qSEAP was increased almost fivefold, doubling total SEAP production. Under optimal conditions for PEI-mediated transfection, transient production of a recombinant chimeric IgG4 encoded on a single vector was enhanced twofold by nocodazole, a final yield of approximately 5 microg mL(-1) achieved at an initial viable cell density of 1 x 10(6) cells mL(-1). The glycosylation of the recombinant antibody at Asn297 was not significantly affected by nocodazole during transient production by this method.