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.     

High‐level protein expression in scalable CHO transient transfection

Chinese hamster ovary cells (CHO) have been extensively utilized as the production platform for therapeutic proteins including monoclonal antibodies in pharmaceutical industry. For early development, it would be advantageous to rapidly produce large amounts of protein in the same cell line; therefore, development of a CHO transient transfection platform with high protein expression level is highly desirable. Here, we describe the development of such a platform in CHO cells. Polyethylenimine (PEI) was used as the transfection reagent. Different media were screened for the best transfection and expression performance, and UltraCHO was chosen as the best performer. DMSO and lithium acetate (LiAc) were discovered to improve CHO transient transfection expression levels significantly. A 14‐day fed‐batch process was successfully developed to further increase production yield. With an optimized transient transfection process, we were able to express monoclonal antibody (Mab) in CHO cells at a high level, averaging 80 mg/L. The process was successfully scaled up to 10 L working volume in a 20 L wave bioreactor. As expected, the Mabs had similar glycosylation patterns in comparison to the Mabs produced from a stably transfected CHO cell line, while in contrast Mabs expressed transiently from HEK293EBNA cells differed. Biotechnol. Bioeng. 2009;103: 542–551. © 2009 Wiley Periodicals, Inc.     

Large-scale Transient Transfection of Mammalian Cells: A Newly Emerging Attractive Option for Recombinant Protein Production

Mammalian expression systems have an undisputed long-standing and very successful history for the generation of recombinant proteins, mainly as biopharmaceuticals. However, for use as ‘tool proteins’ in, e.g. assay development and screening, for structure elucidation and as antigens these expression systems were generally regarded as being cumbersome, tedious and expensive. This bias has largely been overcome with the very recent development of large-scale transient transfection (LST) approaches. Especially the HEK.EBNA expression system described here has contributed significantly to this success. The simplicity and speed of this approach compares well with expression trials using the widely applied Baculovirus/insect cell system. In addition, proteins generated in mammalian cells are usually correctly folded, fully processed and functionally active.     

Endoplasmic reticulum‐directed recombinant mRNA displays subcellular localization equal to endogenous mRNA during transient expression in CHO cells

When expressing pharmaceutical recombinant proteins in mammalian cells, the protein is commonly directed through the secretory pathway, in a signal peptide‐dependent manner, to acquire specific post‐translational modifications and to facilitate secretion into the culture medium. One key premise for this is the direction of the mRNA encoding the recombinant protein to the surface of the endoplasmic reticulum (ER) for subsequent protein translocation into the secretory pathway. To evaluate the efficiency of this process in Chinese hamster ovary (CHO) cells, the subcellular localization of recombinant mRNA encoding the therapeutic proteins, erythropoietin (EPO) and Rituximab, was determined. The results show that ER‐directed recombinant mRNAs exhibited an efficient recruitment to the ER when compared to an endogenous ER‐directed mRNA, with no cytoplasmic translation of ER‐directed recombinant proteins observed. These observations indicate that the recombinant mRNA, encoding ER‐directed proteins, follows the same distribution pattern as endogenous mRNA directed towards the ER. Furthermore, the previous established fractionation method proves to be an efficient tool to study not only recombinant mRNA localization, but also recombinant protein trafficking between the ER and cytosol in CHO cells.     

Sde2 is an intron‐specific pre‐mRNA splicing regulator activated by ubiquitin‐like processing

The expression of intron‐containing genes in eukaryotes requires generation of protein‐coding messenger RNAs (mRNAs) via RNA splicing, whereby the spliceosome removes non‐coding introns from pre‐mRNAs and joins exons. Spliceosomes must ensure accurate removal of highly diverse introns. We show that Sde2 is a ubiquitin‐fold‐containing splicing regulator that supports splicing of selected pre‐mRNAs in an intron‐specific manner in Schizosaccharomyces pombe. Both fission yeast and human Sde2 are translated as inactive precursor proteins harbouring the ubiquitin‐fold domain linked through an invariant GGKGG motif to a C‐terminal domain (referred to as Sde2‐C). Precursor processing after the first di‐glycine motif by the ubiquitin‐specific proteases Ubp5 and Ubp15 generates a short‐lived activated Sde2‐C fragment with an N‐terminal lysine residue, which subsequently gets incorporated into spliceosomes. Absence of Sde2 or defects in Sde2 activation both result in inefficient excision of selected introns from a subset of pre‐mRNAs. Sde2 facilitates spliceosomal association of Cactin/Cay1, with a functional link between Sde2 and Cactin further supported by genetic interactions and pre‐mRNA splicing assays. These findings suggest that ubiquitin‐like processing of Sde2 into a short‐lived activated form may function as a checkpoint to ensure proper splicing of certain pre‐mRNAs in fission yeast.     

Model‐based investigation of intracellular processes determining antibody Fc‐glycosylation under mild hypothermia

Despite the positive effects of mild hypothermic conditions on monoclonal antibody (mAb) productivity (q_mAb) during mammalian cell culture, the impact of reduced culture temperature on mAb Fc‐glycosylation and the mechanism behind changes in the glycan composition are not fully established. The lack of knowledge about the regulation of dynamic intracellular processes under mild hypothermia restricts bioprocess optimization. To address this issue, a mathematical model that quantitatively describes Chinese hamster ovary (CHO) cell behavior and metabolism, mAb synthesis and mAb N‐linked glycosylation profile before and after the induction of mild hypothermia is constructed. Results from this study show that the model is capable of representing experimental results well in all of the aspects mentioned above, including the N‐linked glycosylation profile of mAb produced under mild hypothermia. Most importantly, comparison between model simulation results for different culture temperatures suggests the reduced rates of nucleotide sugar donor production and galactosyltransferase (GalT) expression to be critical contributing factors that determine the variation in Fc‐glycan profiles between physiological and mild hypothermic conditions in stable CHO transfectants. This is then confirmed using experimental measurements of GalT expression levels, thereby closing the loop between the experimental and the computational system. The identification of bottlenecks within CHO cell metabolism under mild hypothermic conditions will aid bioprocess optimization, for example, by tailoring feeding strategies to improve NSD production, or manipulating the expression of specific glycosyltransferases through cell line engineering. Biotechnol. Bioeng. 2017;114: 1570–1582. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals Inc.     

Identification of cell culture conditions to control N‐glycosylation site‐occupancy of recombinant glycoproteins expressed in CHO cells

The effect of different cell culture conditions on N‐glycosylation site‐occupancy has been elucidated for two different recombinant glycoproteins expressed in Chinese hamster ovary (CHO) cells, recombinant human tissue plasminogen activator (t‐PA) and a recombinant enzyme (glycoprotein 2—GP2). Both molecules contain a N‐glycosylation site that is variably occupied. Different environmental factors that affect the site‐occupancy (the degree of occupied sites) of these molecules were identified. Supplementing the culture medium with additional manganese or iron increased the fraction of fully occupied t‐PA (type I t‐PA) by approximately 2.5–4%. Decreasing the cultivation temperature from 37 to 33°C or 31°C gradually increased site‐occupancy of t‐PA up to 4%. The addition of a specific productivity enhancer, butyrate, further increased site‐occupancy by an additional 1% under each cultivation temperature tested. In addition, the thyroid hormones triiodothyronine and thyroxine increased site‐occupancy of t‐PA compared to control conditions by about 2%. In contrast, the addition of relevant nucleoside precursor molecules involved in N‐glycan biosynthesis (e.g., uridine, guanosine, mannose) either had no effect or slightly reduced site‐occupancy. For the recombinant enzyme (GP2), it was discovered that culture pH and the timing of butyrate addition can be used to control N‐glycan site‐occupancy within a specific range. An increase in culture pH correlated with a decrease in site‐occupancy. Similarly, delaying the timing for butyrate addition also decreased site‐occupancy of this molecule. These results highlight the importance of understanding how cell culture conditions and media components can affect the product quality of recombinant glycoproteins expressed in mammalian cell cultures. Furthermore, the identification of relevant factors will enable one to control product quality attributes, specifically N‐glycan site‐occupancy, within a specific range when applied appropriately. Biotechnol. Bioeng. 2009;103: 1164–1175. © 2009 Wiley Periodicals, Inc.     

High yield process for the production of active human α‐galactosidase a in CHO‐K1 cells through lentivirus transgenesis

Fabry disease is an X‐linked recessive disorder caused by a deficiency in lysosomal α‐Galactosidase A. Currently, two enzyme replacement therapies (ERT) are available. However, access to orphan drugs continues to be limited by their high price. Selection of adequate high‐expression systems still constitutes a challenge for alleviating the cost of treatments. Several strategies have been implemented, with varying success, trying to optimize the production process of recombinant human α‐Galactosidase A (rhαGAL) in Chinese hamster ovary (CHO‐K1) cells. Herein, we describe for the first time the application of a strategy based on third‐generation lentiviral particles (LP) transduction of suspension CHO‐K1 cells to obtain high‐producing rhαGAL clones (3.5 to 59.4 pg cell^?1 d^?1). After two purification steps, the active enzyme was recovered (2.4 × 10⁶ U mg^?1) with 98% purity and 60% overall yield. Michaelis‐Menten analysis demonstrated that rhαGAL was capable of hydrolyzing the synthetic substrate 4MU‐α‐Gal at a comparable rate to Fabrazyme®, the current CHO‐derived ERT available for Fabry disease. In addition, rhαGAL presented the same mannose‐6‐phosphate (M6P) content, about 40% higher acid sialic amount and 33% reduced content of the immunogenic type of sialic acid (Neu5Gc) than the corresponding ones for Fabrazyme®. In comparison with other rhαGAL production processes reported to date, our approach achieves the highest rhαGAL productivity preserving adequate activity and glycosylation pattern. Even more, considering the improved glycosylation characteristics of rhαGAL, which might provide advantages regarding pharmacokinetics, our enzyme could be postulated as a promising alternative for therapeutic use in Fabry disease. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1334–1345, 2017     

Metabolic Rates,Growth Phase,and mRNA Levels Influence Cell-Specific Antibody Production Levels from In Vitro-Cultured Mammalian Cells at Sub-Physiological Temperatures

Previous work has shown that recombinant protein yield can be improved from in vitro-cultured mammalian cells by culturing at sub-physiological temperatures, although this effect is cell line and product dependent. The mechanism(s) by which low temperature leads to enhanced product yield are currently unknown; however, recent reports suggest that increased mRNA levels at sub-physiological temperatures may be largely responsible for this. Here, we have investigated whether low-temperature cultivation of cell lines selected for antibody production at 37°C leads to changes in heavy- and light-chain mRNA levels and if this is reflected in antibody yields. Low-temperature in vitro culturing resulted in reduced viable cell concentration, prolonged cell viability, a reduction in metabolite consumption and production, cell cycle arrest in both CHO and NS0 cells, and changes in the levels of heavy- and light-chain mRNA. Despite increases in the level of heavy- and light-chain mRNA upon culturing at 32°C in our model CHO cell line, this did not result in increased total product yield; however, changes in cell-specific yields were observed that reflected the metabolic rate of glucose utilization and changes in mRNA levels.     

Effect of culture temperature on erythropoietin production and glycosylation in a perfusion culture of recombinant CHO cells

To investigate the effect of culture temperature on erythropoietin (EPO) production and glycosylation in recombinant Chinese hamster ovary (CHO) cells, we cultivated CHO cells using a perfusion bioreactor. Cells were cultivated at 37 degrees C until viable cell concentration reached 1 x 10(7) cells/mL, and then culture temperature was shifted to 25 degrees C, 28 degrees C, 30 degrees C, 32 degrees C, 37 degrees C (control), respectively. Lowering culture temperature suppressed cell growth but was beneficial to maintain high cell viability for a longer period. In a control culture at 37 degrees C, cell viability gradually decreased and fell below 80% on day 18 while it remained over 90% throughout the culture at low culture temperature. The cumulative EPO production and specific EPO productivity, q(EPO), increased at low culture temperature and were the highest at 32 degrees C and 30 degrees C, respectively. Interestingly, the cumulative EPO production at culture temperature below 32 degrees C was not as high as the cumulative EPO production at 32 degrees C although the q(EPO) at culture temperature below 32 degrees C was comparable or even higher than the q(EPO) at 32 degrees C. This implies that the beneficial effect of lowering culture temperature below 32 degrees C on q(EPO) is outweighed by its detrimental effect on the integral of viable cells. The glycosylation of EPO was evaluated by isoelectric focusing, normal phase HPLC and anion exchange chromatography analyses. The quality of EPO at 32 degrees C in regard to acidic isoforms, antennary structures and sialylated N-linked glycans was comparable to that at 37 degrees C. However, at culture temperatures below 32 degrees C, the proportions of acidic isoforms, tetra-antennary structures and tetra-sialylated N-linked glycans were further reduced, suggesting that lowering culture temperature below 32 degrees C negatively affect the quality of EPO. Thus, taken together, cell culture at 32 degrees C turned out to be the most satisfactory since it showed the highest cumulative EPO production, and moreover, EPO quality at 32 degrees C was not deteriorated as obtained at 37 degrees C.     

Rapid protein production using CHO stable transfection pools

During early preclinical development of therapeutic proteins, representative materials are often required for process development, such as for pharmacokinetic/pharmacodynamic studies in animals, formulation design, and analytical assay development. To rapidly generate large amounts of representative materials, transient transfection is commonly used. Because of the typical low yields with transient transfection, especially in CHO cells, here we describe an alternative strategy using stable transfection pool technology. Using stable transfection pools, gram quantities of monoclonal antibody (Mab) can be generated within 2 months post‐transfection. Expression levels for monoclonal antibodies can be achieved ranging from 100 mg/L to over 1000 mg/L. This methodology was successfully scaled up to a 200 L scale using disposable bioreactor technology for ease of rapid implementation. When fluorescence‐activated cell sorting was implemented to enrich the transfection pools for high producers, the productivity could be improved by about three‐fold. We also found that an optimal production time window exists to achieve the highest yield because the transfection pools were not stable and productivity generally decreased over length in culture. The introduction of Universal chromatin‐opening elements elements into the expression vectors led to significant productivity improvement. The glycan distribution of the Mab product generated from the stable transfection pools was comparable to that from the clonal stable cell lines. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Aerobic expression of Vitreoscilla hemoglobin improves the growth performance of CHO‐K1 cells

Inefficient carbon metabolism is a relevant issue during the culture of mammalian cells for the production of biopharmaceuticals. Therefore, cell engineering strategies to improve the metabolic and growth performance of cell lines are needed. The expression of Vitreoscilla stercoraria hemoglobin (VHb) has been shown to significantly reduce overflow metabolism and improve the aerobic growth of bacteria. However, the effects of VHb on mammalian cells have been rarely studied. Here, the impact of VHb on growth and lactate accumulation during CHO‐K1 cell culture was investigated. For this purpose, CHO‐K1 cells were transfected with plasmids carrying the vgb or gfp gene to express VHb or green fluorescence protein (GFP), respectively. VHb expression increased the specific growth rate and biomass yields on glucose and glutamine by 60 %, and reduced the amount of lactate produced per cell by 40 %, compared to the GFP‐expression controls. Immunofluorescence microscopy showed that VHb is distributed in the cytoplasm and organelles, which support the hypothesis that VHb could serve as an oxygen carrier, enhancing aerobic respiration. These results are useful for the development of better producing cell lines for industrial applications.     

Evoked Acetylcholine Release Expressed in Neuroblastoma Cells by Transfection of Mediatophore cDNA

Abstract: Transmitter release was elicited in two ways from cultured cells filled with acetylcholine: (a) in a biochemical assay by successive addition of a calcium ionophore and calcium and (b) electrophysiologically, by electrical stimulation of individual cells and real-time recording with an embryonic Xenopus myocyte. Glioma C6-Bu-1 cells were found to be competent for Ca²⁺-dependent and quantal release. In contrast, no release could be elicited from mouse neuroblastoma N18TG-2 cells. However, acetylcholine release could be restored when N18TG-2 cells were transfected with a plasmid coding for mediatophore. Mediatophore is a protein of nerve terminal membranes purified from the Torpedo electric organ on the basis of its acetylcholine-releasing capacity. The transfected N18TG-2 cells expressed Torpedo mediatophore in their plasma membrane. In response to an electrical stimulus, they generated in the myocyte evoked currents that were curare sensitive and calcium dependent and displayed discrete amplitude levels, like in naturally occurring synapses.     

Studies on mRNA Electroporation of Immature and Mature Dendritic Cells: Effects on their Immunogenic Potential

Previous studies have shown that mRNA-electroporated dendritic cells (DCs) are able to process and present tumor-associated antigens, leading to the activation of tumor-specific T cells in vitro and in vivo. However, the optimal maturation state of antigen loading and half-life of the mRNA-translated protein product and its immunogenic epitopes are significant parameters, which needs to be clarified in order to establish an effective electroporation protocol. In addition, despite extensive experimental investigations and their widespread application in research and clinical environments, little is known of the extent to which the immunological properties of DCs are influenced by electrical fields of critical strengths. We found that the mRNA transfection of DCs after maturation with short and low-voltage square-wave electrical pulses resulted in higher level of antigen expression and viability in addition to higher T-cell stimulatory ability compared to transfection of DCs prior to maturation. Mature mRNA-electroporated DCs showed long-lived expression of EGFP and were able to stimulate influenza matrix protein M1 (M1)-specific T cells up to 24 h after electroporation. However, when DCs were subjected to increasing electrical pulses the level of transgene expression was four-fold upregulated, equipping these DCs to be more potent in inducing M1-specific T cells. Also, the application of long electrical pulses induced further upregulation of HLA-DR, CD80, and CD86 expression in mature DCs, but did not promote phenotypic or functional maturation in immature DCs. These findings support the concept of mRNA transfection of DCs after maturation and also highlight the possibility to use long electrical pulses for further improvement of the immune responses by mRNA-transfected DCs.