Expression of furin-linked Fab fragments against anthrax toxin in a single mammalian expression vector

Human anti-recombinant protective antigen (rPA) Fab genes were previously cloned from single B cells of a donor immunized with anthrax vaccine using fluorescence activated cell sorting with fluorescein labeled rPA and single-cell PCR. The light and heavy chains were sub-cloned individually into mammalian expression vectors pSecTag2B or pEXPR44, respectively, and expressed in the same CHOK1 cells. Alternatively, the same heavy and light chains were linked together, using PCR, with an in-frame sequence coding for a furin cleavage site. This construct was cloned into pSecTag2B and expressed in CHOK1 cells. Once expressed, the individual chains combined in vivo to form a Fab fragment which was purified as a single protein when either method was utilized. The human Fab antibodies produced by this technique were functional when tested in Western blots using the recombinant PA antigen as the target.     

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.     

Higher expression of Fab antibody fragments in a CHO cell line at reduced temperature

A chimeric Fab was expressed in Chinese hamster ovary cells under the control of the CMV promoter in a two-stage production process. Cells were first grown to 90% confluence at 37 degrees C in a proliferation phase, followed by a production phase at either 37 degrees C or 28 degrees C. Medium supplemented with serum and medium free from serum was tested in the production phase at both temperatures. Comparison of Fab expression revealed that reducing the temperature to 28 degrees C resulted in a 14-fold increase in product yield when cells were cultivated in serum-containing medium, and in a 38-fold increase in product yield when serum-free medium was applied.     

Expression and purification strategies for the production of single-chain antibody and T-cell receptor fragments inE. coli

This work describes protocols for the production of single-chain antibody and T-cell receptor fragments inE. coli. A choice of methods is given for the purification of the recombinant fragments that rely on the use of either immunoaffinity or metal chelate affinity chromatography. The TCR fragments may have to be denatured and refolded before the fragments attain their proper conformation.     

Codon engineering for improved antibody expression in mammalian cells

While well established in bacterial hosts, the effect of coding sequence variation on protein expression in mammalian systems is poorly characterized outside of viral proteins or proteins from distant phylogenetic families. The potential impact is substantial given the extensive use of mammalian expression systems in research and manufacturing of protein biotherapeutics. We are studying the effect of codon engineering on expression of recombinant antibodies with an emphasis on developing manufacturing cell lines. CNTO 888, a human mAb specific for human MCP-1, was obtained by antibody phage display in collaboration with MorphoSys AG. The isolated DNA sequence of the antibody was biased towards bacterial codons, reflecting the engineering of the Fab library for phage display expression in Escherichia coli. We compared the expression of CNTO 888 containing the parental V-region sequences with two engineered coding variants. In the native codon exchanged (NCE) variant, the V-region codons were replaced with those used in naturally derived human antibody genes. In the human codon optimized (HCO) variant the V-region codons were those used at the highest frequency based on a human codon usage table. The antibody expression levels from stable transfections in mammalian host cells were measured. The HCO codon variant of CNTO 888 yielded the highest expressing cell lines and the highest average expression for the screened populations. This had a significant positive effect on the process to generate a CNTO 888 production cell line and indicates the potential to improve antibody expression in mammalian expression systems by codon engineering.     

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.     

Advancements in mammalian cell transient gene expression (TGE) technology for accelerated production of biologics

Transient gene expression (TGE) in animal cell cultures has been used for almost 30 years to produce milligrams and grams of recombinant proteins, virus-like particles and viral vectors, mainly for research purposes. The need to increase the amount of product has led to a scale-up of TGE protocols. Moreover, product quality and process reproducibility are also of major importance, especially when TGE is employed for the preparation of clinical lots. This work gives an overview of the different technologies that are available for TGE and how they can be combined, depending on each application. Then, a critical assessment of the challenges of large-scale transient transfection follows, focusing on suspension cell cultures transfected with polyethylenimine (PEI), which is the most widely used methodology for transfection. Finally, emerging opportunities for transient transfection arising from gene therapy, personalized medicine and vaccine development are reviewed.     

Optimizing the transient transfection process of HEK-293 suspension cells for protein production by nucleotide ratio monitoring

Large scale, transient gene expression (TGE) is highly dependent of the physiological status of a cell line. Therefore, intracellular nucleotide pools and ratios were used for identifying and monitoring the optimal status of a suspension cell line used for TGE. The transfection efficiency upon polyethyleneimine (PEI)-mediated transient gene delivery into HEK-293 cells cultured in suspension was investigated to understand the effect of different culture and transfection conditions as well as the significance of the culture age and the quality of the cell line used. Based on two different bicistronic model plasmids expressing the human erythropoietin gene (rHuEPO) in the first position and green fluorescent protein as reporter gene in the second position and vice versa, a completely serum-free transient transfection process was established. The process makes use of a 1:1 mixture of a special calcium-free DMEM and the FreeStyle™ 293 Expression Medium. Maximum transfectability was achieved by adjusting the ratio for complex formation to one mass part of DNA and three parts of PEI corresponding to an N/P (nitrogen residues/DNA phosphates) ratio of 23 representing a minimum amount of DNA for the polycation-mediated gene delivery. Applying this method, maximum transfectabilities between 70 and 96 % and a rHuEPO concentration of 1.6 μg mL⁻¹ 72 h post transfection were reached, when rHuEPO gene was expressed from the first position of the bicistronic mRNA. This corresponded to 10 % of the total protein concentration in the cell-free supernatant of the cultures in protein-free medium. Up to 30 % higher transfectabilities were found for cells of early passages compared to those from late passages under protein-free culture conditions. In contrast, when the same cells were propagated in serum-containing medium, higher transfectabilities were found for late-passage cells, while up to 40 % lower transfectabilities were observed for early-passage cells. Nucleotide pools were measured during all cell cultivations and the nucleoside triphosphate/uridine ratios were calculated. These ‘nucleotide ratios’ changed in an age-dependent manner and could be used to distinguish early- from late-passage cells. The observed effects were also dependent on the presence of serum in the culture. Nucleotide ratios were shown being applied to investigate the optimal passage number of cultured cell lines for achieving a maximum productivity in cultures used for transient gene expression. Furthermore, these nucleotide ratios proved to be different for transfected and untransfected cells, providing a high potential tool to monitor the status of transfection under various culture conditions.     

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.     

Addressing a whole bioprocess in real-time using an optical biosensor-formation,recovery and purification of antibody fragments from a recombinant<Emphasis Type="Italic"> E. coli</Emphasis> host

The use of biosensor technology is described to address in real-time the production and subsequent purification of a bioactive recombinant protein product. The product, D1.3 Fv antibody fragment, was expressed in Escherichia coli and purified via two process routes, one for extracellular and one for intracellular product material. The cells were harvested by centrifugation in a solid bowl CARR Powerfuge and stored at –70°C. Clarification of the supernatant was performed by depth filtration, followed by affinity chromatography for final purification of the extracellular product. To purify the intracellular product the harvested cells were resuspended and homogenised. Removal of debris in the CARR Powerfuge was followed by depth filtration and affinity chromatography. In this work we have shown the rapid determination of bioactive product levels, and the impact this has on improved accountability and confidence is demonstrated in process mass balances on the product using the data acquired during process operation.     

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.     

Establishment of a signal peptide with cross-species compatibility for functional antibody expression in both Escherichia coli and Chinese hamster ovary cells

Signal peptides are short peptides located at the N-terminus of secreted proteins. They characteristically have three domains; a basic region at the N-terminus (n-region), a central hydrophobic core (h-region) and a carboxy-terminal cleavage region (c-region). Although hundreds of different signal peptides have been identified, it has not been completely understood how their features enable signal peptides to influence protein expression. Antibody-derived signal peptides are often used to prepare recombinant antibodies expressed by eukaryotic cells, especially Chinese hamster ovary (CHO) cells. However, when prokaryotic Escherichia coli (E. coli) are utilized in drug discovery processes, such as for phage display selection or antibody humanization, signal peptides have been selected separately due to the differences in the expression systems between the species. In this study, we successfully established a signal peptide that enables a functional antibody to be expressed in both prokaryotic and eukaryotic cells by focusing on the importance of having an Ala residue in the c-region of the signal sequence. We found that changing Ser to Ala at only two positions significantly augmented the anti-HER2 antigen binding fragment (Fab) expression in E. coli. In addition, this altered signal peptide also retained the ability to express functional anti-HER2 antibody in CHO cells. Taken together, the present findings indicate that the signal peptide can promote functional antibody expression in both prokaryotic E. coli and eukaryotic CHO cells. This finding will contribute to the understanding of signal peptides and accelerate therapeutic antibody research.     

Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture

Transient expression of recombinant proteins in mammalian cell culture in a 100-L scale requires a large quantity of plasmid that is very labour intensive to achieve with shake flask cultures and commercially available plasmid purification kits. In this paper we describe a process for plasmid production in 100-mg scale. The fermentation is carried out in a 4-L fed-batch culture with a minimal medium. The detection of the end of batch and triggering the exponential (0.1 h(-1)) feed profile was unattended and controlled by Multi-fermenter Control System. A restricted specific growth rate in fed-batch culture increased the specific plasmid yield compared to batch cultures with minimal and rich media. This together with high biomass concentration (68-107 g L(-1) wet weight) achieves high volumetric yields of plasmid (95-277 mg L(-1) depending on the construct). The purification process consisted of alkaline lysis, lysate clarification and ultrafiltration, two-phase extraction with Triton X-114 for endotoxin removal, anion-exchange chromatography as a polishing step, ultrafiltration and sterile filtration. Both fermentation and purification processes were used without optimisation for production of four plasmids yielding from 39 to 163 mg of plasmids with endotoxin content of 2.5 EU mg(-1) or less.     

A pipeline for the production of antibody fragments for structural studies using transient expression in HEK 293T cells

We describe a pipeline for the rapid production of recombinant Fabs derived from mouse monoclonal antibodies suitable for use in structural studies. The pipeline is exemplified by the production of three Fabs derived from the monoclonal antibodies OX108 (anti-CD200 receptor), OX117 and OX119 (anti-SIRPgamma). Heavy and light chain variable domains were inserted into separate expression vectors containing resident constant regions using In-Fusion PCR cloning. Following transient co-expression in HEK 293T cells, secreted Fab fragments were purified by metal chelate chromatography and gel filtration using an automated procedure with yields of up to 4mg/L of cell culture. Following crystallization trials, diffracting crystals were obtained for the recombinant Fabs of OX108 and OX117, and their structures solved to 2.3A and 2.4A, respectively.     

Plastid targeting and transient expression of rat liver ATP: citrate lyase in pea protoplasts

Protoplasts isolated from pea leaves (Pisum sativum L. cv. Hurst Greenshaft) were electroporated in the presence of plasmid pDR#1, which contains the rat liver ATP:citrate lyase gene fused to a duplex 35S cauliflower mosaic virus promoter with a transit peptide sequence of the Rubisco small subunit. The level of enzyme expression and viability of protoplasts were both influenced by polyethylene glycol treatment before electroporation. Under the optimised electroporation conditions, an average increase of ATP:citrate lyase activity of 14% was observed in the transfected cells after 24 h, with a similar magnitude of change in the abundance of the corresponding mRNA. Immunoblot analysis confirmed the correct expression and targeting of ATP:citrate lyase protein in the chloroplasts of pea protoplasts. These results provide a basis for the establishment of a procedure for targeting heterologous protein into pea plastids in the presence of a transit peptide. Received: 14 June 1996 / Revision received: 24 November 1996 / Accepted: 4 January 1997     

Efficient production of bispecific IgG of different isotypes and species of origin in single mammalian cells

Bispecific IgG production in single host cells has been a much sought-after goal to support the clinical development of these complex molecules. Current routes to single cell production of bispecific IgG include engineering heavy chains for heterodimerization and redesign of Fab arms for selective pairing of cognate heavy and light chains. Here, we describe novel designs to facilitate selective Fab arm assembly in conjunction with previously described knobs-into-holes mutations for preferential heavy chain heterodimerization. The top Fab designs for selective pairing, namely variants v10 and v11, support near quantitative assembly of bispecific IgG in single cells for multiple different antibody pairs as judged by high-resolution mass spectrometry. Single-cell and in vitro-assembled bispecific IgG have comparable physical, in vitro biological and in vivo pharmacokinetics properties. Efficient single-cell production of bispecific IgG was demonstrated for human IgG₁, IgG₂ and IgG₄ thereby allowing the heavy chain isotype to be tailored for specific therapeutic applications. Additionally, a reverse chimeric bispecific IgG_2a with humanized variable domains and mouse constant domains was generated for preclinical proof-of-concept studies in mice. Efficient production of a bispecific IgG in stably transfected mammalian (CHO) cells was shown. Individual clones with stable titer and bispecific IgG composition for >120 days were readily identified. Such long-term cell line stability is needed for commercial manufacture of bispecific IgG. The single-cell bispecific IgG designs developed here may be broadly applicable to biotechnology research, including screening bispecific IgG panels, and to support clinical development.     

Functional expression and display of an antibody Fab fragment in Escherichia coli: study of vector designs and culture conditions

Several different vector designs are currently being used to display and express Fab molecules in Escherichia coli, but their relative efficiency in phage display and protein expression cannot be compared from the published data. We systematically investigated which vector design most effectively displays and expresses Fab molecules in E. coli using, as a model system, a human Fab against tetanus toxoid (tt). Three different vectors were used in this study: pFab1 where the VL-CL and VH-CH1 genes were driven by two promoters in two separate expression cassettes, and pFab2 and pFab3 that both contain one dicistronic expression cassette with two translation initiation sites and either VH-CH1 before VL-CL or VL-CL before VH-CH1, respectively. The display of tt-Fab on the surface of phage and the expression of tt-Fab protein in E. coli were compared for the aforementioned vectors. Our results showed that the pFab3 vector was most effective in Fab display. A 10-fold increase in the expression of secreted Fab was observed in pFab3 when compared with vectors pFab1 and pFab2. Further experiments were conducted using pFab3 to optimize expression levels using different strains of E. coli and various culture conditions. The highest expression of tt-Fab was obtained using the pFab3 vector in host strain JM105 with an induction temperature at 37 degrees C and IPTG concentration of 0.1 mM.     

Construction of a dual-tag system for gene expression, protein affinity purification and fusion protein processing

An E. coli vector system was constructed which allows the expression of fusion genes via a l-rhamnose-inducible promotor. The corresponding fusion proteins consist of the maltose-binding protein and a His-tag sequence for affinity purification, the Saccharomyces cerevisiae Smt3 protein for protein processing by proteolytic cleavage and the protein of interest. The Smt3 gene was codon-optimized for expression in E. coli. In a second rhamnose-inducible vector, the S. cerevisiae Ulp1 protease gene for processing Smt3 fusion proteins was fused in the same way to maltose-binding protein and His-tag sequence but without the Smt3 gene. The enhanced green fluorescent protein (eGFP) was used as reporter and protein of interest. Both fusion proteins (MalE-6xHis-Smt3-eGFP and MalE-6xHis-Ulp1) were efficiently produced in E. coli and separately purified by amylose resin. After proteolytic cleavage the products were applied to a Ni-NTA column to remove protease and tags. Pure eGFP protein was obtained in the flow-through of the column in a yield of around 35% of the crude cell extract.     

Codon optimization of bacterial luciferase (lux) for expression in mammalian cells

Expression of the bacterial luciferase (lux) system in mammalian cells would culminate in a new generation of bioreporters for in vivo monitoring and diagnostics technology. Past efforts to express bacterial luciferase in mammalian cells have resulted in only modest gains due in part to low overall expression of the bacterial genes. To optimize expression, we have designed and synthesized codon-optimized versions of the luxA and luxB genes from Photorhabdus luminsecens. To evaluate these genes in vivo, stable HEK293 cell lines were created harboring wild type luxA and luxB (WTA/WTB), codon-optimized luxA and wild type luxB (COA/WTB), and codon-optimized versions of both luxA and luxB genes (COA/COB). Although mRNA levels within these clones remained approximately equal, LuxA protein levels increased significantly after codon optimization. On average, bioluminescence levels were increased by more than six-fold [5×10⁵ vs 2.9×10⁶ relative light units (RLU)/mg total protein] with the codon-optimized luxA and wild type luxB. Bioluminescence was further enhanced upon expression of both optimized genes (2.7×10⁷ RLU/mg total protein). These results show promise toward the potential development of an autonomous light generating lux reporter system in mammalian cells