Semicontinuous bioreactor production of a recombinant human therapeutic protein using a chemically inducible viral amplicon expression system in transgenic plant cell suspension cultures

Plant cell culture is an alternative for the production of recombinant human therapeutic proteins because of improved product safety, lower production cost, and capability for eukaryotic post‐translational modification. In this study, bioreactor production of recombinant human alpha‐1‐antitrypsin (rAAT) glycoprotein using a chemically inducible Cucumber mosaic virus (CMV) viral amplicon expression system in transgenic Nicotiana benthamiana cell culture is presented. Optimization of a chemically inducible plant cell culture requires evaluation of effects of timing of induction (TOI) and concentration of inducer (COI) on protein productivity and protein quality (biological functionality). To determine the optimal TOI, the oxygen uptake rate (OUR) of the plant cell culture was chosen as a physiological indicator for inducing maximum rAAT expression. Effects of COI on rAAT production were investigated using a semicontinuous culture, which enables the distinction between effects of growth rate and effects of inducer concentration. An optimized semicontinuous bioreactor operation was further proposed to maximize the recombinant protein production. The results demonstrated that the transgenic plant cells, transformed with the inducible viral amplicon expression system, maintain higher OUR and exhibit lower extracellular protease activity and lower total phenolics concentration in the optimized semicontinuous bioreactor process than in a traditional batch bioreactor operation, resulting in a 25‐fold increase in extracellular functional rAAT (603 µg/L) and a higher ratio of functional rAAT to total rAAT (85–90%). Surprisingly, sustained rAAT production and steady state, long‐term bioreactor operation is possible following chemical induction and establishment of the viral amplicons. Biotechnol. Bioeng. 2010; 106: 408–421. © 2010 Wiley Periodicals, Inc.     

A chemically inducible cucumber mosaic virus amplicon system for expression of heterologous proteins in plant tissues

A novel cucumber mosaic virus inducible viral amplicon (CMViva) expression system has been developed that allows for tightly regulated chemically inducible expression of heterologous genes in plant hosts. Transient production of recombinant α₁-antitrypsin (rAAT), a human blood protein, was demonstrated in Nicotiana benthamiana leaves. The highest production levels were obtained by co-infiltrating leaves with Agrobacterium tumefaciens cells containing CMViva carrying the AAT gene and A. tumefaciens cells carrying a binary vector constitutively expressing the gene silencing suppressor p19. Accumulation of up to thirty-fold more rAAT was observed in leaves (24 mg per 100 g leaf tissue) when compared with the expression levels observed using the cauliflower mosaic virus (CaMV) 35S promoter. Significantly, 70% of the rAAT produced using the CMViva expression system was found to be biologically active, a 170-fold increase in functional protein compared with the CaMV 35S expression system.     

High-level transient production of a heterologous protein in plants by optimizing induction of a chemically inducible viral amplicon expression system

We have demonstrated that the method of chemical induction using a chemically inducible viral amplicon expression system can be optimized to increase expression of a heterologous protein in plants. A cucumber mosaic virus inducible viral amplicon (CMViva) expression system was used to transiently produce a recombinant human blood protein, alpha-1-antitrypsin (AAT), by co-infiltrating intact and detached Nicotiana benthamiana leaves with two Agrobacterium tumefaciens strains, one containing the CMViva expression cassette carrying the AAT gene and the other containing a binary vector carrying the gene silencing suppressor p19. Infiltrated plants were induced by either topical applications or pressure injections and inducer was applied at either a single or multiple time points. Applying induction solution every 2 days via topical application resulted in increasing maximum levels of biologically functional rAAT from 0.71% to 1.3% of the total soluble protein (TSP) in detached plant leaves, a 1.8-fold improvement. Multiple applications of induction solution via pressure injection into intact leaves resulted in maximum levels of biologically functional rAAT being elevated 3-fold up to 2.4% of TSP compared to 0.8% of TSP when using the conventional method of a single topical application, and expression levels remained high 6 days post-induction. Overall production of rAAT in intact leaves was found to have a maximum level of 5.8% of TSP or 390 mg rAAT per kg leaf tissue when applying multiple injections of chemical induction solution.     

Optimization of the bioprocessing conditions for scale‐up of transient production of a heterologous protein in plants using a chemically inducible viral amplicon expression system

Use of transient expression for the rapid, large‐scale production of recombinant proteins in plants requires optimization of existing methods to facilitate scale‐up of the process. We have demonstrated that the techniques used for agroinfiltration and induction greatly impact transient production levels of heterologous protein. A Cucumber mosaic virus inducible viral amplicon (CMViva) expression system was used to transiently produce recombinant alpha‐1‐antitrypsin (rAAT) by co‐infiltrating harvested Nicotiana benthamiana leaves with two Agrobacterium tumefaciens strains, one containing the CMViva expression cassette carrying the AAT gene and the other containing a binary vector carrying the gene silencing suppressor p19. Harvested leaves were both infiltrated and induced by either pressure or vacuum infiltration. Using the vacuum technique for both processes, maximum levels of functional and total rAAT were elevated by (190 ± 8.7)% and (290 ± 7.5)%, respectively, over levels achieved when using the pressure technique for both processes. The bioprocessing conditions for vacuum infiltration and induction were optimized and resulted in maximum rAAT production when using an A. tumefaciens concentration at OD₆₀₀ of 0.5 and a 0.25‐min vacuum infiltration, and multiple 1‐min vacuum inductions further increased production 25% and resulted in maximum levels of functional and total rAAT at (2.6 ± 0.09)% and (4.1 ± 0.29)% of the total soluble protein, respectively, or (90 ± 1.7) and (140 ± 10) mg per kg fresh weight leaf tissue at 6 days post‐induction. Use of harvested plant tissue with vacuum infiltration and induction demonstrates a bioprocessing route that is fully amenable to scale‐up. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

A cyclical semicontinuous process for production of human alpha 1-antitrypsin using metabolically induced plant cell suspension cultures

Transgenic rice suspension cultures were utilized to produce a human therapeutic protein, recombinant alpha(1)-antitrypsin (rAAT), in a cyclical, semicontinuous operation. Recombinant protein production was induced by removing the carbon source from the cell culture medium. The transgenic rice cells secreted the rAAT into the medium, and therefore medium exchanges could be performed for consecutive growth and protein expression phases. The process consisted of three cycles over a 25-28 day period, with growth phases lasting 4-6 days each and protein expression phases lasting 2.5-5 days each. Biomass and sugar concentrations, oxygen uptake rate, cell viability, culture pH, total extracellular protein, and active rAAT were measured throughout the cyclical process. The data profiles were reproducible between separate cyclical runs where, following each induction period, cell growth and viability could be reestablished once sucrose was added back to the culture. Volumetric productivities ranged from 3 to 12 mg active rAAT/(L day) for individual cycles with overall volumetric productivities of 4.5 and 7.7 mg active rAAT/(L day).     

Production of human alpha-1-antitrypsin from transgenic rice cell culture in a membrane bioreactor

Transgenic plant cell cultures offer a number of advantages over alternative host expression systems, but so far relatively low product concentrations have been achieved. In this study, transgenic rice cells are used in a two-compartment membrane bioreactor (CELLine 350, Integra Biosciences) for the production of recombinant alpha-1-antitrypsin (rAAT). Expression of rAAT is controlled by the rice alpha-amylase (RAmy3D) promoter, which is induced in the absence of sugar. The extracellular product is retained in the bioreactor's relatively small cell compartment, thereby increasing product concentration. Due to the packed nature of the cell aggregates in the cell compartment, a clarified product solution can be withdrawn from the bioreactor. Active rAAT reached levels of 100-247 mg/L (4-10% of the total extracellular protein) in the cell compartment at 5-6 days postinduction, and multiple inductions of the RAmy3D promoter were demonstrated.     

Bioluminescence imaging after HSV amplicon vector delivery into brain

BACKGROUND: Firefly luciferase (Fluc) has routinely been used to quantitate and analyze gene expression in vitro by measuring the photons emitted after the addition of ATP and luciferin to a test sample. It is now possible to replace luminometer-based analysis of luciferase activity and measure luciferase activity delivered by viral vectors directly in live animals over time using digital imaging techniques. METHODS: An HSV amplicon vector expressing Fluc cDNA from an inducible promoter was delivered to cells in culture and into the mouse brain. In culture, expression of Fluc was measured after induction in a dose-dependent manner by a biochemical assay, and then confirmed by Western blot analysis and digital imaging. The vectors were then stereotactically injected into the mouse brain and Fluc expression measured non-invasively using bioluminescence imaging. RESULTS: Rapamycin-mediated induction of Fluc from an HSV amplicon vector in culture resulted in dose-dependent expression of Fluc when measured using a luminometer and by digital analysis. In mouse cortex, a single injection of an HSV amplicon vector (2 microl, 1x10(8) transducing units (t.u.)/ml) expressing Fluc from a viral promoter (CMV) was sufficient to detect robust luciferase activity for at least 1 week. Similarly, an HSV amplicon vector expressing Fluc under an inducible promoter was also detectable in the mouse cortex after a single dose (2 microl, 1x10(8) t.u./ml) for up to 5 days, with no detectable signal in the uninduced state. CONCLUSIONS: This HSV amplicon vector-based system allows for fast, non-invasive, semi-quantitative analysis of gene expression in the brain.     

Bioreactor production of human alpha(1)-antitrypsin using metabolically regulated plant cell cultures

Transgenic rice cell cultures, capable of producing recombinant human alpha(1)-antitrypsin (rAAT), were scaled up from shake flasks to a 5-L bioreactor. The maximum specific growth rates (mu(max)) observed from two bioreactor runs were 0.40 day(-1) (doubling time of 1.7 days) and 0.47 day(-1) (doubling time of 1.5 days), and the maximum specific oxygen uptake rates were 0.78 and 0.84 mmol O(2)/(g dw h). Using a metabolically regulated rice alpha-amylase (RAmy3D) promoter, signal peptide, and terminator, sugar deprivation turned on rAAT expression, and rAAT was secreted into the culture medium. After 1 day of culture in sugar-free medium, there was still continued biomass growth, oxygen consumption, and viability. Extracellular concentrations of 51 and 40 mg active rAAT/L were reached 1.7 and 2.5 days, respectively, after induction in a sugar-free medium. Volumetric productivities for two batch cultures were 7.3 and 4.6 mg rAAT/(L day), and specific productivities were 3.2 and 1.6 mg rAAT/(g dw day). Several different molecular weight bands of immunoreactive rAAT were observed on immunoblots.     

Production of functional human α1-antitrypsin by plant cell culture

Recombinant human α₁-antitrypsin (rAAT) was expressed and secreted from transgenic rice cell suspension cultures in its biologically active form. This was accomplished by transforming rice callus tissues with an expression vector, p3D-AAT, containing the cDNA for mature human AAT protein. Regulated expression and secretion of rAAT from this vector was achieved using the promoter, signal peptide, and terminator from a rice α-amylase gene Amy3D. The Amy3D gene of rice is tightly controlled by simple sugars such as sucrose. It was possible, therefore, to induce the expression of the rAAT by removing sucrose from the cultured media or by allowing the rice suspension cells to deplete sucrose catabolically. Although transgenic rice cell produced a heterogeneous population of the rAAT molecules, they had the same N-terminal amino acids as those found in serum-derived (native) AAT from humans. This result indicates that the rice signal peptidase recognizes and cleaves the novel sequence between the Amy3D signal peptide and the first amino acid of the mature human AAT. The highest molecular weight band seen on Western blots (AAT top band) was found to have the correct C-terminal amino acid sequence and normal elastase binding activity. Staining with biotin-concanavalin A and avidin horseradish peroxidase confirmed the glycosylation of the rAAT, albeit to a lesser extent than that observed with native AAT. The rAAT, purified by immunoaffinity chromatography, had the same association rate constant for porcine pancreatic elastase as the native AAT. Thermostability studies revealed that the rAAT and native AAT decayed at the same rate, suggesting that the rAAT is correctly folded. The productivity of rice suspension cells expressing rAAT was 4.6–5.7 mg/g dry cell. Taken together, these results support the use of rice cell culture as a promising new expression system for production of biologically active recombinant proteins. Received: 18 January 1999 / Received revision: 26 April 1999 / Accepted: 1 May 1999     

Effects of sugar concentration on recombinant human alpha(1)-antitrypsin production by genetically engineered rice cell

Productivity of recombinant human alpha(1)-antitrypsin (rAAT) with a genetically engineered rice cell using an inducible promoter has been studied by batch-wise and continuous production. A simple model explained the effect of proteases released from the disrupted cells on the rAAT degradation. Glucose concentration in the medium significantly affected the rAAT productivity in the continuous production, because the rAAT was induced by sugar depletion. When the fresh medium containing 5mM glucose was supplied to the continuous bioreactor, induction time was long and the productivity was low, indicating that the glucose concentration in the cells was high enough as to repress the promoter. When the glucose concentration in the fresh medium was reduced to 0.5mM, total amount of rAAT produced in 70h cultivation reached 6.7-7.6mg/g-dry cell, which was two times larger than the control medium without glucose.     

Genetic manipulation of stationary-phase genes to enhance recombinant protein production in Escherichia coli

Genetic manipulation of the host strain, by which cell physiology could be modulated, was exploited to enhance recombinant protein production in Escherichia coli. The effects of an inactivated stationary-phase gene (rmf or katF) on recombinant protein production in strains with two different expression systems (the pH-inducible and the lac promoters) were investigated. An improvement of recombinant protein production in the katF mutant at low growth rates was observed for both expression systems. A fourfold and a 30% increase in the volumetric recombinant protein activity were observed for the pH-inducible and the lac promoter system, respectively. The effect of the rmf mutation, on the other hand, depends on the expression system. A twofold increase in the volumetric recombinant protein activity was found for the pH-inducible promoter system, but there was no improvement for the lac promoter system. Improvement in culture performance for slow-growing cultures may have an impact on the design strategy of the host/vector system used in fed-batch cultures, where the specific growth rate is usually slow. The information may also be useful for developing optimal host/vector gene expression systems for recombinant protein production. (c) 1996 John Wiley & Sons, Inc.     

Production of a secreted glycoprotein from an inducible promoter system in a perfusion bioreactor

The primary advantage of an inducible promoter expression system is that production of the recombinant protein can be biochemically controlled, allowing for the separation of unique growth and production phases of the culture. During the growth phase, the culture is rapidly grown to high cell density prior to induction without the extra metabolic burden of exogenous protein production, thus minimizing the nonproductive period of the culture. Induction of the culture at high cell density ensures that the volumetric production will be maximized. In this work, we have demonstrated the feasibility of overexpressing a reporter glycoprotein from the inducible MMTV promoter in recombinant Chinese hamster ovary (CHO) cells cultured in a high cell density perfusion bioreactor system. Retention of suspension-adapted CHO cells was achieved by inclined sedimentation. To maximize volumetric production of the culture, we have demonstrated that high cell density must be achieved prior to induction. This operating scheme resulted in a 10-fold increase in volumetric titer over the low density induction culture, corresponding directly to a 10-fold increase in viable cell density during the highly productive period of the culture. The amount of glycoprotein produced in this high cell density induction culture during 26 days was 84-fold greater than that produced in a week long batch bioreactor. Long-term perfusion cultures of the recombinant cell line showed a production instability, a phenomenon that is currently being investigated.     

Expression of modified gene encoding functional human α-1-antitrypsin protein in transgenic tomato plants

Transgenic plants offer promising alternative for large scale, sustainable production of safe, functional, recombinant proteins of therapeutic and industrial importance. Here, we report the expression of biologically active human alpha-1-antitrypsin in transgenic tomato plants. The 1,182 bp cDNA sequence of human AAT was strategically designed, modified and synthesized to adopt codon usage pattern of dicot plants, elimination of mRNA destabilizing sequences and modifications around 5' and 3' flanking regions of the gene to achieve high-level regulated expression in dicot plants. The native signal peptide sequence was substituted with modified signal peptide sequence of tobacco (Nicotiana tabacum) pathogenesis related protein PR1a, sweet potato (Ipomoea batatas) sporamineA and with dicot-preferred native signal peptide sequence of AAT gene. A dicot preferred translation initiation context sequence, 38 bp alfalfa mosaic virus untranslated region were incorporated at 5' while an endoplasmic reticulum retention signal (KDEL) was incorporated at 3' end of the gene. The modified gene was synthesized by PCR based method using overlapping oligonucleotides. Tomato plants were genetically engineered by nuclear transformation with Agrobacterium tumefaciens harbouring three different constructs pPAK, pSAK and pNAK having modified AAT gene with different signal peptide sequences under the control of CaMV35S duplicated enhancer promoter. Promising transgenic plants expressing recombinant AAT protein upto 1.55% of total soluble leaf protein has been developed and characterized. Plant-expressed recombinant AAT protein with molecular mass of around approximately 50 kDa was biologically active, showing high specific activity and efficient inhibition of elastase activity. The enzymatic deglycosylation established proper glycosylation of the plant-expressed recombinant AAT protein in contrast to unglycosylated rAAT expressed in E. coli ( approximately 45 kDa). Our results demonstrate feasibility for high-level expression of biologically active, glycosylated human alpha-1-antitrypsin in transgenic tomato plants.     

RNA recovery and detection of mRNA by RT-PCR from preserved prokaryotic samples

Levan fructotransferase (LFTase) from Arthrobacter ureafaciens K2032 was expressed with N-terminal fusion of a LacZ-derived secretion motif (TMITNSSSVP) using the lac promoter system in recombinant Escherichia coli JM109 [pUDF-A81]. In flask cultures, recombinant enzyme activity was detected in culture media, and sequence analysis of N-terminal residues showed that about 40% of the extracellular recombinant LFTase had an authentic N-terminus. In a fed-batch bioreactor containing recombinant E. coli at high cell concentrations (OD(600)>200), the extracellular LFTase accumulated to 46000 U ml(-1) (approximately 2.0 g l(-1)) which was almost 40% of total (intra- and extracellular) recombinant LFTase. The synthesized recombinant enzyme was secreted soon after gene expression was induced by IPTG. Prolonged high secretion caused cell lysis and growth inhibition during the production phase in fed-batch cultures. When lactose was added by continuous feed mode, the secretion of recombinant LFTase and hence the cell lysis were significantly delayed in spite of the increased synthesis level. Therefore the induced cell culture of recombinant E. coli could grow up to a much higher cell concentration with continuing recombinant enzyme synthesis. In the case of the controlled feed of lactose, the maximum activities (U ml(-1)) of total and extracellular LFTase were nearly 100% and 70% higher, respectively.     

Optimization of tetracycline-responsive recombinant protein production and effect on cell growth and ER stress in mammalian cells

The inducible T-REx system and other inducible expression systems have been developed in order to control the expression levels of recombinant protein in mammalian cells. In order to study the effects of heterologous protein expression on mammalian host behavior, the gene for recombinant Human transferrin (hTf) was integrated into HEK-293 cells and expressed under the control of the T-REx inducible technology (293-TetR-Hyg-hTf) or using a constitutive promoter (293-CMV-hTf). A number of inducible clones with variable expression levels were identified for the T-REx system with levels of hTf for the high expressing clones nearly double those obtained using the constitutive cytomegalovirus (CMV) promoter. The level of transferrin produced was found to increase proportionately with tetracycline concentration between 0 and 1 mug/mL with no significant increases in transferrin production above 1 mug/mL. As a result, the optimal induction time and tetracycline concentrations were determined to be the day of plating and 1 mug/mL, respectively. Interestingly, the cells induced to express transferrin, 293-TetR-Hyg-hTf, exhibited lower viable cell densities and percent viabilities than the uninduced cultures for multiple clonal isolates. In addition, the induction of transferrin expression was found to cause an increase in the expression of the ER-stress gene, BiP, that was not observed in the uninduced cells. However, both uninduced and induced cell lines containing the hTf gene exhibited longer survival in culture than the control cells, possibly as a result of the positive effects of hTf on cell survival. Taken together, these results suggest that the high level expression of complex proteins in mammalian cells can limit the viable cell densities of cells in culture as a result of cellular stresses caused by generating proteins that may be difficult to fold or are otherwise toxic to cells. The application of inducible systems such as the T-REx technology will allow us to optimize protein production while limiting the negative effects that result from these cellular stresses.     

High level of expression of recombinant human granulocyte-macrophage colony stimulating factor in transgenic rice cell suspension culture

Recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) has been previously produced in tobacco cell suspension cultures. However, the amount of hGM-CSF accumulated in the culture medium dropped quickly from its maximum of 150 microg/L at 5 d after incubation. To overcome this problem, we sought an expression system in which heterologous gene expression could be induced at high levels. We selected a rice amylase expression system in which the promoter Ramy3D is induced to express recombinant protein by sucrose starvation. This induction system was found to give good yield of recombinant hGM-CSF in transgenic rice cell suspension culture and protease activity of this culture medium was low compared to that of tobacco culture system.     

Characterization of a pH-inducible promoter system for high-level expression of recombinant proteins in Escherichia coli

A pH-inducible promoter system was characterized and its potential applicability in recombinant protein production was evaluated using a plasmid construct, pSM552-545C(-), in which the promoter and activator coding sequences of the cad operon were inserted into the upstream region of a lacZ' reporter gene. Graded gene expression levels with respect to culture pH between 8.0 and 5.5 were observed and the induction range can be as high as 200-fold. The effects of several cultivation parameters, including pH, temperature, induction cell density, and inoculum size, were systematically examined. The practical application of this expression system to high level production of recombinant proteins was successfully demonstrated using a rich medium, superbroth. An extremely high recombinant protein productivity at a value of approximately 1.4 g/L with a specific expression level as high as 35% of total cellular protein can be obtained in a simple batch cultivation. The behavior of this expression system was further investigated using chemostat cultures. An uncommon relationship between the volumetric or specific recombinant protein activity and the dilution rate, with a maximal activity at a dilution rate of approximately 0.4 h(-1)was observed. (c) 1995 John Wiley & Sons, Inc.     

Overexpression of cloned genes using recombinant Escherichia coli regulated by a T7 promoter: II. Two-stage continuous cultures and model simulations

A two-stage culture strategy was studied for continuous high-level production of a foreign protein in the chemically inducible T7 expression system. The first stage is dedicated to the maintenance of plasmid-bearing cells and the second stage to the target protein synthesis by induction of cells coming from the first stage. On entering the second stage, recombinant cells undergo a gradual induction of the target gene expression. These plasmid-bearing cells experience dynamic changes in intracellular compositions and specific growth rates with their individual residence times. Therefore, the overall cultural characteristics in the production stage are really averages of the contributions from the various cells with different residence times. The behavior of the two-stage culture is described by a model, which accounts for dynamic variations of cell growth and protein synthesis rates with cell residence times. Model simulations were compared with experimental results at a variety of operating conditions such as inducer concentration and dilution rate. This model is useful for understanding the behavior of two-stage continuous cultures. (c) 1993 John Wiley & Sons, Inc.     

The use of elements of the E. coli Ntr-system for the design of an optimized recombinant expression system for high cell density cultivations.

The inducible glnA promoter 2 of the E. coli glutamine synthetase gene is suitable as an expression unit for the production of recombinant proteins at low and high cell densities. It is active when the concentration of ammonium as the sole nitrogen source in the culture medium is below 1 mM. This nitrogen regulatory system was optimized by introduction of expression cassettes consisting of additional elements of the ntr-system. These artificial constructions result in enhanced recombinant gene expression in the production phase. Furthermore, the basic recombinant protein level during the growth phase is reduced due to a tighter promoter control. A three- to four-fold higher accumulation of chloramphenicol-acetyltransferase (as reporter protein) and of anti-EGF-receptor miniantibodies was achieved by increasing the amount of the final regulator molecule NtrC approximately P via plasmidal co-expression of the ntrC gene. The introduction of a modified glnA promoter 1 inverse to glnAp2 lowered the basic activity of glnAp2 to about one half. It is assumed that under nitrogen excess conditions sigma 70-RNA polymerase binds at glnAp1 and thereby prevents most of the binding of sigma 54-RNA polymerase at glnAp2. The optimized expression systems were successfully applied in low and high cell density cultivations. In the fed-batch phase of high cell density cultivations recombinant protein formation was induced through external nitrogen limitation under FIA-controlled concentration of glucose as carbon source.