Plant factories for the production of monoclonal antibodies

Like animal cells, plant cells bear mechanisms for protein synthesis and posttranslational modification (glycosylation and phosphorylation) that allow them to be seriously considered as factories for therapeutic proteins, including antibodies, with the development of biotechnology. The plant platform for monoclonal antibody production is an attractive approach due to its flexibility, speed, scalability, low cost of production, and lack of contamination risk from animal-derived pathogens. Contemporary production approaches for therapeutic proteins rely on transgenic plants that are obtained via the stable transformation of plant cells as well as the transient (temporary) expression of foreign proteins. In this review, we discuss present-day approaches for monoclonal antibody production in plants (MAPP), features of carbohydrate composition, and methods for the humanization of the MAPP carbohydrate profile. MAPPs that have successfully passed preclinical studies and may be promising for use in clinical practice are presented here. Perspectives on using MAPPs are determined by analyzing their economic benefits and production rates, which are especially important in personalized cancer therapy as well as in cases of bioterrorism and pandemics.     

A single-use purification process for the production of a monoclonal antibody produced in a PER.C6 human cell line

Advances in single-use technologies can enable greater speed, flexibility, and a smaller footprint for multi-product production facilities, such as at a contract manufacturer. Recent efforts in the area of cell line and media optimization have resulted in bioreactor productivities that exceed 8 g/L in fed-batch processes or 25 g/L in high-density cell culture processes. In combination with the development of single-use stirred tank bioreactors with larger working volumes, these intensified upstream processes can now be fit into a single-use manufacturing setting. Contrary to these upstream advances, downstream single-use technologies have been slower to follow, mostly limited by low capacity, high cost, and poor scalability. In this study we describe a downstream process based solely on single-use technologies that meets the challenges posed by expression of a mAb (IgG(1)) in a high-density suspension culture of PER.C6 cells. The cell culture harvest was clarified by enhanced cell settling (ECS) and depth filtration. Precipitation was used for crude purification of the mAb. A high capacity chromatographic membrane was then used in bind/elute mode, followed by two membranes in flow-through (FT) mode for polishing. A proof of concept of the entire disposable process was completed for two different scales of the purification train.     

Novel orbital shake bioreactors for transient production of CHO derived IgGs

Large-scale transient gene expression in mammalian cells is being developed for the rapid production of recombinant proteins for biochemical and preclinical studies. Here, the scalability of transient production of a recombinant human antibody in Chinese hamster ovary (CHO) cells was demonstrated in orbitally shaken disposable bioreactors at scales from 50 mL to 50 L. First, a small-scale multiparameter approach was developed to optimize the poly(ethylenimine)-mediated transfection in 50 mL shake tubes. This study confirmed the benefit, both in terms of extended cell culture viability and increased product yield, of mild hypothermic cultivation conditions for transient gene expression in CHO cells. Second, the scalability of the process was demonstrated in disposable shake bioreactors having nominal volumes of 5, 20, and 50 L with final antibody yields between 30 and 60 mg L(-1). Thus, the combination of transient gene expression with disposable shake bioreactors allows for rapid and cost-effective production of recombinant proteins in CHO cells.     

Techno-economic analysis of a transient plant-based platform for monoclonal antibody production

Plant-based biomanufacturing of therapeutic proteins is a relatively new platform with a small number of commercial-scale facilities, but offers advantages of linear scalability, reduced upstream complexity, reduced time to market, and potentially lower capital and operating costs. In this study we present a detailed process simulation model for a large-scale new “greenfield” biomanufacturing facility that uses transient agroinfiltration of Nicotiana benthamiana plants grown hydroponically indoors under light-emitting diode lighting for the production of a monoclonal antibody. The model was used to evaluate the total capital investment, annual operating cost, and cost of goods sold as a function of mAb expression level in the plant (g mAb/kg fresh weight of the plant) and production capacity (kg mAb/year). For the Base Case design scenario (300 kg mAb/year, 1 g mAb/kg fresh weight, and 65% recovery in downstream processing), the model predicts a total capital investment of $122 million dollars and cost of goods sold of $121/g including depreciation. Compared with traditional biomanufacturing platforms that use mammalian cells grown in bioreactors, the model predicts significant reductions in capital investment and >50% reduction in cost of goods compared with published values at similar production scales. The simulation model can be modified or adapted by others to assess the profitability of alternative designs, implement different process assumptions, and help guide process development and optimization.     

EB66 cell line,a duck embryonic stem cell-derived substrate for the industrial production of therapeutic monoclonal antibodies with enhanced ADCC activity

Monoclonal antibodies (mAbs) represent the fastest growing class of therapeutic proteins. The increasing demand for mAb manufacturing and the associated high production costs call for the pharmaceutical industry to improve its current production processes or develop more efficient alternative production platforms. The experimental control of IgG fucosylation to enhance antibody dependent cell cytotoxicity (ADCC) activity constitutes one of the promising strategies to improve the efficacy of monoclonal antibodies and to potentially reduce the therapeutic cost. We report here that the EB66 cell line derived from duck embryonic stem cells can be efficiently genetically engineered to produce mAbs at yields beyond a 1 g/L, as suspension cells grown in serum-free culture media. EB66 cells display additional attractive grown characteristics such as a very short population doubling time of 12 to 14 hours, a capacity to reach very high cell density (> 30 million cells/mL) and a unique metabolic profile resulting in low ammonium and lactate accumulation and low glutamine consumption, even at high cell densities. Furthermore, mAbs produced on EB66 cells display a naturally reduced fucose content resulting in strongly enhanced ADCC activity. The EB66 cells have therefore the potential to evolve as a novel cellular platform for the production of high potency therapeutic antibodies.     

Viral vectors for the expression of proteins in plants

The use of plant viral vectors for the transient expression of heterologous proteins offers a useful tool for the large-scale production of proteins of industrial importance, such as antibodies and vaccine antigens. In recent years, advances have been made both in the development of first-generation vectors (that employ the 'full virus') and second-generation ('deconstructed virus') vectors. For example, vectors based around the 'full virus' strategy can now be used to express long polypeptides (at least 140 amino acids long) as fusions to the coat protein. In addition, a new generation of vectors was engineered to have a reactogenic amino acid exposed on the surface of the virus, allowing easy chemical conjugation of (separately produced) proteins to the viral surface. This approach is being used to develop new vaccines in the form of antigens coupled to a plant viral surface. Prototypes of industrial processes that require high-yield production, rapid scale-up, and fast manufacturing have been recently developed using the 'deconstructed virus' approach (magnifection). This process, which relies on Agrobacterium as a vector to deliver DNA copies of one or more viral RNA replicons to plant cells, has been shown to work with numerous proteins, including full immunoglobulin G antibodies. Other advances in this area have looked at the development of inducible viral systems and the use of viral vectors to produce nanoscale materials for modular assembly.     

Antibody production by molecular farming in plants

"Molecular farming" is the production of pharmaceutical proteins in transgenic plants and has great potential for the production of therapeutic anti-cancer antibodies and recombinant therapeutic proteins. Plants make fully functional recombinant human or animal antibodies. Cultivating transgenic plants on an agricultural scale will produce almost unlimited supplies of recombinant proteins for uses in medicine. Combinatorial library technology is a key tool for the generation and optimisation of therapeutic antibodies ahead of their expression in plants. Optimised antibody expression can be rapidly verified using transient expression assays in plants before creation of transgenic suspension cells or plant lines. Subcellular targeting signals that increase expression levels and optimise protein stability can be identified and exploited using transient expression to create high expresser plant lines. When high expresser lines have been selected, the final step is the development of efficient purification methods to retrieve functional antibody. Antibody production on an industrial scale is then possible using plant suspension cell culture in fermenters, or by the propagation of stably transformed plant lines in the field. Recombinant proteins can be produced either in whole plants or in seeds and tubers, which can be used for the long-term storage of both the protein and its production system. The review will discuss these developments and how we are moving toward the molecular farming of therapeutic antibodies becoming an economic and clinical reality.     

EB66 cell line,a duck embryonic stem cell-derived substrate for the industrial production of therapeutic monoclonal antibodies with enhanced ADCC activity

Monoclonal antibodies (mAbs) represent the fastest growing class of therapeutic proteins. The increasing demand for mAb manufacturing and the associated high production costs call for the pharmaceutical industry to improve its current production processes or develop more efficient alternative production platforms. The experimental control of IgG fucosylation to enhance antibody dependent cell cytotoxicity (ADCC) activity constitutes one of the promising strategies to improve the efficacy of monoclonal antibodies and to potentially reduce the therapeutic cost. We report here that the EB66 cell line derived from duck embryonic stem cells can be efficiently genetically engineered to produce mAbs at yields beyond a 1 g/L, as suspension cells grown in serum-free culture media. EB66 cells display additional attractive growth characteristics such as a very short population doubling time of 12–14 h, a capacity to reach very high cell density (>30 million cells/mL) and a unique metabolic profile resulting in low ammonium and lactate accumulation and low glutamine consumption, even at high cell densities. Furthermore, mAbs produced on EB66 cells display a naturally reduced fucose content resulting in strongly enhanced ADCC activity. The EB66 cells have therefore the potential to evolve as a novel cellular platform for the production of high potency therapeutic antibodies.Key words: duck, embryonic stem cells, monoclonal antibody, fucose, ADCC     

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.     

Identification of monoclonal antibodies specific for the T cell receptor complex by Fc receptor-mediated CTL lysis

Monoclonal antibodies (mAb) directed at the T cell receptor complex (TcR) on cloned T cells have generally been identified by their ability to inhibit the clone's antigen-specific function. Because such inhibition is highly dependent on antibody concentration and affinity, detection of anti-clonotypic antibodies to murine alloreactive T cells has been very difficult. In this report, an alternative method is described on the basis of the ability of antibodies specific for the TcR complex to activate T cells in an antigen-independent manner. The assay is based upon the observation that soluble antibodies to human T3 promote lysis of irrelevant, Fc receptor-positive targets by a human CTL line. By using this approach, an anti-TcR mAb has been identified among a panel of murine mAb generated against an alloreactive CTL clone. Induction of lysis by soluble anti-TcR mAb has been shown to require both the expression of Fc receptors on the target cell and conjugate formation between the effector and the target cell. This assay provides a screening procedure that is much more sensitive than inhibition of function, and it preferentially detects antibodies specific for cell surface molecules involved in T cell activation.     

Plant-made trastuzumab (herceptin) inhibits HER2/Neu+ cell proliferation and retards tumor growth

Background

Plant biotechnology provides a valuable contribution to global health, in part because it can decrease the cost of pharmaceutical products. Breast cancer can now be successfully treated by a humanized monoclonal antibody (mAb), trastuzumab (Herceptin). A course of treatment, however, is expensive and requires repeated administrations of the mAb. Here we used an Agrobacterium-mediated transient expression system to produce trastuzumab in plant cells.

Methodology/Principal Findings

We describe the cloning and expression of gene constructs in Nicotiana benthamiana plants using intron-optimized Tobacco mosaic virus- and Potato virus X-based vectors encoding, respectively, the heavy and light chains of trastuzumab. Full-size antibodies extracted and purified from plant tissues were tested for functionality and specificity by (i) binding to HER2/neu on the surface of a human mammary gland adenocarcinoma cell line, SK-BR-3, in fluorescence-activated cell sorting assay and (ii) testing the in vitro and in vivo inhibition of HER-2-expressing cancer cell proliferation. We show that plant-made trastuzumab (PMT) bound to the Her2/neu oncoprotein of SK-BR-3 cells and efficiently inhibited SK-BR-3 cell proliferation. Furthermore, mouse intraperitoneal PMT administration retarded the growth of xenografted tumors derived from human ovarian cancer SKOV3 Her2+ cells.

Conclusions/Significance

We conclude that PMT is active in suppression of cell proliferation and tumor growth.     

Biomanufacturing of protective antibodies and other therapeutics in edible plant tissues for oral applications

Although plant expression systems used for production of therapeutic proteins have the advantage of being scalable at a low price, the downstream processing necessary to obtain pure therapeutic molecules is as expensive as for the traditional Chinese hamster ovary (CHO) platforms. However, when edible plant tissues (EPTs) are used, there is no need for exhaustive purification, because they can be delivered orally as partially purified formulations that are safe for consumption. This economic benefit is especially interesting when high doses of recombinant proteins are required throughout the treatment/prophylaxis period, as is the case for antibodies used for oral passive immunization (OPI). The secretory IgA (SIgA) antibodies, which are highly abundant in the digestive tract and mucosal secretions, and thus the first choice for OPI, have only been successfully produced in plant expression systems. Here, we cover most of the up‐to‐date examples of EPT‐produced pharmaceuticals, including two examples of SIgA aimed at oral delivery. We describe the benefits and drawbacks of delivering partially purified formulations and discuss a number of practical considerations and criteria to take into account when using plant expression systems, such as subcellular targeting, protein degradation, glycosylation patterns and downstream strategies, all crucial for improved yield, high quality and low cost of the final product.     

Site‐specific proteolytic degradation of IgG monoclonal antibodies expressed in tobacco plants

Plants are promising hosts for the production of monoclonal antibodies (mAbs). However, proteolytic degradation of antibodies produced both in stable transgenic plants and using transient expression systems is still a major issue for efficient high‐yield recombinant protein accumulation. In this work, we have performed a detailed study of the degradation profiles of two human IgG1 mAbs produced in plants: an anti‐HIV mAb 2G12 and a tumour‐targeting mAb H10. Even though they use different light chains (κ and λ, respectively), the fragmentation pattern of both antibodies was similar. The majority of Ig fragments result from proteolytic degradation, but there are only a limited number of plant proteolytic cleavage events in the immunoglobulin light and heavy chains. All of the cleavage sites identified were in the proximity of interdomain regions and occurred at each interdomain site, with the exception of the V_L/C_L interface in mAb H10 λ light chain. Cleavage site sequences were analysed, and residue patterns characteristic of proteolytic enzymes substrates were identified. The results of this work help to define common degradation events in plant‐produced mAbs and raise the possibility of predicting antibody degradation patterns ‘a priori’ and designing novel stabilization strategies by site‐specific mutagenesis.     

Effects of passage number on growth and productivity of hybridoma secreting MRSA anti-PBP2a monoclonal antibodies

Monoclonal antibodies (mAb) are high added value glycoproteins recommended for immunotherapy, diagnosis, and also for the treatment of bacterial infections resistant to multiple drugs such as Methicillin Resistant Staphylococcus aureus (MRSA). In addition to environmental conditions related to cell cultures, the intrinsic characteristics of hybridoma cells, like the secretion stability of monoclonal antibodies by the cells through successive subcultures, are relevant for the characterization of cell lines related to the productivity of mAb. The rate of mAb production differs significantly between different cell lines and different passage numbers, and it is an important variable in characterization of cell lines. In order to find a more robust, faster-growing, and higher-productivity cell line of hybridoma, cultivations in 24-well plates were performed in different subculture periods, or cell passages (P), of hybridoma cells producing MRSA anti-PBP2a monoclonal antibodies [MRSA-antiPBP2a (mAb)]. The objective of this study was to study the effects of cell growth and production of MRSA-antiPBP2a mAb secreted by murine hybridoma cells grown in different passages as well as determine the which passages the hybridomas can be cultivated without harming their growth and productivity. So, cell growth profiles of hybridomas secreting MRSA-antiPBP2a (mAb) and the production of MRSA-antiPBP2a mAb in different subculture periods or cell passages (P) were studied. Cell growth tests, monoclonal antibody productivity, and metabolite characteristics revealed substantial differences in those cells kept between P10 and P50. Similarities in the secretion of monoclonal antibody, growth, and metabolic profiles, were noted in the MRSA-antiPBP2a mAb producing hybridoma cells kept between P10 and P20. Also, glucose consumption (g/L) and lactate production (g/L) in the latter cell cultures were monitored daily through biochemical analyzer. As of P30, it was observed a 4.4 times reduction in productivity, a 13 % reduction in metabolic yield, and a significant change in cell growth. Secretion of MRSA-antiPBP2a mAb should be obtained through the culture of hybridomas up to P20 in order to keep its stability.     

Monoclonal antibody production using a new supermacroporous cryogel bioreactor

A supermacroporous cryogel bioreactor has been developed to culture hybridoma cells for long-term continuous production of monoclonal antibodies (mAb). Hybridoma clone M2139, secreting antibodies against J1 epitope (GERGAAGIAGPK; amino acids, 551-564) of collagen type II, are immobilized in the porous bed matrix of a cryogel column (10 mL bed volume). The cells got attached to the matrix within 48 h after inoculation and grew as a confluent sheet inside the cryogel matrix. Cells were in the lag phase for 15 days and secreted mAb into the circulation medium. Glucose consumption and lactic acid production were also monitored, and during the exponential phase (approximately 20 days), the hybridoma cell line consumed 0.75 mM day-1 glucose, produced 2.48 mM day-1 lactic acid, and produced 6.5 microg mL-1 day-1 mAb during the exponential phase. The mAb concentration reached 130 microg mL-1 after continuous run of the cryogel column for 36 days. The yield of the mAb after purification was 67.5 mg L-1, which was three times greater than the mAb yield obtained from T-flask batch cultivation. Even after the exchange of medium reservoir, cells in the cryogel column were still active and had relatively stable mAb production for an extended period of time. The bioreactor was operated continuously for 55 days without any contamination. The results from ELISA as well as arthritis experiments demonstrate that the antibodies secreted by cells grown on the cryogel column did not differ from antibodies purified from the cells grown in commercial CL-1000 culture flasks. Thus, supermacroporous cryogels can be useful as a supporting material for productive hybridoma cell culture. Cells were found to be viable inside the porous matrix of the cryogel during the study period and secreted antibodies continuously. The antibodies thus obtained from the cryogel reactor were found to be functionally active in vivo, as demonstrated by their capacity to induce arthritis in mice.     

The role of protein kinase C in transmembrane signaling by the T cell antigen receptor complex. Effects of stimulation with soluble or immobilized CD3 antibodies

Phorbol esters, such as phorbol myristate acetate (PMA), are known to be potent co-stimulants with calcium ionophores for activation of T lymphocytes. The most extensively studied intracellular effect of PMA is its ability to activate the cytoplasmic enzyme protein kinase C (pkC). Herein, we examined the role of pkC activation during T cell activation. During physiologic activation, this enzyme is activated by diacylglycerol which is generated through the hydrolysis of polyphosphoinositides. Therefore, we studied the activation of T lymphocytes induced by a synthetic diacylglycerol, dioctanoylglycerol. In contrast to PMA, this compound can be metabolized in T cells and presumably more closely mimics physiologic activation of pkC. Dioctanoylglycerol together with reagents that induce increases in intracellular free Ca2+ concentration, Ca2+ ionophores, or anti-cluster designation (CD)3 monoclonal antibodies (mAb) were able to induce interleukin 2 receptor expression and proliferation of T lymphocytes. Previous studies have demonstrated that the stimulation of T cells via the CD3/T cell antigen receptor complex by mAb against CD3 leads to an increase in cytoplasmic free Ca2+ and to an activation of pkC. Paradoxically, however, soluble CD3 antibodies do not cause proliferation of resting purified T cells. Inasmuch as immobilization of CD3 mAb has been shown to influence the agonist properties of such antibodies, we compared the ability of soluble and immobilized CD3 mAb to activate pkC. We demonstrated herein that soluble CD3 mAb cause only a very transient activation of pkC in the T cell leukemic line Jurkat. This pkC activation is markedly prolonged when Jurkat cells are stimulated with immobilized rather than soluble CD3 antibodies. These studies suggest that activation of pkC plays a major role in T cell activation and that the activation of pkC is influenced by the form in which CD3 mAb is presented to T cells.     

Serum-free transfection of CHO cells with chemically defined transfection systems and investigation of their potential for transient and stable transfection

The generation of transgenic cell lines is acquired by facilitating the uptake and integration of DNA. Unfortunately, most of the systems generating stable expression systems are cost and time-consuming and transient expression is optimized to generate milligram amounts of the recombinant protein. Therefore we improved and compared two transfection systems, one based on cationic liposomes consisting of DOTAP/DOPE and the second one on polyethylenimine (PEI). Both systems have been used as chemically defined transfection systems in combination with serum-free cultivated host cell line. At first we had determined the toxicity and ideal ratio of DNA to PEI followed by determination of the optimal transfection conditions in order to achieve maximum transfection efficiency. We then directly compared DOTAP/DOPE and PEI in transient transfection experiments using enhanced green fluorescence protein (EGFP) and a human monoclonal antibody, mAb 2F5, as a model protein. The results which were achieved in case of EGFP were more than 15% transfectants at a viability of 85%. Despite the fact that expression of the mAb was found negligible we used both techniques to generate stable mAb 2F5 expressing cell lines that underwent several cycles of screening and amplification with methotrexate, and resulted in cell lines with similar volumetric production titers. These experiments serve to demonstrate the potential of stable cell lines even in case where the transient systems did not show satisfying results.     

Development of GRAS strains for nutraceutical production using systems and synthetic biology approaches: advances and prospects

Nutraceuticals are food substances with medical and health benefits for humans. Limited by complicated procedures, high cost, low yield, insufficient raw materials, resource waste, and environment pollution, chemical synthesis and extraction are being replaced by microbial synthesis of nutraceuticals. Many microbial strains that are generally regarded as safe (GRAS) have been identified and developed for the synthesis of nutraceuticals, and significant nutraceutical production by these strains has been achieved. In this review, we systematically summarize recent advances in nutraceutical research in terms of physiological effects on health, potential applications, drawbacks of traditional production processes, characteristics of production strains, and progress in microbial fermentation. Recent advances in systems and synthetic biology techniques have enabled comprehensive understanding of GRAS strains and its wider applications. Thus, these microbial strains are promising cell factories for the commercial production of nutraceuticals.