Transient Expression of Tetrameric Recombinant Human Butyrylcholinesterase in Nicotiana benthamiana.

To optimize the expression, extraction and purification of plant-derived tetrameric recombinant human butyrylcholinesterase (prBChE), we describe the development and use of plant viral amplicon-based gene expression system; Tobacco Mosaic Virus (TMV) RNA-based overexpression vector (TRBO) to express enzymatically active FLAG-tagged plant made recombinant butyrylcholinesterase (rBChE) in Nicotiana benthamiana leaves using transient agroinfiltration. Two gene expression cassettes were designed to express the recombinant protein in either the ER or to the apoplastic compartment. Leaf homogenization was used to isolate ER-retained recombinant butyrylcholinesterase (prBChE-ER) while apoplast-targeted rBChE was isolated by either leaf homogenization (prBChE) or vacuum-extraction of apoplastic wash fluid (prBChE-AWF). rBChE from apoplast wash fluid had a higher specific activity but lower enzyme yield than leaf homogenate. To optimize the isolation and purification of total recombinant protein from leaf homogenates, an acidic extraction buffer was used. The acidic extraction buffer yielded >95% enzymatically active tetrameric rBChE as verified by Coomassie stained and native gel electrophoresis. Furthermore, when compared to human butyrylcholinesterase, the prBChE was found to be similar in terms of tetramerization and enzyme kinetics. The N-linked glycan profile of purified prBChE-ER was found to be mostly high mannose structures while the N-linked glycans on prBChE-AWF were primarily complex. The glycan profile of the prBChE leaf homogenates showed a mixture of high mannose, complex and paucimannose type N-glycans. These findings demonstrate the ability of plants to produce rBChE that is enzymatically active and whose oligomeric state is comparable to mammalian butyrylcholinesterase. The process of plant made rBChE tetramerization and strategies for improving its pharmacokinetics properties are also discussed

Semicontinuous Bioreactor Production of Recombinant Butyrylcholinesterase in Transgenic Rice Cell Suspension Cultures.

An active and tetrameric form of recombinant butyrylcholinesterase (BChE), a large and complex human enzyme, was produced via semicontinuous operation in a transgenic rice cell suspension culture. After transformation of rice callus and screening of transformants, the cultures were scaled up from culture flask to a lab scale bioreactor. The bioreactor was operated through two phases each of growth and expression. The cells were able to produce BChE during both expression phases, with a maximum yield of 1.6 mg BChE/L of culture during the second expression phase. Cells successfully regrew during a 5-day growth phase. A combination of activity assays and Western blot analysis indicated production of an active and fully assembled tetramer of BChE

A novel plant cell culture platform for semicontinous production of recombinant proteins: Butyrylcholinesterase as a case study

In this paper we describe a novel biomanufacturing production platform that utilizes transgenic rice cell suspension cultures for efficient semicontinuous cell culture (SCC) production of recombinant proteins. The production platform utilizes a metabolically regulated promoter, a secretion signal peptide that enables secretion out of the cell for ease of recovery/purification, coupled with an efficient semicontinuous operational strategy that allows independent optimization of growth and production phases. In addition, long term operation (up several months1) is possible by maintaining viable biomass within the bioreactor, thereby reducing the need for long seed trains, as well as minimizing turn-around time, CIP and SIP operations, chemicals and energy. This platform offers a number of advantages over traditional methods for production of recombinant therapeutic proteins that use E. coli, yeast or mammalian cell cultures, while still retaining the ability to meet cGMP regulatory requirements under well-controlled, reproducible production conditions. Traditional methods for production of biologics use genetically modified E. coli, yeast, insect or mammalian cell cultures in bioreactor systems. For applications where a human therapeutic protein (monoclonal antibodies, vaccines, bioscavengers, replacement biologics) produced under strict cGMP conditions are required, plant cell cultures offer a number of advantages over currently used bioreactor-based systems, including low risk of contamination by mammalian viruses, blood-borne pathogens, prions or bacterial endotoxins or mycoplasma, the ability to perform complex glycosylation, ease of culturing compared with other higher eukaryotic hosts, the ability to target the product to the extracellular medium, and the ability to grow in simple, low cost, chemically defined and animal component-free medium. In this paper we describe the specific characteristics of the rice cell suspension culture that make them particularly useful for continuous operation and superior to other hosts including their slow death rates, growth in small aggregates, limited secretome, and robustness under culture conditions. In addition, the regulatory pathway for plant-based recombinant biologics for human therapeutic use has now been established. ElelysoTM, produced in carrot cell suspension in batch culture by Protalix Biotherapeutics and Pfizer, Inc. for treatment of Gaucher disease was approved by the FDA in May 20122, 3. The transgenic rice cell culture system is operated in a cyclical, semicontinuous operation as shown in Figure 1. Note that gravity sedimentation within the bioreactor can be used to separate the plant cell aggregates from the liquid phase in Steps 3 and 6, and that the product collected in Step 6 can be purified either using a batch downstream strategy or collected to feed a continuous downstream process. Results will be presented for semicontinuous production of butyrylcholinesterase, a bioscavenger for organophosphorus nerve agents such as sarin, using the metabolically regulated transgenic rice cell culture in 5 L bioreactors

Direct Potable Reuse for the City of San Diego, California

The objective of this project is to design a direct potable reuse facility that is capable of supplementing drinking water sources to the city of San Diego, CA, while minimizing the amount of chemicals added throughout the process. The proposed facility is designed to accommodate 72 million gallons of wastewater per day and treat the influent using numerous pieces of equipment that remove particles based on particle diameter or mass. In addition to using filtration medium to remove particulates, aeration tanks containing activated sludge promote biological digestion while continuously stirred tank reactors irradiated with ultraviolet light and injected with hydrogen peroxide disinfect and oxidize biological and chemical constituents. The economic analysis estimates the proposed facility to cost $557 million dollars and have a lifetime of 25 years. Additionally, the net present value after the 25-year lifetime of the facility at an interest rate of 9$2 billion. Due to the need of increasing the water supply for expanding populations and anticipation of future droughts, the proposed design is recommended for implementation. However, a pilot-plant or demonstration facility should be performed due to the sensitivity of the equipment in the facility to the influent water quality

Transient Expression of Tetrameric Recombinant Human Butyrylcholinesterase in Nicotiana benthamiana.

To optimize the expression, extraction and purification of plant-derived tetrameric recombinant human butyrylcholinesterase (prBChE), we describe the development and use of plant viral amplicon-based gene expression system; Tobacco Mosaic Virus (TMV) RNA-based overexpression vector (TRBO) to express enzymatically active FLAG-tagged plant made recombinant butyrylcholinesterase (rBChE) in Nicotiana benthamiana leaves using transient agroinfiltration. Two gene expression cassettes were designed to express the recombinant protein in either the ER or to the apoplastic compartment. Leaf homogenization was used to isolate ER-retained recombinant butyrylcholinesterase (prBChE-ER) while apoplast-targeted rBChE was isolated by either leaf homogenization (prBChE) or vacuum-extraction of apoplastic wash fluid (prBChE-AWF). rBChE from apoplast wash fluid had a higher specific activity but lower enzyme yield than leaf homogenate. To optimize the isolation and purification of total recombinant protein from leaf homogenates, an acidic extraction buffer was used. The acidic extraction buffer yielded >95% enzymatically active tetrameric rBChE as verified by Coomassie stained and native gel electrophoresis. Furthermore, when compared to human butyrylcholinesterase, the prBChE was found to be similar in terms of tetramerization and enzyme kinetics. The N-linked glycan profile of purified prBChE-ER was found to be mostly high mannose structures while the N-linked glycans on prBChE-AWF were primarily complex. The glycan profile of the prBChE leaf homogenates showed a mixture of high mannose, complex and paucimannose type N-glycans. These findings demonstrate the ability of plants to produce rBChE that is enzymatically active and whose oligomeric state is comparable to mammalian butyrylcholinesterase. The process of plant made rBChE tetramerization and strategies for improving its pharmacokinetics properties are also discussed