Plant cell culture platforms for production of bioscavengers for biodefense

There is a critical need for flexible, rapid, cost effective biomanufacturing platforms for medical countermeasures. Our team has developed plant cell culture-based manufacturing platforms for production of recombinant protein bioscavengers against organophosphate (OP) nerve agents and anthrax toxins using both stable transgenic cell cultures for known chemical and biological threats, as well as transient production for rapid response to new and/or unanticipated threats. Plant cells offer several advantages over other hosts for production of medical countermeasures, particularly their ability to produce complex biologics and perform post-translational modification, inherent biosafety since they don\u27t harbor or propagate mammalian viruses thereby simplifying and/or eliminating viral clearance steps required for mammalian production systems. Plant cells are robust, have minimal nutrient requirements (grow in simple, chemically defined media containing sucrose, salts and plant hormones), and are relatively insensitive to changes in environmental conditions. These characteristics, robustness of upstream cultivation/use and reduced downstream purification requirements, make plant cells an ideal choice for field-deployable production of medical countermeasures. Here we present results for the production of functional recombinant butyrylcholinesterase (BChE), an OP nerve agent bioscavenger, in transgenic rice cell suspension cultures in different bioreactor configurations, and transient production of a bioscavenger against an anthrax toxin in N. benthamiana cell cultures. Techno-economic models for scaled-up versions of these plant cell culture production systems will also be presented

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

Marion County Heritage Map Project

With the soft opening of Pasaquan, Marion County’s visionary art environment, in summer 2016, and its grand opening by Columbus State University in fall 2016, the Marion County Chamber of Commerce requested that the Columbus Community Geography Center partner to develop a heritage tour map of the county for welcome centers across the state. This small rural county of 8,700 residences, 60% white, 30% black and 7.5% Hispanic. One fifth of the population live below the poverty level. It recently lost several hundred jobs with the closure of the local chicken processing plant. Plans to launch a tourism program is understood to be of great importance to the community. CSU’s Dr. Amanda Rees, Professor of Geography, and Professor Chuck Lawson, Department of Art, College of the Arts joined forces to create a heritage tour map of Buena Vista and Marion County. Geography students ran a community workshop to identify twenty-one county and city heritage sites for inclusion. They researched and wrote short descriptions for the map and extended histories for an accompanying web page to be accessed from the map with a QR code. Students also produced an accurate map of each site and the major roads and other primary physical features of the county and city. Graphic design students then received the map text and GIS maps of the county and the city. Students designed three “roughs” of the map for external review. The first review included Marion County leaders, state tourism representatives, and several faculty in art, GIS and geography. The roughs were then refined and presented again to a group of reviewers. This project proved to be a good fit for CSU’s QEP “Real World Problems Solving” project in its testing phase in spring 2016. This interdisciplinary “service learning” project offered high impact educational practices, fieldwork, student-led heritage workshop in Marion County, critical feedback from community members on writing, and design. This interdisciplinary project was aligned with CSU’s mission to support alternative pedagogical approaches to address the needs of millennial learners

Technoeconomic analysis of semicontinuous bioreactor production of biopharmaceuticals in transgenic rice cell suspension cultures.

Biopharmaceutical protein production using transgenic plant cell bioreactor processes offers advantages over microbial and mammalian cell culture platforms in its ability to produce complex biologics with simple chemically defined media and reduced biosafety concerns. A disadvantage of plant cells from a traditional batch bioprocessing perspective is their slow growth rate which has motivated us to develop semicontinuous and/or perfusion processes. Although the economic benefits of plant cell culture bioprocesses are often mentioned in the literature, to our knowledge no rigorous technoeconomic models or analyses have been published. Here we present technoeconomic models in SuperPro Designer® for the large-scale production of recombinant butyrylcholinesterase (BChE), a prophylactic/therapeutic bioscavenger against organophosphate nerve agent poisoning, in inducible transgenic rice cell suspension cultures. The base facility designed to produce 25 kg BChE per year utilizing two-stage semicontinuous bioreactor operation manufactures a single 400 mg dose of BChE for $263. Semicontinuous operation scenarios result in 4-11% reduction over traditional two-stage batch operation scenarios. In addition to providing a simulation tool that will be useful to the plant-made pharmaceutical community, the model also provides a computational framework that can be used for other semicontinuous or batch bioreactor-based processes

Robust, Quantitative Analysis of Proteins using Peptide Immunoreagents, in Vitro Translation, and an Ultrasensitive Acoustic Resonant Sensor

A major benefit of proteomic and genomic data is the potential for developing thousands of novel diagnostic and analytical tests of cells, tissues, and clinical samples. Monoclonal antibody technologies, phage display and mRNA display, are methods that could be used to generate affinity ligands against each member of the proteome. Increasingly, the challenge is not ligand generation, rather the analysis and affinity rank-ordering of the many ligands generated by these methods. Here, we developed a quantitative method to analyze protein interactions using in vitro translated ligands. In this assay, in vitro translated ligands generate a signal by simultaneously binding to a target immobilized on a magnetic bead and to a sensor surface in a commercial acoustic sensing device. We then normalize the binding of each ligand with its relative translation efficiency in order to rank-order the different ligands. We demonstrate the method with peptides directed against the cancer marker Bcl-x_L. Our method has 4- to 10-fold higher sensitivity, using 100-fold less protein and 5-fold less antibody per sample, as compared directly with ELISA. Additionally, all analysis can be conducted in complex mixtures at physiological ionic strength. Lastly, we demonstrate the ability to use peptides as ultrahigh affinity reagents that function in complex matrices, as would be needed in diagnostic applications

Effects of Kifunensine on Production and N-Glycosylation Modification of Butyrylcholinesterase in a Transgenic Rice Cell Culture Bioreactor.

The production and N-glycosylation of recombinant human butyrylcholinesterase (BChE), a model highly glycosylated therapeutic protein, in a transgenic rice cell suspension culture treated with kifunensine, a strong α-mannosidase I inhibitor, was studied in a 5 L bioreactor. A media exchange was performed at day 7 of cultivation by removing spent sugar-rich medium (NB+S) and adding fresh sugar-free (NB-S) medium to induce the rice α-amylase 3D (RAmy3D) promoter to produce rice recombinant human BChE (rrBChE). Using a 1.25X-concentrated sugar-free medium together with an 80% reduced working volume during the media exchange led to a total active rrBChE production level of 79 ± 2 µg (g FW)-1 or 7.5 ± 0.4 mg L-1 in the presence of kifunensine, which was 1.5-times higher than our previous bioreactor runs using normal sugar-free (NB-S) media with no kifunensine treatment. Importantly, the amount of secreted active rrBChE in culture medium was enhanced in the presence of kifunensine, comprising 44% of the total active rrBChE at day 5 following induction. Coomassie-stained SDS-PAGE gel and Western blot analyses revealed different electrophoretic migration of purified rrBChE bands with and without kifunensine treatment, which was attributed to different N-glycoforms. N-Glycosylation analysis showed substantially increased oligomannose glycans (Man5/6/7/8) in rrBChE treated with kifunensine compared to controls. However, the mass-transfer limitation of kifunensine was likely the major reason for incomplete inhibition of α-mannosidase I in this bioreactor study

Effects of Kifunensine on Production and N-Glycosylation Modification of Butyrylcholinesterase in a Transgenic Rice Cell Culture Bioreactor.

The production and N-glycosylation of recombinant human butyrylcholinesterase (BChE), a model highly glycosylated therapeutic protein, in a transgenic rice cell suspension culture treated with kifunensine, a strong α-mannosidase I inhibitor, was studied in a 5 L bioreactor. A media exchange was performed at day 7 of cultivation by removing spent sugar-rich medium (NB+S) and adding fresh sugar-free (NB-S) medium to induce the rice α-amylase 3D (RAmy3D) promoter to produce rice recombinant human BChE (rrBChE). Using a 1.25X-concentrated sugar-free medium together with an 80% reduced working volume during the media exchange led to a total active rrBChE production level of 79 ± 2 µg (g FW)-1 or 7.5 ± 0.4 mg L-1 in the presence of kifunensine, which was 1.5-times higher than our previous bioreactor runs using normal sugar-free (NB-S) media with no kifunensine treatment. Importantly, the amount of secreted active rrBChE in culture medium was enhanced in the presence of kifunensine, comprising 44% of the total active rrBChE at day 5 following induction. Coomassie-stained SDS-PAGE gel and Western blot analyses revealed different electrophoretic migration of purified rrBChE bands with and without kifunensine treatment, which was attributed to different N-glycoforms. N-Glycosylation analysis showed substantially increased oligomannose glycans (Man5/6/7/8) in rrBChE treated with kifunensine compared to controls. However, the mass-transfer limitation of kifunensine was likely the major reason for incomplete inhibition of α-mannosidase I in this bioreactor study

Purification, characterization, and N-glycosylation of recombinant butyrylcholinesterase from transgenic rice cell suspension cultures.

Recombinant butyrylcholinesterase produced in a metabolically regulated transgenic rice cell culture (rrBChE) was purified to produce a highly pure (95%), active form of enzyme. The developed downstream process uses common manufacturing friendly operations including tangential flow filtration, anion-exchange chromatography, and affinity chromatography to obtain a process recovery of 42% active rrBChE. The purified rrBChE was then characterized to confirm its comparability to the native human form of the molecule (hBChE). The recombinant and native enzyme demonstrated comparable enzymatic behavior and had an identical amino acid sequence. However, rrBChE differs in that it contains plant-type complex N-glycans, including an α-1,3 linked core fucose, and a β-1,2 xylose, and lacking a terminal sialic acid. Despite this difference, rrBChE is demonstrated to be an effective stoichiometric bioscavenger for five different organophosphorous nerve agents in vitro. Together, the efficient downstream processing scheme and functionality of rrBChE confirm its promise as a cost-effective alternative to hBChE for prophylactic and therapeutic use