A Novel Plant Cell Culture Platform For Semicontinous Production Of Recombinant Proteins: Butyrylcholinesterase As A Case Study
Abstract
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.