Bioprocess Engineering Approaches for Production and Purification of Recombinant Plant Made Human Butyrylcholinesterase

摘要

Butyrylcholinesterase (BChE) is a homotetramer glycosylated serum serine hydrolase shown to protect animals against lethal amounts of cholinesterase-inhibiting organophosphate nerve agents. The supply of plasma-derived butyrylcholinesterase (hBChE) is constrained by the availability human blood which contributes to its high cost, more than $10,000/treatment. Limitations such as cost and availability necessitate the development of expression platforms capable of large-scale, low-cost production of a fully active and efficacious recombinant BChE (rBChE). Transient expression in Nicotiana benthamiana plants represents a rapid production platform for recombinant protein therapeutics. In contrast to transgenic plants which take several months for development, production of therapeutic proteins takes place in a week timeframe making this technology very powerful for rapid response.;In this work, different viral expression vectors were tested for efficient rBChE production, and a Tobacco Mosaic Virus (TMV) based expression vector (TRBO) gave a good production level, around 20 mg rBChE/kg FW. The production level was four-fold higher than that came from a Cucumber Mosaic Virus (CMV) based expression vector (CMVar) or expression vector driven by constitutive 25S promoter from Cauliflower Mosaic Virus (CaMV). Kinetics of production of rBChE was evaluated and found that production was peaked at 6 days post infiltration in intact Nicotiana benthamiana plants. O the other hand, two-fold production (40 mg/kg FW) was found when infiltrating harvested leaf tissue and the production increased up to 12 days post infiltration. Different viral gene silencing suppressors were examined to increase production of rBChE and p19 from Tomato Bushy Stunt Virus (TBSV) was found to increase the production of rBChE in harvested leaf tissue by five-fold compared to rBChE levels when no viral gene silencing suppressor was used.;Development of an effective downstream process for purifying rBChE is critical to study its properties. The extraction buffer was optimized to reduce native plant protein extraction while maintaining rBChE activity, and acidic citric buffered saline (pH 4.0) was found to have six-fold less native proteins compared to Tris buffered saline (TBS) extract with a 10 mg/kg FW yield. Tangential flow filtration (TFF) was used to concentrate the extract and an immunoaffinity chromatography based on 3X FLAG tag was used to purify rBChE to homogeneity. Although the protein preparation was pure (>95%) after the immunoaffinity chromatography, the yield was low (<15%) due to proteolytically cleavage. An improved downstream process was designed based on TFF, ion exchange chromatography (DEAE-Sepharose) and Hupresin (a pseudo-affinity resin based on binding to a BChE inhibitor) and yielded highly pure rBChE with a 2.5-fold improvement in yield.;Enzyme kinetics of the pure rBChE were studied at different concentrations of substrate and the data were fitted to a well-known kinetic model for BChE enzyme and found to agree with human BChE. Thermal inactivation was monitored for the pure rBChE over a temperature range of 4°C-95°C. The overall and site-specific N-glycosylation analyses were performed for pure rBChE protein and showed that heterogeneous glycan structures. Oligomerization of the pure rBChE was studied by performing denaturing PAGE under reducing and non-reducing conditions, native non-denaturing PAGE, and Size-Exclusion Chromatography-Multi-Angle Light Scattering (SEC-MALS). In contrast to the hBChE, rBChE was not found to have lower tetramer percentage (∼30%). This work demonstrates that plants are capable to help meet the global demand for the next generation of safe, efficacious and affordable biopharmaceuticals.