No abstract

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.

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.

Producing recombinant proteins in transgenic plant cell suspension cultures in bioreactors provides controllability, reproducibility, scalability, and low‐cost production, although low yields remain the major challenge. The studies on scaling‐up to pilot‐scale bioreactors, especially in conventional stainless‐steel stirred tank bioreactors (STB), to produce recombinant proteins in plant cell suspension cultures are very limited. In this study, we scaled‐up the production of rice recombinant butyrylcholinesterase (rrBChE), a complex hydrolase enzyme that can be used to prophylactically and therapeutically treat against organophosphorus nerve agents and pesticide exposure, from metabolically regulated transgenic rice cell suspension cultures in a 40‐L pilot‐scale STB. Employing cyclical operation together with a simplified‐process operation (controlling gas sparging rate rather than dissolved oxygen and allowing natural sugar depletion) identified in lab‐scale (5 L) bioreactor studies, we found a consistent maximum total active rrBChE production level of 46–58 µg/g fresh weight in four cycles over 82 days of semicontinuous operation. Additionally, maintaining the overall volumetric oxygen mass transfer coefficient (kLa) in the pilot‐scale STB to be equivalent to the lab‐scale STB improves the maximum total active rrBChE production level and the maximum volumetric productivity to 85 µg/g fresh weight and 387 µg L−1 day−1, respectively, which are comparable to the lab‐scale culture. Here, we demonstrate pilot‐scale bioreactor performance using a metabolically regulated transgenic rice cell culture for long‐term, reproducible, and sustained production of rrBChE.

No abstract

Transgenic plant cell suspension culture is a promising platform for recombinant protein production. Rice cell suspension culture is one of the systems that has been developed due to its unique metabolically-regulated promoter, rice alpha-amylase 3D ( RAmy3D ), that is up-regulated in sugar-deprived medium. Using the RAmy3D promoter system in transgenic rice cell suspensions results in two phases of the culture, the growth phase and the induction phase. Conventionally, medium exchange is performed to remove residual sugar and induce recombinant protein. In this work, a simplified production process is demonstrated in a 5-L bioreactor, including reduction of sugar concentration in the initial culture medium, elimination of the media exchange operation, and uncontrolled dissolved oxygen (DO) with constant aeration. The simplified method significantly improves the accumulation level of a recombinant protein, protein purity, and productivity compared to the conventional method. This method also reduces costs associated with material and labor. • The method of simplified bioreactor processing includes single-stage culture, uncontrolled dissolved oxygen (DO) but controlled inlet air flowrate, and lower (50% reduction) initial sucrose concentration in the culture medium. • This method improves recombinant protein production level and productivity compared to the conventional method. • This method reduces material and labor costs.

Biopharmaceutical protein production using transgenic plant cell bioreactor processes offers advantages over microbial and mammalian cell culture platforms due to the ability to produce complex biologics, use of simple chemically-defined, animal component-free media, robustness of host cells, and biosafety. 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 techno-economic models or analyses have been published. Here we present techno-economic 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.

We propose a method for analyzing the variability in smooth, possibly nonlinear, functionals associated with a set of product production trajectories measured under different experimental conditions. The key challenge is to make meaningful inference on these parameters across different experimental conditions when only a limited number of measurements in time are collected for each treatment, and when there are only a few, or no, replicates available. For this purpose we adopt a modeling approach by representing the production trajectories in a B-spline basis, and develop a bootstrap-based inference procedure for the parameters of interest, also accounting for multiple comparisons. The methodology is applied to study two types of quantities of interest - "time to harvest" and "maximal productivity" in the context of an experiment on the production of certain recombinant proteins under laboratory conditions. We complement the findings by extensive numerical experiments that look into the effects of different types of bootstrap procedures and associated schemes for computing p-values for tests of hypotheses.