Semicontinuous bioreactor production of a recombinant human therapeutic protein using a chemically inducible viral amplicon expression system in transgenic plant cell suspension cultures

Abstract

Plant cell culture is an alternative for the production of recombinant human therapeutic proteins because of improved product safety, lower production cost, and capability for eukaryotic post-translational modification. In this study, bioreactor production of recombinant human alpha-1-antitrypsin (rAAT) glycoprotein using a chemically inducible Cucumber mosaic virus (CMV) viral amplicon expression system in transgenic Nicotiana benthamiana cell culture is presented. Optimization of a chemically inducible plant cell culture requires evaluation of effects of timing of induction (TOI) and concentration of inducer (COI) on protein productivity and protein quality (biological functionality). To determine the optimal TOI, the oxygen uptake rate (OUR) of the plant cell culture was chosen as a physiological indicator for inducing maximum rAAT expression. Effects of COI on rAAT production were investigated using a semicontinuous culture, which enables the distinction between effects of growth rate and effects of inducer concentration. An optimized semicontinuous bioreactor operation was further proposed to maximize the recombinant protein production. The results demonstrated that the transgenic plant cells, transformed with the inducible viral amplicon expression system, maintain higher OUR and exhibit lower extracellular protease activity and lower total phenolics concentration in the optimized semicontinuous bioreactor process than in a traditional batch bioreactor operation, resulting in a 25-fold increase in extracellular functional rAAT (603 microg/L) and a higher ratio of functional rAAT to total rAAT (85-90%). Surprisingly, sustained rAAT production and steady state, long-term bioreactor operation is possible following chemical induction and establishment of the viral amplicons.

Therapeutically Important Proteins From In Vitro Plant Tissue Culture Systems

“…Many of the remedial strategies listed in Table 2 for minimising proteolytic digestion inside plant tissues are also beneficial when protein losses occur in the culture medium. Additional techniques based on the optimisation of medium conditions such as pH and rapid or semi/ continuous harvesting of product have also been successful [38,55,82]. The benefits of using hydrophilic/hydrophobic and charged surfaces such as glass, plastic and stainless steel to suppress adsorption depend on the protein characteristics and medium properties [80,83].…”

A transgenic plant cell‐suspension system for expression of epitopes on chimeric Bamboo mosaic virus particles

“…To overcome the pitfalls of the whole‐plant system and the methodological problems growing cells in vitro , plant cell‐suspension culture system provides an alternative approach for conveniently producing recombinant proteins on a large scale (Xu et al ., ). The efficacy of plant viral‐based vectors has been previously demonstrated by expressing recombinant proteins in a cell‐suspension culture (Dohi et al ., ; Hefferon and Fan, ; Huang et al ., , ; Larsen and Curtis, ; Zhang and Mason, ). Cell‐suspension culture has several advantages over whole‐plant systems, such as high reproducibility and simple maintenance of aseptic conditions (Hellwig et al ., ; Mustafa et al ., ).…”

Evaluation of different modes of operation for the production ofSpirulinasp.

“…Semi‐continuous cultivations have several advantages compared with batch and continuous processes. Semi‐continuous processes involve periodically replacing part of the microalgae culture medium with fresh culture medium (dilution) . Such cultivation methods can be used for larger scale biomass production while maintaining a high microorganism growth rate.…”