Production of mouse interleukin-12 is greater in tobacco hairy roots grown in a mist reactor than in an airlift reactor

Abstract

We compared the growth and productivity of a tobacco line of hairy roots that produces murine interleukin 12 (mIL-12) grown in three different culture systems: shake flasks, an airlift reactor, and a scalable mist reactor. Of the total mIL-12 produced by cultures grown in shake flasks ( approximately 434.8 microg L(-1)), almost 21% was recovered from the medium. In contrast to roots harvested from shake flasks and the mist reactor, roots were not uniformly distributed in the airlift reactor. Roots formed a dense ring around the wall of the reactor and surrounding the central rising column of fine aeration bubbles. Root quality was also better in both the shake flasks and mist reactor than in the airlift reactor. There were more pockets of dark roots in the airlift reactor suggesting some of the roots were nutrient starved. Although the best root growth (7 g DW L(-1)) was in the shake flasks, both reactors produced about the same, but less dry mass, nearly 5 g DW L(-1). Total mIL-12 concentration was highest in the mist reactor at 5.3 microg g(-1) FW, but productivity, 31 microg g(-1) FW day(-1) was highest in shake flasks. Roots grown in the mist reactor produced about 49.5% more mIL-12 than roots grown in the airlift reactor. Protease activity in the media increased steadily during culture of the roots in all three systems. The comparisons of protease activity, protein and mIL-12 levels done in the shake flask system suggest that the increase in proteases associated with progression into stationary phase is most detrimental to mIL-12 concentration. This is the first description of the design and operation of a scalable version of a mist bioreactor that uses a plastic bag. This also the first report of reasonable production levels of functional mIL-12, or any protein, produced by hairy roots grown in a mist reactor. Results will prove useful for further optimization and scale-up studies of plant-produced therapeutic proteins.