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

ARTICLE

Production of Mouse Interleukin-12 Is Greater inTobacco Hairy Roots Grown in a Mist ReactorThan in an Airlift Reactor

Chunzhao Liu,1,2 Melissa J. Towler,3 Giuliana Medrano,1 Carole L. Cramer,1

Pamela J. Weathers1,3

1Arkansas Biosciences Institute, Arkansas State University, State University,

Arkansas 72467-0639; telephone: 508-831-5196; fax: 508-831-5936; e-mail: [email protected] Key Laboratory of Biochemical Engineering, Institute of Process Engineering,

Chinese Academy of Sciences, Beijing, PR China3Worcester Polytechnic Institute, Worcester, Massachusetts 01609

Received 12 June 2008; revision received 23 September 2008; accepted 23 September 2008

Published online 29 September 2008 in Wiley InterScience (www.interscience.wiley.c
om). DOI 10.1002/bit.22154
ABSTRACT: We compared the growth and productivity of atobacco line of hairy roots that produces murine interleukin12 (mIL-12) grown in three different culture systems: shakeflasks, an airlift reactor, and a scalable mist reactor. Of thetotal mIL-12 produced by cultures grown in shake flasks(�434.8 mg L�1), almost 21% was recovered from themedium. In contrast to roots harvested from shake flasksand the mist reactor, roots were not uniformly distributed inthe airlift reactor. Roots formed a dense ring around the wallof the reactor and surrounding the central rising column offine aeration bubbles. Root quality was also better in boththe shake flasks and mist reactor than in the airlift reactor.There were more pockets of dark roots in the airlift reactorsuggesting some of the roots were nutrient starved. Althoughthe 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 highestin the mist reactor at 5.3 mg g�1 FW, but productivity,31 mg g�1 FW day�1 was highest in shake flasks. Roots grownin the mist reactor produced about 49.5% more mIL-12 thanroots grown in the airlift reactor. Protease activity in themedia increased steadily during culture of the roots in allthree systems. The comparisons of protease activity, proteinand mIL-12 levels done in the shake flask system suggest thatthe increase in proteases associated with progression intostationary phase is most detrimental to mIL-12 concentra-tion. This is the first description of the design and operationof a scalable version of a mist bioreactor that uses a plasticbag. This also the first report of reasonable production levelsof functional mIL-12, or any protein, produced by hairyroots grown in a mist reactor. Results will prove useful forfurther optimization and scale-up studies of plant-producedtherapeutic proteins.

Biotechnol. Bioeng. 2009;102: 1074–1086.

� 2008 Wiley Periodicals, Inc.

Correspondence to: P.J. Weathers

Contract grant sponsor: Arkansas Biosciences Institute

1074 Biotechnology and Bioengineering, Vol. 102, No. 4, March 1, 2009

KEYWORDS: mist reactor; interleukin; tobacco; airliftreactor; cytokine

Introduction

Plants offer a low cost approach for the production ofcomplex foreign proteins including industrial enzymes,therapeutic enzymes and proteins, vaccine antigens, andimmuno-modulators (Fisher et al., 2004; Sharp and Doran,2001). Additional benefits include the ability of plants toprovide complex eukaryotic protein processing, a reducedrisk of contamination by animal and human pathogens andviruses, and the potential of plants to meet large scaleproduction needs without the initial capitalization costs ofmammalian cell culture-based bioproduction (Cramer et al.,1999; Fisher et al., 2004). In addition to whole plants, invitro transformed plant cells and tissues grown in bio-reactors offer a highly controlled environment for increasingproduct yield, quality and consistency, especially for highvalue proteins, and may be the only reasonable method forproducing some products (Doran 2000).

The type of bioreactor used for culturing plant cells andtissues can affect production of plant-produced transgenicproteins. A key consideration of plant-based production ofany product in bioreactors is product accumulation in boththe plant cells or tissues and the surrounding culturemedium. Whereas secretion of product into the culturemedium offers putative ease of purification, yields arejeopardized by the presence of proteases and adsorption toculture vessel walls (Doran 2006).

� 2008 Wiley Periodicals, Inc.

Hairy roots offer a unique fast growing plant system forproduction of both small molecules and proteins (Flores andMedina-Bolivar, 1995; Wongsamuth and Doran, 1997;Zhang et al., 2005). In addition, the large scale culture oflarge amounts of hairy root biomass has still been onlymarginally successful for scaling up to 10,000 L mainlybecause these large scale liquid systems have not beenparticularly productive in terms of biomass L�1. Indeed thelargest reported systems are airlift ‘‘balloon’’ shaped bubblereactors (10,000 L) that at the more productive smaller scale(3 L working volume) are reported to produce at best about180 g FW L�1 (Sivakumar et al., 2005). When these arescaled up to 1,000 L the best reported yields are those ofChoi et al. (2006) at 108 g L�1. We define this as a packingfraction, a¼ 0.108 (0.11), where a is g FW L�1 (Wyslouzilet al., 1997). For most biological processes, either theproductivity of the end product, for example, a therapeuticprotein, must be extremely high, or a lot more biomass isneeded, �50% of the working volume of the reactor, ora� 0.5 for cost effective production.

Higher yields of root biomass can be achieved in gas-phase reactors (Huang and Chou, 2006; Ramakrishnan andCurtis, 2004) and scaling efforts have been successful upto 14 L with a final a¼ 0.75 after 25 days of culture(Ramakrishnan and Curtis, 2004). This was only achieved,however, if the reactor was initiated in a liquid bubble mode,and then shifted to a trickle bed mode for the next 8–9 daysto enhance aeration. Oxygen enrichment the last 5 days ofculture was required to obtain the final high yield ofbiomass.

Most of the work with hairy roots in reactors has focusedon secondary metabolites and not proteins. Proteins offeradditional challenges with the need to oxygenate roots inliquid adding shear forces that can degrade secretedproteins. Use of a gas-phase reactor like a mist reactor(Liu et al., 1999; Towler et al., 2006; Weathers and Giles,1988; Weathers and Wyslouzil, 2000) may help to alleviateexternal degradation of any secreted protein while stillenhancing biomass yield.

In the current studies, we have used transgenic tobaccolines that express a mammalian immunomodulator,interleukin 12 (IL-12), a complex 70 kDa heterodimeric(p35 and p40 subunits) glycoprotein cytokine that hasproved challenging to produce. Previous reports ofbioproduction of mammalian IL-12 in plants (Gutierrez-Ortega et al., 2004, 2005; Kwon et al., 2003) yielded IL-12 atvery low levels and demonstrated only partial IL-12 activity.In contrast, the Cramer/Dolan laboratory has developedtobacco lines that yielded high levels of murine IL-12 (mIL-12) that show equivalent bioactivity to animal cell-derivedmIL-12 in both in vitro assays and in mouse vaccinationstudies (Liu, 2006; Liu et al., 2008). Some of their highestproducing tobacco lines were further transformed withAgrobacterium rhizogenes to produce hairy roots that alsoproduced IL-12 at high levels (Liu, 2006; Liu et al., 2008).

Here we compare the growth and productivity of atobacco line of hairy roots that produces mIL-12 when

grown in three different culture systems: shake flasks, anairlift reactor, and a mist reactor. This is the first report ofreasonable production levels of functional mIL-12, or anyprotein, produced by hairy roots grown in a mist reactor.

Materials and Methods

Clone Origin and Culture Conditions

Hairy roots were established using A. rhizogenes ATCC15834 to transform Nicotiana tabacum cv. Xanthi plantsexpressing high levels of mIL-12 as previously described(Liu et al., 2008). The transgene expressed in this lineconsisted of the double-enhanced 35S promoter (Lam et al.,1989), a TEV translational enhancer (Carrington and Freed,1990), sequences encoding a single-chain form of murine IL-12 (IL-12p40 subunit:(Gly3Ser)4 linker: Dp35 subunit;Lieschke et al., 1997; provided by R. Mulligan, HarvardMedical School), and a pAnos terminator provided by thetransformation/expression vector pBIB-Kan (Becker, 1990).Roots were maintained in 250 mL Erlenmeyer flaskscontaining 50 mL Gamborg’s B5 medium (Gamborget al., 1968) with 3% (w/v) sucrose, pH 5.8, on a rotaryshaker at 100 rpm under continuous cool white fluorescentroom lights at <2 mmol m�2 s�1, and 23–258C. All shakeflask experiments were conducted using these conditionswith inoculum at 8 g FW L�1.

Airlift Reactor System

An airlift reactor (Shibata Hario, Tokyo, Japan) filled with2 L of B5 medium and 30 g L�1 sucrose and inoculated with16 g fresh weight of the tobacco hairy roots was used formIL-12 production. Humidified air was passed through a0.2 mm Whatman filter (Fisher Scientific, Pittsburgh, PA)and blown into the medium at a rate of 0.2 L min�1. Thebioreactor culture was maintained at 258C for 14 days.

Mist Reactor System

The mist reactor used in these studies is a scalable redesignbased on earlier versions (Chatterjee et al., 1997; Kim et al.,2001; Towler et al., 2007). The redesigned nutrient mistbioreactor (Fig. 1) now has a flexible, inexpensive, trans-parent growth chamber (3 mil autoclaveable plastic culturebag), along with a SonoTek Broad Band UltrasonicGenerator (06-05108) with an ultrasonic atomizing nozzlewith a conical tip (8700-120), Velcro constriction band, gasinlet, medium reservoir, timer, and peristaltic pump. Theflexible culture bag portion of this bioreactor system isconstructed from either transparent polypropylene sheets orlarge transparent autoclave bags (VWR, Cat# 14220-042)cut to the desired dimensions (29 cm� 65 cm), withprepared areas for the insertion of liquid and gas entry andexit ports (Fig. 1). Culture bag side seams are double heat-sealed and the liquid inlet, the excised top of a 1 gal

Liu et al.: IL-12 From Roots Grown in a Mist Reactor 1075

Biotechnology and Bioengineering

Figure 1. Mist reactor with 4 L working volume culture bag. Left: Photo of reactor layout. Right: Schematic of reactor system.

polypropylene Nalgene bottle (Fisher Scientific, Cat#6-443B) with shoulder in place, is inserted into the bag (Fig. 1).The culture bag is kept attached to the bottleneck using 1 or2 silicone O-rings (#039, McMaster Carr, Atlanta, GA)which grip into the outer screw threads. A silicone O-ringinside the Nalgene bottle cap seals the cap to the excisedthreaded shouldered bottle top that has the bag connected toit. The bottom of the culture bag is then heat-sealed. Theliquid exit port is inserted and sealed with gaskets into thebottom of the culture bag (Fig. 1). A port for inlet gas andanother for the sonic misting head is cut into the Nalgenebottle cap and the mister unit is inserted and sealed withanother O-ring between the cap and the mister. The outerportion of the mister unit is sealed to the cap with aquariumsafe silicone adhesive. The Velcro constriction band isrequired to artificially compact the growth chamber toenhance capture of mist droplets as predicted by the nutrientmist deposition model for hairy root culture developed byWyslouzil et al. (1997) and recently empirically verified byTowler et al. (2007). The minimum restriction is 1.1 cm, butas roots grow the constriction is opened thereby enabling theageotropic roots to fill the bag above and below the Velcroband. Root inoculum is seated inside the culture bag justabove the Velcro constriction, which is about half way up the1–4 L working volume of the bag. Although the reactor bagcan accommodate up to a 4 L root bed, for this study, theworking root bed volume was 2 L. For purposes ofvolumetric productivity calculations, the working liquid


volume of the reactor was taken equal to the 2 L recirculatedvolume of culture medium.

The mister head is not autoclaveable and thus, is sterilizedas follows. Briefly, the mister head is washed with tap waterto remove any large debris, dried at 608C, wrapped inaluminum foil, and heated again at 608C for 12 h. Followingthis heating step, the mister head is heated at 1158C for 2 h.Before being inserted into the mist bioreactor, the misterhead is cooled in a laminar flow hood. The Weathers lab hasalready demonstrated that this protocol is effective bysuccessfully conducting >30 mist reactor runs of hairy rootsthat have remained axenic until harvest for up to 40-dayculture runs (unpublished results).

Assays

Fresh mass was measured immediately after root harvest anddry mass was obtained after drying the roots at 608C for 48 h.Conductivity and pH were measured in media from culturesthat were harvested every few days using a seven easyconductivity (Mettler-Toledo, Columbus, OH) and pH(Oakton Instruments, Vernon Hills, IL) meters. Nitrate,ammonium, phosphate and residual sugars were assayedusing the methods described by Kim et al. (2003), Towleret al. (2007), and Dubois et al. (1956). Total soluble proteinin the biomass and medium was measured using aCoomassie Protein Assay Kit (Pierce, Rockford, IL) with

bovine serum albumin as the standard. Protease activity inthe medium was measured using the method of Studdertet al. (1997) with modifications. A 600-mL aliquot ofmedium was mixed with 400 mL of 3% azocasein (Sigma–Aldrich Chemical, St. Louis, MO) solution in sterile PBSbuffer (content in g L�1: NaCl 8, KCl 0.2, Na2HPO4

1.44, KH2PO4 0.24) and incubated for 1 day at 258C. Thereaction was stopped by addition of 500 mL of cold 20% (w/v) trichloroacetic acid (Sigma–Aldrich Chemical). Thesamples were left at 48C for 60 min and then centrifuged at14,000g for 10 min. The supernatant absorbance was read at340 nm. One unit of protease activity (U) was defined as theamount of enzyme that produced an increase of 1 in opticaldensity per day under the above conditions.

The amount of mIL-12 was measured in roots and rootculture medium with a heterodimer-specific mIL-12enzyme-linked immunoabsorbant assay (ELISA) (R&DSystems, Minneapolis, MN) described in detail in Liuet al. (2008). To assay mIL-12 in root tissue, roots wereground in extraction buffer (100 mM Tris base, 100 mMascorbate, 150 mM NaCl, 4 mM EDTA, 2.5% PVP-40, 0.1%Tween 20, pH 7.0) at a 1:2 (w/v) ratio and cell-free extractswere analyzed by the ELISA. To assay mIL-12 in the culturemedium, the collected medium samples from the timecourse of root culture were analyzed by the ELISA. ThisELISA used antibodies specific for mIL-12 p70 product (doesnot detect disassembled IL-12 subunits) and thus providesquantification of conformationally intact bioactive mIL-12(Liu et al., 2008). For Western immunoblots, proteins frommedia fractions were resolved on a NuPAGE 10% Bis-Trisgel (Invitrogen, Carlsbad, CA) under reducing conditions(50 mM DTT) and blotted onto 0.2 mm nitrocellulosemembranes (Bio-Rad, Hercules, CA). For immunoblotdetection of the p70 heterodimer and the p40 and p35subunits, the primary antibody used was a polyclonalgoat anti-mouse IL-12 antibody (R&D Systems) at 1:5,000dilution; alkaline phosphatase-conjugated rabbit anti-goatIgG (Sigma–Aldrich Chemical) served as the secondarydetection antibody. Detection was carried out using theCDP-Star (Roche, Nutley, NJ) and Nitroblock II (TROPIX,Bedford, MA) system in accordance with manufacturers’procedures. For control plant-derived mIL-12, the cytokinewas used after purification from leaves of Nicotianabenthamiana transiently expressing the mIL-12 gene cons-truct (Medrano et al., in press). This transient expressionsystem yields abundant plant-made mIL-12 in leavesharvested 67 h after vector infiltrations and thus providesa profile of mIL-12 p70 and p40 distribution consideredrepresentative of product within the tissue (i.e., time fordegradation is minimized).

Effects of Ultrasonics Versus Spargingon b-Galactosidase Activity

A solution of b-galactosidase (Sigma–Aldrich Chemical)in Z-buffer (0.06M Na2HPO4�7H2O, 0.04 M

NaH2PO4�H2O, 0.01M KCl, 0.001 M MgSO4, 0.05 M b-mercaptoethanol, pH 7.0), at 1 mg mL�1 (11.2 unitsmg�1 ¼ 0.0112 units mL�1), pH 7.0 was completelyrecycled, twice, through the SonoTek ultrasonic mistinghead at power settings ranging from 0 to 4.5 W. Afterentirely passing the enzyme solution (50 mL, at25 mL min�1) through the misting head twice at one powersetting, the power was increased by 0.5 watts and the processrepeated with the same solution of enzyme. The spargedcontrol was 50 mL b-galactosidase solution in a large glasstest tube (ID¼ 3.4 cm) bubbled with 800 mL min�1 roomair through a sintered metal sparger. The stirred control was48 mL b-galactosidase solution in a 100 mL glass beaker on astir plate. b-galactosidase activity was assayed using themethod of Miller (1972).

Statistical Analysis

Experiments and assays were run at least three times exceptfor the bioreactors that were each run twice. Data wereaveraged and experimental variation shown as standarderror.

Results

Before growing mIL-12 producing roots in the bioreactors,both growth and mIL-12 productivity in shake flasks weremeasured. This gave a basis for comparison with growth andmIL-12 production in the bioreactors. Media constituentswere also measured including pH, total sugars, nitrate,ammonium, phosphate, and conductivity. These data willfacilitate scaling beyond the 2 L reactor scale that was used inthis study.

Growth, mIL-12 Production and Nutrient ConsumptionKinetics of N. tabacum Hairy Roots in Shake Flasks

Hairy root cultures of N. tabacum were initiated with8 g FW L�1 and grown in 250 mL shake flasks for 16 days.Shake flask cultures (3 flasks per time point) were harvestedevery 2–3 days for collection of roots and media. Rootsbegan log growth at about 6 days, growing rapidly untilday 12 at which time growth ceased. Dry mass of the rootsincreased from 0.45 g L�1 to a final DW of about 7 g L�1 withbiomass productivity of 5.74 g FW L�1 day�1 (Fig. 2,Table I). The average specific growth rate, m, was 0.19 day�1.

The level of mIL-12 was measured in both the culturemedium and in the roots (Fig. 3). Most of the product(�360 mg L�1) was produced and remained within theroots; maximum concentration was at day 14 (Fig. 3). Of thetotal mIL-12 produced by the culture (�435 mg L�1),however, almost 21% of the mIL-12 was recovered from themedia (Fig. 3). Although the maximum extracellular con-centration was achieved at day 12, the maximum rate ofproduction was between days 6 and 9 and this correlated to



Figure 2. Growth kinetics of hairy roots of Nicotiana tabacum cv. Xanthi in

shake flasks.Figure 3. Kinetics of mIL-12 production by N. tabacum hairy roots in shake

flasks over 16 days.

mid-log phase of growth for both secreted and root retainedmIL-12. After day 14, the intracellular mIL-12 decreased.Recovery of the extracellular mIL-12, however, began todecrease after day 12, suggesting that entry into stationaryphase impacts both mIL-12 production and mIL-12stability.

Total soluble protein concentration in the roots wasinitially high at �900 mg g�1 FW at day 3, but decreased bynearly 50% by day 14 (Fig. 4). Protein secretion into themedium began later at day 6 and increased until day 16 to�30 mg L�1. The mIL-12 content of the roots representedabout 0.7% (w/w) of total soluble protein at day 14, the

Table I. Comparisons of some yields of plant produced IL-12.

Transformed plant

species

Production

tissue or cells

IL-12

type

IL-12 yield

(mg g�1 FW)

IL-12

producti

(mg L�1 da

N. tabacum cv.

Havana

Cell suspensions Human �3.1a 35 without

ND 140 with g

Lycopersicon

esculentum

cv. Tanksley

Plant leaves 7.3 NA

Fruit Murine 3.4 NA

Nicotiana tabacum

cv. Xanthii

Plant leaves Human 0.04 NA

Plant leaves Murine 25–40 NA

Hairy roots 33b ND

Hairy roots

after 14 days

Shake flasks Murine 4.9 31

Airlift reactor 3.8 14

Mist reactor 5.3 22

ND, not determined; NA, not applicable to whole plant field production.aCalculated from DW data in Kwon et al. (2003) with a DW assumption obThese high levels of mIL-12 in the N. tabacum hairy roots were not sustai


point at which the mIL-12 was highest in the roots (Figs. 3and 4). On the other hand, the highest content of mIL-12 inthe medium was 0.5% of the extracellular protein, butpeaked instead at day 12.

The culture media was analyzed for pH, total sugars,nitrate, ammonium, phosphate, and conductivity (Fig. 5).Conductivity dropped with root growth (Fig. 5A) andshowed an inverse linear correlation with biomass (Fig. 5B).As expected the pH rose slightly during the 16 days ofgrowth (Fig. 5C). When the main N, P, and C nutrients weremeasured in the culture medium, it was observed thatammonium dropped rapidly almost to zero by day 6, while

vity

y�1)

Growth yield

(g FW L�1)

Biomass

productivity

(g FW L�1 day�1) Final a References

gelatin ND ND ND Kwon et al. (2003)

elatin

NA NA NA Gutierrez-Ortega

et al. (2005)

NA NA NA Gutierrez-Ortega

et al. (2004)

NA NA NA Liu et al. (2008)

ND ND ND

80.4 5.74 0.08 This study

47.3 3.37 0.05

67.8 4.84 0.07

f 10% of FW. Data taken from day 5 when IL-12 was at its highest yield.ned beyond the first few subcultures.

Figure 4. Total protein production in roots and culture medium of N. tabacum

hairy roots grown in shake flasks.

nitrate dropped slower and was not depleted until betweendays 12 and 14 (Fig. 5D). Interestingly ammonium depletioncorresponds to the peak in mIL-12 secretion while nitratedepletion correlates with the peak of mIL-12 in the roots.Phosphate also dropped rapidly and was no longer

Figure 5. Conductivity, pH, and nutrient composition of medium from shake flask cu

conductivity to biomass; (C) pH; (D) residual sugars, KNO3, (NH4)2SO4, and NaH2PO4 H2O.

detectable at about day 9. The sugar concentration decreasedsteadily until about day 14; this point correlates with thedepletion of all the nitrogen and phosphate in the mediumsuggesting that carbon uptake halted once these otheressential nutrients were exhausted (Fig. 5D). Root growthalso ceased between days 12 and 14 (Fig. 2).

Root Growth and mIL-12 Production in the Airlift andthe Mist Reactors

The hairy roots were cultivated in the 2 L bag mist bio-reactor, as well as in a 2 L airlift bioreactor and 250-mL shakeflasks, in order to evaluate the performance of root growthand mIL-12 production in the bag mist bioreactor (Fig. 6).In contrast to roots harvested from shake flasks and the mistreactor, roots were not uniformly distributed in the airliftreactor (Fig. 6; compare B and H to E). Roots formed adense ring around the wall of the reactor and surroundingthe central rising column of fine aeration bubbles. Rootquality was also better in both the shake flasks and mistreactor than in the airlift reactor (Fig. 6; compare C and Ito F). There were more pockets of dark, likely phenolicroots in the airlift reactor suggesting some regions of thebiomass were nutrient starved. Although the best root

ltures of N. tabacum mIL-12 producing hairy roots. A: conductivity; (B) relationship of



Figure 6. A view of root cultures from the three different culture systems after 14 days. Both reactors were 2 L working volume; shake flasks each contained 50 mL.

growth (7 g DW L�1) was in the shake flasks, both reactorsproduced about the same amount of dry mass, nearly5 g DW L�1 (Fig. 7). Biomass productivity was 3.37 and4.84 g FW L�1 day�1 in the airlift and the mist reactors,

Figure 7. Comparison of growth and mIL-12 production in the three culture

systems. Biomass yields of N. tabacum hairy roots cultured in shake flasks, and in the

2 L airlift reactor, and the 2 L mist reactor are presented as g dry weight per L. The

mIL-12 levels in root mass and culture media were determined by ELISA.


respectively (Table I). The total mIL-12 concentration wasalso highest in shake flasks. In the reactors, however, rootsgrown in the mist reactor produced about 49.5% more mIL-12 per liter than roots grown in the airlift reactor (Fig. 7).

Protein Concentration in the Medium andProtease Activity

One of the concerns with secreted proteins is the presence ofdamaging proteases in the culture medium of plant cultures.Protease activity was therefore measured in the culturemedium to determine how it might be affecting the level ofextracellular mIL-12. In shake flask cultures, total proteaseactivity in the media increased steadily during log-phasegrowth, with accelerated accumulation of proteases in themedia observed during the stationary phase (Fig. 8A).Comparison of mIL-12 levels and protease activity measuredin the media suggest that increases in extracellular proteasesassociated with stationary phase are detrimental to con-centrations of mIL-12. Media ‘‘steady state’’ levels of mIL-12reflect both synthesis/secretion and degradation and thusthe decline in mIL-12 levels between days 12 and 16 may alsoreflect a decrease in mIL-12 synthesis as root growth ceases(impacts of cell growth on the 35S promoter used to drive

Figure 8. Relationship of protease activity and extracellular mIL-12 concentra-

tion and quality. A: Levels of protease activity are compared to IL-12 concentrations in

medium from shake flask cultures (data also presented in Fig. 3); dashed lines bracket

the log phase determined in Figures 2 and 5A. B: Western blot of proteins from medium

of mIL-12-expressing hairy root cultures from airlift, flask, mist cultures at day 14.

Proteins were resolved by PAGE under reducing conditions, transferred to nitrocel-

lulose membrane, and visualized using a polyclonal anti-mIL-12 antibody. Purified

plant-derived mIl-12 was used as a positive control. The positions of the two bands 70

and 40 kDa (corresponding to mIL-12 p70 full length and p40 subunits, respectively) are

indicated on the left.

mIL-12 in hairy roots is not known). Together these datasuggested that cultures should be harvested at day 12 (i.e.,prior to onset of stationary phase) to obtain maximumconcentrations of secreted mIL-12.

The extracellular protease activity in shake flask cultureswas compared to that measured in media recovered at finalharvest (day 14) of the airlift and mist reactor runs. Theprotease activity levels in media (day 14) from the airlift andmist reactors averaged 264 and 356 U L�1, respectively.These levels were comparable to protease activity in shakeflask culture media (Fig. 8A), which were 357 U L�1 atday 14 (480 U L�1 at day 16). To assess whether proteaseactivity or other culture conditions significantly impactedmIL-12 quality, we compared mIL-12 from media of thefinal harvest of shake flasks and the two bioreactor systemsby western immunoblot analysis (Fig. 8B). As described in

Liu et al. (2008), the mIL-12 products recovered from rootor leaves expressing the single-chain mIL-12 construct asvisualized on Western blots comprise a 70 kDa band whenresolved under non-reducing conditions and both a p70form (full-length product) and p40 bands under reducingconditions. Subsequent analyses demonstrated that thelinker between the p40 and p35 subunits is susceptible tocleavage in tobacco but that the subunits are linked by adisulphide bond and retain activity (Liu et al., 2008). Wetherefore visualized mIL-12 products from media of thethree production systems using PAGE under reducingconditions and compared them to purified tobacco-derivedmIL-12 produced in a transient expression system(Medrano et al., in press). The p70 product is present inthe media of all three culture systems and the ratio of p40–p70 is comparable (Fig. 8B) suggesting that, although overallmIL-12 concentration in the media of these systems differs,the quality of the product is similar.

Effects of Sparging and Ultrasonics on Protein Activity

Proteins readily denature at the interface between gas andliquid in culture media and the airlift reactor spargingsystem creates a lot of small bubbles. In contrast, little isknown about how a flow-through ultrasonics system affectsprotein stability, so some simple experiments were done todetermine how each system might affect protein stability.

First the ELISA assay was used to compare the effect of airsparging to sonic misting on the mIL-12 in spent culturemedium that also contained small bits of roots. After 4 hthere was a 43% decrease in mIL-12 in the sparged media,but the mIL-12 that was repeatedly passed through the sonicmister (2 min on, 28 min off) actually increased 28%.Control medium that remained in a beaker on the benchduring this test also increased slightly in mIL-12 content by14%. These increases were attributed to enhanced extractionof intracellular mIL-12 from the root material remaining inthe medium.

Because these simple tests used complex media thatcontained proteases, mIL-12, and other undefined mediaconstituents as well as nutrients, it was decided to use a wellstudied model enzyme, b-galactosidase, and measure itsactivity in buffer over 24 h in response to the threeaforementioned treatments. After 24 h, the activity of b-galactosidase remained rather constant in the ultrasonicallytreated solution declining by <5%. The activity of both thestirred and sparged enzyme solutions declined 20% and27%, respectively (data not shown). Although these resultsmight suggest that the enzyme solution is somewhat stableto ultrasonics, this experiment was designed to mimic theactual misting cycle used in the reactor runs and did notexpose the protein solution to much ultrasonic energy. Onlya small proportion, 50 mL, of a 1 L solution is passedthrough the misting head every time the mister is turned on(25 mL min�1 for 2 min), so misting for 24 h �2,400 mL oftotal volumetric throughput. The enzyme is on average,



thus, exposed to ultrasonics only 2.4 times each day. A b-galactosidase solution was further subjected to a moreintensive exposure to ultrasonic energy while also determin-ing the effect of increasing the power input by passing asmaller volume of the enzyme solution through the mistinghead twice, but at five successive and increasing powersettings. Neither the power intensity nor repeated passageof the solution through the misting head decreased b-galactosidase activity (data not shown). These experimentssuggested that ultrasonics probably does not adversely affectthe stability of proteins in culture media in the mist reactor,a conclusion further substantiated by the Western analysis(Fig. 8B).

Discussion

In this comparison of growth and mIL-12 production intobacco hairy roots, for the first time the design andoperation of a scalable version of a mist bioreactor that usesa plastic bag is described. The utility of this system has alsobeen demonstrated for production of a mIL-12 cytokineproduced using hairy roots grown in three culture systems:shake flasks, an airlift reactor, and a mist reactor.

Earlier work with the mist reactor showed the benefits ofgrowing hairy roots in mist compared to other reactors:healthier roots than in liquid systems (Towler et al., 2006,2007; Wyslouzil et al., 2000), higher yields of secondaryproducts (Kim et al., 2001), no oxygen limitations even indensely packed root beds (Weathers et al., 1999), and growthrates equal to or greater than in shake flasks (Towler et al.,2007). Serious limitations, however, existed: designs wereprone to breakdown, all were not scalable, droplet deliverywas tightly coupled to gas flow rate, and volumetricthroughput was low (Table II). In addition for growthchamber volumes much beyond 0.1 L, a supporting trelliswas required to obtain reasonably high growth rates; thereactor was best operated in a hybrid mode beginning inliquid until roots attached to the trellis and initiated goodgrowth after which misting commenced (Kim et al., 2002,2003). The newer design described in this report hasminimized or eliminated the earlier problems.

The mist bioreactor functions nonintuitively comparedto traditional liquid-based reactors; better growth isachieved in dense root beds versus loosely packed beds(Towler et al., 2007). Because beds of roots act like fibrousfilters in catching mist droplets, mist droplet size must besmall (�10 mm diameter) to penetrate dense root beds foradequate mass transfer of nutrients into roots growing in theinterior of the bed. When the droplet size is too large, then asthe density of the root bed mass (a) increases, O2 becomeslimiting and either growth slows, or the air supply must beincreased or enriched with O2 (Weathers et al., 2008). Incontrast, if the droplet size is small, but a is too low (Kimet al., 2002; Towler et al., 2007), then the growth rate is poorbecause the roots are not capturing an adequate amount ofdroplets and thus, liquid nutrients. Keeping these critical


features of the mist deposition model in mind, a morescalable mist bioreactor was designed and constructed asdescribed here. Although the mist droplet diameter isslightly greater in the current design compared to earlierversions, comparable growth rates were obtained for theYUT16 clone of Artemisia annua grown for the same time inthe same medium which suggested that the slight increase inaverage droplet size may not be detrimental (Table II).

Preliminary studies showed that the headspace distancebetween root bed and mister must be at least 20 cm to ensureuniform media distribution (data not shown). Furthermore,physical testing of the Sono Tek misting head showed that aspray diameter of up to 50 cm is attainable, yielding a liquiddelivery of about 2 L cm�1 of depth. Gas and liquid deliveryare also no longer coupled to one another in the new designso vvm is easily altered and independent of liquid flow rate.Liquid throughput via the new misting head is up to 4 L h�1

thereby enabling considerable volumetric scalability beyondearlier mist reactor designs. Studies related to the scale-up ofthis new mist reactor will be summarized in a later report.

Use of a plastic bag as a root growth chamber builds onconcepts conceived by others (Hsiao et al., 1999; Medina-Bolivar and Cramer, 2004) and enables creation of anartificially dense root bed inoculum which increases theprobability of droplet capture and enhanced root growth.The Velcro constriction further provides not only a seat forthe inoculum, but also aids in funneling the bag wall therebyclustering the roots to enhance droplet capture. Moredensely clustered roots act as a fibrous filter catching mediadroplets. The more media droplets caught, the better thegrowth per the study using the mini mist reactor in Towleret al. (2007). The aspect ratio of the fully inflated bag is 1.3,and the minimum constriction diameter at the Velcro neckis 1.1 cm in order to maintain Re< 10 so the mist depositionmodel is not violated (Wyslouzil et al., 1997). The Velcroconstriction also funnels the passage of mist through theroot mass, not around it, thereby permitting capture ofenough media to sustain a high growth rate.

Root growth in the three culture systems was comparableto that of other reported protein-producing tobacco hairyroots grown in shake flasks and airlift reactors (Table I).Although low packing densities were reported for all thesystems in Table I, higher biomass yields and growth ratesshould be achievable after optimization. For example, priorwork with A. annua hairy roots showed that increasing thecarbon content of the medium from 3% to 5% significantlyincreased growth rates of hairy roots in a mist reactor(Towler et al., 2007). Likewise, optimization of inorganicnutrients, especially nitrogen needed for over-productionof transgenic proteins, should also significantly improvegrowth.

Our research has focused on gas-phase (mist) reactors,because this environment reduces the gas-exchange limita-tions and the high shear conditions normally found inliquid-phase reactors. Unlike growth in liquid systems, rootsgrown in mist reactors are not oxygen limited (Weatherset al., 1999) even at high bed densities (Kim et al., 2001), and

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the production of secondary metabolites is often greater inmist reactors than in liquid phase reactors (Bais et al., 2002;Kim et al., 2001). The concentration of intracellular mIL-12(4.7 mg g FW�1) in the medium of roots grown in the lowcost gas-phase mist reactor was >60% greater than that(2.9 mg g FW�1) in the liquid-phase airlift reactor. Althoughsome dark probably phenolic containing roots wereobserved in the center part of the low-density portion ofthe root beds harvested from the liquid-phase airlift reactor,none was found in the high-density portion of the root bedsfrom the mist bioreactor. This indicated that roots in theairlift reactor were likely oxygen limited, and this may havelimited mIL-12 biosynthesis. These results further demon-strate that a gas-phase mist reactor not only offers significantadvantages over a liquid-phase reactor for production ofsecondary metabolites, but also for production of engi-neered proteins from hairy root cultures.

The maximum concentration for a human IL-12 (hIL-12)produced in tobacco cell suspensions was about 3.1 mg g�1

FW after 5 days of cell culture and corresponded to a midloggrowth phase. More than 80% of hIL-12 was secreted intothe medium with less than 20% remaining in the cells (Kwonet al. 2003). Although the cells continued vigorous growth,the concentration of total and secreted concentration of IL-12 dropped precipitously to about 10 mg g�1 FW, while theintracellular levels increased to about 55 mg g�1 FW at day11, well into stationary phase. The concentration of mIL-12in the media of shake flasks, airlift reactor, and mist reactorwas 434, 196, and 308 mg L�1, respectively. Compared toother plant production systems (see review by Curtis, 2006),these are still rather low. For example, a-interferon has beenproduced in Lemna at 600 mg L�1, while a number ofantibodies have been reported to be produced by tobacco atconcentrations ranging from 1.6 to 200 mg L�1 (Curtis,2006). The highest transgenic plant-produced protein todate is still likely the B.t. insecticidal protein at 46% of totalsoluble protein (DeCosa et al., 2001). Again optimization ofthe N. tabacum culture will surely lead to higher mIL-12production.

Gelatin (Kwon et al., 2003; Lee et al., 2002) and otherprotease limiting constituents added to the mediumincluding BSA (James et al., 2000), bacitracin (Sharp andDoran, 1999), and PVP (Liu and Lee, 1999) have beenproposed to act as substrates for protease thereby limitingdamage to the desired secreted protein product. Theaddition of gelatin resulted in a fourfold increase inrecovery of hIL-12 in the medium at day 5 (Kwon et al.,2003). Unfortunately, the addition of gelatin, an animal by-product, is not reasonable for plant-produced pharmaceu-tical proteins because it obviates one of the main advantagesof using plants to produce therapeutic proteins, the absenceof any animal-based products. Likewise, addition of anti-biotics like bacitracin is also not permitted.

Interleukin-12 is a potent immunomodulator withpotential pharmaceutical applications as an anti-cancertherapeutic and vaccine adjuvant (reviewed in Salem et al.,2006; Trinchieri, 1994). Its ability to direct immunogenicity


toward cell-mediated immunity is particularly advantageousfor vaccines targeting intracellular disease agents (e.g.,viruses). Effective, scalable production systems, however,remain a key challenge for this complex heterodimericglycoprotein requiring eukaryotic bioproduction. As anactive cytokine, large-scale field growth of IL-12-expressingplants may be challenging from both a regulatory and publicperception perspective. Thus, demonstration of a scalablereactor system that supports productive yields of thiscomplex cytokine at commercially viable levels is significant.IL-12 is produced effectively throughout logarithmic growthof the roots. Onset of stationary phase, however, showed aprecipitous drop in secreted IL-12, likely the result of theincreasing protease levels in the medium (Fig. 8A).

Protease is not the only possible protein antagonistinvolved in this study. Each type of bioreactor posedadditional challenges for protein stability. In the airlift re-actor air sparging of the medium produced a large quantityof small bubbles. Proteins are susceptible to denaturation atair–liquid interfaces. In the mist reactor, the culture mediumwas repeatedly passed through an ultrasonic mister subject-ing secreted proteins to possible degradation by ultrasonicenergy. By using b-galactosidase as a model, it wasdetermined that there was no detrimental effect of ultrasonicenergy on enzyme activity. Indeed these results are con-sistent with those by Chisti (2003) who showed thatultrasound in a sonobioreactor did not affect microbial cellviability, glucose uptake rate, or antibody production. Ourstudy, therefore, shows that repeated passage of a proteinsolution through the mist bioreactor sonic sprayer isunlikely to result in protein degradation or loss of activity.

Analysis of the medium constituents for all of the culturesin this study showed that by days 10 and 13 the roots becamenutrient limited for both phosphorus and nitrogen andentered stationary phase with about 30% of the originalsugars remaining in the medium. Although mediumoptimization was out of the purview of this study, it isanticipated that both growth and mIL-12 productivity canbe significantly improved by optimization studies. Althoughneither of the bioreactors used in this study yielded moreroot growth nor mIL-12 than the unscalable shake flasks,they still provided comparable yields to the results obtainedin suspension cultures (Kwon et al., 2003). Further, neitherof these reactor systems nor the culture medium for shakeflask cultures has yet been optimized for productivity; thatprocess will surely increase both growth and mIL-12 levels.

The application of scalable low-cost or disposable bio-reactors operating with cultivation bags made from plasticfilm will clearly contribute to additional savings in cost andwill exploit the potential of plant cell-based bioprocessing.The superiority of low-cost and disposable bioreactorspossessing a gas-permeable cultivation bag of plastic filmwas effectively proved in a number of plant cell suspensioncultivations (Terrier et al., 2007). The current mist bio-reactor constructed using a plastic culture bag and aSonoTek ultrasonic mister has some key design featuresthat are different from our earlier mist reactor versions

including: independently controlled gas and mist flow,flexible culture bag of potentially variable geometry, anadjustable constriction device, and greater mist throughputvolume with only slightly larger median droplet diameter(�18 mm). These features make the mist reactor quitescaleable, easy to use, and low in capital costs.

In conclusion this study has shown that mIL-12 can beproduced at significant levels in not only shake flasks, butalso in scalable bioreactors. Better production was observedin a mist reactor than in an airlift reactor. This is the firstdemonstration of successful production of a therapeuticprotein in a mist bioreactor with potential for largescale applications. These results provide a frameworkwhereby IL-12s and similar cytokines can be producedusing hairy root cultures and, once optimized for growthand production, successfully scaled up for production inbioreactors for pharmaceutical, veterinary, poultry or live-stock applications.

Nomenclature

DW
dry weight
FW
fresh weight
Re
Reynold’s number
V
working volume of reactor
vvm
volume of air per volume of reactor volume per minute
Greek symbols

a
packing fraction
m
average specific growth rate
Support was provided in part by the Arkansas Biosciences Institute,

the major research component of the Arkansas Tobacco Settlement

Proceeds Act of 2000.

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