A Bio Reactor System Based on a Novel Oxygen Transfer Method

8/3/2019 A Bio Reactor System Based on a Novel Oxygen Transfer Method

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68 BioProcess International J UNE 2008

I N S EARCH OF A N EW O 2 T RANSFER M ETHOD

Most process development scientists inbiotechnology use 125- to 250-mLshaker flasks to screen production cellline candidates by mammalian cellsuspension culture. It is a usefulmethod; however, we have observedthat the effective volume is limited to30–60 mL. Beyond that workingvolume limit, a sudden collapse of cellculture is often observed, which wehypothesized was due to a lack of O 2.

We designed a frustoconical–bottom shaker vessel (Photo 1) to offermore air-containing inner space. Theshape is of a cone with its pointed endtruncated parallel to its base. Whenusing a 3-L cell culture workingvolume, we achieved an extremelyhigh cell density for CHO cell culture.

Measuring the O 2 level, we found thatit is always 60 –100% within theworking volume, even with celldensities larger than 7% packed-cellvolume (~15–20 × 10 –6 cells/mL). Bysimply changing (or reversing) theclassical shaker flask’s bottom into afrustoconical bottom, we hadsurprisingly enhanced our CHO cellculture ( 5).

We then hypothesized that theremust be a superior O 2 transfer

mechanism operating in thefrustoconical-bottom culture vessel. Onan orbital shaker platform, culturemedia appears to swirl in a motionresembling that created by a river’sstrong current. By using a high-speedcamera, we were stunned to find that

this action was captured as a tiltedsurface of media repetitively sweepingthe air-exposed walls of the bioreactorvessel. The culture medium develops atilted and fast-moving surface thatclimbs one side of the bioreactor at anygiven time, giving the illusion that the

medium is swirling in place.An interaction occurs between the

air-exposed smooth surface of thevessel and the culture medium inside. This interaction is accomplishedthrough force generated by the speedof the medium current and thesmoothness of the wall surface. Asmedia travels around an air-exposedvessel surface, it generates microscopicbubbles that enter the culture asdissolved oxygen (DO), which is

defined as microscopic bubbles of oxygen scattered among the watermolecules. So generation of thosemicroscopic bubbles is the key elementto transform “air” oxygen intodissolved oxygen. Medium interactionwith the bioreactor’s smooth inner wall

must generate microscopic air bubbles(Figure 1).

We designed three methods to testthe mechanism of this novel O 2 transfermethod. Figure 3 shows a 150-mLworking volume culture vessel with thefrustoconical bottom on a shaker

platform with an orbital shaking speedof 100 rpm. As a control, we tested thesame vessel with a tube bubblingdevice providing 3 L/min airf low. Table1 clearly shows superior O 2 transferspeed for this culture vessel.

We also tested the vessel wall andmedium current interaction by using arotating plastic bottle containing 0.5 Lof culture medium at 50 rpm (Figure 3,Panel A). The control was the sametype of plastic bottle containing 0.5 L

culture medium at an airflow rate of 3L/min (Panel B). Results clearlydemonstrate superior O 2 transfer speedfor the rotating bottle method (Table2).

As a final demonstration, weassembled a rolling wheel dipped into

Table 1: Results from a dissolved oxygen time course experiment showthe O 2 transfer speed of the 150-mL frustoconical-bottomed culturevessel on a shaker platform with a 100-rpm orbital shaking speed.

Time (minutes) 0 30 60 90

DO (%) for 150-mL vessels on shaker at100 rpm (Panel A)

45 75 92 100

DO (%) for 150-mL vessels with air-tube

bubbling method (Panel B)

45 52 55 75

Figure 1: Hypothesis of dissolved oxygen generation; the smooth-surfaced vessel wall interactswith the current of medium to create microscopic bubbles, thus providing a theoretical foundationfor this novel O 2 transfer method.

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Table 2: Results from dissolved oxygen time course experiment

Time (minutes) 0 10 20 30 40 50 60 70

DO (%) for rotatingbottle (Panel A)

58 90 90 90 90 90 90 90

DO (%) for airsparging (Panel B)

58 62 69 74 78 82 82 84

Figure 2: Small frustoconical-bottom culture vessels on a shakerplatform; one is simply on an orbital shaking platform, and the other issupplied with air through an air pump. The tilted surface of the culturemedium is clearly visible.

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Figure 3: Rolling plastic culture vessels on a rotating apparatus; one issimply rotated on one axis, and the other is supplied with air through anair pump.

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70 BioProcess International J UNE 2008

a bath of culture medium. Similarresults obtained this way (data not

shown), support our hypothesis andinteraction theory ( 6). Those experiments showed that

interaction of a vessel wall (with acertain smoothness) and a current of medium (with a certain force)significantly increased cell culture O 2levels, thus establishing a novel O 2transfer method ( 6). However, no directevidence shows O 2 transfer establishedthrough the creation of microscopicbubbles by means of that interaction. To effect the rapid increase in DO,microscopic air bubbles wouldprobably be transferred quickly intothe culture medium. Thus, wehypothesize that the interactioncreates microscopic bubbles, which isthe theoretical foundation of this novelO2 transfer method (Figure 1). If that isthe case, then the smooth-surfacednature of the wall must play animportant role. We went on to testdifferent materials and found that acertain smoothness of ethylene vinyl

acetate (EVA) plastic material worksbest for O 2 transfer — and thus for

plastic bag bioreactor production.We believe that this novel O 2transfer method is the meansunderlying the Wave bioreactor’s O 2transfer mechanism ( 6). The wavecreation increasing a culture medium’sO2 transfer surface area might play aminor role in O 2 transfer, but it isprimarily the smooth surface of thevessel wall and its interaction with acurrent of medium that increases O 2transfer ( 3, 4, 6). The larger the surfacearea the medium sweeps, the more O 2

is transferred into it.We tested the rocking motion by

using a Wave plastic bag comparedwith a round-type EVA plastic bag onan orbital shaker, and we found thatthe round bag had double the O 2transfer speed of the Wave bag. Thissuggests that two-dimensional“sweeping” of culture medium along asmooth-surfaced vessel wall doubledO2 transfer compared with one-dimensional medium rocking against avessel wall. If you wanted to improvethe O 2 transfer of a Wave bioreactor,you could simply modify it bychanging the one-dimensional rockingmotion into a multiple-dimensionshaking motion or a two-dimensionalrocking motion and increase mediacontact with the vessel surface. Such achange would improve its O 2 transfer

by providing a mechanicallyadvantaged mixing motion.

U SING THE N EW METHOD IN P LASTIC -B AG BIOREACTORS We used the novel O 2 transfer methodto design and construct two types of plastic-bag bioreactors. We use our 50-L “Current” suspension culture vesselto scale up CHO cell culture in serum-free suspension medium (Photo 2),routinely reaching 5% packed cell

volume (~15 × 10–6

cells/mL) in fed-batch mode. The second type of bioreactor is a

rotating plastic bag specialized formicrocarrier-based VERO and MDCK cell culture (Photo 3). We find that therotating bag or bottle method is goodfor reducing shear force and mixing-motion–related cell damage onmicrocarrier surface-anchored cells,thus maintaining cellular integrity. Italso makes cell transfer possiblebetween individual microcarriers by

Photo 2: 50-L working volume cell culture vessels for high-density suspension CHO cell culture withair supplied using a simple air pump

Photo 3: Rotating plastic-bag bioreactorswithout shear force perform well for microcarriercell culture and maintain the integrity of cellsanchored on microcarrier surfaces.

Photo 4: With the rotating plastic-bagbioreactor, cellular integrity is well maintainedwhile cells are anchored to the microcarriersurface, even without the presence of serum.

Photo 5: Current single-use bioreactor system (5-L, 50-L, and 150-L working volumes) with a control

tower is close to commercialization stage.



J UNE 2008 BioProcess International 71

minimizing mixing motion anddisturbance.

AmProtein has set up threecontract-service facilities using thesebioreactors. Our mammalianexpression vector and cell linetechnology are available to partner

companies for cost-effectiveproduction of preclinical materials.We’re currently developing a controltower to control pH, DO, temperature,and both orbital shaking and rotatingspeed (Photo 5).

Our Current bioreactor also featuressimplicity of ambient air supply, adisposable plastic bag to eliminateproduction downtime, and pH controlthrough both air flow regulation andCO2 supply. There is no interferencefrom foaming, little or no shear force,and no cell damage related to airbubbles bursting. The orbital shakingmotion imparts superior mixingrelative to rocking methods.

Throughout its working volumerange, the Current bioreactor’s shakingmotion carries with it a mechanicaladvantage far greater than that of arocking platform, exemplified by earlydevelopment stage difficulties withrockers. Rocking motion attempts to

transfer the complete mass of a systemto one side and then return it back tothe other. So it requires very strongcomponents, rockers, and bags — andit consumes large amounts of energycompared with smooth orbital shakingand relatively smooth two-dimensional

rocking. Another advantage lies inindependent intellectual property anda freedom to operate commercially.

R EFERENCES

1 Wang D, et al. The Bioreactor: APowerful Tool for Large-Scale Culture of Animal Cells. Curr. Pharm. Biotechnol. 6(5)2005: 397–403.

2 Warnock JN, Al-Rubeai M. Systems forthe Production of Bi opharmaceuticals fromAnimal Cells. Biotechnol. Appl. Biochem. 45,2006: 1–12.

3 Singh V. Method for Culturing CellsUsing Wave-Induced Agitation. US Patent No.6,190,913; 20 Februar y 2001.

4 Singh V. Disposable PerfusionBioreactor for Cell Culture. US Patent No.6,544,788; 8 April 2003.

5 Hui M. Suspension Culture Vessels.WIPO Patent No. WO 2006/138143; 28December 2006; www.wipo.int/pctdb/en/wo.jsp?wo=2006138143.

6 Hui M. A Method to Increase Dissolved

Oxygen in a Culture Vessel. WIPO Patent No.WO 2007/142664; 13 December 2007; www.wipo.int/pctdb/en/wo.jsp?wo=2007142664.

7 Gupta A, Rao G. A Study of Oxygen Transfer in Shake Flasks Using a Non-Invasive Oxygen Sensor. Biotechnol. Bioeng.84, 2003: 351–358.

8 Kermis H, et al. Dual ExcitationRatiometric Fluorescent pH Sensor for Non-Invasive Bioprocessing: Development andApplication. Biotech. Prog. 18, 2002:1047–1053.

Corresponding authors Qian Jia is a professional senior engineer for New DrugR&D Co. Ltd. of NCPC and coordinator of the AmProtein Alliance, 355 North LantanaStreet #220, Camarillo, CA 93010; 1-805-807-3362, fax 1-424-644-0053;[email protected], www.amprotein.com. Huicheng Li is vice president, and Mizhou Hui is founder and CSO of

AmProtein Corporation. Ni Hui is chief engineer of Harbin BioengineeringCorporation. Aziz Joudi is an associatescientist, and Gilbert Rishton is a senior advisor for AmProtein Corporation. Lei Baois an associate scientist, Ming Shi is anassociate scientist, and Xiuqin Zhang isassistant director of Harbin BioengineeringCorporation. Li Luanfeng is an engineer for New Drug R&D Co. Ltd. of NCPC. Jiangyun Xu is CEO of AmProtein-China. And Guoqing Leng is CEO of HarbinBioengineering Corporation.

E XPERIMENTAL M ETHODS AND M ATERIALS

We bought paper-patch DO and pHsensors (Photo 6, developed by Dr. Rao’slaboratory at the University of Maryland)from Fluorometrix Corporation (www.fluorometrix.com) ( 7, 8). We tested themusing a carbonated beverage as anegative control because of its low O 2content. The positive control was

increased gassing with an air spargingstone. To further normalize, we used a DOprobe directly from an Applikon (www.applikon-bio.com) bioreactor. All controlsshowed that the paper-patch DO sensor responded to both low and high DO levels.

We measured pH using both a paper-patch pH sensor and a portable pH meter (Photo6). Control was achieved with Hepes non–CO 2-dependent buffer from Sigma (www.sial.com) and through airflow regulation to deplete CO 2. We measured glucose levelsusing glucose strips and a one-touch glucose meter (Photo 6). Cell density wasmeasured with a hemocytometer, and cell biomass was measured with PCV tubes,both from VWR Scientific Products (www.vwrsp.com).

We used CHO cell lines expressing 60–160 pg/cell/day of anti-CD52, anti-EGFR, TNFR2-Fc, EPO, and HBsAg for our bioreactor testing. AMP B001 basal growth medium and

AMP F001 feed medium were used for serum-free suspension cell culture. We also usedVERO and MDCK cells from the American Type Culture Collection (www.atcc.org) formicrocarrier Cytodex 1 cell culture in our rotating plastic-bag bioreactors. During allculture processes, an air pump and air flow meter from VWR were used for O 2 supply.

Photo 6: Experimental setup for testingdissolved oxygen, pH, and glucose

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A Bio Reactor System Based on a Novel Oxygen Transfer Method