Disposable bioreactor systems are

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1 Oxygen Mss Trnsfer Correltion for Rocking- Motion Biorector System Erin Shughnessey, Armin Opitz, nd Jck Prior Disposble biorector systems re technologies commonly used in bioprocessing. They provide costeffective contmintion control nd llow more flexible fcility lyouts thn do stinless steel lterntives. One of the most populr types of single-use biorectors uses rocking pltform in plce of trditionl shft nd gittor ssembly to erte nd mix cell culture mteril within presterilized, single-use plstic bg (1). The system studied here is the RedyToProcess WAVE 25 biorector (GE Helthcre Life Sciences). In contrst to conventionl stirred tnks, rocking-motion biorector systems do not rely on oxygen sprgers for culture ertion or gittors for mixing. Insted, the lrge surfce-to-volume rtio nd rocking motion of these biorectors generte surfce ertion tht meets the oxygen demnds of mmmlin cell cultures often exceeding those delivered by stirred tnks (2). Rocking-motion biorector systems lso hve been shown to improve fluid turnover nd minimize sher forces in comprison with stirred tnks (3). Such flow conditions reportedly produce better cell suspension nd reduce risk of cell dmge nd thus hve enbled rocking-motion biorectors to rech higher mximum cell densities thn trditionl spinner flsks (2). However, reltive to the extensively reserched stirred-tnk, this new technology wrrnts further chrcteriztion. Previous studies of rocking-motion bicorectors hd nrrowly defined focus for the process conditions tested. Often, emphsis ws plced on smll rnge of set points pproprite for culturing specific type of cell. Furthermore, the different biorector system designs studied my explin the oxygen mss trnsfer coefficient results tht cnnot be compred dequtely. Fetured Report Figure 1: Exmple of dissolved oxygen (DO) profile over the course of one test: (1) stop rocking nd compress to eliminte hedspce ir; (2) inflte with nitrogen; (3) strt rocking; (4) stop rocking nd compress to eliminte hedspce nitrogen; (5) inflte with ir; (6) strting rocking t predetermined test conditions. DO (% Sturtion) Time (min) For exmple, Singh studied oxygen trnsfer in 2-L bg t 5% fill on system with rocking cpcity of 5 4 rpm nd 5 1, nd the mximum observed ws 4 h 1 (2). Mikol et l. exmined WAVE biorector with 1-L bg filled to 5% volume with phosphte-buffered sline (PBS) nd UCON ntifom t 3 C (4). When the system rocked t 1 ngle, ws linerly dependent on rocking rtes up to 25 rpm but incresed by fctor of four when the rocking rte ws 4 rpm. Westbrook et l. found tht with the sme bg size nd liquid volume heted to 28 C ws liner function of rocking rtes from 2 to 4 rpm with ngle constnt t 1 (5). Oxygen mss trnsfer ws negtive liner function of working volume in 1-L bg filled with 1-, 3-, nd 5-L volumes t mximum gittion. The vlue lso ws wekly dependent on overly flow rte. In study involving 5-L WAVE biorector with 2-L working volume, Yuk et l. (6) reported tht correlted positively with REPRINT WITH PERMISSION ONLY June (6)i BioProcess Interntionl 1

2 Tble 1: Operting set points by bg size Input Vlues Prmeters Units 5-L Bg 2-L Bg 1-L Bg Rocking rte RPM Rocking ngle Degrees Working volume % of totl bg volume Tble 2: WAVE 25 biorector length specifictions per try size Try Size Length GE Product Code Try 1.43 m Try 2.48 m Try 5.61 m Equtions: Eqution 1 Eqution 2 C [ t ] = k (C* C) L ln ( C* C C* C ) = k (t t ) L Eqution 3 k L = C (Fill frction) α (Rocking rte) β (Angle) γ (Try length) δ Eqution 4 ) = ln(c) + α ln(fill frction) + β ln(rpm) + γ ln(angle) + δ ln(l) k L = Eqution 5 (Rocking rte) 3 (Angle) (Try length) (Fill frction) 1.5 rocking rte nd rocking ngle, s supported by the observtions of Westbrook et l. (5). Yuk et l. (6) identified rocking rte nd ngle s the most significnt fctors ffecting. However, it ws reported tht overly flow rte hd no observble effect. Previous reserch hs noted generl trends of oxygen mss trnsfer in rocking-motion biorectors. Mention of specific vlues, however, often is limited to nrrow rnge of process conditions tht were of interest for culturing specific cell line. The gol of this study ws to complete wide-rnging survey of in the WAVE 25 system (GE Helthcre Life Sciences) operting t vrious process conditions, from which quntittive trends in oxygen trnsfer could be described. Mterils nd Methods We instlled the WAVE 25 system ccording to mnufcturer recommendtions nd secured bgs on the trys such tht they would not come loose upon infltion. During experiments, filter heter ws clipped to the overly outlet of the bg. A CBCU gs controller (GE Helthcre) regulted overly gses. Air nd nitrogen shred the sme inlet port on the CBCU, so the individul gs lines were disconnected nd swpped s needed. Ech bg ws filled to different volume with simulted culture medium composed of 1 PBS nd Pluronic F-68 (Dow Chemicl Compny) t concentrtion of 1 g/l. We connected dissolved oxygen (DO) sensor to the corresponding opticl membrne on the underside of the bg. The DO opticl probe ws clibrted by entering the clibrtion constnts on the bg lbel into UNICORN system control softwre (GE Helthcre Life Sciences) running on the connected computer. The probe ws set to the minimum reding cycle of 1 seconds, s indicted by the supplier. To prepre for experimenttion, we secured the bg to the rocker nd semi-inflted it with ir. As the bg continued to inflte, it ws filled with volume of cell-free, simulted culture medium through seprte port nd heted it to 37 C. The rocker ws turned on, nd the medium ws left to equilibrte for t lest two hours before testing. Experiments were conducted using 5-L bg on Try 5, 2-L bg on Try 2, nd 1-L bg on Try 1. We conducted series of tests on ech bg size t two different working volumes. Tests with the sme ngle were performed in succession, nd the rocking rtes were ordered rndomly within ech constnt ngle group. Results from preliminry tests supported the findings of Westbrook et l. nd Yuk et l. (5, 6): Overly flow rte ws not significnt fctor in determining the vlue of given the rnge of flow rtes typiclly used for mmmlin cell culture. Thus, overly flow rte ws not considered further nd ws kept constnt t.3 stndrd liters per minute (SLPM) for the reminder of the experiments. Tble 1 outlines the operting set points tested on ech bg size. Oxygen is trnsferred between the liquid nd gs phses in response to difference between liquid sturtion concentrtion nd current liquid concentrtion t given point in time. The rte t which oxygen trnsfers between these two phses depends on the mgnitude of tht concentrtion difference s well s. Eqution 1 describes this reltionship, where C is the DO concentrtion t time t, nd C* is the theoreticl sturtion concentrtion. 2 BioProcess Interntionl 16(6)i June 218 Fetured Report

3 Tble 3: Summry of fit Prmeter Although C nd C* cn be mesured using DO probe, the vlue of cnnot be mesured directly. By integrting Eqution 1 from time t to t, the vlue of cn be evluted by determining the slope of line fitted to the left-hnd side (LHS) of Eqution 2 over time. We pplied the dynmic gs-in, gs-out method with bg compression for determintion s previously implemented by Yuk et l. for the study of rocking-motion biorector (6). To fcilitte oxygen trnsfer out of the medium, we opened port nd compressed the bg to force out the hedspce ir. The bg ws reinflted with nitrogen t 3 SLPM while the rocker ws sttionry. Once the bg ws firm, we reduced the nitrogen flow rte nd initited the rocking motion. Under these conditions, the DO content of the medium decresed. Following deoxygention, we pused the rocker nd compressed the bg gin to force out hedspce nitrogen. The bg ws reinflted with ir t 3 SLPM until firm. Finlly, we set the overly flow rte to.3 SLPM nd strted the rocking motion t specified rte nd ngle. Figure 1 shows smple DO profile observed during the course of the test, with the numbered regions distinguishing the different phses of the test. Fetured Report Vlue R squred.928 R squred djusted.926 Root men squred error.194 Men of response 2.71 Observtions 168 AICc 66.5 BIC 48.3 Figure 2: Exmple of the left-hnd side (LHS) of Eqution 2 bsed on n exmple dt set nd the resulting slope determining k L. LHS.2.4 LHS fit Experimentl dt Time (min) Tble 4: Prmeter estimtes Term Estimte Stndrd Error t Rtio Prob > t Intercept <.1* ln(fill frction) <.1* ln(rpm) <.1* ln(ngle) <.1* ln(length) <.1* Figure 3: Averge k L nd rocking rte by ngle (columns) nd bg size (rows). K L Fill frction Angle Rocking Rte (RPM) Bg Size (m 3 ) DO runtime dt were processed in MATLAB softwre. The vlue of the LHS of Eqution 2 ws clculted bsed on the DO mesurement t ech time point, nd the resulting vector ws plotted over time. A constrined lest squres model ws used to pproximte the slope of the LHS versus time plot, nd this vlue ws tken s. Figure 2 shows plot of the LHS of the eqution bsed on the experimentl dt nd the right-hnd side of Eqution 2 nd the resulting. Results nd Discussion Figure 3 shows the verge or ech combintion of set points (63 in totl). The vlue of with respect to rocking rte is plotted by ngle (columns) nd bg size (rows). Different working volumes re indicted by mrker color nd reported in the legend s fill frction. Error brs represent one stndrd devition from the men. In generl, incresed with rocking rte over the rnge of set points tested in the two bg sizes. This behvior is expected becuse incresing gittion cuses lrger wves nd thus ir June (6)i BioProcess Interntionl 3

4 Figure 4: Actul nd predicted k L plot Figure 5: Residul ) by predicted ) plot L Bg Legend 2-L Bg 5-L Bg.4.2 Bg Size (m 3 ): ln(k L ) Actul Residul ) ) Predicted (P<.1 Rsq =.93 RTMSE =.1942) ) Predicted Figure 6: Digrm of uninflted bg proportions (not to scle) reltive to the xis of rocking shown in red. 5 L Length 1 L 2 L entrinment. The effect of ngle ws less pronounced, lthough incresing ngle did hve sizeble effect on when operting t higher rocking rtes. The vlues for lower working volume were greter thn those for higher working volumes given the sme rocking rte nd ngle. The vlues were reltively similr in the 2-L nd 5-L bgs when the working volumes in ech were 5%. The reltionship between nd vrious process prmeters ws nlyzed using JMP (SAS Institute). A liner-regression sttisticl model ws fitted to ll obtined vlues. The vlues were ssumed to be independent, despite the potentil for intrbg correltion between vlues given tht repeted vlues were clculted from experiments on the sme bg. As previously noted, results from the oxygen trnsfer experiments indicted strong dependence of on rocking rte s well s modest dependence on ngle. Fill volume lso hd significnt effect on s greter vlues were observed for the lower fill in ech bg. Figure 4 shows plot of the ctul vlues versus the model predictions. The influence of bg geometry on lso ws considered. Figure 6 shows reltive bg dimensions. It ws observed during the experiments tht fluid flow occurs predominntly in the length direction becuse the system is not bffled nd rocks only in one dimension. In this wy, the width hs lesser role in dictting the physicl phenomen tht occur in the bg. Therefore, the only dimension tken into ccount for the model ws the length. However, becuse the length of the bg decreses fter it is filled nd inflted, the try s length ws better description of the chrcteristic length ffecting wve-ction phenomen. Tble 2 shows the mnufcturer s specifictions for try length. Note tht ll experiments conducted in this study used Try 1 for the 1-L bg The power lw expression in Eqution 3 ws developed to nlyze the reltionship between nd the four relevnt prmeters: fill frction, rocking rte, rocking ngle, nd try length. The distribution of residuls versus predicted for the bove model ws curved, so nturl log trnsformtion ws used to improve the linerity of the model. A lest squres nlysis ws used to estimte the constnts (C,, b, g, d) in Eqution 4. As Tble 3 shows, n R 2 vlue of.93 ws observed between nd the selected prmeters. The t-vlues indicte tht the reltionship between nd these prmeters is sttisticlly significnt (Tble 4). Figure 5 shows residuls, nd Eqution 5 defines the prediction expression, where the response vrible is (h 1 ), nd the fctors re rocking rte (rpm), ngle ( ), try length (m), nd fill frction (dimensionless). Bsed on tht model, rocking rte hs the strongest effect on. Chnges in ngle hve more modest effect. The vlue is inversely relted to fill frction, consistent with trditionl biorectors. Bsed on the four fundmentl prmeters, this model cn predict with 87% of 4 BioProcess Interntionl 16(6)i June 218 Fetured Report

5 the cses within 3% ccurcy of the mesurement in either of the two bg sizes tested. Becuse is relted to try length, oxygen trnsfer behvior in 1-L bg cn differ depending on whether it is operted on Try 1 or Try 2. Significnce of Bg Geometry Figure 6 shows the xis of rocking in reltion to the three bg sizes. Two blue plstic cylinders re built into ech bg so tht it cn be clipped onto the corresponding try t opposite ends. Ech bg size hs distinct proportions nd thus distinct physicl spce where mss trnsfer occurs. The 1-L nd 2-L bgs re similr in length. However, they form noticebly different shpes when inflted. During setup, we observed tht the uninflted 2-L bg ws tut when properly clipped onto the try. Thus, when it ws inflted, the 2-L did not expnd upwrd very much nd remined reltively flt. By contrst, the 1-L nd 5-L bgs (not pictured) were loose in the middle fter being secured to the try, so they becme rounded nd blloon-like when inflted. Consequently, the 2-L bg ws nrrower t ech end where the wve crshes. It is possible tht this physicl shpe directed crshing wves downwrd upon themselves rther thn upwrd through the hedspce, thus ltering the mechnism of oxygen entrinment in the 2-L bg with respect to the other two sizes. We believe tht this geometric distinction is better cptured by the try length thn the bg size, so the try length prmeter ws used in the model. Sttisticl Model for Rocking-Motion Rectors Experiments were performed to chrcterize the oxygen mss trnsfer behvior of RedyToProcess WAVE 25 system. The vlue of ws investiged in response to inputs for rocking rte, rocking ngle, working volume, nd bg dimensions. It ws observed tht incresed with rocking rte, wheres the reltionship between nd ngle ws less pronounced. The vlue of decresed with greter working volume. Clculted vlues were used to develop sttisticl model for prediction bsed on four relevnt process prmeters. A stndrd lest squres nlysis ws used to identify correltion (R 2 =.93) between the clculted vlues nd the set points for fill frction, rocking rte, rocking ngle, nd try length. The sttisticl model suggests tht is negtively correlted with fill frction nd positively correlted with rocking rte, ngle, nd try length. With the incresing populrity of disposble rocking-type biorector systems, this model offers useful tool for trnsltion of trditionl cell-expnsion processes into this new technology by providing n estimte of given different combintions of set points. The results lso hve shown tht when operting t 5% of bg s volume, the mss trnsfer is pproximtely the sme regrdless of bg size. This simplifies seed trin expnsion becuse lrger bg, if used t 5% fill volume, would operte with similr s the previous step. References 1 Levine HL, et l. Efficient, Flexible Fcilities for the 21st Century. Bioprocess Intl. 1(11) 212: Singh V. Disposble Biorector for Cell Culture Using Wve-Induced Agittion. Cytotechnol. 3(1 3) 1999: Öncül AA, et l. Chrcteriztion of Flow Conditions in 2 L nd 2 L Wve Biorectors Using Computtionl Fluid Dynmics. Biotechnol Prog. 26(1) 21: 11 11; doi:1.12/ btpr Mikol M, Seto J, Amnullh A. Evlution of Novel Wve Biorector Cellbg for Aerobic Yest Cultivtion. Bioprocess Biosyst. Eng. 3(4) 27: ; doi:1.17/ s y. 5 Westbrook A, et l. Appliction of Two-Dimensionl Disposble Rocking Biorector to Bcteril Cultivtion for Recombinnt Protein Production. Biochem. Eng. J. 88, 214: Yuk IH, et l. Overcoming Chllenges in Wve Biorectors Without Feedbck Controls for ph nd Dissolved Oxygen. Biotechnol. Prog. 27(5) 211: ; doi: 1.12/btpr.659. c Erin Shughnessey ws co-op student during this work. Corresponding uthor Armin Opitz is principl process engineer, nd Jck Prior is senior director t the Mnufcturing Science group in Globl Mnufcturing Science nd Technology, Snofi, 1 Reserch Drive, Westborough, MA 1581; rmin.opitz@snofi.com. Conflict of Interest The reserch presented in this mnuscript is originl nd hs not been submitted previously or concurrently for publiction in ny other journl. The uthors hve no conflicts of interest to declre To shre this in PDF or professionlly printed formt, contct Jill Kleth; kleth@mossbergco.com; Fetured Report June (6)i BioProcess Interntionl 5