Journal of Biotechnology, 21 (1991) 173-186

173

© 1991 Elsevier Science Publishers B.V. All rights reserved 0168-1656/91/$03.50 ADONIS 016816569100152W BIOTEC 00682

Enzyme sensor-FIA-system for on-line monitoring of glucose, lactate and glutamine in animal cell cultures R. Renneberg 1, G. Trott-Kriegeskorte 2, M. Lietz 2, V. Jiiger 2, M. Pawlowa 1, G. Kaiser ~, U. Wollenberger 1, F. Schubert ~, R. Wagner 2, R.D. Schmid 2 and F.W. Scheller i Zentralinstitut fiir Molekularbiologie, Berlin-Buch, F.R.G. and 2 GBF-Gesellschaft fiir Biotechnologische Forschung mbH, Braunschweig, F.R. G.

(Received 18 February 1991; revision accepted 18 June 1991)

Summary Enzyme sensors for glucose, lactate and glutamine were connected via flow-injection analysis (FIA) devices to two different bioprocesses. They were used for on-line process control of perfused bioreactor systems containing mammalian cell lines producing a monoclonal antibody and recombinant interleukin-2. The biosensor system gives direct access to important process data which can be used as control parameters for long term cell cultivation systems. FIA; Enzyme sensors for glucose; Lactate; Glutamine; On-line bioreactor control; Hybridoma cell culture; Recombinant B H K cells

Introduction Enzyme sensors have recently started to demonstrate their potential in biotechnological process control. Flow injection analysis (FIA) (Ruzicka and Hansen, Correspondence to: R.D. Schmid, Bereich Enzymtechnologie, Gesellschaft fiir Biotechnologische

Forschung m.b.H., Mascheroder Weg I, D-W-3300 Braunschweig, F.R.G. Abbreviations: BHK, baby hamster kidney; IgG, immunoglobuline; IL-2, interleukin-2; FIA, flow

injection analysis; GOD, glucose oxidase; LOD, lactate oxidase; SCE, saturated calomel electrode.

174

1988) has proven to be applicable to a wide variety of analytical problems. The advantages of both FIA and enzyme sensors can be exploited for effective bioprocess monitoring (Schmid and Kiinnecke, 1990). Recently, several methods for on-line monitoring of microbial bioprocesses have been established such as a gas diffusion FIA for ethanol production during the growth of Saccharomyces cerevisiae with immobilized alcohol oxidase (Kiinnecke and Schmid, 1990), or a FIA system connected to a column filled with immobilized glucose oxidase prepared for on-line glucose determination (Garn et al., 1989). Additionally, Liidi et al. (1990) studied the on-line determination of glucose during batch cultures of yeast cells under industrialconditions. There are as yet only a few examples of the application of biosensors in cell culture techniques with the exception of cell mass estimation (Geahel et al., 1989; for review see Merten, 1988). The first attempt to control mammalian cell culture using enzyme sensors for glucose, L-lactate and L-glutamine was described by Romette and Cooney (1987). But there were no data available on the control of real bioprocesses. In this report we present on-line measurements of D-glucose, L-lactate and L-glutamine concentrations in media during long term cultivation of mammalian cells in stirred bioreactors.

Materials and Methods

Enzyme membranes Commercial glucose oxidase (GOD) and lactate oxidase (LOD) membranes were obtained from the Central Institute of Molecular Biology, Berlin-Buch (Schubert et al., 1988; Nentwig et al., 1985) containing 10 U GOD per cm 2 or 4-5 U LOD per cm 2 immobilized in polyurethane. Glutaminase from Sigma (13.8 U cm -2) and a novel highly specific glutamate oxidase (0.7 U cm -2) isolated from Streptomyces violascens by B6hmer et al. (1989) were coimmobilized in a gelatine membrane. Glutaminase splits glutamine into L-glutamate and ammonia. L-glutamate is subsequently oxidized by glutamate oxidase to 2-oxoglutarate (Renneberg et al., 1989; Trott-Kriegeskorte et al., 1989). All chemicals were purchased from Merck (Darmstadt, F.R.G.).

Biosensor systems For the three enzyme sensors the hydrogen peroxide formed was controlled amperometrically at + 0.6 V vs SCE using flow-through measuring cells equipped with platinum working electrodes and silver/silver chloride reference electrodes. The electrodes were connected to a modified commercial Glucometer GKM-01 (Zentrum for wissenschaftlichen Ger~itebau, Berlin), allowing the determination of both current-time curves and their first derivative (di/dt, Fig. 4). The signals were recorded on a chart recorder (Kipp and Zonen, Delft, The Netherlands). The three different enzyme sensors were coupled with a miniaturized modular multi-

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177 channel FIA system developed at GBF, Braunschweig (Schmid and Ktinnecke, 1990; see Fig. 2). An Alphatronic PT 8105 timer was obtained from Alphatronic (Stutensee, F.R.G.). Cell lines, media and bioreactors A 1.4-1-membrane bioreactor (Fig. 1) was used in both bioprocesses for bubblefree aeration as described previously (Lehmann et al., 1988). For production of rat IgG~, a rat-mouse hetero-hybridoma cell line (187.1, ATCC HB58) was cultivated in a batch process using serum-free medium as described by J~iger et al. (1988). For the constitutive production of interleukin-2 (IL-2) a recombinant baby hamster kidney cell line (BHK 21 pSVIL2) was used (Conradt et al., 1989). Cells were cultivated in a perfused process with internal microfiltration (double membrane stirrer system as shown in Fig. 1) using a protein-free medium formulation as described by Lucki-Lange and Wagner (1990). Immunoglobulin concentrations were determined by using a standard sandwich ELISA. IL-2 activity was tested by the [3H]thymidine incorporation assay using the IL-2 dependent murine CTLL as described (Gillis et al., 1978) and employing an internal laboratory standard preparation for comparison. The specific activity of the purified IL-2 amounts to 107 U mg-~ (Conradt et al., 1985). The three different enzyme sensors were coupled via the FIA with the bioreactors (Fig. 2). The glucose and lactate flow-through sensors used the same buffer (phosphate buffer, pH 7.0). Since the pH-optimum for determination of glutamine was found to be at pH 5.5 the glutamine sensor was supplied by separate FIA channels (see Fig. 2). For the control of on-line data coming from the biosensor, free amino acids were also quantified off-line by means of a reversed-phase HPLC system with pre-column derivatisation with o-phthaldialdehyde (OPA, Serva, Heidelberg; Ryli et al., 1990; Larsen and West, 1981). Glucose and lactate contents were determined with YSI 27A glucose and lactate analyzers (YSI, Yellow Springs, OH). For determination of total cell number, nuclei were fixed and stained with 0.1% cristal violet in 0.1 mol 1-~ citrate and subsequently counted by means of a hemocytometer. The proportion of dead cells was estimated by trypan blue exclusion (Boehringer, Mannheim). All chemicals not specified were purchased from Merck, Darmstadt, F.R.G.

Results and Discussion

FIA-enzyme sensor system The hydrogen peroxide formation for all enzyme sensors was followed amperometrically at +0.6 V. No interference was observed from media constituents at

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this potential. The optimum flow rate was determined to be 1.2 ml min -~, the dispersion rate 3.7 and the sample volume 44 ~I. To extend the linear range of the sensors an additional diluting chamber with a mixing stirrer was used, achieving an overall dilution of 13. Under these conditions the calibration curves were linear for glucose up to 30 mmol 1-t (Fig. 3a), for lactate up to 20 mmol 1-~ (Fig. 3b), and for glutamine up to 15 mmol 1-~ ( Fig. 3c) in the culture media. The detection limit was 0.025 mmol 1-i for all three substances. The coefficients of variation for calibration solutions were determined to be lower than 2%, whereas for samples to be between 3 and 5%. The signal to noise ratio was 5 : 1. All three enzyme sensors worked at room

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Fig. 5. Cultivationof hybridomacells. Growthcurveand productionof the monoclonalantibody. temperature during the entire cultivation time of 11 and 22 d, respectively, without exchange of the enzyme membranes. No fouling was observed due to the addition of 1 mmol 1-l sodium azide to all buffers to prevent microbial growth and inhibit traces of catalase in the GOD-membrane. Comparison of all the data measured on-line with the FIA-coupled enzyme sensors and measured off-line with commercial enzyme sensors (for glucose and lactate, Figs. 6a, b and 8a, b, respectively) and HPLC (Figs. 6c and 8c, respectively) showed a good agreement for the enzyme sensor data. In general, the off-line values were slightly higher, probably due to different sample handling methods: samples for off-line measurement were collected and frozen for 12 and 23 d, respectively, and analyzed together. In contrast, off-line HPLC data for glutamine were up to 20% higher than the on-line sensor values. However, this effect was also observed when glutamine calibration solutions were determined with HPLC. Recalculation of the HPLC-data excludes the discrepancies. To highlight this problem if, however, we decided to publish the data directly given by the HPLC-integrator. On-line process monitoring Two different bioprocesses were controlled by the 3-sensor-system (Fig. 2): a batch system for the production of monoclonal antibodies by rat-mouse hybridoma cells and a perfusion system with continuous medium exchange for the production of interleukin 2 (IL-2) by BHK pSVIL2 cells (see Fig. 1). Both processes showed characteristic, conventional growth curves correlated with a linear production of the monoclonal antibody (Fig. 5) and the recombinant protein (Fig. 7) as described by J~iger et al. (1988) and Ryll et al. (1990), respectively. The FIA-system was coupled directly on-line via an exchangeable sterile filter (Millex GV, Millipore, Molsheim, France) to the harvest line of the bioreactor (Fig. 2). This simple connection was made possible by two important process preconditions. Firstly, serum- or protein-free media were used resulting in low

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cuttivation time [d ] Fig. 6. Data obtained for batch culture of hybridoma cells: comparison of on-line and off-line data for glucose (a), glutamine (b), and lactate (c); data for productivity(mAb) and viable cell number see Fig 5. protein contents in the supernatant, and secondly perfusion was based on an internal microfiltration system so that the harvest was free of cells or cell debris. After passing the FIA-sensor system the samples were not recirculated into the bioreactor. Therefore, strict sterility of the sensors was not necessary. Off-line samples were analysed only every 12 h because substrate and metabolite concentrations changed very slowly. After calibration of the system for glucose, glutamine and lactate the harvest stream of the bioreactor was usually connected via a sterile filter with the FIA-sensor. Twenty samples were taken within 30 min. As demonstrated in Figs. 6 and 8 the signals showed a high accuracy.

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Problems caused by air bubbles in the system were prevented by an additional air-bubble trap. For sampling of media with higher protein content different filter units with small volumes but prolonged lifetime are used to prevent clogging. There are several advantages of the systems described here in comparison with the results obtained from other authors (Romette, 1987; Romette and Cooney, 1987), which are essentially due to improved enzyme sensors. Instead of 5 ml sample volume (diluted 5 times) our system needed only 44/~1 samples which were diluted 13 times. On the basis of a 5-fold dilution in previous works, it was calculated that Romette (1987) was only able to determine in a linear scale restricted to glucose, lactate and glutamine concentration of 25 mmol 1- ~, 5 mmol 1-t and 1 mmol 1-1, respectively. However, the concentration variation of glucose, lactate and glutamine in cell culture supernatants was much higher and could be covered by the sytems described here.

Conclusions

The philosophy of FIA is to measure with a high frequency. In the case of animal cell culture, however, the alterations in medium constitution occur very slowly in contrast to microbial processes (Liidi et al., 1990). Therefore, extreme conditions for the FIA-coupled enzyme sensors as in fast growing microbial cultures were not tested. The stability of the enzyme sensors would allow a fulltime operation if the enzyme membranes are calibrated from time to time with respect to their activity. Anyway, the high frequency of FIA-obtained signals allows a

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184 direct feed-back control for the m a i n n u t r i e n t s in the future. Additionally, instead of H 2 0 2 control as used in our biosensors, the m e a s u r e m e n t of O 2 - c o n s u m p t i o n will be m o r e a d v a n t a g e o u s d u e to the a b s e n c e of i n t e r f e r e n c e , t h o u g h this raises new problems, which have to be solved, related to the different 0 2 levels in the samples. F u r t h e r m o r e , i m m u n o s e n s o r s should be developed to m o n i t o r i m p o r t a n t h i g h - m o l e c u l a r weight products of a n i m a l cells.

Acknowledgements This work was m a d e possible by the G e r m a n - G e r m a n A g r e e m e n t for scientifictechnical cooperation. W e would like to t h a n k Detlev H a n i s c h a n d H e n n i n g Schilling for their i n p u t to F I A design a n d work, J o a c h i m H a m m e r for H P L C a m i n o acid analysis of the samples.

References B/Shiner, A., Miiller, A., Passarge, M., Liebs, P., Honeck, H. and M~iller, H.G. (1989) A novel L-glutamate oxidase from Streptomyces endus. Eur. J. Biochem. 182, 327-332. Conradt, H.S., Geyer, R., Hoppe, J., Grotjahn, L., Plessing, A. and Mohr, H. (1985) Structure of the major carbohydrates of natural human interleukin-2. Eur. J. Biochem. 153, 255-261. Conradt, H.S., Nimtz, M., Dittmar, K.E.J., Lindenmaier, W., Hoppe, J. and Hauser, H. (1989) Expression of human interleukin-2 in recombinant baby hamster kidney, Ltk-, and Chinese hamster ovary cells. J. Biol. Chem. 264, 17368-17373. Garn, M.B., Gisin, M., Gross, H., King, P., Schmidt, W. and Thommen, E. (1989) Extensive flow-injection dilution for on-line sample pretreatment. Anal. Chim. Acta 207, 225-231. Geahel, I., Dordet, Y., Duval, D., Dufan, A.F. and Hache (1989) On line cell density estimation by spectrocolorimetry. In: Spier, R.E., Griffiths, J.B., Stephenne, J. and Crooy, P.J., (Eds.), Advances in Animal Cell Biology and Technology for Bioprocesses, Butterworths, pp. 134-135. Gillis, S., Ferm, M.M., Ou, W. and Smith, K.A. (1978) T Cell Growth Factor: Parameters of Production and a Quantitative Microassay for Activity. J. Immunol. 129, 2027-2032. J~iger, V., Lehmann, J. and Friedl, P. (1988) Serum-free growth medium for the cultivation of a wide spectrum of mammalian cells in stirred bioreactors. Cytotechnology 1,319-329. Kiinnecke, W. and Schmid, R.D. (1990) Development of a gas FIA system for on-line monitoring of ethanol. J. Biotechnol. 14, 127-140. Larsen, B.R. and West, F.G. (1981) A method of quantitative amino acid analysis using pre-column o-phthaldialdehyde derivatisation and high performance liquid chromatography. J. Chromatogr. Sci. 19, 259-265. Lehmann, J., Vorlop, J. and Biintemeyer, H. (1988) Bubble-free reactors and their development for continuous culture with cell recycle. In: Spier, R.E. and Griffiths, J.B., (Eds.), Animal Cell Biotechnology Vol. 3, Academic Press, pp. 220-237. Lucki-Lange, M. and Wagner, R. (1990) Conditions for the production of recombinant IL-2 in stirred suspension culture using a protein-free medium. In: Spier, R.E., Griffiths, J.B. and Meigner, B. (Eds.), Production of Biologicals from Animal Cells in Culture. Butterworth-Heinemann, Oxford, pp. 180-185. Liidi, H., Garn, M.S., Bataillard, P. and Widmer, H.M. (1990) Flow injection analysis and biosensors: applications for biotechnology and environmental control. J. Biotechnol. 14, 71-79. Merten, O.W. (1988) Sensors for the Control of Mammalian Cell Processes. In: Spier, R.E. and Griffiths, J.B., (Eds.), Animal Cell Biotechnology, Academic Press Vol. 3, pp. 74-139.

185 Nentwig, J., Scheller, F., Weise, H., Heinrich, G., Kirstein, D., Becker, M., Hinmann, P. and Pfeiffer, D. (1985) Laminierte Membran und Verfahren zu ihrer Herstellung. German Patent DD 279587. Renneberg, R., Trott-Kriegeskorte, G., Pawlowa, M., Schubert, F., Hammer, J., Jiiger, V., Wagner, R., Schmid., R.D. and Scheller, F (1989) Enzyme sensors for biotechnological processes and products. GBF-Monographs Vol. 13, pp. 59-66. Romette, J.L. (1987) Mammalian cell culture process control: sampling and sensing. BGF-Monographs, Vol. I0, pp. 81-86. Romette, J.L. and Cooney, C.L. (1987) L-glutamine electrode for on-line mammalian cell culture process control. Anal. Lett. 20, 1069-1081. Ruzicka, J. and Hansen, E.H. (1988) Flow injection analysis, 2nd edit., John Wiley and Sons, New York. Ryll, T., Lucki-Lange, M., J~iger, V. and Wagner, R. (1990) Production of recombinant human interleukin 2 with BHK cells in a hollow fibre and a stirred tank reactor with protein-free medium. J. Biotechnol. 14, 377-392. Schmid, R.D. and Kiinnecke, W. (1990) Flow injection analysis (FIA) based on enzymes or antibodiesapplication in the life sciences, J. Biotechnol. 14, 3-31. Schubert, F., Scheller, F., Hanke, G., Hauptmann, B. and KiJhnel, S. (1988) Verfahren zur Schnellbestimmung von Lactat in VoUblut. German Patent DD 3149367. Trott-Kriegeskorte, G., Renneberg, R., Pawlowa, M., Schubert, F., Hammer, J., J~iger, V., Wagner, R., Schmid, R.D. and Scheller, F.W. (1989) Enzymsensoren fiir die Prozesskontrolle von Zellkulturen. GBF-Monographs Vol. 13, pp. 67-70.